Quantitative Plating:Paseurization PDF

Preview

Summary

Download Quantitative Plating:Paseurization PDF

Description

Quantitative Plating/Pasteurization Purpose: The purpose of this lab is to observe the effects of pasteurization and to learn about quantitative plating by using various dilutions of raw and treated milk samples, and determining the number of viable cells/mL of sample.

Procedure: 1. Obtain two tubes of raw milk 2. Pasteurize one tube in 63°C water bath for 30 minutes and cool 3. Perform serial dilutions and plate both the pasteurized and the raw untreated milk 1. Label all five petri plates (10-3, 10-4, 10-5, 10-6, 10-7) and the three dilution bottles (10-2, 10-4, 10-6) before beginning dilutions 2. Fill each dilution bottle with 99mL of water 3. Add one mL of the pasteurized milk to the first bottle labeled 10-2 (this will give you the 10-2 dilution) 4. Using a new pipette, measure 1 mL from the 10-2 dilution, and add that to the 10-4 dilution bottle (this will give you the negative 10-4 dilution) 5. Using a new pipette, measure 1 mL from the 10-4 dilution, and add that to the 10-6 dilution bottle (this will give you the negative 10-6 dilution) 6. Using a new pipette, measure 0.1 mL from the 10-6 dilution, and add it to the center of 10-7 plate 7. Using the same pipette, take 1.0 mL from the 10-6 dilution, and add it to the center of the 10-6 plate

8. Using the same pipette, take 0.1 mL from the 10-4 dilution, and add it to the center of the 10-5 plate 9. Using the same pipette, take 1.0 mL from the 10-4 dilution, and add it to the center of the 10-4 plate 10. Using the same pipette, take 0.1 mL from the 10-2 dilution, and add it to the center of the 10-3 plate 11. Repeat this process using the raw/unpasteurized milk 4. Pour in molten agar to each petri plate, gently swirl, and let solidify 5. Once plates are solidified, invert and incubate 30°C

Results:

Pasteurized Milk Sample Dilution 10-3 10-4 10-5 10-6 10-7 Sample Calculation:

CFU Count on Plate TNTC TNTC 6 25 5

CFU/mL of Original Sample TNTC TNTC 6x105 2.5x107 5x107

CFU/mL = 1/10-6 x 25 =106 x 25 =2.5x107 Average Calculation: Cannot take average due to low CFU count

Unpasteurized/Raw Milk Sample Dilution 10-3

CFU Count on Plate TNTC

CFU/mL of Original Sample TNTC

10-4 10-5 10-6 10-7 Sample Calculation:

TNTC TNTC 133 13

TNTC TNTC 1.33x108 1.3x108

CFU/mL = 1/10-6 x 133 =106 x 133 =1.33x108

Average Calculation: Cannot take an average due to low CFU count

Discussion/Conclusions: Quantitative plating works by performing serial dilutions to figure out approximately how many microbes are in the original microbe- dense sample. While this is a relatively easy, low-cost method, it only gives us a low estimation of the number of microbes due to many organisms not being able to grow in culture media. Also, as suggested in our lab data, there is a lot of room for human error and contamination, thus compromising the results. When comparing the 133 CFU count to the accepted value (105), our value of 1.3x108 is much larger, so our milk did not meet acceptable limits for the raw/unpasteurized milk sample. Our data shows our numbers to be outrageously higher than the accepted values. Pasteurization is the process of heating a liquid below the boiling point to destroy microorganisms. Pasteurizing is an easy way to kill harmful organisms and pathogens within food to make it safe for consumption. Also, because the liquid isn’t pasteurized at high temperatures, the nutrients in the food are able to remain intact, while most of that “bad” is removed. We were unable to compare our pasteurized milk data with the accepted value because we could not get an accurate average due to the CFU

count being so low. Though we were unable to get an accurate average, the data still suggests our numbers were much higher than the accepted value of 2x104. Overall, although the milk samples don’t meet the acceptable limit, pasteurization did reduce the number of CFU per milliliter compared to the raw milk sample. The experiment’s results of not meeting the acceptable limits of CFU per milliliter is directly tied to errors made while performing the lab. One cause of error could be from adding extra sample to the first water flasks, messing with the dilutions of the following flasks, thus resulting in “TNTC” samples. Another cause of error could be from contamination in some way, whether it was human contamination, or contamination from the pipettes. This could be why the results show the more diluted sample has more colonies than the less diluted samples, and therefore, it is not possible to accurately estimate the CFU/ml of the undiluted milk sample. Ultimately, it is very important to take high precautions and to be extremely careful during the plating process to prevent contamination....