Microbial Testing for E. coli and Fecal Coliforms

Note:  Instruments are calibrated before each use.

BE SURE THE WATER BATH TEMP IS 35^oC USING THERMOMETER, NOT BATH DISPLAY.

The flame is used to sterilize the forceps (tweezers) used to handle the filters.  Be careful around the flame and let the forceps cool for at least 30 seconds before using them.  Dip the forceps in the beaker of ethanol and flame every time before moving a filter.  If the flame needs more alcohol or is out, find a Stream Team Manager for help.

1.  Invert an ampule of m-Coliblue24 (in the foil bag) two or three times to mix well. 

2.  Take two Petri dishes for each sample and label the tops with the sample number, date, and amount of sample used (2 mL and 10 mL for each sample) using a Sharpie.  This procedure will be repeated for a 2 mL and 10 mL Petri dish for each sample.  Pour contents of the m-Coliblue ampule evenly over the sterile absorbent pad in each sterile Petri dish.  Replace the lid once ampule is poured to prevent contamination. 

3.  Carefully unwrap the corner of a new sterile membrane filter. 

Remove the membrane filter from the package using flamed

 forceps.  Make sure to separate the filter from the blue backing, which

should be discarded with the packaging.  A new funnel must be used

 each sample day.  Place the filter in the funnel, grid side up.  Use

Hyclone® water to make sure the filter is wet before sample is added.

4.  Shake sample well to mix thoroughly.  Using the 1 mL pipetter, place 2 mL of sample into funnel.  For the second Petri dish, 10 mL of the sample will be pipetted into the funnel. Use a new pipette tip for each sample. 

5.  Turn on peristaltic pump to operate filter, being sure the large flask is placed at the end of the out-flow tube.  

-  To operate the pump, set the first button to forward, the second button to fast, and the dial to 10.  If running one sample be sure flow

 valve is only pumping from the correct side; if running two samples

 be sure flow valve is open for both.

6.  When all of the fluid from the sample is pulled out of the funnel rinse the funnel walls with approximately 20mL of Hyclone ® water. 

7.  When water is no longer in funnel, turn off the

vacuum (or the valve if second sample is still filtering) and lift off the top of the funnel.  Using sterile (flamed) forceps transfer the filter to the previously prepared Petri dish, grid side up.    Replace the lid.

- Using a rolling motion to place the filter on the pad to prevent air bubbles from being trapped between the two.  Be sure the filter is touching the pad.

8.  Turn the dish upside down and place in

 a Whirl-Pak bag.  Close the bag and place it

 in the water bath at a temperature of 35 deg. C for 24 hours. 

9.  Petri dishes will be removed after 24 hours to examine the filters for colony growth.  Red colonies are total coliforms, and blue colonies are E. coli. 

-  If colonies are too numerous to count (i.e. > 200) we note this to be sure to dilute samples from that location in the future.

The Petri dishes will be disposed of by depositing 2 ml of 15% bleach solution into each dish, bagging the dishes, and treating as normal refuse.