Bacteria Testing Protocol

SDCK LABS BACTERIA ANALYSIS: WATER SAMPLES INTRODUCTION The overall procedure of bacterial analysis includes diluting the sample, transferring either the Colisure or Enterolert reagent into the sample, pouring the sample into an IDEXX tray, sealing the IDEXX tray, and incubating the sealed IDEXX tray. There are two incubators in the lab. One is set at 35°C and another at 41°C. Samples mixed with the Colisure reagent should be placed in the 35°C incubator. Samples that are mixed with the Enterolert reagent should be placed in the 41°C incubator. All samples should be prepared ahead of time placed in the incubators all at once because opening the incubators causes temperature fluctuations. Both incubators should be within one(1) degree of the desired temperature. MATERIALS 1 1 1 1 1 2 2 2 2 2 1 1 2 2 1 1 1

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gallon distilled water bottle of sanitizing solution (alcohol hand sanitizer or anti-bacterial soap) roll of paper towels 100ml whirlpack water sample 100ml whirlpack BLANK (reagent and distilled water) reagent capsules - Colisure - Enterolert - 120 ml sterile plastic specimen containers - sterile 10ml plastic pipettes - pipette bulbs - IDEXX Quanti-Tray®/2000 sample trays - Sealer - silicon feeder for sealer - incubators - thermometers - long wave UV lamp @ 365nm - autoclave - disposable 2ml plastic pipette

***IDEXX anti-foam if possible. PROCEDURE I. PREPARING WORK AREA

1) The two incubators need to be set to 41°C(Entero) and the other to 35°C(E.Coli). 2) Wash hands with anti-bacterial soap or hand sanitizer. 3) Clean the work area with sanitizing solution. 4) Place sterile paper towels down covering the work area. 5) Have sterile pipette (still sealed), pipette bulb, specimen container, 100ml whirlpack water sample, and reagents out and ready to process. 6) Turn on the Sealer and let it warm up for at least 15 minutes. II. PREPARING SAMPLE 5) Label trays with... a. Date b. Site ID c. Temperature of Incubation d. Name of Lab Technician e. Lab Location f. Dilution g. Colisure Grid or Enterolert Grid Colisure Grid for Total Coliform and E-Coli(Figure 1) Large Cells

Small Cells

MPN (most probable number)

Total Coliform E. Coli (24 hours) Figure 1: Example grid label written directly onto the trays using the Colisure reagent. Grid is used to count the number of positive and negative cells at the end of the incubation period. Positive cells are then then matched to the IDEXX Quanti-Tray®/2000 MPN Table(step 23).

Enterolert Grid for Enterococci(Figure 2) Large Cells Enteroco cci (24 hours)

Small Cells

MPN (most probable number)

Figure 2: Example grid label written directly onto the trays using the Enterolert reagent. Grid is used to count the number of positive and negative cells at the end of the incubation period. Positive cells are then then matched to the IDEXX Quanti-Tray®/2000 MPN Table(step 24).

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Open sterile specimen container and add approximately 20 ml of distilled water. 7) Empty one reagent capsule into the specimen container by cracking it directly above the container. See example video 1 below. Example Video 1 @ 2 minutes: http://www.idexx.com/water/colilert/colilert300_en.jsp 8) Gently swirl the specimen container to dissolve the reagent completely. 9) Next take the 100ml whirlpack water sample and gently twirl the whirlpack to homogenize the sample. You can also squeeze the bottom of the whirlpack gently with your hand. 10) Open the sterile plastic pipette and attach bulb. 12) Using the pipette extract 10ml from the homogenized 100ml whirlpack water sample. Release the 10ml into the specimen container. 13) Fill the specimen container up to the 100ml mark as accurately as possible with distilled water. You can do this by using the disposable 2ml plastic pipette. Minimize bubbles and froth, otherwise the tray will not seal correctly. 14) Open IDEXX tray. Hold tray with left hand, while cells are facing up, and with your right hand pull on the white tab. 15) With the tray open, have partner pour the 100ml solution from the specimen container into the tray. 16) Place tray into silicon feeder by matching up the cell sizes. Make sure the notch in the feeder is situated towards the bottom right of the small cells on the tray. 17) With the white plastic facing up, the feeder should be inserted into the sealer with the small cells going in first. This will ensure that the sample solution in the tray does not spill out. 18) Repeat steps 5-17 to prepare all trays for all sampling sites. All trays should be placed into the incubators at the same time! 19) Finally, once all trays have been prepared, record onto each tray (where previous information has been recorded) the ‘time of the start of incubation’!!! 20) Place trays into their corresponding incubators. - Colisure reagent trays into the incubator set to 35°C. These will incubate for a period no less the 24 hours. - Enterolert reagent trays into the incubator set to 41°C. These will incubate for a period no less the 24 hours. *Do not stack trays more then ten high!!! Be careful not to block air circulation in the incubators!!! III. POST-INCUBATION: Recording the Results (24 hours later) 21) Turn on UV lamp to allow for a 10min warm-up.

22) Remove the trays form the incubators and add to the record the time and date of removal on each tray. 23) Recording and interpretation of the results for the Colisure trays are as follows. These results must be interpreted within 24 hours after the incubation period. a. A positive signal for the total coliform will have a magenta color. Count the number of large cells and small cells that have turned magenta. Record onto the grid along the Total Coliform row(see figure 1). b. A positive signal for the E-Coli will have, in addition to the magenta color, a fluorescent blue color. Any cells that do not have the magenta color and fluoresces does not count as a positive! Using the UV-lamp @ 365nm carefully shine the light onto the tray by holding it a few inches above the cells. Make sure that the lamp is not directed at anyone’s eyes! Count the number of large cells and small cells that fluoresce blue. Record onto the grid along the E-Coli row(see figure 1). c. Refer to the IDEXX Quanti-Tray®/2000 MPN Table and record the MPN number in the last column of the grid. Link below. http://www.idexx.com/water/refs/096323501.pdf

24) Recording and interpretation of the results for the Enterolert trays are as follows. These results must be interpreted within 4 hours after the incubation period. a. A positive signal for the Enterococci will have a fluorescent blue color. Using the UVlamp @ 365nm carefully shine the light onto the tray by holding it a few inches above the cells. Make sure that the lamp is not directed at anyone’s eyes! Count the number of large cells and small cells that fluoresce blue. Record onto the grid along the Enterococci row(see figure 2). b. Refer to the IDEXX Quanti-Tray®/2000 MPN Table and record the MPN numbers in the last column of the grid. Link below. http://www.idexx.com/water/refs/096323501.pdf Clean-up: To be determined. For now simply store the trays in a cool container with a lid.