Chapter 8 Microbiological Sampling Overall goal is to

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

Quality assurance project plan (QAPP)

Sampling Plan

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 109 cells/cm 3 0– 1 1– 5 5 – 10 10 – 15 15 - 20 11 12 5 2 2 120 96 41 34 13

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

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

Sample Processing and Storage for Soil • Store samples at 40 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

Bacterial fractionation vs. in situ lysis for recovery of DNA from soil Issue Bacterial Fractionation In situ Lysis Yield of DNA 1 -5 ug/g 1 -20 ug/g Representative? Less representative, sorption More representative Source of DNA Only bacteria Mostly bacteria Shearing Less shearing More shearing Fragment size 50 kb 25 kb Humic contamination Less contaminated More contaminated Method ease Slow, laborious Faster, less laborious Which method is preferred?

Analysis for microorganisms 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 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.

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 1000 L sample VIRADEL – virus adsorption-elution Viruses stick through a combination of electrostatic and hydrophobic interactions. Positively charged filters Help we’re stuck!!! Negatively charged filters – adjust p. H to 3. 5 where viruses become positively charged.

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

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

Water sample processing - viruses - step 1, collect and filter a 100 to 1000 L 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

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

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

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

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

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

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