MICRO LAB

It was also expected that there would be little to no chloroforms n the milk that had been pastured as compared to the raw milk which had not been pastured. To test the water sample we performed an MAN (most probable number) test and a series of tests including an indolent production test, Methyl-red test, Vogues-Proteases test and a citrate utilization test.

This series of tests is commonly referred to as the Iambic. To test the milk we performed both a standard plate count test and a chloroform plate count test.After conducting all tests on the water samples it was found that fecal chloroforms were not found in the bottled or tap water but were found in the other water samples and that one f these water samples was confirmed to contain Escherichia coli specifically. After performing all tests on the milk samples it was found that all samples with the exception of whole milk contained microbes with raw milk containing the largest number of microbes. It was also found that raw milk was the only milk sample that contained chloroforms.Introduction Water is known to serve as a mode of transmission for pathogens. This is why tests are conducted on our water to detect the presence of indicator organisms which may be linked to pathogenic organisms (Littleton, 2010).

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Specifically, eater is tested for the presence of chloroforms which are bacteria (often referred to as enteric bacteria due to being from the Intercontinental family of bacteria) commonly found in the intestines of birds and mammals. This type of bacteria does not produce endosperm and ferment lactose which produces acid and gas within forty-eight hours of testing.Fecal contamination of water can contribute to dangerous pathogens being in water sources. Fecal chloroforms indicate that the water may contain pathogens found in the intestines such as E. Coli which is often used in determining whether water is safe for human consumption or not Littleton, 2010). A multiple tube fermentation test, commonly known as a most probable number (MAN) test is a common way to detect chloroforms in water by injecting lactose broths with a sample of the water sample being tested, if bubbling or color change appear, it can be assumed that chloroforms are present in the water sample.

To confirm whether or not the water sample contains chloroforms, a sample of the water is then placed on EMBED agar which contains lactose, and then performing a gram stain to retest for fermentation of lactose. To differentiate between the different bacteria of the Intercontinental family, series of tests called the Iambic are performed on the water samples and determine whether or not the bacteria is a fecal chloroform (Littleton, 2010).Due to the many nutrients in milk, it makes it an ideal place for microbes to grow which increases the risk of microbes growing at a fast rate causing spoilage of the milk – increasing the risk of spreading disease.

Like water, milk can contain dangerous pathogens that are due to fecal contamination (Littleton, 2010). While milk is pastured to reduce pathogenic microbes which cause spoilage and disease, bastardization does not sterilize milk. Two tests can be performed on milk samples to determine if the milk contains bacteria and if the bacteria found in the milk are fecal chloroforms or not.One is a standard plate count which will allow bacterial Scuffs to be counted and the other is a chloroform plate count which will determine whether the bacteria found in the standard plate count are chloroforms (Littleton, 2010). I hypothesized that there would be a presence of microbes which could possibly be chloroforms in the fresh water sources but not in the tap or bottled water. I hypothesized this due to the fact that bottles and tap water are homicidally treated or filtered while fresh water sources receive no filtration or chemical treatments.Fresh water is also exposed to the environment and is more likely to have been contaminated by the organisms in the environment.

I also hypothesized that most milk samples would contain a certain number of bacteria but no chloroforms (due to the fact that bastardization does not sterilize or kill off all the bacteria entirely but only makes it safe for human consumption) and that raw milk would contain the greatest amount of bacteria and possibly contain chloroforms due to raw milk being unappreciated.Materials and Methods Quantifications of Microbes in Water First Lab session 10 ml pipette and pump (green) 1. 0 ml pipette and pump (blue) 5 double-strength lactose fermentation broths with Durham tube 10 single- strength lactose fermentation broths with Durham tube Water sample Olivine’s eosin methyl blue (EMBED) agar plate Second Lab Session 1 lactose fermentation broth with Durham tube 1 TTS slant Third Lab Session Gram stain reagents 2 MRS.. Broths 1 Trenton broth 1 Simmons citrate agar slant Fourth Lab Session Ampoule of VIA (alpha-naphtha) reagent Ampoule of BP (potassium hydroxide) reagentAmpoule of indolent (Kava’s) reagent Four lab sessions were needed to test the water samples for fecal chloroforms. Each group was assigned a different water sample from either bottled water, Lake Byron, Campus Creek, Tap water, or Littleton Lake.

The sample our group was assigned was the Campus Creek water sample. For the first lab session we began by labeling 5 double-strength and 10 single-strength lactose broths with our group information, section number and which sample we were testing. We labeled five of the single-strength tubes 1. Ml and the other five 0.

1 ml.We then hook up the water sample vigorously and then transferred mm’ of the water to each of the five double-strength lactose broths, 1. Ml of the water to the single- strength lactose labeled 1. Mi, and 0.

1 ml of the water to the single-strength lactose labeled 0. Ml. After transferring the water samples to the broths, the tubes were placed in the incubator at 37 co for 24 hours. After 24 hours the tubes were examined to see if any of the tubes had bubbles (indicating gas production) and if there was a change in color from red to yellow (indicating production of acid).To confirm gas-producing bacteria were chloroforms, we chose one positive be and inoculated an EMBED plate and incubated the inverted plate at ICC for an additional 24 hours and then at room temperature until the next lab session. For the second lab session we observed the EMBED plate looking for colonies with a metallic sheen or glossy sheen.

We then selected a suspected chloroform colony and inoculated a TTS slant and a lactose broth and labeled each with our group name, section and type of water sample.We then incubated the tubes at ICC for 24 hours and then at room temperature until the next lab session. For the third lab session we observed the lactose broth for acid and gas production. We then selected a colony from the TTS slant and performed a gram stain. Next, we labeled one Trenton broth, one citrate slant, and two MRS..

Broths. One MRS.. Broth was labeled MR.

. And the other was labeled UP. Each tube was then inoculated with a confirmed chloroform (gram negative, non-endosperm forming rod that fermented lactose).

For the fourth and final lab session we performed an Iambic test.For the methyl red test we added 5 drops of methyl red to the culture and shook it gently, a change in color of liquid to a red color would indicate a positive reaction and no color change indicates a negative reaction. For the Vogues-Proteases test we added 1 ampoule of VIA reagent and 1 ampoule of BP reagent and agitated the tube vigorously to cause the butadiene to be oxidized to action. A positive test is indicated by the media turning red within 30 minutes, if the media does not turn red this indicates a negative test.We then examined the citrate slant, a bright blue color would indicate the organism utilized the citrate as its source of carbon (positive test) and a green color would indicate a negative test. Lastly, we examined the Trenton broths and added 1 ampoule of indolent reagent to the broth and did not agitate the tubes. A red ring at the top of the tube indicates a positive test and a beige/yellow ring indicates a negative test.

We then recorded all results of the experiment in our lab manuals.Quantification of Microbes in Milk Standard Plate Count 2 sterile Mimi water blanks 1. Ml pipette and pump 6 Petri dishes 2 ml bottles of Tropic Soy Agar (TTS) Milk sample Chloroform Plate Count Milk sample and dilutions from standard plate count 1 . Mil pipette with pump 4 Petri dishes 1 ml bottle of molten Violet Red Bile Agar (VERA) Each group was given a different sample of either half and half, whole milk, housecoat milk, butter milk or raw milk to test for microbes. The first experiment performed was a standard plate count.We started the experiment by labeling the bottom of each Petri dish with one of the following dilutions: 1; 1:10; 1:100; 1: 1,000; 1:100,000. We also labeled one of the water blanks A and the other B. We then aseptically pipettes 1.

0 ml of our milk sample (chocolate milk) into bottle A and agitated vigorously, then drew and released some of the dilution several times to flush the pipette. From bottle A, we then pipettes 1. Ml into the plate labeled 1:100 and 0. 1 into the plate labeled 1:1 ,000. Next, we pipettes 1. Ml of milk dilution from bottle A into bottle B and agitated vigorously.

From bottle B, we pipettes 1. Ml into the plate labeled and 0. 1 ml into the plate labeled We then aseptically poured enough TTS into each plate to cover the bottom of the plate and swirled the plate gently and then gave the plates time to cool and solidify. The plates were then incubated at 37 co for 48 hours and then at room temperature until the next lab session. At the next lab session the plates were observed and all surface and subsurface colonies were counted and the Scuffs were recorded.Any plate with 300 or more colonies was recorded as TNT or too numerous to count. The second experiment performed was a chloroform plate count.

We started the experiment by labeling the bottom of each dish with one of the following dilutions: 1; 1:10; 1:100; 1:1000. We then aseptically pipettes 1. Ml of milk sample into the plate labeled 1 and O. Mil of milk sample into the plate labeled 1:10.

Next we pipettes 1. Ml from bottle A into the plate labeled 1: 100 and 0. 1 ml into the plate labeled 1:1000.We then poured VERA into each plate to cover the bottom and swirled the plates gently. The plates were then allowed to cool and solidify. After the VERA solidified, we poured an additional layer of VERA on the surface of the solidified VERA in each plate to encourage anaerobic fermentation by the chloroform bacteria. We incubated the plates upside down at ICC for 48 hours and then at room temperature until our next lab session. At the next lab session we observed the plates and counted all surface and subsurface colonies and recorded the number of Scuffs.

Plates with more than 300 colonies were recorded as TNT. Lastly, we performed a gram stain on a suspected chloroform colony. Results The results of the MAN test of the water samples showed that all water samples except the bottled water and tap water samples tested positive for chloroforms (see Table 1).

Since bottle and tap water tested negative for chloroforms Iambic tests were not performed on these samples, instead two samples from campus creek and Lake Byron were used for the Iambic tests.The results of the Iambic tests showed that one sample of Lake Byron tested negative for indolent production, positive for methyl red reaction, negative for the UP test, and positive for the citrate slant. The other sample from Lake Byron tested positive for indolent reduction, positive for MR..

Reaction, negative for UP test and negative for the citrate slant. The sample from Littleton Lake tested negative for indolent production, positive for MR.. Reaction, negative for UP test and positive for the citrate slant.One of the samples from Campus Creek tested positive for indolent production, positive for MR.

. Reaction, negative for UP test, and positive for the citrate slant. Our sample (the second sample from Campus Creek) tested positive for indolent production, positive for MR.. Reaction, negative for UP test, and negative for the citrate slant indicating that our sample from Campus Creek contained the LIFO Escherichia coli (see Table 2). Table 1 Results of MAN Tests on Water Samples Water sample ADS lactose broth (ml) AS lactose broth (1. Ml) AS lactose broth (0.

Ml) MAN Confirmed Campus Creek 5 >1600 :telethon Lake aka Byron rap water Bottled water Table 2 Iambic Test Results Indolent MR.. Citrate Lake Byron Littleton Lake After performing the standard plate count, results showed that half and half milk contained 50 total bacteria/ml of milk, whole milk Coifs were TFTP, chocolate milk contained 39 total bacteria/ml of milk, buttermilk contained 7,700,000 total acetate/ml of milk, and raw milk contained the most bacteria at 36,000,000 total bacteria/ml of milk (see Table 3).After performing the chloroform plate count, results showed that the only milk sample to contain chloroforms was the raw milk sample (see Table 3). Table 3 Standard Plate Count and Chloroform Plate Count Results Type of Milk Standard Count Chloroform Count Half and Half 50 ETC Whole Milk TFTP Chocolate Milk 39 gutter Milk 7,700,000 Raw Milk 9,000 Discussion After performing the MAN test on the water samples it was found that the fresh water sample sources tested positive for chloroforms while the samples of tap water ND bottled water tested negative for fecal chloroforms.

These results were predicted in my hypothesis due to tap water and bottled water being chemically treated and filtered while lake and creek water are not normally treated or filtered. My hypothesis is fully supported by the results we received from the MAN test. Due to the fact that no chloroforms were found in bottled nor in tap water, two groups performed Iambic tests on the Lake Byron and Campus Creek samples. The only water sample with results consistent with E. Coli after the Iambic tests was my groups sample from the Campus Creek.After performing the standard plate count tests and chloroform count tests on the milk samples, the results showed that all the samples with the exception of whole milk contained bacteria but that none of the samples of milk contained chloroforms with the exception of raw milk. This is also consistent with what I anticipated in my hypothesis.

I hypothesized that there would be a lower number or no microbes/chloroforms in the milk samples that were pastured than in the raw milk (due to raw milk being unappreciated). Raw milk had the highest standard plate count and was the only sample to contain chloroforms.