Chapter 8 - Microbiological Sampling Overall goal is to recover material representative of the subsurface environment being studied. Objectives Be familiar with: 1) Elements of a QAPP 2) Soil sampling techniques for surface soils and subsurface samples 3) Soil storage and processing 4) Microbial (bacteria, fungi) recovery from soil samples 5) Microbial (virus, bacteria, protozoa) recovery from water samples 6) Approaches used for analysis of recovered microbes
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Chapter 8 - Microbiological Sampling Overall goal is to recover material representative of the subsurface environment being studied. Objectives Be familiar.
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Chapter 8 - Microbiological Sampling
Overall goal is to recover material representative of the subsurface environment being studied.
Objectives
Be familiar with:
1) Elements of a QAPP
2) Soil sampling techniques for surface soils and subsurface samples
3) Soil storage and processing
4) Microbial (bacteria, fungi) recovery from soil samples
5) Microbial (virus, bacteria, protozoa) recovery from water samples
6) Approaches used for analysis of recovered microbes
Collection and storage specifications for a QAPP
1) Sampling strategies: Number and type ofsamples, locations, depths, times,intervals.
2) Sampling methods: Specific techniquesand equipment to be used.
3) Sample storage: Types of containers,preservation methods, maximum holdingtimes.
The QAPP plan normally also includes details onthe proposed microbial analysis to be conductedon the soil samples.
Quality assurance project plan (QAPP)
D) Systematic grid
B) TransectA) Simple random
C) Two-stage
Sampling Plan
Sampling approaches
Surface soils • Shovel or hand-auger • Sterile technique
• Store samples at 40C• Process samples as quickly as possible
Surface soils- air dry and sieve through a 2 mm mesh- microbial communities remain essentially intact for 3 weeks
Subsurface samples- perform analyses immediately under sterile conditions (if
not possible place samples in dry ice and ship overnight to lab for analysis next day)
Analysis for microorganisms 1. bacteria - cultural assay (choose culture medium carefully) - direct counts
- antibodies - extraction and analysis of nucleic acids
Bacterial fractionation vs. in situ lysis for recovery of DNA from soil
Issue Bacterial Fractionation In situ Lysis
Yield of DNA
Representative?
Source of DNA
Shearing
Fragment size
Humic contamination
Method ease
1-5 ug/g
Less representative, sorption
Only bacteria
Less shearing
50 kb
Less contaminated
Slow, laborious
1-20 ug/g
More representative
Mostly bacteria
More shearing
25 kb
More contaminated
Faster, less laborious
Issue Bacterial Fractionation In situ Lysis
Yield of DNA
Representative?
Source of DNA
Shearing
Fragment size
Humic contamination
Method ease
1-5 ug/g
Less representative, sorption
Only bacteria
Less shearing
50 kb
Less contaminated
Slow, laborious
1-20 ug/g
More representative
Mostly bacteria
More shearing
25 kb
More contaminated
Faster, less laborious
Which method is preferred?
2. fungi from soil
Hyphae - a soil washing methodology is used wherein a fine spray of water is used to tease apart soil aggregates and separate the heavy particles from the fines. The heavy particles are then examined under a microscope for the presence of hyphae.
Spores - a soil sample is washed in boxes containing sieving meshes of increasing size. Spores are enumerated by plating successive washes. This washing procedure separates spores from hyphae.