1. 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

2. Quality assurance project plan (QAPP)

3. Sampling Plan

4. Sampling approaches Surface soils Shovel or hand-auger Sterile technique Rhizosphere – a special case Distance from root surface (mm) Number of types Distinguishable Estmated frequency 10 9 cells/cm 3 0 – 1 1 – 5 5 – 10 10 – 15 15 - 20 11 12 5 2 120 96 41 34 13

5. Subsurface unsaturated zone Hand-auger simple cheap 0 – 20 ft Hollow stem auger split spoon sampling push-tube sampling 20 – 100 ft Air drilling and coring hundreds of meters

6. Subsurface saturated zone Shallow depth, hollow-stem auger combined with: - split-spoon sampling - push-tube sampling Deep subsurface - mud rotary coring Groundwater Flushing/sterile sampling

7. Sample Processing and Storage for Soil Store samples at 4 0 C 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

8. Bacterial fractionation vs. in situ lysis for recovery of DNA from soil IssueBacterial FractionationIn 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 IssueBacterial FractionationIn 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?

9. 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. Analysis for microorganisms

10. 3. viruses (soil and biosolids) To detect viruses in samples containing solids, it is first necessary to remove or desorb the virus particles from the solid surfaces. Once removed, the virus particles are enumerated using cell culture. Analysis for microorganisms

11. Water Sample Processing – viruses Sampling procedures are easier than for soil because water samples are more homogeneous. – step 1, collect and filter a 100 to 1000L sample VIRADEL – virus adsorption-elution Viruses stick through a combination of electrostatic and hydrophobic interactions. Help were stuck!!! Positively charged filters Negatively charged filters – adjust pH to 3.5 where viruses become positively charged.

13. Water sample processing - viruses - step 1, collect and filter a 100 to 1000L sample - step 2, elute the viruses from the filter

14. Water sample processing - viruses – step 1, collect and filter a 100 to 1000L sample - step 2, elute the viruses from the filter - step 3, reconcentrate the sample

15. Water sample processing - viruses - step 1, collect and filter a 100 to 1000L sample - step 2, elute the viruses from the filter - step 3, reconcentrate the sample - step 4, assay using cell culture or PCR or ICC-PCR

16. Water sample processing - bacteria - step 1, collect sample (1 to 100 ml) using a 0.2 um filter - step 2, use MPN analysis* or use membrane filtration technique* or dilution plating * Commonly used for analysis of pathogens in water

17. Water sample processing - protozoa step 1, collect and filter 100 to >1000 L sample

18. Water sample processing - protozoa step 1, collect and filter 100 to >1000 L sample step 2, elute protozoa from filter

20. Water sample processing - protozoa step 1, collect and filter 100 to >1000 L sample step 2, elute protozoa from filter step 3, centrifuge with a Percoll-sucrose gradient and collect supernatant

21. Water sample processing - protozoa step 1, collect and filter 100 to >1000 L sample step 2, elute protozoa from filter step 3, centrifuge with a Percoll-sucrose gradient and collect supernatant step 4, stain with antibody

22. Water sample processing - protozoa step 1, collect and filter 100 to >1000 L sample step 2, elute protozoa from filter step 3, centrifuge with a Percoll-sucrose gradient and collect supernatant step 4, stain with antibody step 5, examine with a microscope Epifluorescence microscopy