Soil Ecology Wiki - User contributions [en]

Stinkwort (Dittrichia graveolens)

2021-05-07T17:29:51Z

Mrhonan:

[[File:Screen Shot 2021-05-03 at 9.37.50 PM.png|thumb||right| Stinkwort (''Dittrichia graveolens'') [1]]]

==Taxonomy==

'''Domain:''' Eukaryota
'''Kingdom:''' Planteae
'''Phylum:''' Tracheophyta
'''Subphylum:''' [[Angiosperms]]
'''Class:''' Magnoliopsida
'''Order:''' Asterales
'''Family:''' Asterceae
'''Subfamily:''' Asteroideae
'''Genus:''' ''Dittrichia''
'''Species:''' ''D. graveolens''

==Description==
''Dittrichia graveolens'' or stinkwort belongs to the Asteraceae family which consist of flowering plants [1]. Stinkwort is an invasive species that has found its way into many areas. It is also considered a noxious weed since it has the potential to harm horticultural crops, natural habitats, ecosytems, humans, and livestock [1,2,4,5]. Annually flowering in the fall stinkwort is erect growing up to 2.5 feet [1,2,3,4,5]. Sticky glandular hairs of the stinkwort give off strong aromatic odor that is easily recognizable [1,2,4]. Stinkwort has flowers that have short yellow rays on the outer edge and a yellow to reddish disk flowers in the center [1,2,4] Stinkwort which is native to the Mediterranean is rapidly invading other areas. It has found its way many other areas including Central Europe, Australia, South Africa, and the United States [1].

[[File:stinkwort PM.png|thumb||left| a. Stinkwort b. zoomed in stinkwort c. flower head d. habitat along a road [3]]]

===Habitat===
Stinkwort can be found in a variety of places in its native area. It is known to be prevalent in riparian woodlands, margins of tidal marshes, [[Vernal Pools|vernal pools]], and alluvial floodplains [1]. However, in areas where stinkwort is not native it grows in disturbed areas such as overgrazed rangelands, roadsides, pastures, wastelands, vineyard edges, gravel mines, levees, washes, and mining sites [1,2].

===Seed Dispersal===
There a few different ways in which stinkwort seeds are dispersed. The fine hairs of the seeds allow for wind dispersal [3]. It also sticks to clothing, wool, hair, and machinery [3]. Stinkwort seeds have high viability. About 90% of the seeds are capable of germination at the time of dispersal [1].

===Negative Impacts===
Stinkwort is not a palatable species so it can cause problems within livestock. If consumed by livestock stinkwort can poison them leading to mortality in some cases [1,4]. The fine hairs of the seed induce pulpy kidney or a fatal bacterium if grazed by livestock [4]. It does not only cause problems in [[animals]], but also humans. When stinkwort is flowered if it is handled by bare skin, it can cause severe dermatitis [4,5].

===Control===
There are several different ways in which stinkwort can be controlled. For smaller areas of invasions mechanical practices can be used. This involves pulling, hoeing, and mowing [1,4]. When hand pulling and hoeing gloves should be worn to avoid dermatitis. Mowing is only a partial control. Mowing should be done late in the season and multiple times [1]. The buds remaining may grow back however which is why this should be conducted more than once [1]. For larger areas herbicides can be used. There is either post or preemergence herbicides. Postemergence herbicides are applied to young plants and target visibly infested areas [1]. Preemergence herbicides are used for larger areas before seeds germinate [1].

===References===
[1] Brownsey, R., Kyser, G.B., DiTomaso, J.M., 2013. Stinkwort is rapidly expanding its range in California. Cal Ag 67, 110–115. https://doi.org/10.3733/ca.v067n02p110

[2] Brownsey, R.N., Kyser, G.B., DiTomaso, J.M., 2014. Growth and phenology of Dittrichia graveolens, a rapidly spreading invasive plant in California. Biol Invasions 16, 43–52. https://doi.org/10.1007/s10530-013-0501-4

[3] Kocián, P., 2015. Dittrichia graveolens (L.) Greuter – a new alien species in Poland. Acta Musei Silesiae, Scientiae Naturales 64, 193–197. https://doi.org/10.1515/cszma-2015-0027

[4] Stinkwort Guide [WWW Document], n.d. . HerbiGuide. URL http://www.herbiguide.com.au/Descriptions/hg_Stinkwort.htm

[5] Thong, H.-Y., Yokota, M., Kardassakis, D., Maibach, H.I., 2007. Allergic contact dermatitis from Dittrichia graveolens (L.) Greuter (stinkwort). Contact Dermatitis 58, 51–53. https://doi.org/10.1111/j.1600-0536.2007.01154.x

Measuring Microbial Communities' Biomass

2021-05-07T17:25:04Z

Mrhonan: /* Indirect */

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
[[File:AgarPM.png|thumb|right|Top view of soil microorganisms nutrient agar in plate [14]]]

Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]]]

The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [9].

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,8].

===Substrate-Induced-Respiration (SIR)===
The SIR technique involves adding a substrate such as glucose to stimulate respiration [1,4]. Glucose os often the most used substrate due to [[microorganisms]] being able to readily utilize it as a carbon source [4]. Depending on the soils physical and chemical [[properties]] the amount of glucose varies [4] Once the substrate is added respiration rapidly increases and remains constant for several hours [4]. The initial maximum respiration is proportional to the size of the original soil microbial biomass [4]. SIR technique is useful for the "measurement of the contribution of bacterial and fungal biomass to substrate-induced CO2 respiration through coupling with antibiotics" [4]. This has been deemed successful is arable grasslands, [[rhizosphere]]-rhizoplane soils, and in plant residue [4].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

[14] N.d. . Stock Photo- Top view soil microorganisms Nutrient agar in plate on black background. URL https://www.123rf.com/photo_122899146_top-view-soil-microorganisms-nutrient-agar-in-plate-on-black-background-.html

Measuring Microbial Communities' Biomass

2021-05-07T17:24:04Z

Mrhonan: /* Agar Plates */

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
[[File:AgarPM.png|thumb|right|Top view of soil microorganisms nutrient agar in plate [14]]]

Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [9].

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]]]

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,8].

===Substrate-Induced-Respiration (SIR)===
The SIR technique involves adding a substrate such as glucose to stimulate respiration [1,4]. Glucose os often the most used substrate due to [[microorganisms]] being able to readily utilize it as a carbon source [4]. Depending on the soils physical and chemical [[properties]] the amount of glucose varies [4] Once the substrate is added respiration rapidly increases and remains constant for several hours [4]. The initial maximum respiration is proportional to the size of the original soil microbial biomass [4]. SIR technique is useful for the "measurement of the contribution of bacterial and fungal biomass to substrate-induced CO2 respiration through coupling with antibiotics" [4]. This has been deemed successful is arable grasslands, [[rhizosphere]]-rhizoplane soils, and in plant residue [4].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

[14] N.d. . Stock Photo- Top view soil microorganisms Nutrient agar in plate on black background. URL https://www.123rf.com/photo_122899146_top-view-soil-microorganisms-nutrient-agar-in-plate-on-black-background-.html

Measuring Microbial Communities' Biomass

2021-05-07T17:22:58Z

Mrhonan: /* References */

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
[[File:AgarPM.png|thumb|right|Top view soil microorganisms nutrient agar in plate [14]]]

Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [9].

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]]]

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,8].

===Substrate-Induced-Respiration (SIR)===
The SIR technique involves adding a substrate such as glucose to stimulate respiration [1,4]. Glucose os often the most used substrate due to [[microorganisms]] being able to readily utilize it as a carbon source [4]. Depending on the soils physical and chemical [[properties]] the amount of glucose varies [4] Once the substrate is added respiration rapidly increases and remains constant for several hours [4]. The initial maximum respiration is proportional to the size of the original soil microbial biomass [4]. SIR technique is useful for the "measurement of the contribution of bacterial and fungal biomass to substrate-induced CO2 respiration through coupling with antibiotics" [4]. This has been deemed successful is arable grasslands, [[rhizosphere]]-rhizoplane soils, and in plant residue [4].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

[14] N.d. . Stock Photo- Top view soil microorganisms Nutrient agar in plate on black background. URL https://www.123rf.com/photo_122899146_top-view-soil-microorganisms-nutrient-agar-in-plate-on-black-background-.html

Measuring Microbial Communities' Biomass

2021-05-07T17:22:40Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
[[File:AgarPM.png|thumb|right|Top view soil microorganisms nutrient agar in plate [14]]]

Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [9].

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]]]

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,8].

===Substrate-Induced-Respiration (SIR)===
The SIR technique involves adding a substrate such as glucose to stimulate respiration [1,4]. Glucose os often the most used substrate due to [[microorganisms]] being able to readily utilize it as a carbon source [4]. Depending on the soils physical and chemical [[properties]] the amount of glucose varies [4] Once the substrate is added respiration rapidly increases and remains constant for several hours [4]. The initial maximum respiration is proportional to the size of the original soil microbial biomass [4]. SIR technique is useful for the "measurement of the contribution of bacterial and fungal biomass to substrate-induced CO2 respiration through coupling with antibiotics" [4]. This has been deemed successful is arable grasslands, [[rhizosphere]]-rhizoplane soils, and in plant residue [4].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

[14] N.d. . Stock Photo- Top view soil microorganisms Nutrient agar in plate on black background. URL https://www.123rf.com/photo_122899146_top-view-soil-microorganisms-nutrient-agar-in-plate-on-black-background-.html

Measuring Microbial Communities' Biomass

2021-05-07T17:21:00Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
[[File:AgarPM.png|thumb|right|Top view soil microorganisms nutrient agar in plate [14]]]

Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [9].

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]]]

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,8].

===Substrate-Induced-Respiration (SIR)===
The SIR technique involves adding a substrate such as glucose to stimulate respiration [1,4]. Glucose os often the most used substrate due to [[microorganisms]] being able to readily utilize it as a carbon source [4]. Depending on the soils physical and chemical [[properties]] the amount of glucose varies [4] Once the substrate is added respiration rapidly increases and remains constant for several hours [4]. The initial maximum respiration is proportional to the size of the original soil microbial biomass [4]. SIR technique is useful for the "measurement of the contribution of bacterial and fungal biomass to substrate-induced CO2 respiration through coupling with antibiotics" [4]. This has been deemed successful is arable grasslands, [[rhizosphere]]-rhizoplane soils, and in plant residue [4].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

Measuring Microbial Communities' Biomass

2021-05-07T17:19:28Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].

[[File:AgarPM.png|thumb|right|Top view soil microorganisms nutrient agar in plate [14]]]

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [9].

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]]]

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,8].

===Substrate-Induced-Respiration (SIR)===
The SIR technique involves adding a substrate such as glucose to stimulate respiration [1,4]. Glucose os often the most used substrate due to [[microorganisms]] being able to readily utilize it as a carbon source [4]. Depending on the soils physical and chemical [[properties]] the amount of glucose varies [4] Once the substrate is added respiration rapidly increases and remains constant for several hours [4]. The initial maximum respiration is proportional to the size of the original soil microbial biomass [4]. SIR technique is useful for the "measurement of the contribution of bacterial and fungal biomass to substrate-induced CO2 respiration through coupling with antibiotics" [4]. This has been deemed successful is arable grasslands, [[rhizosphere]]-rhizoplane soils, and in plant residue [4].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

File:AgarPM.png

2021-05-07T17:17:42Z

Mrhonan:

Measuring Microbial Communities' Biomass

2021-05-07T17:05:57Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [9].

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]]]

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,8].

===Substrate-Induced-Respiration (SIR)===
The SIR technique involves adding a substrate such as glucose to stimulate respiration [1,4]. Glucose os often the most used substrate due to [[microorganisms]] being able to readily utilize it as a carbon source [4]. Depending on the soils physical and chemical [[properties]] the amount of glucose varies [4] Once the substrate is added respiration rapidly increases and remains constant for several hours [4]. The initial maximum respiration is proportional to the size of the original soil microbial biomass [4]. SIR technique is useful for the "measurement of the contribution of bacterial and fungal biomass to substrate-induced CO2 respiration through coupling with antibiotics" [4]. This has been deemed successful is arable grasslands, [[rhizosphere]]-rhizoplane soils, and in plant residue [4].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

Measuring Microbial Communities' Biomass

2021-05-07T17:05:02Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [9].

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]]]

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,8].

===Substrate-Induced-Respiration (SIR)===
The SIR technique involves adding a substrate such as glucose to stimulate respiration [1,4]. Glucose os often the most used substrate due to [[microorganisms]] being able to readily utilize it as a carbon source [4]. Depending on the soils physical and chemical [[properties]] the amount of glucose varies [4] Once the substrate is added respiration rapidly increases and remains constant for several hours [4]. The initial maximum respiration is proportional to the size of the original soil microbial biomass [4]. SIR technique is useful for the "measurement of the contribution of bacterial and fungal biomass to substrate-induced CO>sub<2>/sub< respiration through coupling with antibiotics" [4]. This has been deemed successful is arable grasslands, [[rhizosphere]]-rhizoplane soils, and in plant residue [4].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

Measuring Microbial Communities' Biomass

2021-05-07T16:40:17Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,5]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,5]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,5]. Once dried the sample can then be put under a microscope to count organisms [3,5].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,13]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This allows for the extraction of naked DNA adsorbs to colloids [7]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [7]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [7]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [7]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [6]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [7]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [7]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [7]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [7].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,10,12]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,10,12]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [11,12]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [12]. This provides evidence of viable and non-viable microbes [12].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [9]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [9].

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[6]]]

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,8]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,8]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,8].

===Substrate-Induced-Respiration (SIR)===
The SIR technique involves adding a substrate

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4] Lin, Q., Brookes, P.C., 1999. An evaluation of the substrate-induced respiration method. Soil Biology and Biochemistry 31, 1969–1983. https://doi.org/10.1016/S0038-0717(99)00120-0

[5]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[6] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[7] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[8] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[9] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[10] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[11] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[12] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[13] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

Stinkwort (Dittrichia graveolens)

2021-05-07T16:06:51Z

Mrhonan: Undo revision 7154 by Mrhonan (talk)

==Taxonomy==

[[File:Screen Shot 2021-05-03 at 9.37.50 PM.png|thumb||right| Stinkwort (''Dittrichia graveolens'') [1]]]

'''Domain:''' Eukaryota
'''Kingdom:''' Planteae
'''Phylum:''' Tracheophyta
'''Subphylum:''' [[Angiosperms]]
'''Class:''' Magnoliopsida
'''Order:''' Asterales
'''Family:''' Asterceae
'''Subfamily:''' Asteroideae
'''Genus:''' ''Dittrichia''
'''Species:''' ''D. graveolens''

==Description==
''Dittrichia graveolens'' or stinkwort belongs to the Asteraceae family which consist of flowering plants [1]. Stinkwort is an invasive species that has found its way into many areas. It is also considered a noxious weed since it has the potential to harm horticultural crops, natural habitats, ecosytems, humans, and livestock [1,2,4,5]. Annually flowering in the fall stinkwort is erect growing up to 2.5 feet [1,2,3,4,5]. Sticky glandular hairs of the stinkwort give off strong aromatic odor that is easily recognizable [1,2,4]. Stinkwort has flowers that have short yellow rays on the outer edge and a yellow to reddish disk flowers in the center [1,2,4] Stinkwort which is native to the Mediterranean is rapidly invading other areas. It has found its way many other areas including Central Europe, Australia, South Africa, and the United States [1].

[[File:stinkwort PM.png|thumb||left| a. Stinkwort b. zoomed in stinkwort c. flower head d. habitat along a road [3]]]

===Habitat===
Stinkwort can be found in a variety of places in its native area. It is known to be prevalent in riparian woodlands, margins of tidal marshes, [[Vernal Pools|vernal pools]], and alluvial floodplains [1]. However, in areas where stinkwort is not native it grows in disturbed areas such as overgrazed rangelands, roadsides, pastures, wastelands, vineyard edges, gravel mines, levees, washes, and mining sites [1,2].

===Seed Dispersal===
There a few different ways in which stinkwort seeds are dispersed. The fine hairs of the seeds allow for wind dispersal [3]. It also sticks to clothing, wool, hair, and machinery [3]. Stinkwort seeds have high viability. About 90% of the seeds are capable of germination at the time of dispersal [1].

===Negative Impacts===
Stinkwort is not a palatable species so it can cause problems within livestock. If consumed by livestock stinkwort can poison them leading to mortality in some cases [1,4]. The fine hairs of the seed induce pulpy kidney or a fatal bacterium if grazed by livestock [4]. It does not only cause problems in [[animals]], but also humans. When stinkwort is flowered if it is handled by bare skin, it can cause severe dermatitis [4,5].

===Control===
There are several different ways in which stinkwort can be controlled. For smaller areas of invasions mechanical practices can be used. This involves pulling, hoeing, and mowing [1,4]. When hand pulling and hoeing gloves should be worn to avoid dermatitis. Mowing is only a partial control. Mowing should be done late in the season and multiple times [1]. The buds remaining may grow back however which is why this should be conducted more than once [1]. For larger areas herbicides can be used. There is either post or preemergence herbicides. Postemergence herbicides are applied to young plants and target visibly infested areas [1]. Preemergence herbicides are used for larger areas before seeds germinate [1].

===References===
[1] Brownsey, R., Kyser, G.B., DiTomaso, J.M., 2013. Stinkwort is rapidly expanding its range in California. Cal Ag 67, 110–115. https://doi.org/10.3733/ca.v067n02p110

[2] Brownsey, R.N., Kyser, G.B., DiTomaso, J.M., 2014. Growth and phenology of Dittrichia graveolens, a rapidly spreading invasive plant in California. Biol Invasions 16, 43–52. https://doi.org/10.1007/s10530-013-0501-4

[3] Kocián, P., 2015. Dittrichia graveolens (L.) Greuter – a new alien species in Poland. Acta Musei Silesiae, Scientiae Naturales 64, 193–197. https://doi.org/10.1515/cszma-2015-0027

[4] Stinkwort Guide [WWW Document], n.d. . HerbiGuide. URL http://www.herbiguide.com.au/Descriptions/hg_Stinkwort.htm

[5] Thong, H.-Y., Yokota, M., Kardassakis, D., Maibach, H.I., 2007. Allergic contact dermatitis from Dittrichia graveolens (L.) Greuter (stinkwort). Contact Dermatitis 58, 51–53. https://doi.org/10.1111/j.1600-0536.2007.01154.x

Stinkwort (Dittrichia graveolens)

2021-05-07T16:06:20Z

Mrhonan: Undo revision 7155 by Mrhonan (talk)

==Taxonomy==

[[File:Screen Shot 2021-05-03 at 9.37.50 PM.png|thumb||right| Stinkwort (''Dittrichia graveolens'') [1]]]

{{Taxonomy
| common_name = Stinkwort
| kingdom = Planteae
| phylum = Tracheophyte
| subphylum = Angiosperms
| class = Magnoliopsida
| order = Asterceae
| family = Asterceae
| genus = Dittrichia
| species = D. Graveolens
}}

==Description==
''Dittrichia graveolens'' or stinkwort belongs to the Asteraceae family which consist of flowering plants [1]. Stinkwort is an invasive species that has found its way into many areas. It is also considered a noxious weed since it has the potential to harm horticultural crops, natural habitats, ecosytems, humans, and livestock [1,2,4,5]. Annually flowering in the fall stinkwort is erect growing up to 2.5 feet [1,2,3,4,5]. Sticky glandular hairs of the stinkwort give off strong aromatic odor that is easily recognizable [1,2,4]. Stinkwort has flowers that have short yellow rays on the outer edge and a yellow to reddish disk flowers in the center [1,2,4] Stinkwort which is native to the Mediterranean is rapidly invading other areas. It has found its way many other areas including Central Europe, Australia, South Africa, and the United States [1].

[[File:stinkwort PM.png|thumb||left| a. Stinkwort b. zoomed in stinkwort c. flower head d. habitat along a road [3]]]

===Habitat===
Stinkwort can be found in a variety of places in its native area. It is known to be prevalent in riparian woodlands, margins of tidal marshes, [[Vernal Pools|vernal pools]], and alluvial floodplains [1]. However, in areas where stinkwort is not native it grows in disturbed areas such as overgrazed rangelands, roadsides, pastures, wastelands, vineyard edges, gravel mines, levees, washes, and mining sites [1,2].

===Seed Dispersal===
There a few different ways in which stinkwort seeds are dispersed. The fine hairs of the seeds allow for wind dispersal [3]. It also sticks to clothing, wool, hair, and machinery [3]. Stinkwort seeds have high viability. About 90% of the seeds are capable of germination at the time of dispersal [1].

===Negative Impacts===
Stinkwort is not a palatable species so it can cause problems within livestock. If consumed by livestock stinkwort can poison them leading to mortality in some cases [1,4]. The fine hairs of the seed induce pulpy kidney or a fatal bacterium if grazed by livestock [4]. It does not only cause problems in [[animals]], but also humans. When stinkwort is flowered if it is handled by bare skin, it can cause severe dermatitis [4,5].

===Control===
There are several different ways in which stinkwort can be controlled. For smaller areas of invasions mechanical practices can be used. This involves pulling, hoeing, and mowing [1,4]. When hand pulling and hoeing gloves should be worn to avoid dermatitis. Mowing is only a partial control. Mowing should be done late in the season and multiple times [1]. The buds remaining may grow back however which is why this should be conducted more than once [1]. For larger areas herbicides can be used. There is either post or preemergence herbicides. Postemergence herbicides are applied to young plants and target visibly infested areas [1]. Preemergence herbicides are used for larger areas before seeds germinate [1].

===References===
[1] Brownsey, R., Kyser, G.B., DiTomaso, J.M., 2013. Stinkwort is rapidly expanding its range in California. Cal Ag 67, 110–115. https://doi.org/10.3733/ca.v067n02p110

[2] Brownsey, R.N., Kyser, G.B., DiTomaso, J.M., 2014. Growth and phenology of Dittrichia graveolens, a rapidly spreading invasive plant in California. Biol Invasions 16, 43–52. https://doi.org/10.1007/s10530-013-0501-4

[3] Kocián, P., 2015. Dittrichia graveolens (L.) Greuter – a new alien species in Poland. Acta Musei Silesiae, Scientiae Naturales 64, 193–197. https://doi.org/10.1515/cszma-2015-0027

[4] Stinkwort Guide [WWW Document], n.d. . HerbiGuide. URL http://www.herbiguide.com.au/Descriptions/hg_Stinkwort.htm

[5] Thong, H.-Y., Yokota, M., Kardassakis, D., Maibach, H.I., 2007. Allergic contact dermatitis from Dittrichia graveolens (L.) Greuter (stinkwort). Contact Dermatitis 58, 51–53. https://doi.org/10.1111/j.1600-0536.2007.01154.x

Stinkwort (Dittrichia graveolens)

2021-05-07T16:05:56Z

Mrhonan: Undo revision 7157 by Mrhonan (talk)

Stinkwort (Dittrichia graveolens)

2021-05-07T16:05:28Z

Mrhonan: Undo revision 7158 by Mrhonan (talk)

Stinkwort (Dittrichia graveolens)

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Mrhonan: Undo revision 7159 by Mrhonan (talk)

Stinkwort (Dittrichia graveolens)

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Mrhonan: Undo revision 7160 by Mrhonan (talk)

Stinkwort (Dittrichia graveolens)

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Mrhonan: Undo revision 7161 by Mrhonan (talk)

Stinkwort (Dittrichia graveolens)

2021-05-07T16:04:14Z

Mrhonan:

==Taxonomy==

[[File:Screen Shot 2021-05-03 at 9.37.50 PM.png|thumb||right| Stinkwort (''Dittrichia graveolens'') [1]]]

{{Taxonomy
| common_name = stinkwort
| kingdom = planteae
| phylum = tracheophyte
| subphylum = angiosperm
| class = Mangoliopsida
| order = asterales
| family = asteraceae
| genus = dittrichia
| species = D. graveolens
}}

==Description==
''Dittrichia graveolens'' or stinkwort belongs to the Asteraceae family which consist of flowering plants [1]. Stinkwort is an invasive species that has found its way into many areas. It is also considered a noxious weed since it has the potential to harm horticultural crops, natural habitats, ecosytems, humans, and livestock [1,2,4,5]. Annually flowering in the fall stinkwort is erect growing up to 2.5 feet [1,2,3,4,5]. Sticky glandular hairs of the stinkwort give off strong aromatic odor that is easily recognizable [1,2,4]. Stinkwort has flowers that have short yellow rays on the outer edge and a yellow to reddish disk flowers in the center [1,2,4] Stinkwort which is native to the Mediterranean is rapidly invading other areas. It has found its way many other areas including Central Europe, Australia, South Africa, and the United States [1].

[[File:stinkwort PM.png|thumb||left| a. Stinkwort b. zoomed in stinkwort c. flower head d. habitat along a road [3]]]

===Habitat===
Stinkwort can be found in a variety of places in its native area. It is known to be prevalent in riparian woodlands, margins of tidal marshes, [[Vernal Pools|vernal pools]], and alluvial floodplains [1]. However, in areas where stinkwort is not native it grows in disturbed areas such as overgrazed rangelands, roadsides, pastures, wastelands, vineyard edges, gravel mines, levees, washes, and mining sites [1,2].

===Seed Dispersal===
There a few different ways in which stinkwort seeds are dispersed. The fine hairs of the seeds allow for wind dispersal [3]. It also sticks to clothing, wool, hair, and machinery [3]. Stinkwort seeds have high viability. About 90% of the seeds are capable of germination at the time of dispersal [1].

===Negative Impacts===
Stinkwort is not a palatable species so it can cause problems within livestock. If consumed by livestock stinkwort can poison them leading to mortality in some cases [1,4]. The fine hairs of the seed induce pulpy kidney or a fatal bacterium if grazed by livestock [4]. It does not only cause problems in [[animals]], but also humans. When stinkwort is flowered if it is handled by bare skin, it can cause severe dermatitis [4,5].

===Control===
There are several different ways in which stinkwort can be controlled. For smaller areas of invasions mechanical practices can be used. This involves pulling, hoeing, and mowing [1,4]. When hand pulling and hoeing gloves should be worn to avoid dermatitis. Mowing is only a partial control. Mowing should be done late in the season and multiple times [1]. The buds remaining may grow back however which is why this should be conducted more than once [1]. For larger areas herbicides can be used. There is either post or preemergence herbicides. Postemergence herbicides are applied to young plants and target visibly infested areas [1]. Preemergence herbicides are used for larger areas before seeds germinate [1].

===References===
[1] Brownsey, R., Kyser, G.B., DiTomaso, J.M., 2013. Stinkwort is rapidly expanding its range in California. Cal Ag 67, 110–115. https://doi.org/10.3733/ca.v067n02p110

[2] Brownsey, R.N., Kyser, G.B., DiTomaso, J.M., 2014. Growth and phenology of Dittrichia graveolens, a rapidly spreading invasive plant in California. Biol Invasions 16, 43–52. https://doi.org/10.1007/s10530-013-0501-4

[3] Kocián, P., 2015. Dittrichia graveolens (L.) Greuter – a new alien species in Poland. Acta Musei Silesiae, Scientiae Naturales 64, 193–197. https://doi.org/10.1515/cszma-2015-0027

[4] Stinkwort Guide [WWW Document], n.d. . HerbiGuide. URL http://www.herbiguide.com.au/Descriptions/hg_Stinkwort.htm

[5] Thong, H.-Y., Yokota, M., Kardassakis, D., Maibach, H.I., 2007. Allergic contact dermatitis from Dittrichia graveolens (L.) Greuter (stinkwort). Contact Dermatitis 58, 51–53. https://doi.org/10.1111/j.1600-0536.2007.01154.x

Stinkwort (Dittrichia graveolens)

2021-05-07T16:00:09Z

Mrhonan:

Stinkwort (Dittrichia graveolens)

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Mrhonan:

Stinkwort (Dittrichia graveolens)

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Mrhonan: /* Taxonomy */

Stinkwort (Dittrichia graveolens)

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Mrhonan:

Stinkwort (Dittrichia graveolens)

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Mrhonan:

Stinkwort (Dittrichia graveolens)

2021-05-07T15:58:04Z

Mrhonan: /* Taxonomy */

Polymerase Chain Reaction (PCR)

2021-05-07T15:39:02Z

Mrhonan: /* Primers */

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for the creation of PCR along with Michael Smith for his work on PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning and end, where the DNA-Polymerase binds and starts synthesis of new DNA strand [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:PCR cyclePM.png|thumb|left|Multiple completed cycles of PCR [7]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. It allows for the understanding of gene expression either spatially or temporally among [[organisms]] [1]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1]. Bacterial cultures do not work well for slow-growing [[microorganisms]] such as mycobacteria and anaerobic bacteria [5]. PCR is able to be done rapidly and effectively making it a common practice across the science community. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Today PCR is widely known for detecting pathogens such as Covid-19.

==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Measuring Microbial Communities' Biomass

2021-05-05T19:57:06Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,4]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,4]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,4]. The soil suspension is then made up to 50ml with 1.5% being agar [3,4]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,4]. Once dried the sample can then be put under a microscope to count organisms [3,4].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,12]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [6]. This allows for the extraction of naked DNA adsorbs to colloids [6]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [6]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [6]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [6]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [5]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [6]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [6]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [6]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [6].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,9,11]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,9,11]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [10,11]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [11]. This provides evidence of viable and non-viable microbes [11].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,8]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [8]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,8]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [8].

[[File:CFEPM.png|thumb|right|1. Soil samples are exposed to chloroform fumigation and extraction. (a).Biomass is assumed to be extracted with equal and complete efficiency 2. A fraction of the soil samples are are incubated. 3. New DNA is present from incubation 4. Relationship between DNA and microbial biomass carbon content of the community.[5]]]

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,7]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,7]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,7].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[5] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[6] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[7] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[8] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[9] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[10] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[11] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[12] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

Measuring Microbial Communities' Biomass

2021-05-05T19:47:51Z

Mrhonan: Undo revision 6952 by Mrhonan (talk)

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,4]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,4]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,4]. The soil suspension is then made up to 50ml with 1.5% being agar [3,4]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,4]. Once dried the sample can then be put under a microscope to count organisms [3,4].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,12]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [6]. This allows for the extraction of naked DNA adsorbs to colloids [6]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [6]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [6]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [6]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [5]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [6]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [6]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [6]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [6].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,9,11]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,9,11]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [10,11]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [11]. This provides evidence of viable and non-viable microbes [11].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,8]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [8]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,8]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [8].

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,7]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,7]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,7].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[5] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[6] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[7] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[8] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[9] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[10] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[11] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[12] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

Measuring Microbial Communities' Biomass

2021-05-05T19:45:55Z

Mrhonan: Undo revision 6941 by Mrhonan (talk)

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,4]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,4]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,4]. The soil suspension is then made up to 50ml with 1.5% being agar [3,4]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,4]. Once dried the sample can then be put under a microscope to count organisms [3,4].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,11]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [5]. This allows for the extraction of naked DNA adsorbs to colloids [5]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [5]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [5]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [5]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [5]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [5]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [5]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [5]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [5].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,8,10]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,8,10]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [9,10]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [10]. This provides evidence of viable and non-viable microbes [10].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,7]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [7]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,7]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [7].

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,6]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,6]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,6].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[5] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[6] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[7] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[8] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[9] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[10] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[11] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

Measuring Microbial Communities' Biomass

2021-05-05T19:37:54Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,4]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,4]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,4]. The soil suspension is then made up to 50ml with 1.5% being agar [3,4]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,4]. Once dried the sample can then be put under a microscope to count organisms [3,4].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,12]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [6]. This allows for the extraction of naked DNA adsorbs to colloids [6]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [6]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [6]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [6]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [5]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [6]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [6]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [6]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [6].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,9,11]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,9,11]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [10,11]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [11]. This provides evidence of viable and non-viable microbes [11].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,8]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [8]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,8]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [8].

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,7]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,7]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,7].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[5] Pold, G., Domeignoz-Horta, L.A., DeAngelis, K.M., 2019. Heavy and wet: evaluating the validity and implications of assumptions made when measuring growth efficiency using 18 O water (preprint). Microbiology. https://doi.org/10.1101/601138

[6] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[7] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[8] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[9] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[10] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[11] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[12] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

Measuring Microbial Communities' Biomass

2021-05-05T19:33:20Z

Mrhonan: /* Chloroform Fumigation and Extraction (CFE) */

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,4]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,4]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,4]. The soil suspension is then made up to 50ml with 1.5% being agar [3,4]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,4]. Once dried the sample can then be put under a microscope to count organisms [3,4].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,11]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [5]. This allows for the extraction of naked DNA adsorbs to colloids [5]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [5]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [5]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [5]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [5]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [5]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [5]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [5]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [5].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,8,10]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,8,10]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [9,10]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [10]. This provides evidence of viable and non-viable microbes [10].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,7]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [7]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,7]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [7].

===Chloroform Fumigation and Extraction (CFE)===
When [[soil pH]] reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,6]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,6]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,6].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[5] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[6] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[7] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[8] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[9] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[10] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[11] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

File:CFEPM.png

2021-05-05T19:31:02Z

Mrhonan:

Polymerase Chain Reaction (PCR)

2021-05-05T19:12:39Z

Mrhonan: /* Uses */

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for the creation of PCR along with Michael Smith for his work on PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:PCR cyclePM.png|thumb|left|Multiple completed cycles of PCR [7]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. It allows for the understanding of gene expression either spatially or temporally among [[organisms]] [1]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1]. Bacterial cultures do not work well for slow-growing [[microorganisms]] such as mycobacteria and anaerobic bacteria [5]. PCR is able to be done rapidly and effectively making it a common practice across the science community. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Today PCR is widely known for detecting pathogens such as Covid-19.

==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T19:10:26Z

Mrhonan: /* Uses */

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for the creation of PCR along with Michael Smith for his work on PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:PCR cyclePM.png|thumb|left|Multiple completed cycles of PCR [7]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. It allows for the understanding of gene expression either spatially or temporally among [[organisms]] [1]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1]. Bacterial cultures do not work well for slow-growing [[microorganisms]] such as mycobacteria and anaerobic bacteria [5] Pathogens are among samples that are able to be seen using PCR [1,6,9]. Today PCR is widely known for detecting pathogens such as Covid-19.

==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T19:04:15Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for the creation of PCR along with Michael Smith for his work on PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:PCR cyclePM.png|thumb|left|Multiple completed cycles of PCR [7]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. It allows for the understanding of gene expression either spatially or temporally among [[organisms]] [1]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1]. Today PCR is widely known for detecting pathogens such as Covid-19.

==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T18:51:49Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:PCR cyclePM.png|thumb|left|Multiple completed cycles of PCR [7]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].

==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T18:51:22Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:PCR cyclePM.png|thumb|leftt|Multiple completed cycles of PCR [7]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].

==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T18:50:59Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:PCR cyclePM.png.png|thumb|leftt|Multiple completed cycles of PCR [7]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].

==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T18:49:21Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:PCR cylce.png|thumb|leftt|Multiple completed cycles of PCR [7]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].

==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

File:PCR cyclePM.png

2021-05-05T18:46:19Z

Mrhonan:

Polymerase Chain Reaction (PCR)

2021-05-05T18:42:31Z

Mrhonan: /* Uses */

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. Pathogens are among samples that are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].

==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T18:41:00Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows a closer look at DNA [8]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. used this method Pathogens among samples are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].
==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T18:38:57Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows scientists to get a closer look at DNA [8]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in [[soil]], [[sand]], and sediment [1]. used this method Pathogens among samples are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].
==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T18:37:46Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6].

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Uses==
PCR is helpful as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows scientists to get a closer look at DNA [8]. Bruce et al. (1992) used this method to study DNA sequences of native bacterial populations in soil, [[sand]], and sediment [1]. used this method Pathogens among samples are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].
==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T18:27:24Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6]

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Uses==
PCR is helpful for scientist as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows scientists to get a closer look at DNA [8]. Pathogens among samples are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].
==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T18:25:37Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC to separate the strands which are held together by hydrogen bonds. This is often done for up to 5 minutes to ensure that both the template and the primer have completely seperated and are single strand only. Taq polymerase is also activated during this stage [5,6].

'''Annealing Stage:''' During the annealing stage the temperature is lowered to about 50oC/ below the specific primers melting temperature so the primers can attach themselves to themselves to single DNA strands to create double-stranded DNA. The temperature in this stage is critical as the wrong temperature can result in primers not binding to template DNA at all or binding at random [5,6]

'''Extension Stage:''' Following the annealing stage comes extension. During this stage DNA polymerase (an enzyme) catalyzes the the synthesis of the new strands of DNA. With the annealed primer DNA polymerase adds complimentary nucleotides complimentary to the unpaired DNA strand [5,6]

These steps are then repeated 20-40 to potentially create millions or billions amount of copied DNA of the target sequence.

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Uses==
PCR is helpful for scientist as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows scientists to get a closer look at DNA [8]. Pathogens among samples are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].
==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T17:48:30Z

Mrhonan:

=Background=
Polymerase chain reaction (PCR) was first created in 1983 by biochemist Kary Mullins [3]. This method was first created as a way to pinpoint certain strands of DNA and create synthetic copies of it in order to examine it better [1,2]. Before PCR studying DNA was more difficult as it was hard to isolate small strands of DNA and study them [2]. Kary Mullins would go on to win the 1993 Nobel Prize in Chemistry for his invention of PCR [2]. Since winning this award in 1993 there have been only been four other years where the Nobel Prize in Chemistry was awarded for a method (1998,2002,2005,2020)[7].
=Definition=
PCR is a method used to amplify a small amount of DNA in order to study it in detail[1]. RNA can also be extracted from samples and converted into complimentary DNA (cDNA) for PCR amplification [6]. Primers are used to identify the location of the DNA in the sample. Enzymes that have defined segments of DNA are utilized to recreate cDNA [6].

==PCR Components==
In order to conduct PCR there are numerous components that are required.
* DNA template or cDNA: These are regions that will be getting amplified [5]
*Two primers: This determines the beginning and end of the DNA template or cDNA being amplified [5]
*Taq polymerase: This copies the region being amplified [5]
*Nucleotides: This is from the DNA-Polymerase for the new DNA [5]
*Buffer: This provides a chemical environment for the DNA-Polymerase [5]

==Primers==
PCR primers are single strands of DNA used to identify the location of the DNA in the sample. This refers to a small set of nucleotides in DNA. The use of primers corresponds with the beginning and end of the DNA fragment to be amplified. They stick to the DNA template at the beginning, where the DNA-Polymerase binds and starts synthesis of new DNA strand. and endpoints [1,2,4,8]. For bacteria and [[archaebacteria]] primers that are ubiquitous to the 16s ribosomal RNA (rRNA) are used [1,2,4,8,9].

==Stages of PCR==
'''Denaturing Stage:''' The denaturing stage is where the double-stranded DNA is heated up anywhere from 94-100oC

[[File:Screen Shot 2021-04-15 at 3.51.44 PM.png|thumb|right|Stages of PCR and the resultant amplification of DNA copies of the target region[2]]]

==Uses==
PCR is helpful for scientist as it recreates small strands of DNA using either DNA or RNA. This is especially helpful in looking at genetic [[ecology]] studies as it allows scientists to get a closer look at DNA [8]. Pathogens among samples are able to be seen using PCR [1,6,9]. Bacterial cultures is the traditional way to sample these, but it usually only accounts for a small amount of microbial biomass [1].
==References==
[1] Bruce, K.D., Hiorns, W.D., Hobman, J.L., Osborn, A.M., Strike, P., Ritchie, D.A., 1992. Amplification of DNA from native populations of [[soil]] bacteria by using the polymerase chain reaction. Applied and Environmental Microbiology 58, 3413–3416. https://doi.org/10.1128/AEM.58.10.3413-3416.1992

[2] Henson, J.M., French, R.C., n.d. THE POLYMERASE CHAIN REACTION AND PLANT DISEASE DIAGNOSIS 30.

[3] Kossakovski, F., n.d. The eccentric scientist behind the ‘gold standard’ COVID-19 test [WWW Document]. National Geographic. URL https://www.nationalgeographic.com/science/article/the-eccentric-scientist-behind-the-gold-standard-covid-19-pcr-test

[4] Picard, C., Ponsonnet, C., Paget, E., Nesme, X., Simonet, P., 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Applied and Environmental Microbiology 58, 2717–2722. https://doi.org/10.1128/AEM.58.9.2717-2722.1992

[5] Rahman, M.T., Uddin, M.S., Sultana, R., Moue, A., Setu, M., 2013. Polymerase Chain Reaction (PCR): A Short Review. Anwer Khan Mod Med Coll J 4, 30–36. https://doi.org/10.3329/akmmcj.v4i1.13682

[6] Schochetman, G., Ou, C.-Y., 2021. Polymerase Chain Reaction 5

[7] The Nobel Prize in Chemistry [WWW Document], n.d. . nobelprize. URL https://www.nobelprize.org/prizes/chemistry/

[8] Tsai, Y.L., Olson, B.H., 1992. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Applied and Environmental Microbiology 58, 754–757. https://doi.org/10.1128/AEM.58.2.754-757.1992

[9] WILSONl, K.H., Blitchington, R.B., Greene, R.C., 1990. Amplification of Bacterial 16S Ribosomal DNA with Polymerase Chain Reaction. J. CLIN. MICROBIOL. 28, 5.

Polymerase Chain Reaction (PCR)

2021-05-05T17:35:56Z

Mrhonan:

Measuring Microbial Communities' Biomass

2021-05-05T02:09:05Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3,4]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3,4]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3,4]. The soil suspension is then made up to 50ml with 1.5% being agar [3,4]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3,4]. Once dried the sample can then be put under a microscope to count organisms [3,4].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [1,11]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [5]. This allows for the extraction of naked DNA adsorbs to colloids [5]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [5]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [5]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [5]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [5]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [5]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [5]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [5]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [5].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1,8,10]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1,8,10]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [9,10]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [10]. This provides evidence of viable and non-viable microbes [10].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,7]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [7]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,7]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [7].

===Chloroform Fumigation and Extraction (CFE)===
When soil pH reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,6]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,6]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,6].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[5] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[6] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[7] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[8] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[9] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[10] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[11] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996

Measuring Microbial Communities' Biomass

2021-05-05T01:59:57Z

Mrhonan:

To sample microbial communities’ biomass there are two approaches. The two approaches include several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involved counting while indirect sampling involves the use of chemicals [1].

==Direct==
===Agar Plates===
Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] in microscopic fields [3]. [[Soil]] samples are first taken at random and sifted through a sieve and then weighed out [3]. Following this the sample is then put into a crucible with sterile distilled water and ground up with a glass rod [3]. The sample is then washed with sterile distilled water with the suspended matter poured into a flask [3]. The soil suspension is then made up to 50ml with 1.5% being agar [3]. Once this is done the flask is shaken and left to rest for a short period. Once rested using a pipette the samples are taken and put on a slide [3]. Once the slide is prepared it is then put into sterile distilled water [3]. Once dried the sample can then be put under a microscope to count organisms [3].

===Extractable DNA===
Torsvik et al (1990) used the extractable DNA to determine the identities of organisms in soil samples [2]. Six 30g soil samples were first prepared. Following this samples were washed with 2% sodium hexametaphosphate to increase the yield of DNA [5]. This allows for the extraction of naked DNA adsorbs to colloids [5]. The suspensions are then stored in a refrigerator and the pellets were stored in isopropanol [5]. Pellets are then centrifuged, suspended in a buffer, and then homogenized to lyse the soil bacteria [5]. Following this the volume is adjusted to 25ml using a buffer and then incubated for one hour [5]. Then 2 mg of proteinase K ml-1 was added and then incubated for another hour [5]. The suspension is then heated to 60oC, sodium dodecyl sulfate was added, and then incubated for five minutes [5]. The lysate was then, KCl was added, refrigerated overnight, and then centrifuged [5]. The supernatants were pooled and purified on a hydroxyapatite (HAP) column [5]. DNA from the pooled fractions were then concentrated by cetylpyridinium bromide precipitation to purify the DNA [5].

===Signature Lipid Biomarkers (SLB)===
This technique involves the measurement of ester-linked polar lipid fatty acids and steroids to find microbial biomass and community structure [1]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [1]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass [10]. When a cell dies the cellular enzymes hydrolyze and release a phosphate group. The remaining lipid is then compared to the ratio of PFLAs to the remaining lipid [10]. This provides evidence of viable and non-viable microbes [10].

==Indirect==
===Chloroform Fumigation and Incubation (CFI)===
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl2) vapor is used to fumigate soil [1,7]. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated [7]. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,7]. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by nonfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [7].

===Chloroform Fumigation and Extraction (CFE)===
When soil pH reaches levels below 5.0 CFI is not well suited to measure microbial biomass [1]. Vance et al (1987) modified the original CFI technique to create the Chloroform fumigation and extraction technique. Like CFI in CFE soil samples are fumigated using CHCl3, but instead of being incubated samples are extracted using .5M potassium sulfate (K2SO4 [1,2,6]. The filtrate of both fumigated and nonfumigated samples are analyzed for total organic carbon (TOC) [1,2,6]. Microbial biomass is then calculated by (TOC [fumigated]-TOC [nonfumigated])/kc [1,2,6].

==References==
[1] Coleman, D.C., Crossley, D.A., Hendrix, P.F., 2007. Fundamentals of soil [[ecology]], 2. ed., [Nachdr.]. ed. Elsevier/Academic Press, Amsterdam.

[2]Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatments on metabolism in soil—V. Soil Biology and Biochemistry 8, 209–213. https://doi.org/10.1016/0038-0717(76)90005-5

[3] Jones, P.C.T., Mollison, J.E., Quenouille, m. H., 1948. A Technique for the Quantitative Estimation of Soil Micro-organisms 54–69. https://doi.org/10.1099/00221287-2-1-54

[4]Olsen, R.A., Bakken, L.R., 1987. Viability of soil bacteria: Optimization of plate-counting technique and comparison between total counts and plate counts within different size groups. Microb Ecol 13, 59–74. https://doi.org/10.1007/BF02014963

[5] Torsvik, V., Goksøyr, J., Daae, F.L., 1990. High [[diversity]] in DNA of soil bacteria. Applied and Environmental Microbiology 56, 782–787. https://doi.org/10.1128/AEM.56.3.782-787.1990

[6] Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry 19, 703–707. https://doi.org/10.1016/0038-0717(87)90052-6

[7] Voroney, R.P., Paul, E.A., 1984. Determination of kC and kNin situ for calibration of the chloroform fumigation-incubation method. Soil Biology and Biochemistry 16, 9–14. https://doi.org/10.1016/0038-0717(84)90117-2

[8] White, D., 1993. In situ measurement of microbial biomass, community structure and nutritional status. Phil. Trans. R. Soc. Lond. A 344, 59–67. https://doi.org/10.1098/rsta.1993.0075

[9] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015. Microbial signature lipid biomarker analysis - an approach that is still preferred, even amid various method modifications. J Appl Microbiol 118, 1251–1263. https://doi.org/10.1111/jam.12798

[10] Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129. https://doi.org/10.1007/s003740050533

[11] Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Applied and environmental microbiology 62, 316–322. https://doi.org/10.1128/AEM.62.2.316-322.1996