# FIELD SAMPLING

# Streamwater sample collection

> Streamwater sampling technique
>
> Streamwater---Bacterial indicators and coliphage
>
> Streamwater---*Cryptosporidium and Giardia*
>
> Streamwater---Enteric viruses

# Ground-water sample collection 

> Ground-water sampling technique.
>
> Ground water---Bacterial indicators and coliphage
>
> Ground water---*Cryptosporidium and Giardia*
>
> Ground water---Enteric viruses

# References

Sampling for microorganisms requires that sampling equipment be clean
and sterile and that attention to sterile technique be followed. For
more details on sampling for microorganisms, the reader is referred to
U.S. Environmental Protection Agency (1978), Myers and Sylvester (1997),
and Francy and others (2000), parts of which are included below.

# Streamwater sample collection 

[Streamwater sampling technique]{.underline}. The spatial and temporal
distribution of microorganisms in surface water can be as variable as
the distribution of suspended sediment because microorganisms are
commonly associated with solid particles.

-   When collecting a sample for microorganisms, follow equal-width
    increment (EWI) or equal-discharge increment (EDI) methods described
    in Edwards and Glysson (1988), whenever possible, to ensure that a
    sample is representative of the flow in the cross section.

Because churn and cone splitters cannot be autoclaved, use a sterile 3-L
bottle to composite subsamples when using EDI and EWI methods. If
possible, composite by collecting subsamples at vertical locations in
the cross section without overfilling the bottle.

-   Collect a sample by the hand-dip method if the stream depth and (or)
    velocity is not sufficient to use depth-width integrating
    techniques, the stream is well mixed, and (or) project objectives do
    not require an EWI or EDI sample.

[Streamwater]{.underline}---[Bacterial indicators and
coliphage.]{.underline} Samplers that may be used to collect streamwater
for bacterial indicators and coliphage include the US-D77TM, US-D95,
US-DH81, and weighted- and open-bottle samplers with autoclavable
Teflon, glass, or polypropylene components.

-   Clean the equipment coming in contact with the water with dilute
    liquinox, rinse it three times with tap and deionized water, and
    sterilize it (preferably by autoclaving).

-   Prepare a separate set of sterile equipment (bottles, nozzles, and
    caps) for sampling at each site.

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-   Collect approximately 800 mL of streamwater for bacterial indicators
    or coliphage.

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-   Leave at least an inch of headspace in the bottle to allow adequate
    mixing and aeration.

-   Process the samples for *E. coli* and (or) enterococci within 6
    hours.

-   For coliphage and *Clostridium perfringens,* send samples (at least
    500 mL) on ice to the qualified laboratory to arrive within 48 hours
    after collection.

For *E. coli* in streamwater, use the mTEC (see mTEC method), modified
mTEC, MI (see MI method), or Colilert (see Colilert Quantitray) methods.
For enterococci, the mEI method (see mEI) is recommended, although the
mE method also may be used. For coliphage, a quantitative assay is
preferred, such as the single-agar layer method (see single-agar layer
method). For *Clostridium perfringens*, use the mCP agar method (see mCP
agar method).

[Streamwater]{.underline}---*[Cryptosporidium and Giardia]{.underline}*.
The standard samplers used in the USGS can be used to collect
streamwater samples for *Cryptosporidium and Giardia*. Special
sterilization procedures, however, are needed for equipment used in the
collection of samples for *Cryptosporidium* and *Giardia*. Autoclaving
is not effective in neutralizing the epitopes on the surfaces of the
oocysts and cysts that will react with the antibodies used for
detection. The following procedure is best done in the office with
dedicated sampling equipment for each site; it may be done in the field
as long as the bleach is stored and disposed of properly.

-   Wash and scrub the equipment with soap and warm tap water to remove
    larger particulates and rinse with deionized water.

-   Submerge the equipment in a vessel containing 12 percent
    hypochlorite solution for 30 minutes.

-   Wash the equipment free of residual sodium hypochlorite with three
    rinses of filter-sterilized water; do not dechlorinate the equipment
    using sodium thiosulfate.

For analysis of water samples for *Cryptosporidium* and *Giardia*, USEPA
Method 1623 is recommended (see Method 1623).

-   Collect 10 L of streamwater for each protozoan pathogen using
    standard sampling techniques. Composite the sample in a 10-L
    cubitainer (Hedwin Corporation, Baltimore, MD) that is presterilized
    by the manufacturer.

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-   Send 10-L samples in a cubitainer to a qualified laboratory to
    arrive within 48 hours after collection

-   The sample does not have to be kept on ice during transport.

[Streamwater]{.underline}---[Enteric viruses]{.underline}. A different
procedure is used to collect samples for enteric viruses than for
bacterial indicators, coliphage, or protozoan pathogens.

-   Because large volumes of water are needed, water samples are
    filtered in the field and EDI or EWI techniques are not possible.

-   Collect a sample from several sections of the cross section, if
    possible, by placing the pump tubing in each vertical section.

The analyzing laboratory should instruct you on how to collect the
sample, provide you with a specially designed sampling apparatus
containing a Virosorb 1MDS filter (Cuno, Meriden, Conn.), and advise on
the quantity of water needed (usually about 100 L of streamwater).

-   Use a specially-designed sampling apparatus to filter the sample on
    site.

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-   Clean the sampler with dilute liquinox, rinse it with tap water and
    then deionized water, sterilize it with 10% household bleach, and
    rinse it with sodium thiosulfate and then sterile deionized water to
    remove any residuals.

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-   Place a Virosorb1MDS filter (Cuno, Meriden, Conn.), that removes
    viruses present in the water by charge interactions, in the sterile
    cartridge housing.

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-   After sampling, keep the 1MDS filter within the cartridge housing on
    ice. Send the cartridge housing and 1MDS filter to a qualified
    laboratory, to arrive within 48 hours after collection, for virus
    elution, concentration, and detection.

Samples should be analyzed by both a molecular technique such as
reverse-transcriptase-polymerase chain reaction (RT-PCR) and a
cell-culture method (U.S. Environmental Protection Agency, 1996). A
method that incorporates both techniques is integrated cell
culture-polymerase chain reaction (ICC-PCR) (Reynolds and others 1996).
Molecular techniques target specific viruses, but cannot be used to
detect the infectious state of a virus; it is usually a presence or
absence method. The cell culture method detects infectious viruses, but
does not identify the type of virus. Cell culture methods require about
2 weeks to complete, whereas RT-PCR results are available in a few days.

# 

# Ground-water sample collection 

[Ground water sampling technique]{.underline}. Collecting ground-water
samples by use of sterile technique requires knowledge of the type of
well, its use, its construction, and its condition. Samples are
collected once purging criteria have been met as described in Koterba
and others (1995).

-   In sampling wells with a dedicated pump, collect the sample directly
    from the tap into a sterile container.

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-   Swab the tap with ethanol and let dry before sampling.

A tap that yields water directly from the well and before entering any
kind of holding tank is preferred. Water collected after treatment is
unsuitable for microbiological analysis.

For wells without in-place pumps, samples should be obtained by use of
the following methods (in descending order from most to least
desirable): (1) a peristaltic or vacuum pump with autoclavable silicon
tubing, (2) a sterile bailer, (3) a chlorine-disinfected pump and
tubing, or (4) a detergent-cleaned pump and tubing. Refer to Myers and
Sylvester (1997) for a detailed discussion of ground-water sampling for
microbiological analysis.

-   If using a detergent-cleaned pump and tubing, collect additional
    field blanks to evaluate the effectiveness of the cleaning
    procedure.

[Ground water]{.underline}---[Bacterial indicators and
coliphage.]{.underline} In sampling ground water for bacterial
indicators and coliphage, simply fill a clean and sterile sample bottle
directly from the tap or line.

-   Use any autoclavable Teflon, glass, or polypropylene sample bottle.

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-   Clean the sample bottle and equipment coming in contact with the
    water with dilute liquinox, rinse it three times with tap and then
    deionized water, and sterilize it by autoclaving or using bleach and
    sodium thiosulfate.

-   Prepare a separate set of sterile equipment for sampling at each
    site.

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-   Collect approximately 800 mL for bacterial indicators. Two 100-mL
    aliquots are recommended plating volumes for ground water.

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-   Leave at least an inch of headspace in the bottle to allow adequate
    mixing and aeration.

-   Process the samples for total coliforms, *E. coli*, and (or)
    enterococci within 6 hours.

-   For *Clostridium perfringens*, send samples on ice to the qualified
    laboratory to arrive within 48 hours after collection. A 500-mL to
    1-L plating volume is recommended.

-   For coliphage, collect from 1-L to 4-L, depending on project
    objectives. Send the sample on ice to the qualified laboratory to
    arrive within 48 hours after collection.

For total coliforms and *E. coli* in ground water, use the mENDO and
NA-MUG (see mENDO and NA-MUG method), the MI (see MI method), or the
Colilert Quantitray or Colilert presence/absence (see the Colilert
system) methods. For enterococci, the mEI method (see mEI) is
recommended, although the mE method may also be used. For coliphage in
ground water, a presence/absence assay is recommended (see two-step
enrichment method). For *Clostridium perfringens*, use the mCP agar
method (see mCP agar method).

[Ground water]{.underline}---*[Cryptosporidium and
Giardia]{.underline}*. In most circumstances, sampling ground water for
*Cryptosporidium and Giardia* is not recommended. If ground water is
under the influence of surface water or suspected to be contaminated
from protozoan pathogens, follow streamwater sampling instructions with
some obvious modifications.

[Ground water]{.underline}---[Enteric viruses]{.underline}. A different
procedure is used to collect samples for enteric viruses than for
bacterial indicators or coliphage.

-   Because large volumes of water are needed, water samples are
    filtered in the field.

-   Collect a sample by attaching the intake line from a specially
    designed sampling apparatus directly to the tap or by inserting the
    intake line down the wellhead.

The analyzing laboratory should instruct you on how to collect the
sample, provide you with a specially designed sampling apparatus
containing a Virosorb 1MDS filter (Cuno, Meriden, Conn.), and advise on
the quantity of water needed (usually about 2,000 L of ground water).

-   Use a specially designed sampling apparatus to filter the sample on
    site.

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-   Clean the sampler with dilute liquinox, rinse it with tap water and
    then deionized water, sterilize it with 10% household bleach, and
    rinse it with sodium thiosulfate and then sterile deionized water to
    remove any residuals.

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```
-   Place a Virosorb1MDS filter (Cuno, Meriden, Conn.), that removes
    viruses present in the water by charge interactions, in the sterile
    cartridge housing.

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```
-   After sampling, keep the 1MDS filter within the cartridge housing on
    ice. Send the cartridge housing and 1MDS filter to a qualified
    laboratory, to arrive within 48 hours after collection, for virus
    elution, concentration, and detection.

Samples should be analyzed by both a molecular technique such as
reverse-transcriptase, polymerase chain reaction (RT-PCR) and a
cell-culture method (U.S. Environmental Protection Agency, 1996). A
method that incorporates both techniques is integrated cell culture
polymerase chain reaction (ICC-PCR) (Reynolds and others 1996).
Molecular techniques target specific viruses but cannot be used to
detect the infectious state of a virus; it is usually a presence or
absence method. The cell culture method detects infectious viruses, but
does not identify the type of virus. Cell culture methods require about
2 weeks to complete, whereas RT-PCR results are available in a few days.

# REFERENCES

Edwards, T.K., and Glysson, G.D., 1988, Field methods for measurement of
fluvial sediment: U.S. Geological Survey Techniques of Water-Resources
Investigations, book 3, chap. C2, 80 p.

Francy, D.S., Myers, D.N., and Helsel, D.R., 2000, Microbiological
monitoring for the U.S. Geological Survey National Water Quality
Assessment Program: U.S. Geological Survey Water-Resources
Investigations Report 00-4018, p. 17-21.

Koterba, M.T., Wilde, F.D., and Lapham, W.W., 1995, Ground-water
data-collection protocols and procedures for the National Water-Quality
Assessment Pro­gram---Collection and documentation of water-quality
samples and related data: U.S. Geological Survey Open-File Report
95-399, 113 p.

Myers, D.N., and Sylvester, M.D., 1997, National field manual for the
collection of water-quality data---Biological indicators: U.S.
Geological Survey Techniques of Water-Resources Investigations, book 9,
chap. A7, 38 p.

Reynolds, K.A., Gerba, C.P., and Pepper, I.L., 1996, Detection of
infectious enteroviruses by an integrated cell culture-PCR procedure:
Applied and Environmental Microbiology, v. 62, no. 4, p. 1424-1427.

U.S. Environmental Protection Agency, 1978, Microbiological methods for
monitoring the environment---Water and wastes: Washington, D.C., U.S.
Environmental Protection Agency, EPA-600/8-78-017, p. 5-31.

U.S. Environmental Protection Agency, 1996, EPA Information Collection
Rule microbial laboratory manual: Washington, D.C., EPA/600/R-95/178.

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