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

• Rhizosphere – a special case

Distance from root surface (mm)

Number of typesDistinguishable

Estmated frequency109 cells/cm3

0 – 11 – 5

5 – 1010 – 1515 - 20

1112522

12096413413

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

Rod inside hollowstem for removingplug

Flight

Removablesampling

barrel

Bit or samplingbarrel Aseptic soil

core

Hinged teeth(paring device)

Hydraulic cylinderpress

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

SwivelHose

Piston pump

Drilling mud and cuttings return pit

Drilling mud supply pit

Drilling mud and cuttings

Drill bit

Drill pipe

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

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.

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.

Recovery and concentration of viruses from sludge

Procedure Purpose

500 - 2000 ml sludge

Adsorb viruses to solids

Adjust to pH 3.50.005M AlCl3

Centrifuge to pelletsolids

Discard supernatant

Resuspend pellet in 10%beef extract

Centrifuge to pellet solids

Discard pellet and filterthrough 0.22 m filter

Assay using cell culture

Elute (desorb)viruses from solids

Remove bacteriaviruses arein supernatant

Analysis for microorganisms

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 we’re stuck!!!Positively charged filters

Negatively charged filters – adjust pH to 3.5 where viruses become positively charged.

Water sample processing - viruses

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

- step 2, elute the viruses from the filter

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

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

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

Cut filter apartDivide filter fibers andplace into 2 buckets

containing 1.5 l of elu tion so lution

Handwash for at least10 m in or until clean

Concentrate eluentvia centrifugation Add 10% buffered

formalin and refrigerate

S pun fi ber fi lter f or concentrat ing protozoa f rom water

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

Less than 1 m l of packed pelle t ism ixed with 20 ml elution solution

(Tween 80, SDS & PBS)

25 m l of percoll-sucrose(sp. G r. 1.10) is layered

under the sam ple

The sam ple is centrifuged Supernatant andinterface is collected

Interface

Sample andelution solution

Percoll-sucroseflotation m edia

Debris

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