Measuring Microbial Communities' Biomass - Revision history

Saigemat: /* Indirect Approaches */

2022-05-05T03:56:47Z

Indirect Approaches

← Older revision		Revision as of 23:56, 4 May 2022
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	[[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]]]		[[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 (CHCl<sub>2</sub>) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl<sub>3</sub> vapor is removed ~~then~~ the soil ~~is incubated~~ [9]. Evolved CO<sub>2</sub> levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/k<sub>c</sub> (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO<sub>2</sub> evolved by nonfumigated soil, and k<sub>c</sub>= fraction of biomass mineralized to CO<sub>2</sub> 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 (k<sub>n</sub>) mineralized to inorganic nitrogen [9].		The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass within soil microbials. Chloroform (CHCl<sub>2</sub>) vapor is used to fumigate soil [1,9]. Once the soil is fumigated, the CHCl<sub>3</sub> vapor is removed before incubating the soil [9]. Evolved CO<sub>2</sub> levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/k<sub>c</sub> (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO<sub>2</sub> evolved by nonfumigated soil, and k<sub>c</sub>= fraction of biomass mineralized to CO<sub>2</sub> during the incubation) biomass can be calculated where kc is a constant [1]. Voroney and Paul (1984) furthered this technique to include labile nitrogen, measuring the fraction of biomass nitrogen (k<sub>n</sub>) mineralized to inorganic nitrogen [9].

	===Chloroform Fumigation and Extraction (CFE)===		===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 CHCl<sub>3</sub>, ~~but~~ instead of ~~being incubated~~ samples are extracted using .5M potassium sulfate (K<sub>2</sub>SO<sub>4</sub> [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])/k<sub>c</sub> [1,2,8].		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. Similar to CFI in CFE, soil samples are fumigated using CHCl<sub>3</sub>; though, instead of incubation, samples are extracted using .5M potassium sulfate (K<sub>2</sub>SO<sub>4</sub> [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])/k<sub>c</sub> [1,2,8].

	===Substrate-Induced-Respiration (SIR)===		===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].		The SIR technique involves adding a substrate, such as glucose, to stimulate respiration [1,4]. Glucose is one of the most commonly used substrates due to [[microorganisms]] to its ability to be utilized as a carbon source [4]. Depending on the physical and chemical [[properties]] of a soil sample, the amount of substrate needed 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==		==References==

Saigemat: /* Direct Approaches */

2022-05-05T03:43:16Z

Direct Approaches

← Older revision		Revision as of 23:43, 4 May 2022
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	===Extractable DNA===		===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].		Torsvik et al (1990) used extractable DNA to determine the identities of organisms within soil samples [1,13]. In this study, six 30g soil samples were prepared. These samples were then were washed with 2% sodium hexametaphosphate to increase the yield of DNA [7]. This step 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)===		===Signature Lipid Biomarkers (SLB)===

Saigemat: /* Direct Approaches */

2022-05-05T03:40:58Z

Direct Approaches

← Older revision		Revision as of 23:40, 4 May 2022
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	[[File:AgarPM.png\|thumb\|right\|Top view of soil microorganisms nutrient agar in plate [14]]]		[[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]] present in microscopic fields [3]. To begin, random samples of [[soil]] are obtained and sifted through a sieve before being weighed out [3,5]. Following this, the sample is placed into a crucible with sterile distilled water and ground up with a glass rod [3,5]. Next, the sample is washed with sterile distilled water, and any remaining suspended matter is poured into a separate 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].		Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] present in microscopic fields [3]. To begin, random samples of [[soil]] are obtained and sifted through a sieve before being weighed out [3,5]. Following this, the sample is placed into a crucible with sterile distilled water and ground up with a glass rod [3,5]. Next, the sample is washed with sterile distilled water, and any remaining suspended matter is poured into a separate flask [3,5]. The soil suspension is then made up to 50ml with 1.5% being agar [3,5]. The sample flask is then shaken and left to rest for a short period. Once rested, a pipette is used to remove the sample solution and place drops onto a glass slide [3]. The prepared slide is then put into sterile distilled water [3,5]. Once dried, the sample can be placed under a microscope to count organisms [3,5].

	===Extractable DNA===		===Extractable DNA===
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	===Signature Lipid Biomarkers (SLB)===		===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].		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 Approaches ==		==Indirect Approaches ==

Saigemat at 03:36, 5 May 2022

2022-05-05T03:36:59Z

← Older revision		Revision as of 23:36, 4 May 2022
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	To ~~sample~~ the biomass of a microbial community, there are two ~~primary~~ approaches incorporating several different methods. These approaches include the use of either direct or indirect sampling techniques [1]. Directing sampling involves counting numerical values, while indirect sampling involves the use of chemicals [1].		To analyze the biomass of a microbial community, there are two types of approaches incorporating several different methods. These approaches include the use of either direct or indirect sampling techniques [1]. Directing sampling involves counting numerical values, while indirect sampling involves the use of chemicals [1].

	==Direct==		==Direct Approaches ==
	===Agar Plates===		===Agar Plates===
	[[File:AgarPM.png\|thumb\|right\|Top view of soil microorganisms nutrient agar in plate [14]]]		[[File:AgarPM.png\|thumb\|right\|Top view of soil microorganisms nutrient agar in plate [14]]]
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	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].		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==		==Indirect Approaches ==
	===Chloroform Fumigation and Incubation (CFI)===		===Chloroform Fumigation and Incubation (CFI)===

Saigemat at 03:35, 5 May 2022

2022-05-05T03:35:20Z

← Older revision		Revision as of 23:35, 4 May 2022
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	To sample the biomass of a microbial community, there are two primary approaches incorporating several different methods. These approaches ~~are~~ either direct or indirect sampling techniques [1]. Directing sampling involves counting numerical values, while indirect sampling involves the use of chemicals [1].		To sample the biomass of a microbial community, there are two primary approaches incorporating several different methods. These approaches include the use of either direct or indirect sampling techniques [1]. Directing sampling involves counting numerical values, while indirect sampling involves the use of chemicals [1].

	==Direct==		==Direct==

Saigemat at 03:34, 5 May 2022

2022-05-05T03:34:55Z

← Older revision		Revision as of 23:34, 4 May 2022
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	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].		To sample the biomass of a microbial community, there are two primary approaches incorporating several different methods. These approaches are either direct or indirect sampling techniques [1]. Directing sampling involves counting numerical values, while indirect sampling involves the use of chemicals [1].

	==Direct==		==Direct==
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	[[File:AgarPM.png\|thumb\|right\|Top view of soil microorganisms nutrient agar in plate [14]]]		[[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].		Outlined by Jones et al (1948) the use of agar plates was used to count [[organisms]] present in microscopic fields [3]. To begin, random samples of [[soil]] are obtained and sifted through a sieve before being weighed out [3,5]. Following this, the sample is placed into a crucible with sterile distilled water and ground up with a glass rod [3,5]. Next, the sample is washed with sterile distilled water, and any remaining suspended matter is poured into a separate 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===		===Extractable DNA===

Mrhonan: /* Indirect */

2021-05-07T17:25:04Z

Indirect

← Older revision		Revision as of 13:25, 7 May 2021
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	==Indirect==		==Indirect==
	===Chloroform Fumigation and Incubation (CFI)===		===Chloroform Fumigation and Incubation (CFI)===
	The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl<sub>2</sub>) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl<sub>3</sub> vapor is removed then the soil is incubated [9]. Evolved CO<sub>2</sub> levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/k<sub>c</sub> (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO<sub>2</sub> evolved by nonfumigated soil, and k<sub>c</sub>= fraction of biomass mineralized to CO<sub>2</sub> 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 (k<sub>n</sub>) 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]]]		[[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 (CHCl<sub>2</sub>) vapor is used to fumigate soil [1,9]. Once the soil is fumigated the CHCl<sub>3</sub> vapor is removed then the soil is incubated [9]. Evolved CO<sub>2</sub> levels are then measured for both fumigated and then unfumigated soil to calculate biomass [1,9]. Using the expression B=F/k<sub>c</sub> (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO<sub>2</sub> evolved by nonfumigated soil, and k<sub>c</sub>= fraction of biomass mineralized to CO<sub>2</sub> 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 (k<sub>n</sub>) mineralized to inorganic nitrogen [9].

	===Chloroform Fumigation and Extraction (CFE)===		===Chloroform Fumigation and Extraction (CFE)===
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	===Substrate-Induced-Respiration (SIR)===		===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].		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==		==References==

Mrhonan: /* Agar Plates */

2021-05-07T17:24:04Z

Agar Plates

← Older revision		Revision as of 13:24, 7 May 2021
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	==Direct==		==Direct==
	===Agar Plates===		===Agar Plates===
	[[File:AgarPM.png\|thumb\|right\|Top view soil microorganisms nutrient agar in plate [14]]]		[[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].		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===		===Extractable DNA===

Mrhonan: /* References */

2021-05-07T17:22:58Z

References

← Older revision		Revision as of 13:22, 7 May 2021
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	[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		[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		[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

Mrhonan at 17:22, 7 May 2021

2021-05-07T17:22:40Z

← Older revision		Revision as of 13:22, 7 May 2021
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	[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		[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