Measuring Microbial Communities' Biomass
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 [2]. Directing sampling involved counting while indirect sampling involves the use of chemicals [2].
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 [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 [6]. 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 [2]. A common biomarker used for this technique are phospholipids fatty acids (PFLAs) [2]. The total number of PFLAs provides quantitative measure of viable or potentially viable biomass []. 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 []. This provides evidence of viable and non-viable microbes [].
Indirect
Chloroform Fumigation and Incubation (CFI)
The Chloroform fumigation and incubation method (CFI) is used to determine organic C biomass in soil microbials. Chloroform (CHCl3) vapor is used to fumigate soil []. Once the soil is fumigated the CHCl3 vapor is removed then the soil is incubated []. Evolved CO2 levels are then measured for both fumigated and then unfumigated soil to calculate biomass []. Using the expression B=F/kc (B=soil biomass C, F= carbon dioxide carbon evolved by fumigated soil minus CO2 evolved by unfumigated soil, and kc= fraction of biomass mineralized to CO2 during the incubation) biomass can be calculated where kc is a constant [2]. Voroney and Paul (1984) then furthered this technique to include labile nitrogen and measured the fraction of biomass nitrogen (kn) mineralized to inorganic nitrogen [].