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)
Collection and storage specifications for a QAPP
1) Sampling strategies: Number and type of
samples, locations, depths, times,
intervals.
2) Sampling methods: Specific techniques
and equipment to be used.
3) Sample storage: Types of containers,
preservation methods, maximum holding
times.
The QAPP plan normally also includes details on
the proposed microbial analysis to be conducted
on the soil samples.
Sampling Plan
A) Simple random
C) Two-stage
B) Transect
D) Systematic grid
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/cm3
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
Rod inside hollow
stem for removing
plug
Hydraulic cylinder
press
• Air drilling and coring
hundreds of meters
Flight
Removable
sampling
barrel
Bit or sampling
barrel
Hinged teeth
(paring device)
Aseptic soil
core
Subsurface saturated zone
• Shallow depth, hollow-stem
auger combined with:
- split-spoon sampling
- push-tube sampling
Hose
Swivel
Piston pump
Drilling mud and cuttings
return pit
• Deep subsurface
- mud rotary coring
Drilling mud and cuttings
Drill pipe
Drill bit
Groundwater
• Flushing/sterile sampling
Drilling mud
supply pit
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
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
Recovery and concentration of viruses from sludge
3. viruses (soil and biosolids)
Procedure
To detect viruses in samples
containing solids, it is first
500 - 2000 ml sludge
necessary to remove or
desorb the virus particles from Adjust to pH 3.5
0.005M AlCl3
the solid surfaces.
Purpose
Adsorb viruses
to solids
Centrifuge to pellet
solids
Once removed, the virus
particles are enumerated
using cell culture.
Discard supernatant
Resuspend pellet in 10%
beef extract
Elute (desorb)
viruses from solids
Centrifuge to pellet solids
Discard pellet and filter
through 0.22 m filter
Assay using cell culture
Remove bacteria
viruses are
in supernatant
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
Help we’re stuck!!!
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 apart
Divide filter fibers and
place into 2 buckets
containing 1.5 l of
elution solution
Handwash for at least
10 min or until clean
Concentrate eluent
via centrifugation
Add 10% buffered
formalin and refrigerate
Spun fiber filter for concentrating protozoa from 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
Sample and
elution solution
Interface
Percoll-sucrose
flotation media
Less than 1 ml of packed pellet is
mixed with 20 ml elution solution
(Tween 80, SDS & PBS)
25 ml of percoll-sucrose
(sp. Gr. 1.10) is layered
under the sample
Debris
The sample is centrifuged
Supernatant and
interface is collected
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
Y Y Y
Y Y
Y
Y Y Y
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