GW
PROTOCOL
SAMPLING,
ANALYSIS, AND REPORTING RESULTS
Samples are enumerated for total coliforms and Escherichia coli (E. coli) in the field by use of the MI method. See Total coliforms and Escherichia
coli using the MI method. For
each well, two 1-L (or two 500-mL) bottles are collected and 100-mL samples are
plated from each bottle—a regular and a replicate. Use the GW sample and replicate
report form to record analytical results from these samples. For coliphage analysis, a 3-L bottle is
filled with ground water by study unit personnel and sent to the Ohio District
Microbiology Laboratory (ODML) for analysis using the two-step, enrichment
method. This method determines the
presence or absence of somatic and F-specific coliphage. A list of recommended and required
quality-control samples for bacteria and coliphage are described in Quality-control samples for
NAWQA microbiology.
Refer to the USGS National
Field Manual, Biological Indicators for detailed instructions on sample collection and analysis,
parts of which are included below.
Equipment and supplies. Refer to the list of Equipment and Supplies. For rinsing the filtration equipment, bring one 250-mL
bottle of sterile, buffered water, PO4/MgCl2
(made by
Clean and sterilize equipment. Wash and
gently scrub equipment (filter funnels, graduated cylinders, 1-L and 3-L sample
bottles) with 0.1 percent liquinox.
Thoroughly rinse the scrubbed items with warm tap water until there is
no sign of any detergent. Then, thoroughly rinse all equipment with deionized
water (DIW). After cleaning, cover the open ends with aluminum foil or kraft
paper and autoclave for 15 minutes at 121o C and 15 lb/in2.
Use heat-sterilizing tape to ensure proper performance of the autoclave.
Prepare a separate set of sterile equipment (filter
funnels, graduated cylinders, and sample bottles) for microbiological sampling
at each site.
Heat incubators.
Heat incubator to 35°C. It takes about 2 hours for
the incubator to reach desired temperature.
Prepare media. Prepare
mixture according directions on dehydrated agar bottle (36.5 grams agar to l
liter deionized water). Heat to boiling
with a stirring rod on a hot plate. Add exactly
100-mL of agar into each dilution bottle.
Autoclave for 15 minutes. Store
dilution bottles at 4°C for up to six months.
Prepare cefsulodin
solution. Add 0.001-g (1mg) of cefsulodin to 1-mL
reagent water (this can be done in a test tube). Sterilize the solution into a labeled sterile
test tube using a 0.22-mm filter and a 5- or 10-cc syringe. Use immediately or store at 4°C for a short period of time (less than 2 hours). Do not save the unused portion.
Prepare agar plates. Label the bottom
of the plates with the medium type and date prepared. The agar can be melted in two ways. Place the 100-mL bottle of MI agar, with the
cap loosened, in an autoclave for 5 minutes or in a beaker with boiling water
on a hot plate until melted. Let the
agar cool until it is warm (50-60°C). Add 0.5-mL
of the freshly-prepared cefsulodin solution to the 100-mL bottle of agar. Gently mix the agar and pour plates.
Store plates upside down in a plastic bag at 1-4°C. Plates are
good for two weeks in the refrigerator.
You must keep the plates chilled at all times.
Well purging. All the wells are existing systems with dedicated
pumps. Purge the well as required for
NAWQA sampling.
Collect bacteria sample. Spray the spigot inside and out with a small amount
of ethanol or isopropyl alcohol. Air dry
the spigot and rinse with STERILE deionized water. Let the tap run for about 10 seconds. For bacteria, fill two 1-L (or two 500-mL)
sterile sample bottles with water directly from the tap leaving at least 2
inches of headspace in each bottle.
After collection,
immediately chill samples in a cooler with ice or in a refrigerator at 1 to
4ºC. SAMPLES NEED TO BE PLATED FOR BACTERIA WITHIN 6 HOURS OF COLLECTION.
Collect coliphage
sample. For coliphage, fill a 3-L sample bottle with
water directly from the tap leaving at least 2 inches of headspace. Place the sample immediately on ice. Please see Sample Submission
for information on how to submit a sample to the ODML. BE SURE TO PROVIDE THE LABORATORY WITH AT LEAST 2-DAYS ADVANCE NOTICE THAT YOU ARE SENDING A sample for COLIPHAGE
analysis. For detailed information on the coliphage method,
see Somatic and F-specific
coliphage using a two-step, enrichment method.
Plating samples for bacteria [Refer to the USGS National
Field Manual, Biological Indicators for detailed membrane-filtration
procedures.]
Label your plates. On the lid of the plate, record the site, date, sample-collection
time, and the volume (or FB for filter blank).
Use the GW sample and replicate
report form for the regular and
replicate samples, and the GW positive and negative
control report form for the
positive and negative control samples (See Quality control section below).
Assemble membrane filtration apparatus and gather
supplies. Place the filter funnel in
a manifold attached to a peristaltic pump. Use a different sterile filter
funnel for each ground-water site.
Filter the filter blank. First, a filter blank (FB), which tests the
sterility of buffered water and filtration equipment, is filtered. This is done
by filtering a 50-100 mL aliquot of sterile, buffered
water, PO4/MgCl2 before filtering the sample. Use the gradations on the side of the filter
funnel to measure a 50-100 mL aliquot of buffered water. If contamination from a FB is found, results
are suspect and may be qualified or not reported. For criteria, see Quality-control samples for
NAWQA microbiology.
Filter the sample. BE SURE TO THOROUGHLY SHAKE THE SAMPLE BEFORE MEASURING THE VOLUME. Measure the 100-mL sample volume using a
graduated cylinder. After filtering the
sample and before removing the membrane, rinse the funnel with
approximately 100 mL of buffered water. This removes any bacteria stuck to the
side of the funnel.
Do not change filter funnels. Filter a procedure blank
(PB), which tests the efficiency of the rinsing procedure, by filtering a
50-100 mL aliquot of sterile buffered water, PO4/MgCl2 before filtering the
second sample. Then, filter the100-mL sample volume
(replicate sample) from the second sample bottle.
Incubate plates.
Invert the plates and incubate for 24 + 2 hours at 35°C. Fill out the
report form.
Count E. coli. All colonies that
are blue under natural light are counted as E. coli. Using
a dissecting microscope or portable illuminated magnifier to count colonies is
highly recommended.
Count total coliforms. All colonies that
fluoresce under long wave UV light are counted as total coliforms including:
·
All colonies that were counted as E. coli. These colonies do not fluoresce
completely but rather fluorescent halos may be observed around the edges. Some E. coli colonies are
non-fluorescent and are to be counted also.
·
Blue/white colonies – total coliforms other than E. coli.
·
Tiny, flat, or pinpoint blue colonies
·
Bright green fluorescent colonies (these are Pseudomonas species
and may indicate the breakdown of the antibiotic in the MI agar)
Disposal. After counting, place all plates in an autoclave bag and autoclave for 30 minutes at 121oC. Dispose of the bag in the trash.
Because only 100-mL sample volumes are
plated, the number of colonies counted on the plate from the 1st
sample bottle is reported as colonies per 100 mL (col/100 mL). The next 100-mL volume (taken from the 2nd
sample bottle) is treated as a sequential replicate sample and
reported in the same manner.
The results from the membrane-filtration tests
are entered into NWIS using the following parameter codes:
90900, Total coliforms, MI method
90901, E. coli, MI method
If counts are not within the ideal range of
20-80 colonies, enter a remark code of “E” and a value qualifier of “k”.
If the filter has a colony count greater than
80, but the colonies are distinct enough to count, report the number and enter
a remark code of “E” and a value qualifier of “k”. If the plate is too numerous
to count (TNTC) and the colonies are not discernable, report a minimum
estimated value. Assume a count of the maximum range of colonies (80 for E.
coli and total coliforms on MI agar) for the smallest sample volume
filtered and report the result as greater than the calculated value per 100 mL.
For example, assume 80 colonies on the 100 mL volume filtered for total
coliforms in GW to get a result of >80 colonies per 100 mL, with a value
qualifier of “k”.
Enter the first sample in QWDATA with the
following codes:
·
Sample medium is “6” for ground water
· Sample type is “7” for replicate sample (All environmental samples will be replicates because replicates are done for every well).
·
Parameter code 99111 (QC sample associated with this environmental
sample) is “30” for replicate sample, or “100” if more than 1 type of field QC sample is collected.
·
Parameter code 99105 (replicate type) is “20”
for sequential
Enter the 2nd sample in QADATA
with the following codes:
·
Sample medium is “S” for ground water
·
Sample type is “7” for replicate
· Parameter code 99105 (replicate type) is “20” for sequential
Coliphage results, provided by the ODML, are
entered into NWIS by the study unit using the following parameter codes:
99332, Coliphage, somatic, E. coli
CN-13 host, 2-step enrichment, record as (1) presence or (2) absence per 1
L.
99335, Coliphage, F-specific, E. coli
F-amp host, 2-step enrichment, record as (1) presence or (2) absence per 1 L.
·
Check the incubators daily to ensure they are
operating at the proper temperature. Use an external digital thermometer that enables
you to take a temperature reading with the incubator closed (see equipment
list). Record the results in a QA/QC
logbook. Criteria for acceptance is 35 + 0.5°C.
·
The gray incubators purchased from Millipore
sometimes respond to a power surge by shutting off. A good practice is to periodically monitor
the incubator temperature with a HOBO temperature recorder for 48 hours
throughout field and laboratory use.
·
Use heat-indicating tape with each autoclave run to ensure it is
operating properly.
·
Test the autoclave with biological indicators quarterly or more often
during heavy use (you can order these from a scientific supplier—Barnstead No.
AY759X7).
·
Before processing samples in the field vehicle, wipe down the area with
dilute (50/50) isopropyl alcohol.
·
After counting, turn the plate 180° and ensure the second count is within 5 percent of the first count.
·
Have a second analyst check calculations for errors.
Quality control
Collect and analyze quality-control samples
at the required frequency. See Quality-control samples for
NAWQA microbiology.
For total coliforms and E. coli, the following QC is required (in addition to the replicate done for every sample and described above):
·
Filter blanks—a 50-100 mL aliquot of sterile buffered water filtered before the first
bottle. Filter blanks are used to
qualify associated environmental sample.
Filter blank data are not stored in NWIS.
·
Procedure blanks—a 50-100 mL aliquot of sterile buffered water filtered after the first
bottle and before the replicate bottle.
Procedure blanks are used to qualify associated environmental
sample. Procedure blank data are not
stored in NWIS.
·
Positive and negative controls can be ordered from the ODML. The controls come in 100-mL aliquots so that
the whole bottle is plated in the same manner as a regular ground-water
sample. Use separate filter funnels for
the positive and negative controls. Use
the GW positive and negative
control report form to record results.
Instructions for analyzing the positive and negative controls are in GW control instructions. Control data are not stored in NWIS at this
time.
Positive controls are used
to ensure that field personnel are able to correctly identify target
colonies. In most GW studies, results
are positive in a small percentage of samples so positive controls are also needed
to maintain proficiency in the method. Two cultures are included in the positive
control:
o E. coli—positive for total coliforms and E.
coli. The colonies fluoresce under long wave UV light and are blue under natural
light.
o
Serratia
marcescens—Positive control for total coliforms, but negative
for E. coli. The colonies fluoresce under long wave UV
light but are not blue.
Negative controls are used
to test the selectivity of the medium (the ability of the medium to inhibit
nontarget growth) or to ensure field personnel are able to correctly
distinguish between target and nontarget growth. Two cultures are included in
the negative control:
o
Pseudomonas sp.—Negative for total coliforms and E.
coli and does not grow on MI. Growth
of this green-fluorescent culture on MI agar suggests that the antibiotic added
to the medium to inhibit nontarget growth may have degraded.
o Providencia alcalifaciens— Negative for total coliforms and E. coli and is able to grow on MI. This culture is used to familiarize the
technician with nontarget growth that may appear on regular-sample plates.
Analyzing positive and
negative controls at regular intervals throughout the sampling period is very
important. Analyze controls at the
beginning and end of the two-week holding time for a batch of plates.
Send the results from
controls to the Ohio District (amgbrady@usgs.gov).
·
Field blanks—A field blank is a 250 mL aliquot of sterile buffered water poured into
a clean and sterile sample bottle and processed like a regular sample. Do not analyze a replicate field blank.
Field blank results are
stored in QADATA with the following codes:
o Sample-medium code is “Q”
for quality-control sample
o Sample-type code is “2”
for blank
o Parameter code 99100
(blank-solution type), is “70” for sterile buffered water PO4/MgCl2 (lot number
is not required)
o Parameter code 99101
(source of blank water) is “80” for
o Parameter code 99102
(blank-sample type) is “100” for field
For coliphage analysis, the following QC is required:
·
Field blanks—Generally not required for GW studies because contamination potential is
very low and coliphage are infrequently detected in ground water. However, if greater than 15 percent of the
samples are positive for coliphage, consider analyzing a field blank. A field blank for coliphage is collected by
pouring about 3-L of sterile water into a clean and sterile 3-L sample
bottle. If needed, contact the Ohio
District (Rebecca Bushon or Donna Francy) for more information on field blanks
for coliphage. Coliphage field blank data are not stored in NWIS.
·
Laboratory matrix spikes—Collect two 3-L bottles of ground water from the
same well—one for each E. coli host.
Send both bottles to the ODML for laboratory spiking. (Be sure to also include the regular 3-L
environmental sample.) BE SURE TO PROVIDE THE LABORATORY WITH AT LEAST 2-DAYS
ADVANCE NOTICE THAT
YOU ARE SENDING A MATRIX SPIKE FOR COLIPHAGE. Coliphage matrix spike data are
not stored in NWIS.
Laboratory matrix spikes
are used to assess the effect of matrix interferences (from a GW type) on
coliphage analysis. Known concentrations
of coliphage are added to 3 L of GW for each host. The 3-L of GW are then aliquoted into three
replicate bottles and each bottle is tested for the presence or absence of
coliphage. For the test to be
acceptable, at least one out of three bottles must be positive for
coliphage.
Updated February 2004