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NUR AQILAH BINTI HAPIZ
(F18B0292)
NUR AZMINA BINTI AZHAR
(F18A0143)
NURFA SHAHIRAH SERIGER BINTI MOHD JENDI SERIGER
(F18A0169)
KHAIRINA SAFI BINTI MOHD MARZUKI
(F18B0047)
SITI NURSOLEHAH BINTI ROZAINAL
(F18A0226)

DISCUSSION

1. What do you understand in the practical ?

As we know, consumers prefer a safe and healthy milk product selection in market. The dairy hygience and sanitation in industry is important things to be considers to prevent milk infection,dirt or any contamination. In this practical, we understand that by use the viable counted method, bacteria is easy to see with naked eye and can be counted which based on the number of colonies formed in petri dish containing specific growth media after the incubation. With this method, industry can improving the hygiene quality of milk. It is useful to monitor milk quality and to help diagnose potential on-farm problems/deficiencies associated with abnormally high counts and poor quality milk. Farmers can be advised about possible hygiene breaches enabling them to correct for leaks, insufficient cleaning or cooling in the milking system or health problems in the herd.

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

However, the viable count method has several disadvantages that the flaw is that it is selective and thus unbalanced. Which bacteria in a mixed population can grow depends on the conditions of the growth conditions, such as the composition and substrate concentration used, as well as other factors such as temperature.

What we understand for this practical is bacteria can be counted by using the plating method, which based on the number of colonies formed in the petri dish containing specific growth media. The viable count method is a direct counting methods.  It is easy to perform and not require specialized equipment. Next, the bacteria growing a colony on a nutrient medium is easy to see with naked eye and easy to be counted after incubation.

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

The Resazurin test is used to determine the quality of raw bulked milk. This is essentially a quick bacteria estimation and can be performed as a 10 minute pass or fail test, a 3 hour triple reading test, or the regular and widely regarded 1 hour test. Resazurin gives milk a distinctive blue colour, and the test is based on the capacity of bacteria in the milk to diminish the blue dye. The quality of the milk is determined by recording the degree of colour change from blue to mauve, purple, pink, and eventually colourless after a specified duration of incubation, or the time required to reduce the dye to a predetermined colour.

There are several method to check for sanitation. First, measure one-tenth of one cc. of 0.05 percent Resazurin (Eastman) dye solution and place in a sterile test tube. Then milk samples will be obtained directly from the weigh can by mixing the milk with 10 cc. a narrow bore-type dipper that has been sanitized into a sterile test tube. Next, incubate the sample at 98°F for one hour in a covered water bath. Lastly, read the samples and record as quickly as possible after the incubation period.

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

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1) Ng Pui Sie (F18A0105)
2) Nur Nazihah Binti Mat Pozian (F18A0268)
3) Khadijah bt Mohammed Faiseol (F18A0256)
4) Siti Nurhidayah Binti Rozali
(F18A0276)
5) Nur Zihan Binti Zakaria (F18A0271)

DISCUSSION
1. What do you understand in the practical?
Milk is easy to spoil since it is a good place for microorganism growth. So, in this practical, we understand the importance of the sanitation test to ensure the food safety of milk for consumption by to optimize storage hygiene and lower the risk of spoilage. Next, we discovered how to determine the proper method of microbial examination in milk by counting the number of bacteria (CFU) per millilitre of sample. The serial dilution technique is suitable for microorganism isolation in the viable count method in the milking process.

2. What is the advantages and disadvantages of this method?
Two advantages of the methods are the equipment required to perform viable plate counts is much cheaper and easily obtainable in every microbiology lab compared to other methods. Next, The number of bacteria in a specific class also possible to be determined, even in mixed populations.
On the other hand, the disadvantages of viable plate counts are extra caution should be taken during dilution and plating to avoid errors because even a minor dilution error can have a significant impact on the final numbers. The colony arises from one cell also have to be expect.

3. Name 1 other method to check for sanitation and elaborate the method.
The other method to check for sanitation is Preliminary incubation count. Preliminary incubation count method is one of the ways in measuring the survival of bacteria in milk, especially detecting bacteria growing in cold environment known as psychrotrophic bacteria. This method can be done by first incubating the milk samples at 55°C for 18 hours before plating on violet red bile agar(VRBA). The plates will then being incubated for approximately 24 hours at 32°C. The preliminary incubation count is measure of hygiene practices of the farm operation and indicator of the shelf life of milk. Ideally, farms should strive for a low preliminary incubation count below 10,000 cfu/mL.

REFERENCES
Tamilee D. Nennich. (May, 2006). Texas Dairy Matters: Milk Quality Tests. Retrieved from http://agrilife.org/texasdairymatters/files/2010/07/5-06-Milk-Quality-Tests-Updated-12-16-1.pdf

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

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By: Selene Reeves and D. M. Amaral-Phillips Printable Version

The goal of any dairy producer is to maximize milk production while producing quality milk. Somatic cell count or SCC is a universal method to determine presence of an infection based on the number of somatic cells within the milk. A low SCC is one measure of milk quality within a herd. However, there may still be evidence of milk quality issues even when SCC is low. The preliminary incubation(PI) count is a unique test that has the ability to detect bacteria that grow in cold environments called psychrotrophic bacteria. By holding milk at 55°F for 18 hours, these bacteria can be detected. If these bacteria are not controlled, milk bonuses can be lost. From a producer standpoint, PI count can be used as a reflection of cleanliness of equipment and cows. Ideally, farms should strive for a low PI (<10,000 cfu/mL). Increases in PI can be linked to the following factors:

Milking Equipment Water Wash Temperature -A major contributor to a high PI count is using wash water that is not hot enough when cleaning milking equipment with detergent. Make sure the wash water temperature is between 155 to 170°F at the start. Drain water when wash water temperature reaches 120°F, if not before.

Acid Sanitation - The last step in equipment sanitation after using hot water and detergent should be an acid wash to prevent bacterial growth over a longer period of time.

Rubber Parts - Rubber parts, i.e. hoses, gaskets, and liners, need to be properly cleaned, air dried and changed on a set schedule as indicated by the manufacturer. Cracks within the rubber parts can house bacteria and lead to a high PI count.

Bulk Tank - The bulk tank should be cleaned after each milk pick-up similar to the milking equipment. Wash water with detergent should start washing at greater than 155°F and conclude before the wash water is at 120°F.

Sanitizers - an iodine or chlorine sanitizer is recommended to use just before milking.

Refrigeration - Poor cooling of milk in a bulk tank will allow psychotropic bacteria to grow. Milk should be down to 40°F within 1 hour of milking for the first milking stored in the bulk tank and down to 36 to 38°F within an additional hour. For subsequent milkings, blend temperature should remain below 45°F and then cooled as for the first milking. Storing milk at temperatures of 33 to 35°F may not completely kill temperature resistant bacteria, but it can significantly slow their growth.

Cleanliness of Udder - Teats should be sanitized then wiped with a clean towel until free of dirt and moisture. Various species of bacteria will grow on the udder without proper teat preparation and drying practices.

Teat Cup Liners - Liners need to be cleaned after each milking and replaced after the recommended number of milkings.

The goal of PI is to detect hygiene practices on a farm. Therefore, a high PI count can be an indication of improper habits on the farm. Proper sanitation practices in accordance with the list above should be applied to potentially lower the PI count.

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Factors that Increase PI Count in Milk | Animal & Food Sciences

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By: Selene Reeves and D. M. Amaral-Phillips Printable Version

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MUHD FAUZAN BIN MOHD NIZAM (F18B0085)
MUHAMAD FARID BIN MOHD ZIN (F18A0073)
NUR SHAFINAZ BINTI ABDUAL MALEK (F18B0163)
NURUL DINI FADLIN BINTI ALIAS (F18A0186)
MIZA SABRINA BINTI MOHD RADZI (F18A0258)

1. What do you understand in the practical?
In this practical, we understand that sanitation is important to ensure milk quality in milk processing technique which is via viable count method.  In this practical, serial dilutions of a sample containing viable microorganisms are plated onto a suitable growth medium. Also via the viable count method, we are able to determine the microbial load of processed and fresh milk by counting the number of colonies and accounting for the dilution factor.

2. What are the advantages and disadvantages of this method?
One of the advantages of the viable count method is the sensitivity of this method only requires counting living bacteria only. The microorganism can be counted easily in any concentration by plating appropriate dilution. In water analysis, bacterial populations are at low density, hence the solution can be concentrated before counting. Besides that, the equipment needed to carry out this method is readily available as they are cheap.

Selective medium is also possible in determining the number of bacteria of a certain class, including mixed populations. Thus, this method is selective and therefore biased becomes the major disadvantage. The composition and pH of the medium are included in the nature of the growth used as well as the conditions that determine which bacteria in a mixed population can grow such as temperature. Since there is no universal set of conditions that permits the growth of all microorganisms, it is impossible to enumerate all microorganisms by a viable count method.

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

The other method is microbial testing because one of the advantages of microbiological testing is to measure the effectiveness of sanitation. As it can vary the collection device, hydro swab, or culture swab depending on where the sampling does. The advantage of microbial testing is it allows us to evaluate the effectiveness of cleaning in niche or hard-to-inspect areas or areas where soil and food tend to accumulate, which could allow for the growth of bacteria.

References
1. Microbiology, V. (n.d.). Viable plate counts. Retrieved October 27, 2021, from https://instr.bact.wisc.edu/book/displayarticle/106.
2.https://www.qualityassurancemag.com/article/how-do-i-know-if-my-equipment-is-really-clean-and-food-safe--sanitation-manager/
3.https://www.sas.upenn.edu/LabManuals/biol275/Table_of_Contents_files/14-Enumeration.pdfhttps://www.sas.upenn.edu/LabManuals/biol275/Table_of_Contents_files/14-Enumeration.pdf

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How do I know if my equipment is really clean and food safe? - Sanitation manager

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Resources for food safety and quality professionals on regulations, processes, practices and industry news.

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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

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1. NUR SYUHADA BINTI SAZALI (F18A0270)

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.

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1. MOHAMMAD ROHAIMI BIN ABDUL AZIZ
(F18A0259)

2. NIK NUR AKMAL ALIAH BINTI ABDUL RASHID
(F18A0261)

3. MOHAMAD IKMAL TAJUDDIN BIN ABDUL HADI (F18A0294)

4. MUHAMMAD SYUKRI AMIR BIN ZAINUDDIN
(F18A0094)

5. AMIRAH FAZILA BINTI JAMALUDIN
(F18A0001)

DISCUSSION

1. What do you understand in the practical

The practical is about how to count the number of bacteria (CFU) per millilitre of sample. What we do understand in this practical is the importance of the test of sanitation to determine the amount of bacteria found in the sample to optimize the hygiene of storage, lessing the risk of spoilage. Then, we do understand the formula used to determine the colony forming unit (CFU) using number of colonies time dilution factor divided by the volume of culture plate.

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

For the advantages of this method, this method will only count the living bacteria that contains in the sample due to it sensitivity. Next, the equipment that is used is cheap and easy to be obtained especially in the lab compared to other method. The medium that is used in this method can help to determine the amount of bacteria in certain class even though it is in a mixed population.
Disadvantage of this method is not adhering to equipment cleaning & sanitisation standards can result into contamination through harmful substances such as milk residues, allergens, microorganisms or chemical residues.

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

Methylene Blue Dye Reduction Test, commonly known as MBRT test is used as a quick method to assess the microbiological quality of raw and pasteurized milk. This test is based on the fact that the blue colour of the dye solution added to the milk get decolourized when the oxygen present in the milk get exhausted due to microbial activity. The sooner the decolourization, more inferior is the bacteriological quality of milk assumed to be. This test is widely used at the dairy reception dock, processing units and milk chilling centres where it is followed as acceptance/rejection criteria for the raw and processed milk

1. Mix the milk sample thoroughly to distribute the fat uniformly.

2. Add 10ml of milk sample in a test tube.

3. Then add 1ml of standard methylene blue solution in this test tube and invert the test tube to mix it properly.

4. After that, place the test tube in a water bath at 37°C (99°F) for 30 minutes, and Cover the bath with a lid.

5. After 30 minutes of incubation observe the sample and check for discoloration, and make subsequent readings at hourly intervals thereafter.

6. After each reading, remove decolorized tubes and then slowly make one complete inversion of remaining tubes.

7. Record reduction time in whole hours between last inversion and decolorization. For example, if the sample were still blue after L 5 hours but was decolorized (white) at the 2.5-hour reading, the methylene blue reduction time would be recorded as 2 hours.

8. Decolorization is considered complete when four-fifths of the color has disappeared.

REFERENCES

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

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

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/

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Methylene Blue Dye Reduction Test for Assessing the Raw Milk Quality | Dairy Knowledge Portal

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Methylene Blue Dye Reduction Test for Assessing the Raw Milk QualityMethylene Blue Dye Reduction Test, commonly known as MBRT test is used as a quick method to assess the microbiological quality of raw and pasteurized milk. This test is based on the fact that the blue colour of the dye solution adde…

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NURUL WAFA BINTI SOBRI (F18A0200)
MOHD FAREEZMAN BIN ZAMZURI (F18B0066)
MUHAMMAD AMEER TAQIUDDIN BIN MD NOOR (F18B0080)
MOHAMAD NABIL BIN RAZALI (F18A0059)
NURUL AWATIF BINTI MOHD YUSRI (F18A0185)

DISCUSSION

QUESTION 1

In this practical, we understand that there is bacterial contamination along the procedures used during the handling of milk. It is important to determine the number of bacteria in the milk to ensure hygienic milk processing and avoid any contamination on the milk.  We can test the sanitation of the milk processing by using the viable count method and it was a common method. By doing this test, we could ensure the food safety of milk for consumption.

QUESTION 2

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

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

2. Miyamoto, T., Murata, Y., Kobayashi, H., Shimoyae, M., Kamikado, H., Noda, N., & Iio, M. (2005). Enumeration of Viable Counts in Raw Milk Using the Automated Fluorescence Microscopic Method. Biocontrol science, 10(4), 147-154.

3. Lumen. (n.d.). Counting Bacteria. Retrieved October 27, 2021, from https://courses.lumenlearning.com/boundless-microbiology/chapter/counting-bacteria/.

4. VPC Viable Plate Count. https://mospace.umsystem.edu/xmlui/bitstream/handle/10355/69341/5-VPC%20Viable%20Plate%20Count.pdf. (n.d.). Retrieved October 27, 2021.