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.

Lab #5 Enumeration of Microorganisms

Lab #5 Enumeration of Microorganisms

Introduction

                  The purpose for this lab was to become comfortable with being able to understand the total viable count for enumerating microorganisms. This experiment will allow you to realize just how many microorganisms are harbored in the environment. Now what I mean by viable count is being based on the ability to place any microorganism in a nutrient beneficial environment to watch it grow in population. To do this we use different types of media that closely resemble the environment in which the microbes thrive best. In order to count the amount of microorganisms in the media, we literally use a counting method that resembles just looking at the plate and counting the large specs on the plates. This is one of the oldest techniques in the books and still is currently being used.

                  After collecting a sample of water we would like to test, we can dilute the sample to help counting the plates later on. The goal is to produce one series of plates of about 30 to 300 colonies that have grown on the plate. Thus, sometimes we have to seriously dilute the water sample down 3 separate times at which each is tenfold.  However, the more you dilute the water the lest accurate the counting will be. Now in order to get them to grow we have to plate them on an agar which is nutrients. Then we use a pour method in which we take 1 mL of our solution and pour it on the plate and use a sterile hockey stick like device to spread the sample on the nutrient filled agar.

Methods and Materials

                  Me being the designated water boy for this lab. I collected a sample of lake water next to my apartment out in BFE for this lab. We had taken this sample and tested it in two different types of media. The first is a Luria-Bertani (LB) Agar (nutrient rich) which has several components such as 10 g tryptone , 5 g yeast extract, 10 g sodium chloride and 15 g agar per liter. The second media is our R2A Agar (low-nutrient) that consist of 0.5 g each of yeast extract, protease petone, casamino acids, glucose, and soluble starch; 0.3 g dipotassium hydrogen phosphate; 0.05 g magnesium sulfate heptahydrate; 0.3 g sodium pyruvate; and 15 g agar per liter.

                  We needed to dilute our sample by placing 0.9 mL of buffer mixed with 0.1 mL of the surface water sample. From there, we placed 0.1 mL of that mixture into a sterile cuvet and mixed it with 0.9 more mL of buffer, repeating this process several more times until we had a 10^-7dilution. We used the first 3 dilutions for our test (10^-1 ,10^-2 , and 10^-3) This was because of the said microbes in freshwater lakes to be 10³ to 10⁵ therefore we only diluted up to 3 times for an accurate reading. We then did 3 samples for each of the diluted samples thus ending with 9 total samples for each media. In order to spread the sample on the plates however, we used an old method of spreading with a glass sterile hockey stick like piece. We stroked the liquid on the agar carefully to not rip the agar and made sure it covered the top layer in the plate. We needed to incubate the samples to allow ample amount of growth. After we waited for the growth, we then counted the CFU in each sample. Below is a sample of what they looked like as they progressed down in the dilution process.

Results

            The results of our CFU's counting are listed below. The charts show how many CFU's were counted at each dilution stage.  

LB CFU’s
Dilutions
10-1	10-2	10-3
83	37	4
TMC	85	1
138	98	21

R2A CFU’s
Dilutions
10-1	10-2	10-3
56	9	1
80	11	16
85	26	6

Analysis

                  Of course when picking a media, you have to pay attention to environmental and physiological factors such as how dirty the water is and where you get it from. You have to also keep in mind on what you aim to find in the sample that you wish to find. Each broth has a better result for certain types of bacteria. LB for the faster growers and R2A for the more diverse results. However, in our data above we seemed to get the best results using the LB agar due to the fungi that takes over the R2A agar. However, each plate of the 3 samples are different per dilution, so it was beneficial to make 3 of each because we may not have had any decent data to show otherwise.

                  Now we had only plated 1 mL of our diluted sample per plate because if we had plated any more than that then we would have flooded the agar and it would have just not absorbed into the agar thus rendering our data, if not enough then we wouldn’t be able to spread over the entire plate. After we incubated the plates upside down to prevent condensation from tampering with our results.  

                  The streak plate technique normally yields higher counts due to the type of bacteria that thrive in freshwater lakes and other surface waters. The pour plate technique could potentially wipe out some aerobic bacteria and kill some heat sensitive bacteria due to the temperature of the agar when its poured as well as the anaerobic conditions that some bacteria like.