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Chapter 8 - Microbiological Sampling

Chapter 8 - Microbiological Sampling. Overall goal is to recover material representative of the subsurface environment being studied. Objectives Be familiar with: Elements of a QAPP Soil sampling techniques for surface soils and subsurface samples Soil storage and processing

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Chapter 8 - Microbiological Sampling

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  1. Chapter 8 - Microbiological Sampling Overall goal is to recover material representative of the subsurface environment being studied. • Objectives • Be familiar with: • Elements of a QAPP • Soil sampling techniques for surface soils and subsurface samples • Soil storage and processing • Microbial (bacteria, fungi) recovery from soil samples • Microbial (virus, bacteria, protozoa) recovery from water samples • 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

  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 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

  8. Bacterial fractionation vs. in situ lysis for recovery of DNA from soil Which method is preferred?

  9. 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.

  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. Help we’re stuck!!! • 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. Positively charged filters Negatively charged filters – adjust pH to 3.5 where viruses become positively charged.

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

  13. 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

  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 - step 4, assay using cell culture or PCR or ICC-PCR

  15. 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

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

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

  18. 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

  19. 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

  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 step 4, stain with antibody step 5, examine with a microscope Epifluorescence microscopy

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