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.
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 35oC. Fecal coliforms are a sub-group of the coliforms that grow at 44.5oC. 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)
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 37.5oC
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
**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!**
C. Interpretation
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: