SW PROTOCOL

SAMPLING, ANALYSIS, AND REPORTING RESULTS

 

Overview

Samples are enumerated for Escherichia coli (E. coli) in the field by use of the modified mTEC method.  See Escherichia coli using the modified mTEC method.   For each sample, a series of volumes are plated to obtain the ideal plate count of 20-80 colonies per plate. A list of recommended and required quality-control samples for bacteria is 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. 

 

Preparations for the field

Equipment and supplies.  Refer to the list of Equipment and Supplies.  For rinsing the filtration equipment and making dilutions, use “saline” buffer made by Ocala (Q373BACT, 250 mL volume and Q421BACT, 99-mL volume) or sterile, buffered water, PO4/MgCl2 made by Ocala (Q453BACT, 250-mL volume) and the 47-mm, 0.45-mm sterile membrane filters (OCALA, Q353BACT).  Modified mTEC medium is purchased as dehydrated agar from Becton-Dickinson (BD), catalog number 214884. 

 

Clean and sterilize equipment.  Wash and gently scrub equipment (filter funnels, graduated cylinders, caps and nozzles, 1-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, caps and nozzles, 1-L sample bottles) for microbiological sampling at each site. 

 

Heat incubators. Heat two incubators or chambers—one at 35°C and another at 44.5°C. It takes about 2 hours for the incubators to reach desired temperatures

 

Prepare modified mTEC plates.  Prepare mixture as directed on the dehydrated medium bottle (45.6 grams agar to 1 liter deionized water).  Heat to boiling with a stirring rod on a hot plate.  Add approximately 100-mL of agar to each dilution bottle.  Autoclave for 15 minutes.  Store dilution bottles at 4°C for up to six months.  Melt agar either for 5 minutes in an autoclave or in a beaker of boiling water on a hotplate until melted.  Be sure to loosen the cap prior to melting.  Allow the agar to cool until it is warm (50-60°C).  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.

 

 

Sample collection

Collect bacteria sample. Because surface-water sampling for NAWQA Source Water Quality Assessments is done near the intake to drinking-water plants, samples for E. coli will be collected using the hand-dip (grab) sampling method.  See Stream water sample collection.  Do not obtain the E. coli sample from churn or cone-splitters, as these devices are not sterile.  Instead, use a separate, sterile, 1-L bottle to collect the sample for E. coli.  Be sure to leave at least 2 inches of headspace in the bottle.

 

After collection, immediately chill samples in a cooler with ice or in a refrigerator at 1 to 4°C.  SAMPLES NEED TO BE PLATED FOR BACTERIA WITHIN 6 HOURS OF COLLEC­TION.

 

Plating samples for bacteria [Refer to the USGS National Field Manual, Biological Indicators for detailed membrane-filtration procedures.] 

 

Label your plates and gather forms.  Decide on appropriate sample volumes to obtain at least one plate in the ideal-count range of 20-80 colonies.  If the bacterial water-quality of the stream is unknown, plate the following sample volumes:  100, 30, 10, 3, 1, 0.3, and 0.1 mL.  For less polluted waters, the three smallest volumes may be omitted.  For more polluted waters, the two highest volumes may be omitted.  As you become more familiar with the water-quality of the stream, the number of sample volumes plated can be reduced.

 

On the lid of the plate, record the site, date, sample-collection time, and the volume (or FB for filter blank and PB for procedure blank).  Use the SW sample and positive control report form for the regular sample and the positive control culture, and the SW sample and replicate report form for the regular and associated replicate samples. (See Quality control section below for information on positive controls and replicates).

 

Assemble membrane filtration apparatus and gather supplies. Place the filter fun­nel in a manifold attached to a peristaltic pump. Use the same filter funnel to plate the series of volumes for one sample. Use a different sterile filter funnel for each surface-water sample.

 

Prepare sample dilutions (if needed).  To accurately deliver sample volumes less than 1.0 mL, you must prepare dilutions of the sample.  For example, for more polluted waters, plate 30- and 10-mL volumes of a 1/100 dilution (these equal 0.3 and 0.1 mL of actual sample volumes, respectively).  To prepare a 1/100 dilution, label a 99-mL bottle of saline buffer as the 1/100 dilution and loosen the bottle cap.  Place the pipet bulb on a 1-mL pipet (kept sterile in the original wrapper).  Shake the sample at least 20 times.  Immediately remove a 1-mL aliquot from the center of the sample and transfer it to the dilution bottle.  If the bottle sits on the counter for more than a few seconds before removing the 1-mL aliquot, you need to shake the bottle again—bacteria settle quickly to the bottom of the sample bottle. 

 

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.  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. Filter the sample in order of smallest to largest sample volume.  Use a graduated cylinder to measure volumes greater than 10 mL. You can also use a 25-mL pipet to measure volumes of 30 mL (go to –5 mL on the pipet).  Use a 10-mL pipet to measure volumes from 2 to 10 mL. Use a 1-mL pipet to measure 1-mL samples volumes. 

 

FOR VOLUMES LESS THAN 10 ML, PUT ABOUT 10-ML OF BUFFER INTO THE FUNNEL BEFORE ADDING THE SAMPLE. This ensures even distribution of the sample across the membrane.

 

FOR ALL VOLUMES, RINSE THE FILTER FUNNEL WITH BUFFER AFTER FILTERING THE SAMPLE AND BEFORE REMOVING THE FILTER. This ensures that the bacteria are washed off the side of the fun­nel adequately.

 

BE SURE TO THOROUGHLY SHAKE THE SAMPLE BEFORE MEASURING THE VOLUME.  If you don’t, you will not get a representative subsample for plating.  If the bottle sits on the counter for more than a few seconds before removing the sample aliquot for plating, you need to shake the bottle again.

 

The three bolded and capitalized steps are VERY IMPORTANT!  

 

Filter the procedure blank (these are included after every tenth sample).  The procedure blank (PB) tests the adequacy of the rinsing procedure.  This is done by filtering a 50-100 mL aliquot of sterile buffered water after filtering the sample, using the same filter funnel.

 

Incubate plates. Invert the plates and incubate for 35°C for 2 hours.  Transfer all plates to an incubator set at 44.5°C for 22 hours. Fill out the report form. 

 

Count E. coli.  Count all red or magenta colonies as E. coli.  Using a dissecting microscope or portable illuminated magnifier to count colonies is highly recommended.

 

Disposal.  After counting, place all plates in an autoclave bag and autoclave for 30 minutes at 121oC. Dispose of the bag in the trash.

 

Calculate concentrations and report results

Calculate the colonies per 100 milliliters (col/100 mL) as described in the USGS National Field Manual, Biological Indicators.

 

 

The results from the membrane-filtration tests are entered into NWIS using the following parameter code:

90902, E. coli on modified mTEC agar, colonies per 100 mL

 

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 and 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 on modified mTEC 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 1 mL volume filtered to get a result of >8,000 colonies per 100 mL, with a value qualifier of “k”.

 

 

Quality assurance

·         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 are 35 ± 0.5°C or 44.5 ± 0.2°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 recorded 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.  

The following QC is required:

·         Filter blanks—a 50-100 mL aliquot of sterile buffered water filtered before the sample. 

Filter blanks are used to qualify associated environmental sample.  Filter blanks results are not stored in NWIS.

 

·         Procedure blanks—a 50-100 mL aliquot of sterile buffered water filtered after the sample.  Include procedure blanks if the analyst is inexperienced in membrane filtration and (or) if water is suspected to have high concentrations of target colonies.  If the procedure blank is positive, this indicates that rinsing techniques may be inadequate.  Procedure blanks are used to qualify associated environmental sample.  Procedure blank results are not stored in NWIS. 

 

·         Positive controls of E. coli are required for surface-water sampling.  They are especially important when first using the method—to assess if a good dilution series is obtained and whether the analyst can correctly identify E. coli.   Positive controls for surface water can be obtained from the Ohio District Microbiology Laboratory (ODML).  The controls come in 100-mL aliquots in dilution bottles, and are plated in the field using 30-, 10-, 3-, and 1-mL sample volumes.  The positive-control bottles must be plated the same day they are received from the ODML.  Use the SW sample and positive control report form to record results.  Instructions for analyzing the positive controls are in SW control instructions.  

 

Positive control results are not stored in NWIS.  Send your results to the Ohio District (amgbrady@usgs.gov). 

 

·         Negative-controls are not recommended for surface-water sampling because the agar is very stable and target colonies are easily discerned from non-target colonies. 

 

·         Field blanks—Pour about 250 mL of sterile buffered water into a clean and sterile sample bottle.   Process as you would any other sample.

 

Field blank results are stored in QADATA with the following codes:

o       Sample-medium code is “Q” for quality control

o       Sample-type code is “2” for blank

o       Parameter code 99100 (blank-solution type), is “70” for sterile buffered water PO4/MgCl2 or “60” for sterile saline buffered water (lot number is not required)

o       Parameter code 99101 (source of blank water) is “80” for Ocala

o       Parameter code 99102 (blank-sample type) is “100” for field

 

·         Replicates—The replicate is collected as a split sequential grab sample.  This means that two bottles are collected and each bottle is plated twice.  Use the SW sample and replicate report form to record results of the regular and replicate samples. 

 

Enter the 1st sample in QWDATA with the following codes:

o       Sample medium is “9” for surface water

o       Sample type is “7” for replicate

o       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

o       Parameter code 99105 (replicate type) is “50” for split sequential

 

Enter the 2nd to 4th samples in QADATA with the following codes:

o       Sample medium is “R” for surface water

o       Sample type is “7” for replicate

o       Parameter code 99105 (replicate type) is “50” for split sequential

 

NOTE: Offset sample times so that splits of the same grab sample are closer in time than splits of different grab samples. For example: 1300, 1301, 1305, and 1306 where 1300 is the 1st environmental sample (grab sample 1, split 1), 1301 is the 2nd sample (grab sample 1, split 2), 1305 is the 3rd sample (grab sample 2, split 1) and 1306 is the 4th sample (grab sample 2, split 2).

 

Updated February 2004