Title Enumeration of Bacteria in Milk Samples and Presumptive Test for Coliforms.

Introduction Quality of milk depends on the number and the types of microorganism present in milk. However, milk can be contaminated at any stage; hence care is to be taken to adopt sanitary practices in each stages of production. The quality of milk can be assessed by enumeration of total bacteria and testing for the presence of coliforms. Objective To count the number of bacteria in milk sample (unpasteurized and pasteurized samples) and do the presumptive test for coliforms. Material Milk samples (sample A pasteurized milk and sample B unpasteurized milk), sterile water, petri plates, pipettes, nutrient agar, incubator, colony counter, beakers, maker pen, test tubes and sticker.

Experiment 1: Enumeration of Bacteria in Milk

Procedures 1. Firstly, procure both milk samples and label as sample A (pasteurized milk) and sample B (unpasteurized milk). 2. Dilute the samples A with sterile distill water for the isolation of microorganism from water. 3. Then, transfer 1mL of the sample A serially in sterile distill water to make dilution of; 1:10, 1:100 and 1:1000. Refer picture below.

4. Transfer 1mL of sample A; from every each dilution ratio (1:10, 1:100 and 1:1000) into petri dish and pour melted and cooled tryptone glucose yeast extract agar medium. 5. Repeat procedures two until four for Sample B. 6. After the agars have been solidified, incubate the plates at 350C for 24-48 hours. 7. Finally collect the result.

Result Sample A 1:10 Colonies Count CFU/mL 286 2.86 X 103 Sample B 1:10 748 7.48 X 103

1:100 117 1.17 X 104

1:1000 18 1.8 X 104

1:100 532 5.32 X 104

1:1000 474 4.74 X 105

Calculation 1. CFU counts for sample A (pasteurized milk) a) 1.0mL dilution of 1 : 10 CFU = = 286 X 10 2.86 X 103 CFU/mL

b) 1.0mL dilution of 1 : 100 CFU = = 117 X 100 1.17 X 104 CFU/mL

c) 1.0mL dilution of 1 : 1000 CFU = = 18 X 1000 1.8 X 104 CFU/mL

2. CFU counts for sample B (unpasteurized milk) a) 1.0mL dilution of 1 : 10 CFU = = 748 X 10 7.48 X 103 CFU/mL

b) 1.0mL dilution of 1 : 100 CFU = = 532 X 100 5.32 X 104 CFU/mL

c) 1.0mL dilution of 1 : 1000 CFU = = 474 X 1000 4.74 X 105 CFU/mL

Conclusion: Objective of the experiment is achieved.

Experiment 2: Presumptive Test for Coliform

Procedures 1. Firstly, transfer 1mL from the highest dilution (1:1000) sample A and B from Experiment 1 into 9mL of MacConkey broth tubes for production of acid and gas. 2. Then mix them together and place a single Durham tube in inverted position inside the broth containing tube and incubate them into incubator for 24 hours. 3. Finally, observe the presence of acid and gas production.

Result

Acid production (yellow) Sample A Sample B Yes Yes

Gas production

No No

Conclusion: The presence an acid product causes a color change Mac Conkey broth agar from purple-violet turn to yellow occurs. No gas has been produce. So the presumptive test for coliforms is negative. No further test should be proceeding.

Discussion MacConkey agar is a differential plating medium recommended for use in the isolation and differentiation of lactose-fermenting organisms. It is selective by the presence of specific inhibitors. Indication for the presence of coliforms bacterial should be presence of acid and gas production in Mac Conkey broth agar but from Experiment 2, only production of acid is presence. After incubate for 24 hours Mac Conkey broth agar change, it color from purple-violet turn to yellow and none gas presence is being observe in Dulham Tube. So the presumptive test for coliforms bacterial is negative and no further test should be processing. Coliform organisms are mainly members of the Escherichia and the Aerobacter genera. They are all aerobic and facultative gram negative non-spore-forming bacilli capable of fermenting lactose with the production of gas. Milk supports the growth of a variety of bacteria including pathogenic one; other types of bacterial that gas-forming and acid production such as; a) Coliform bacteria b) Clostridium peifringens c) Clostridium butyricum It is important to be able to detect microorganisms in food, in particular pathogenic microorganisms or genetically modified microorganisms.

Questions 1. Explain why it is not advisable to thaw and the refreeze food products without having cooked them. 2. Would detection of E. Coli in meat be indicative of contamination or spoilage of the product? Explain. Answers 1. Refreezing is generally not being advised because every each freeze-thaw cycle negatively impacts the quality of the food. The cell walls of food become damaged and leak, so the food will become soft and furthermore, the process does not destroy the entire microorganism. 2. Detection of E. Coli in meat indicative of contaminated of food because E. Coli does not grow on the surface of the meat but it is reaching toward the food by the contaminated. E. Coli is a pathogenic bacteria and it can cause the death.