Protocols: IDEXX Laboratory Analysis

SaniPath Laboratory Protocols(IDEXX).pptx

Important note on microbiological assays:

Be sure to use 100 mls of the sterile water or PBS used for washing items or making dilutions as a negative control in each assay. This is especially important if you are purchasing bottled water from a vendor, rather than using lab-prepared filtered or autoclaved water, as bottled water is not always sterile.

I. Introduction

Coliforms are a group of bacteria that are normal inhabitants of the intestinal tract of humans and animals. Historically, they were defined as the group of facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas and acid formation within 48 hours at 35^oC. Fecal coliforms are a sub-group of the coliforms that grow at 44.5^oC. It is actually more accurate to refer to them as “thermotolerant coliforms” because there are some non-fecal sources of these bacteria. One specific fecal coliform of interest is E. coli because it is almost exclusively fecal in origin. (There are some reports of E. coli detection in pristine tropical waters that suggest non-fecal sources.) While some strains of E. coli are harmless, other types of E. coli, such as the enteropathogenic and enterohemorrhagic strains, can cause severe disease in humans. The presence of fecal coliforms, and especially E. coli, in water is considered an indication of fecal contamination. However, the absence of fecal coliforms does not guarantee that the water is free of pathogens. Some pathogens, such as viruses, tend to survive longer than fecal coliforms and may still be present in water after the fecal coliforms have died off.

IDEXX Colilert Quanti-Tray system.

The IDEXX Quanti-Tray system is a testing technique that determines the most probable number (MPN) of coliforms in a water sample based on predetermined statistical parameters. One hundred mL of water (or, for highly contaminated samples, 100mL of a diluted water sample) are mixed with the Colilert reagent, poured into the Quanti-Tray and sealed. The compartments in the tray each contain a specific volume of water + reagent, similar to using a test tube. The tray is incubated (at 37.5^oC for total coliforms and 44.5^oC for fecal coliforms) for 18 to 24 hours. During this time, the target organisms will interact with the reagent, causing compartments that contain at least one coliform to turn yellow, while those that do not contain any coliforms will remain clear. Small and large cells are counted and the MPN is determined according to the IDEXX table found at the end of this hand-out. Wells that contain at least one E. coli will turn yellow and fluoresce under UV light. We will also examine the Quanti-Tray under UV light to look for fluorescence in order to determine the MPN of E. coli in our samples.

The Colilert reagent in the Quanti-Tray system uses “patented Defined Substrate Technology® (DST®) to simultaneously detect total coliforms and E. coli. Two nutrient-indicators, ONPG and MUG, are the major sources of carbon in Colilert and can be metabolized by the coliform enzyme β-galactosidase and the E. coli enzyme β-glucuronidase, respectively. As coliforms grow in Colilert, they use β-galactosidase to metabolize ONPG and change it from colorless to yellow. E. coli use β-glucuronidase to metabolize MUG and create fluorescence. Since most non-coliforms do not have these enzymes, they are unable to grow and interfere. The few non-coliforms that do have these enzymes are selectively suppressed by Colilert's specifically formulated matrix.” (Description from IDEXX website)

II. Equipment and Supplies

1. IDEXX Colilert

· WhirlPak bags containing samples or 100mL flasks/bottles

· PBS (Phosphate Buffered Saline)

· Sterile pipets

· Sterile graduated cylinders

· IDEXX Quanti-Tray

· Colilert-24 Reagent (blue and white box)

· IDEXX sealer

· Rubber tray (red one only)

· Sharpie Marker

· 125mL Erlenmeyer flasks (orange cap)

· Incubators at 37.5^oC

III. Procedure

1. IDEXX Quanti-tray Method for fecal coliforms and E. coli

A. Sample dilution

You will process multiple dilutions of your environmental samples. Follow the SaniPath dilution protocol. We might adjust the dilutions after the first round of sampling. The total volume of water processed must be 100 ml! That means, for example, for a 1 ml sample volume, you must mix the 1 ml of your sample water with 99 ml of PBS to give a total volume of 100 ml.

B. IDEXX Processing procedure

Turn on the IDEXX sealer. It will take 15 minutes or so to warm up. Using a Sharpie, label the backs of the IDEXX trays with your initials, sample ID, dilution, volume of sample per 100 ml, date, and time.
Open the reagent packet and tap into the Erlenmeyer flask with your final dilution to be tested. Close the cap to avoid contamination while you prepare your sample.

**Note: adding less than 100ml to each tray will result in unfilled wells and an invalid test result. Adding more than 100ml will cause the liquid to overflow the tray during sealing, potentially damaging the sealer. It is important that you measure carefully!**

Swirl the flask with the sample until the reagent has dissolved. Allow foam to settle completely.
Without touching the inside of the tray, pull the tab to open the top of the tray, flexing the plastic to allow a gap between the plastic and the paper back. Be careful not to tear the paper back or tab. Have your partner pour in the sample. If there are bubbles in any of the cells, gently “flick” the back of the try with your finger so that the bubbles float to the top of the tray. Tiny bubbles are not a problem, but try to get rid of any large bubbles or foam.
Place the sample tray face down (paper side up) in the rubber tray so that the compartments align.
Gently push the tray into the IDEXX sealer. When the sealer senses the tray, it will automatically pull the tray in, and you can stop pushing. Do not try to force the tray through the sealer.
The tray will come out the back of the sealer. Do not pull it out until the sealer has stopped.
Place the tray in the incubator at 37°C.
Record the date and time that the sample was placed in the incubator and your name (Lab Operator) on the laboratory form.

C. Interpretation

You must return to the lab 24 hours after you have processed your sample to examine your trays.
Examine the Quanti-Tray under UV light and record the number of small and large cells that fluoresce (light blue). Use the MPN chart to determine the concentration of E. coli in your sample.

Count the number of small and large cells that fluoresce

Example: 32 large wells and 5 small wells (MPN count is 57.3)

Recommended SaniPath dilutions to be plated by Sample type:

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