The viable plate count method is a count of viable or live cells. It is based on the principle that viable cells replicate and give rise to visible colonies when incubated under suitable conditions for the specimen. (OpenStax CNX, 2018) A measured amount of liquid culture is inoculated onto a plate. The plate is incubated and the colonies that result are counted. The results are usually expressed as colony-forming units per millilitre (CFU/mL). This method has been done in many industries such as food, medical, aquatic, and research laboratories for the routine determination of cell numbers. However, the viable plate count method has its own advantages and disadvantages. 

The first advantages of the viable plate count method are only for counting living, which is often an important issue. Any concentration of microorganisms can be easily counted when the appropriate dilution is plated. It is even possible to concentrate a solution before counting such as water analysis, where bacterial populations are usually at low density. Secondly, a viable cell count allows identifying the number of actively growing/dividing cells in a sample. It relies on bacteria growing a colony on a nutrient medium. Thirdly, the colony becomes visible to the naked eye and the number of colonies on a plate can be counted after incubation. No need to use other equipment to count the colonies. Fourthly, the equipment necessary for performing viable plate counts is readily available in any microbiology lab and cheaper compared to the other methods. Finally, by using a selective medium it is possible to determine the number of bacteria of a certain class, even in mixed populations.

Next, the first disadvantage of viable plate count is the assumption that each colony arises from one cell. There are species where cells grow together in clusters. Secondly, even one error in dilution can have large effects on the final numbers. Great care must also be taken during dilution and plating to avoid errors. Thirdly, if the incubation period used is too short, some colonies may be missed. The rate at which bacteria give rise to an observable colony can also vary. Fourthly, the temperature of incubation and medium conditions must also be optimized to achieve the largest colonies possible so that they are easily counted. Finally, this technique takes time depending on the organism. One day to several weeks might be necessary to determine the number of CFUs that were present when the experiment started.

QUESTION 3

Thus, we are suggesting other methods to check for sanitation which is an automated fluorescence microscopic method. An automated fluorescence microscopic method was developed for the measurement of viable bacterial counts with a blue light-emitting diode for the detection and counting of viable bacterial cells having esterase activity. It was applied to the detection and enumeration of viable bacteria in milk. This method was fast and more accurate compare to the conventional method. In the automated analysis of viable bacteria, bacterial cells were recovered from milk after the treatments with EDTA and Triton X-100, stained with 6-CFDA, and the stained cells were counted with the automated microscopic method. Many esterified fluorescent dyes remain nonfluorescent until digested by intracellular enzymes, where a fluorescent product is released. Cells become fluorescent only after substrate cleavage by functional cytoplasmic enzymes and product retention by intact membranes. It is necessary to treat the milk sample with EDTA and Triton X-100 to avoid the interference of casein micelle and fat in milk. Salmonella Enteritidis expressing the enhanced green fluorescent protein (S. Enteritidis-EGFP) was used to clarify the relationship between the fluorescent bacterial counts by the automated microscopic method and the viable counts by the conventional plating method. Viable counts by the plating method and the counts of 6-CFDA stained bacterial cells by the automated microscopic method were determined by using various raw milk samples. However, the only disadvantage by using this method is human error. By using microscopes in a long time, it would lead to the inability to distinguish between dead bacterial cells and living cells.

REFERENCES

Significance of Hygiene in the Dairy Industry : Practicing the Basic Hygiene Standards. (2020, January 9). Retrieved from Lindstorm : https://lindstromgroup.com/in/article/significance-of-hygiene-in-the-dairy-industry-practicing-the-basic-hygiene-standards/

 

2. NUR NAZURAH MARYAM BINTI ROSLAN (F18A0281)

3. NURIN JAZLINA BINTI MOHAMAD FAUZI (F18A0282)

4. NURUL ADZHA BINTI HAJI HAMIZI (F18B0178)

5. NUR ALYANI BINTI MOHAMAD AMIN (F18B0133)

 

DISCUSSION

1. What do you understand in the practical? 

                We understand that this practical is to check the presence of microorganism growth in food generally. This practical also shows method that can be use to test the food hygiene to make sure it is safe to be consume. For example, milk, as milk is a highly perishable food and contained many nutrients, a strict hygienic practice is very important in the milk industry. Thus, from this practical, we learn viable count methods for the sanitation of milk processing.

2. What is the advantages and disadvantages of this method? 

The advantages of this method are the technique is simple and easy as it can be counted by naked eyes and this method is sensitive and only counting living bacteria. Any microbiology lab will have the essential equipment to run this method. This method also is cheap in comparison to other methods. Besides, even in mixed populations, it is possible to determine the number of bacteria of a specific group by using a selective medium.

However, the disadvantages of this method are this test assumes that each colony arises from one cell. Great caution must be taken during dilution and plating to avoid errors and the time it takes for bacteria to form a visible colony can also be varied. The temperature of incubation and medium conditions must also be optimized to achieve the largest colonies possible so that they are easily counted. Last but not least, this method is time consuming. 

3. Name 1 other method to check for sanitation and elaborate the method.

The methylene blue reduction test (MBRT) is another standard milk quality test that is also very easy to conduct. The mixture of methylene blue and milk is initially blue in colour. The blue colour fades to white when the oxygen level in the milk decreases due to bacterial activity. The time it would take for the colour to change indicates the amount of bacterial contamination in the milk sample. The faster milk becomes a different colour, the more contaminated it is. The MBRT test can be used to grade milk, which can help the milk processor decide whether or not to process it further.

 

REFERENCES

Microbiology, V. (n.d.). Viable plate counts. Retrieved October 27, 2021, from https://instr.bact.wisc.edu/book/displayarticle/106. 

Milk harvesting and hygiene - CSIRO publishing. (n.d.). Retrieved October 27, 2021, from https://www.publish.csiro.au/ebook/chapter/9780643107403_Chapter14. 

Boundless microbiology. Lumen. (n.d.). Retrieved October 27, 2021, from https://courses.lumenlearning.com/boundless-microbiology/chapter/counting-bacteria/#:~:text=A%20viable%20cell%20count%20allows%20one%20to%20identify,of%20colonies%20on%20a%20plate%20can%20be%20counted. 

Dairy knowledge portal. Methylene Blue Dye Reduction Test for Assessing the Raw Milk Quality | Dairy Knowledge Portal. (n.d.). Retrieved October 27, 2021, from https://www.dairyknowledge.in/content/methylene-blue-dye-reduction-test-assessing-raw-milk-quality. 

 

1. NURATHIRAH BT A RAHMAN (F18A0168)
2. NUR SHAHIDA NAFISAH BINTI BUHAIRA
(F18A0164)
3. NUR HANIS BT ROMLI
(F18A0149)
4. NUR FARIZAH BINTI MOHAMED SABERI
(F18B0308)
5. SITI NORFAIRAH BINTI AMRAN
(F18A0275)

DISCUSSION

1. What do you understand in the practical?

Milk has become the most consumed food all around the world. Somehow, milk is easy to spoil since it is a good place for microorganism growth. In this practical, we learn the importance to ensure milk quality in milk processing techniques to prolong the shelf life. Other than that, we learned to determine the correct method of microbial examination in milk using the viable count method. In the milking process, the serial dilution technique is suitable for microorganism isolation in the viable count method.

2. What are the advantages and disadvantages of this method?

The advantage of this method is firstly, it easy to count the number of bacteria because the dilution reduced a dense culture of cells. Secondly, using this method the equipment used is easier to get and cheap. Next, the microbial spread/mix evenly in the diluent.
However, the disadvantages of this method are this test of serial dilution method is first, it need to incubation period is needed at least 1 day. Secondly, the miscalculation for dilution will affect the final CFU. Next, this method also can risk contamination. And lastly, the result of colonies may be too small to see.

3. Name 1 other method to check for sanitation and elaborate the method

The other method to check sanitation is Polymerase Chain Reaction (PCR) analysis. This technique is used to amplify small segments of DNA. Polymerase Chain Reaction (PCR) is a sensitive molecular method to identify bacteria in food contamination. PCR test is highly accurate, sensitive, durable, fast, and it can even be automated. 

The advantage of using PCR analysis using a rapid filtration technique is this technique simple and rapid. This technique only takes around 8 hours to go from collecting a milk sample for enrichment to viewing the electropherogram. Next, PCR capable of amplifying DNA fragments corresponding to serotypic antigens and virulent factors of E. coli O157:H7 would allow for unambiguous detection and identification (Hu et al. 1999). Furthermore, real-time PCR methods can remove the requirement for gel electrophoresis and staining by simultaneously amplifying and detecting amplicons (Bellin et al.2001; Hein et al. 2001). The limitations of these techniques are becoming less cost-effective when performed with a multi-organism. Moreover, the cost of using these techniques is expensive due to supply costs, machinery fees and training expenses.

Method-
Firstly, sample collection from raw milk of different milch animals and pasteurized milk. Then, for milk inoculation, the culture of E. coli O157:H7 was mixed in 100 ml LB broth and permitted to grow at 37°C for 3 hours for nondividing cels dilution. Following that, serial dilutions were performed, and the number of bacteria in each dilution was assessed by plating on LB broth. Then, incubated in an orbital mechanical environment shaker at 160 rpm for 24 hours. Next, preparation of DNA sample from milk. A 045-m membrane filter (HA type, 13 mm diameter) was used to filter 5ml of broth. Then, the tube was heated to 95°C for 10 minutes to lyse the cells. After that, ten microlitres of the lysates were submitted to PCR amplification. For this PCR analysis, primers used in this study were ECPAL-GGCAATTGCGGCATGTTCTTCC-3' and ECPAL-R 5'-CCGCGTGACCTTCTACGGTGAC-3'. The ECPAL-L and ECPAL-R primer pairs are specific for all Escherichia coli strains and it will amplify a 280-bp control segment from the exeC gene. Denaturation at 94°C for 10 minutes was followed by 40 cycles of denaturation at 94°C for 30 seconds and polymerization at 68°C for 90 seconds, followed by a final extension at 72°C for 5 minutes. Following amplification, 10 l of the PCR reaction was electrophoretically separated on agarose (2%) gel and visualized with ethidium Bromide.




REFERENCES

Ben-David, A., & Davidson, C. E. (2014). Estimation method for serial dilution experiments. Journal of Microbiological Methods, 107, 214–221. https://doi.org/10.1016/j.mimet.2014.08.023

Cheptsov, V., Tsypina, S., Minaev, N., Yusupov, V., & Chichkov, B. (2018). New Microorganism Isolation Techniques With Emphasis on Laser Printing. International Journal of Bioprinting, 5(1). https://doi.org/10.18063/ijb.v5i1.165

Kaag, C. S., & Croft, V. F. (1990). Online Fulltext Article Retrieval. Technical Services Quarterly, 7(2), 29–41. https://doi.org/10.1300/j124v07n02_04

Wu, S. J., & Kado, C. (2004). Preparation of milk samples for PCR analysis using a rapid filtration technique. Journal of Applied Microbiology, 96(6), 1342–1346. https://doi.org/10.1111/j.1365-2672.2004.02259.x

R. A. Ledford, G. F. Senyk, E. Kotsides, and E. T. Wolff. (1983). Preliminary Incubation Method for Detection of Coliforms in Commercially Processed Milk Samples. Journal of Dairy Science 66:1607-1610. Retrieved from https://www.journalofdairyscience.org/article/S0022-0302(83)81981-X/pdf

Selene Reeves and D. M. Amaral-Phillips. (n.d.). Factors that Increase PI Count in Milk. Retrieved from https://afs.ca.uky.edu/content/factors-increase-pi-count-milk

References :

1. Enumeration of microorganisms. (n.d.). Retrieved October 27, 2021, from https://www.sas.upenn.edu/LabManuals/biol275/Table_of_Contents_files/14-Enumeration.pdf

2. W.D. Barrett, H. Rutan, J.A. Keenan, The Resazurin Test—Its Use and Practicability as Applied to the Quality Control of Raw Milk, Journal of Dairy Science, Volume 20, Issue 11, 1937, Pages 705-709, ISSN 0022-0302. Retrieved from https://www.sciencedirect.com/science/article/pii/S002203023795731X