Lab #6 Coliform Determination by the Membrane Filter Technique

Lab #6 Coliform Determination by the Membrane Filter Technique

Introduction

            In this lab, you might notice some similarities from lab 5 however, we test a waste stream instead of surface water. The purpose of lab 6 was to become familiar with the membrane filter techniques for plate counting total and fecal coliform bacteria. The difference between fecal and total coliform is simple. Fecal coliform is primarily a form of total coliform that consist of bacteria that rest generally in the intestines of an animal as well as the feces. Generally fecal coliform range from 100,000 to about 1 million per one milliliter of solution. Which is about 10 percent of the total coliform. “Total coliforms include bacteria that are found in the soil, in water that has been influenced by surface water, and in human or animal waste.”(Department of Health)

Methods & Materials

            The materials that were used for this lab included a pipette/ plastic pipette tips, a sample of waste water collected by the TA,  Phosphate Buffered Saline Solution (PBS), about 8 cuvets, a total of 12 dishes, a filter apparatus with a pump, tweezers, filters and some fire to spice things up a bit. That’s hot! TISSSS

            Let’s start this thang. Using your 8 cuvets, we put 9 mL of the PPS buffer solution in the last 8 cuvets. Then of course we diluted each from the first cuvet of straight wastewater. These dilutions were made in order to reduce the coliform counts to roughly 20-80 colonies on each culture dish. We had used the 10^-1 , 10^-2 , and the 10^-3 solutions to maximized our results. We then made culture dishes while we had an assigned member start the preparation of the filter apparatus. An ampule of M-Endo is squeezed into each culture dish as to completely cover the pad along the bottom of the dish, the filter apparatus is utilized to pull each dilution through the filter.

For the filter to be used we needed sterile tweezers, carefully removing the filter from the sterile package and placing onto the filter apparatus with the grid side facing up. Next, the top of the apparatus is set in place and 0.1 mL of the 10^-1 dilution and 9.9 mL of PBS are pipetted in. The suction pump is turned on and the liquid is completely pulled through the filter. Then, DI water is sprayed onto the sides in order to wash down any extra bacteria that did not make it through the filter. Once the water is completely pulled through, the pump is turned off and the filter is removed again with sterile tweezers and is placed onto the M-Endo saturated pad inside of the culture dish. It is important for no air bubbles to become trapped underneath the filter, therefore the filter is rolled onto the pad like a band aide on the skin. Repeating this twice for each solution. Having 6 in total. Now incubate for 22 to 26 hours to allow the coliform colonies to grow. Now count and now you are all done J

Results

            Our results are as followed for our group as well as one others. Each number is a colony

	Total Coliform			Fecal Coliform
	10^-1	10^-2	10^-3	10^-3	10^-4	10^-5
Sample 1	200	45	9	1	0	0
Sample 2	250	56	10^-3	0	0	0

Example:

Cfu/mL=(200)/((10^-1)*0.1)…. = 20000

Analysis

            If you are looking above, our data for our particular group was for total coliform. The Fecal coliform was another groups data as a comparison. We only diluted the waste water sample slightly therefore our numbers are clearly much higher as well as it being total instead of fecal. However, given the idea that fecal is supposed to be 10 percent of total. Clearly something went wrong here because most of the fecal was zero. Therefor we can say our data is invalid. I believe however our total coliform representation is decently close to what it could be.

Citations

 "Department of Health." Coliform Bacteria in Drinking Water Supplies. New York State Department of Health,, n.d. Web. 30 Sept. 2016.