sop bacteria

Galveston Bay Foundation

Enterococcus

Standard Operating Procedure

9/4/2012

Table of Contents

Section Page1.0 Test Method.........................................................................................2

2.0 Applicable Matrix.................................................................................2

3.0 Detection Limits...................................................................................2

4.0 Scope and Application.........................................................................2

5.0 Summary of Test Method.....................................................................2

6.0 Interferences.......................................................................................2

7.0 Safety..................................................................................................2

8.0 Equipment and Supplies......................................................................3

9.0 Reagents and Standards......................................................................3

10.0 Sample Collection, Handling, and Preservation ..................................3

11.0 Quality Control.....................................................................................3

12.0 Procedure............................................................................................3

13.0 Data Reduction, Reporting, and Validation..........................................4

14.0 Method Performance...........................................................................5

15.0 Corrective Actions for Out-of-Control Data..........................................5

16.0 Pollution Prevention and Waste Management.....................................5

17.0 References...........................................................................................5

Revision 0 9/12 Page 1

1.0 Identification of Test MethodThis Standard Operating Procedure is for the Enterolert™ test (Enterolert) using Enterolert™ and Quanti-Tray®. Enterolert™ is based on IDEXX’s Defined Substrate Technology®, and is used for the detection of enterococci such as E. faecium and E. faecalis in fresh and marine water.

2.0 Applicable Matrix Aqueous (non-potable water).

3.0 Detection Limit and Method Reporting Limit The Enterolert™ test should detect one (1) enterococcus in 100 ml of water within 24 hours.

4.0 Scope and ApplicationThis method covers the quantification of enterococcus in non-potable environmental water samples that are monitored for enterococcus levels.

5.0 Summary of Test Method The Enterolert medium, based on IDEXX’s Defined Substrate Technology® is used for the detection of enterococci such as E. faecium and E faecalis in fresh and marine water. When enterococci utilize their β-glucosidase enzyme to metabolize Enterolert’s nutrient-indicator, 4-methylum-belliferyl-β-D-glucoside, the sample fluoresces under a UV (365-366 nm) lamp. Enterolert™ detects enterococci at 1 /100 ml within 24 hours incubation at 41°C.

6.0 InterferencesMarine waters or samples with excessive turbidity or organic content (causing background color) should be diluted at least ten fold to prevent interference in interpreting results. (TCEQ SWQM protocol).

7.0 Safety

The MSDS sheet of each reagent is kept on file in the lab and is available to all analysts involved in chemical analysis.Disposable gloves and lab coats should be worn when performing analysis or working with reagents. Report all injuries and major spills to the Supervisor.

8.0 Equipment and Supplies IDEXX Quanti-Tray Sealer w/97 well insert 41 ± 0.5C incubator 10-50ºC thermometer, Teflon coated, NIST traceable, with 0.5ºC

divisions Long-wave UV Lamp (365-366 nm)


IDEXX Quanti-Tray 10 ml pipets – sterile, disposable Negative Comparator (Sterile water + Enterolert incubated for 24

hours)

Note: Do not dilute samples in buffered water. The Enterolert reagent is buffered.

9.0 Media and Standards IDEXX Enterolert™ medium

10.0 Sample Collection, Handling, and Preservation Samples to be analyzed for enterococcus must be held at < 6°C and

arrive at the laboratory within 6 hours of collection. Samples must be analyzed within 2 hours of arriving at the

laboratory. Samples that arrive after the 6-hour time limit will not be analyzed. Samples are collected only in WhirlPaks treated with sodium

thiosulfate.

11.0 Quality Control Perform a sterility check on each lot of media prior to first use. Note: IDEXX only guarantees that Enterolert™ media is

enterococcus free, therefore the term “sterility check” does not strictly apply. This check should be performed to determine that the media is enterococcus free and free of autofluorescence only and recorded as such.

Record the temperature of the incubators before placing trays on shelves and when removing trays.

Test UV lamp for proper fluorescence monthly using an IDEXX Colilert® comparator.

Check the Quanti-Tray® Sealer for performance monthly. Pour 100 ml of colored water into a Quanti-Tray® and seal as usual. There should be no color observed outside any well.

Perform bacteriological duplicate analyses on 10% of samples tested and at least once per set of samples received if possible.

Perform sterility check on commercially prepared sterile dilution water as stated in the QA Manual

12.0 ProcedureThe standard test volume for surface waters is 10 ml diluted with 90 ml sterile DDI water to 100 ml total volume. Sample dilutions must be used within 20 minutes of preparation.

Turn on Quanti-Tray® Sealer 10-15 minutes before starting analysis. The sealer is ready to use when the green light comes on.

Record the time sample is analyzed on the worksheet.


Prepare 1:10 dilution: Pipet 10 ml of sample into a 90 ml sterile dilution blank for a total

volume of 100 ml. Shake well to dissolve reagent. Place the rubber insert on the input shelf with the large cutout

facing away from the Sealer Label the Quanti-Tray/2000 with the sample number and volume. Confirm that reagent has dissolved completely. If not, shake dilution

again. Carefully pour the entire contents of the sample into a sterile

Quanti-Tray®. Lightly tap the small wells to release trapped air. Put the Quanti-Tray® onto the rubber insert, well side down, with

the open end facing away from the sealer, making sure that the tray is properly seated in the rubber insert.

Slide the Quanti-Tray® into the sealer until the motor begins to draw it in.

Remove the Quanti-Tray® from the back of the sealer. Repeat the above steps for all dilutions Place the Quanti-Tray/2000 in a 41.0°C ± 0.5°C incubator, well

side down, within 30 minutes of adding reagent. Do not stack more than 6 high.

Incubate for 24 hours @ 41.0 ± 0.5C Turn sealer off when not in use.

Do not read samples before 24 hours. Samples can be incubated up to 28 hours.

After 28 hours, the following guidelines apply:Lack of fluorescence is a VALID NEGATIVE TEST and can be reported as such. Fluorescence (positive result) is NOT VALID and should not be reported.

Detect fluorescence using the IDEXX viewing box and UV Lamp. Blue fluorescence is positive for enterococcus.

Record the number of positive wells on the worksheet Record date and time analysis is complete.

13.0 Data Reduction, Reporting, and Validation Determine the MPN for each sample using the IDEXX MPN tables or

the IDEXX MPN Generator. Multiply each result by the dilution factor. Round the final result to 2 significant figures. Report results as enterococcus MPN/100 ml.


14.0 Method Performance and Estimation of Uncertainty Enterolert™, based on IDEXX’s Defined Substrate Technology®,

detects and confirms enterococcus in water at the level of 1 /100 ml in 24 hours at 41°C.

Estimation of Uncertainty – The Lab defers to IDEXX documentation cited at IDEXX.com/water. There are many technical papers and approvals available there that document the false positive and false negative statistics of the Enterolert™ medium.

Calculate precision of duplicate analyses for each different type of sample examined, according to the procedure in Standard Methods, 21st edition, 9020B.8.b. The Precision Criterion is set with the first 15 samples received. If the range of routine duplicates is greater than the set criterion, there is greater than 99% probability that the laboratory variability is excessive. Determine if increased imprecision is acceptable; if not, discard all analytical results since the last precision check.

Note: If a sufficient number of samples (15) are not received to calculate a precision criterion, use the default criterion stated in the Clean Rivers Program QAPP.

15.0 Contingencies for Handling out of Control Data Data will not be recorded if incubator temperature is discovered

out-of-range (41.0± 0.5°C). Upon sample receipt, if the samples are not properly cooled, they

will be invalidated.16.0 Pollution Prevention and Waste Management

All Quanti-Trays® that have any positive wells (fluorescing) must be placed in a sealed container and taken offsite to be sterilized in a 30 minute autoclave cycle and disposed of in an appropriate manner.

17.0 References “Standard Methods for the Examination of Water and Wastewater”:

21st Edition, American Public Health Association, 2005. Enterolert™ Test Insert, IDEXX Laboratories, Inc. ASTM Standards, volume 11, section 11.02, 2006
