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Key Topics
- Requirement
- Solution
- 1. Identify the different reasons for microbiological testing of the named product.
- B. Explain the approach you would take in determining the organisms you would include in the test regime. [20 marks]
- C. Identify and characterize the organisms you would test for, with your rationale for their inclusion and an indication of when you would test.
- D. Produce outline microbiological laboratory procedures for three of the organisms you have selected, including a pathogen, a quality indicator and an indicator of poor hygiene practice
- REFERENCES:
Requirement
Microbiological Assessment of Ready-to-eat salad
Solution
1. Identify the different reasons for microbiological testing of the named product.
The Health Protection Agency (HPA) aims to prevent and reduce the consequences of infections. HPA examines the food from Local and Port Health Authorities for helping to safeguard the health of the consumer. Hence, the food samples are submitted for monitoring and surveillance and then tested further for investigations. RTEs refer to the food that is intended for direct consumption, and it can be raw, cooked and can be taken without any treatment. Due to the ready-for-consumption and presence of diverse nutrients, they are considered as ‘potentially hazardous' as they have the high probability of enhancing the growth of pathogens. The various components present in the named products include cows' milk, wheat flour, asparagus, egg, cheese, butter, salmon, onion, corn flour, mustard, rapeseed oil, etc. The various reasons for the microbiological testing of the named product are:
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Support the growth of pathogens and this growth increases when the food items are exposed to high temperatures.
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This increases the chances of food poisoning and formation of toxins taken place when exposed to high temperature.
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Any source of microbiological contamination on RTEs leads to a grave food safety hazard.
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The fishes and seafood can easily be contaminated due to the presence of pathogens like as Vibrio cholera, Salmonella, E. coli, Shigella, and Listeria either by poor hygiene or human activity while processing or producing the food. This leads to foodborne illness and related infections. ("Food Safety: What you should know", 2015)
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The microbes and pathogens can deteriorate the nutritional value of the food items making them substandard. The microbiological testing and related guidelines can be beneficial in assessing the quality as well as the quality of food. (Guidelines for Assessing the Microbiological Safety of Ready-to-Eat Foods Placed on the Market, 2009)
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The various criteria related to the food type like pH, water activity, storage temperature, packaging, shelf life, etc. can be assessed using the microbiological testing.
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The measures of rate of growth of micro-organisms can be determined. Hence the microbiological testing aids in determining the rapid and accurate detection of microbiological hazards.
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Laboratory methods allow rapid identification and quantification of the microbiological hazards were enhancing the ability to scrutinize and examine contamination throughout the food chain.
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The microbiological testing defines the acceptability of the made product.
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Apart from testing the quality, the microbiological testing helps to minimize the risk towards the heat of consumers.
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The test help to differentiate the products apart from other products presents in the market that does not fulfil the specifications.
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The test, therefore, helps to control the overall performance of the product.
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The gravity of the harm that can be done to the human system along with its impact can easily be estimated form the testing.
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The items and products are periodically inspected for checking the quality of the product.
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Further, a detailed consideration can be done on withdrawal of the product from the market in case it does not comply with the specifications.
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B. Explain the approach you would take in determining the organisms you would include in the test regime. [20 marks]
The food items present in the named RTE product along with their sources are mentioned below:
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The Cow’s milk has been the source of various items used in the named RTE Salad namely, Single cream, Taw Valley Cheddar Cheese, Unsalted Butter and Full fat soft cheese.
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Channel Islands' Cows' Milk, Pasteurised Free Range Egg, Rapeseed Oil are the items present in RTE Salad that is Fat-enriched.
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Spices like white pepper and black pepper have also been used in this RTE Salad.
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Dijon Mustard, Corn flour, Poached and Smoked Salmon, Asparagus, Fortified Wheat Flour are some other items present in the RTE Salad that is enriched with components like proteins, vitamins, minerals, etc.
The three major components for determining the microbiological criteria:
1. Aerobic colony count (ACC)
The total number of bacteria that are grown in a suitable culture medium is given by the term Aerobic Colony Count (ACC). ACC is used as an indicator that can reflect the quality of the food but cannot contribute towards a safety assessment of RTE food directly. Additionally, the ACC is beneficial in determining the general quality and, therefore, the shelf life of the food. Hence, these counts can help in deciding the potential problems related to storage and handling of the RTE product. ("Centre for Food Safety - Frequently Asked Questions - Microbiological Guidelines for Ready-to-eat Food", n.d.)
2. Hygiene indicator organisms – E. coli and Enterobacteriaceae
E. coli is a generally used faecal indicator organism. The mere presence of E. coli in food determines the direct or indirect faecal contamination. If the E. coli is present is the considerable amount of food, then it suggest the lack of cleanliness while processing, handling and storage of the product. For example presence of Enterobacteriaceae indicates the insufficient cooking of the food or post-processing contamination. Enterobacteriaceae can be explained as a large group of bacteria that are related either biochemically and genetically and are used to assess the hygiene and quality status of the product.
Enterobacteriaceae is responsible for the formation of histamine in food items like scombroid fish and some cheeses, incase these items are not processed properly with utmost care. ("Learn about the Microbiological Guidelines for Ready-to-eat Food", 2016) Generally, the hygiene indicator of contamination suggests some handling problem like undercooked food, poor quality of components used, etc. The hygiene indicator factor of an organism is directly proportional to the population of the pathogens.
3. Specific foodborne pathogens
The specific foodborne pathogens are the bacteria that are responsible for food poisoning. Pathogens primarily include Salmonella, E.coli 0157, Campylobacter, Clostridium perfringens, Clostridium botulinum, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes.
The symptoms along with the pathogens responsible for causing them are listed as mentioned below:
Additionally, the categorization can be done as follow:
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Category 1: The food items included in this category are RTEs and included the components that are thoroughly cooked while the preparing the final product and do not undergo any processing. The examples include soups, fish, seafood, etc.
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Category 2: These foods include few components that have been cooked, but they are processed or handled during the preparation process. It also includes the food items assembled from RTE foods but are not cooked. For example burgers, sandwich, etc.
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Category 3: These food items include the food items in which high colony counts is expected due to a considerable population of microbes related to these items. Example: Examples of foods in this category are fresh fruits or vegetables, fermented foods, chicken salad, deli meats, tubule, all kind of sprouts and cultured dairy, etc.
Test Microbiological Quality
Satisfactory Marginal Unsatisfactory Potentiality
Categories of microbiological qualityOn the basis of aerobic colony counts, indicator organisms, pathogens, four categories of microbiological quality have been assigned. These include satisfactory, marginal, unsatisfactory and potentially hazardous.
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Satisfactory results are the indication of good microbiological quality. Due to which no actions are required as there are no food quality concerns.
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Marginal results represent the borderline quality where the quality is within limits of the acceptable microbiological quality, but it also indicates some possible hygiene problems related to the preparation of the food. Action required include appropriate re-sampling of the product. Organizations regularly yielding borderline microbiological results should investigate their food handling process and related controls.
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Unsatisfactory results include the results outside of the acceptable microbiological limits and hence indicate poor hygiene or low-quality food handling practices being followed in the premises. Action required involving further sampling, including the testing of other foods used or processed in the food premises and an investigation, should be undertaken for determining whether the food handling practices, and controls along with the hygiene practices are adequate or not.
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Potentially Hazardous results are the levels that might be responsible for causing food borne illness and other related problems. Hence, immediate remedial actions must be initiated. Action required to be taken- Sufficient consideration that should be given to either the withdrawal of any of the substandard food item available for sale or to the distribution process If applicable, a recall action can be indicated.
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Further, a detailed examination of food production or handling practices must be done for determining the source and gravity of the problem so that adequate actions can be taken.
C. Identify and characterize the organisms you would test for, with your rationale for their inclusion and an indication of when you would test.
The following are some microbes considered harmful for the humans by the pathogenicity of organisms. Adequate precautions must be taken in case of they are identified in any RTE.
Salmonella:
Salmonella comes from Gram-negative species, flagellated facultative anaerobic bacilli that are characterized by O, H, and Vi antigens. There are around 2,700 serovars of Salmonella bacteria. The pathogenic salmonella that is ingested via the RTE salad goes through the gastric acid barrier where it enters the mucosa of the large and small intestine where toxins are produced. Invasion of epithelial cells further stimulates the proinflammatory cytokines release thereby inducing an inflammatory reaction. This acute inflammatory reaction causes diarrhea and sometimes leads to ulceration causing severe damage to the mucosa. The bacteria then spread from the intestines causing serious systemic disease (enteric fever) that requires prompt antibiotic treatment. Additionally, Salmonella bacteria cause considerable losses of livestock.
The presence of this organism is the indication of low-level food preparation and poor handling practices like inadequate cooking or cross contamination. Salmonellosis must be considered during any acute diarrheal or febrile illness without any evident reason. The diagnosis of salmonellosis can be done by bacteriologically isolating the organisms from the RTE. Biochemical tests should be performed in the laboratory for identifying the genus Salmonella and the serologic testing for identifying the serologic type. Therefore, there is need to protect the processed foods from contaminating; training should be provided in hygienic practices for all food-handling personnel in premises, food processing plants, etc. (Giannella, 1996)Staphylococcus:
S aureus and S intermedius are coagulase positive. Other than that all the staphylococci are coagulase negative. They are usually tolerant to salt and often hemolytic.
S aureus expresses various potential virulence aspects.
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(1) Surface proteins responsible for promoting the colonization of host tissues.
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(2) Factors that are inhibiting the phagocytosis-like capsule, immunoglobulin binding protein A.
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(3) Toxins were responsible for damaging the host tissues and thereby causing the disease symptoms. Coagulase-negative staphylococci are relatively less virulent and hence express fewer virulence factors.
The diagnosis is done by tests performed in the colonies. Various factors can be used for identification of S aureus like the tests for clumping factor, thermostable deoxyribonuclease and hemolysin. (Foster, 1996)
Vibrio cholera
The transmission of Cholera is usually via the fecal-oral route. Vibrios are acid-sensitive therefore most of them are died in the stomach itself. The rest that is survived colonize in the small bowel where secretion of cholera enterotoxin (also known as “choleragen”). This potent toxin gets bound to the plasma membrane of intestinal epithelial cells where it releases a subunit and gives rise to the production of cyclic adenosine 51-monophosphate (cAMP). This increasing level of intracellular cAMP secretes the high level of electrolytes and water into the intestinal lumen of humans.
The diagnosis is usually recommended in case of choric case of watery diarrhea. A wet mount of the liquid faeces is tested microscopically. Other methods include
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1. Culture of stool or rectal swab samples of RTE on the TCBS agar and related selective and non-selective media culture
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2. Slide agglutination test of colonies along with specific antiserum;
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3. Fermentation tests (also called as oxidase positive); and
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4. Peptone broth enrichment followed by the fluorescent antibody tests, culture, or retrospective serologic diagnosis.
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Recently, various genetically-based rapid techniques have been recommended for using specialized laboratories like the polymerase chain reaction (PCR). (Finkelstein, 1996)
Campylobacter:
Campylobacter is usually found in the intestinal tract of wild as well as the domesticated animals. These are easily found in raw meats and poultry and have the high probability of being present in eggs, raw milk and natural water. In the ready-to-eat foods, the presence of Campylobacter is an indicator of undercooked food or cross-contamination owing to poor hygiene practices, especially while handling of the raw as well as the cooked animal products. ("rapid microbiology » Salmonella Detection and Identification Methods", n.d.)
D. Produce outline microbiological laboratory procedures for three of the organisms you have selected, including a pathogen, a quality indicator and an indicator of poor hygiene practice
Answer.
The Salmonella can be identified as the pathogenic organism
A.) Requirements:
Equipment
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Erlenmeyer flasks (500 ml) etc. sterile for the pre-enrichment
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Food and faeces samples
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Disposable inoculation loops (1 µl and ten µl)
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Bunsen burner
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Plastic Petri dishes (9 cm diameter) sterile
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Balance
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Wood Spatulas Media
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Incubators at 37 ºC and 41.5ºC
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Pipettes for 0.1 ml (e.g. 1 ml pipettes)
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225 ml Buffered peptone water
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Nutrient agar plates Bacterial strains
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10 ml Tetrathionate broth (Müller-Kauffmann)
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10 ml Rappaport Vassiliadis soy peptone broth
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Xylose Lysine Desoxycholate (XLD) agar plates
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Brilliant Green (BGA) agar plates
Day 1: Non-selective pre-enrichment
25g of the RTE food sample was weighed out using a sterile wooden spatula and was put in an Erlenmeyer flask. To this around 225 mL of Buffered Peptone Water is added for obtaining 1 part of the sample in 9 part of buffer solution and mix thoroughly. This was incubated at the temperature of 37 ºC overnight for around 16-20 hours.
Day 2: Preparation of selective enrichment (I) and (II)
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Step 1: 1 ml of the pre-enrichment was transferred using a pipette to 10 ml Tetrathionate broth (MüllerKaufmann).
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Step 2: 0.1 ml of the pre-enrichment was transferred with a pipette to 10 ml Rappaport-Vassiliadis soy peptone (RVS) broth.
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The tube one was incubated at 37.0ºC ± 0.5ºC and tube two at 41.5ºC ± 0.5ºC overnight for around 18-24 hours.
Day 3: Spread on selective agar plates
A 10 µl loop filled from the inoculated was spread, and then incubation was done on Tetrathionate broth (I) and RVS broth (II) on XLD along with the BGA agar plates and then incubated at approximately 37ºC overnight for around 18-24 hours. (Gelinski, Martin, Destro, Landgraf & Franco, 2002)
Day 4: Sub-cultivation of Salmonella suspect colonies
The XLD plates were read: A Salmonella colony usually adopts somewhat transparent zone with reddish colour containing a black centre, a pink-red zone may be seen in the media surrounding the colonies. A typical Salmonella growth was marked positive on XLD on the record sheets.
The BGA plates were read: A Salmonella colony usually adopts a red or pink colour of the medium. The colonies adopt a grey-reddish or pink and slightly convex nature. The typical Salmonella growth was marked positive on the BGA the record sheets.
On the extremely swarmed plates, the colonies usually give a tan appearances against their green foundation. Now these dark settlements or the red and pink colonies are focused. The dark settlements and red color indicate H2S- positive and the one without color indicates H2S-negative. In some cases, the edge of these states may turn yellow in next 24 hours and might turn to red. Similarly, the pink settlements indicate H2S-positive and without color indicate H2S negative. While the Absence of agglutination was inferred as the absence of Salmonella. (Hendriksen, 2003)
Quality marker:
E.Coli is considered as a quality indicator while the nature of RTE is surveyed. By utilizing the standard culture techniques, the bacterial determination can take place in two folds. The procedure involves taking 10gm of the food sample from the 85 mL of the Buffered Peptone Water. The solution is diluted and made available for suspension while using the BPW. The solution is dissected for E Coli by using T.B.X. medium. These vaccinated plates are then incubated at 30 °C for approximately 24 to 48 hours and around 44 °C for exactly 24h separately
The undercooking of food, or cross-contamination from the crude meat and poultry or storing the food at the poor temperature and controlling of time. The pathogens pollute the food or enter the human body via treating the human sewage to dirt or when the sewage water is used for harvesting the crops. These activities can be harmful and further worsen the case if the nourishment via the nutrients is misused while handling or food processing. These are some scenarios that increase the cases of pathogens thereby contaminating the food.
One way of handling the ready-to-eat food items is to scatter the allocate into the sterile water and vaccination of various miniaturized scale litres of the test into tubes that contain 2.5 ml of Trypticase Soy Broth. The development was then brooded at 35oC, for 3 to 5 hours and then was vaccinated to various media cultures. The MacConkey agar without the gem violet can easily be streaked while the control purposes. The strips of Gelatin can then be added to the first development. The enterobacteriacae can then be sub-cultured to the blood agar, and the further affirmations can also be done.
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Place Your OrderREFERENCES:
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Centre for Food Safety - Frequently Asked Questions - Microbiological Guidelines for Ready-to-eat Food. Cfs.gov.hk. Retrieved 15 March 2016, from http://www.cfs.gov.hk/english/faq/faq_11.html
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Finkelstein, R. (1996). Cholera, Vibrio cholerae O1 and O139, and Other Pathogenic Vibrios. University Of Texas Medical Branch At Galveston. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK8407/
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Food Safety: What you should know. (2015). South-East Asia Regional Office. Retrieved 15 March 2016, from http://www.searo.who.int/entity/world_health_day/2015/whd-what-you-should-know/en/
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Foster, T. (1996). Staphylococcus. University Of Texas Medical Branch At Galveston. Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK8448/
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Gelinski, J., Martin, G., Destro, M., Landgraf, M., & Franco, B. (2002). Rapid detection of Salmonella in foods using a combination of SPRINT TM,MSRV TM and Salmonella Latex TestTM. Revista Brasileira De Ciências Farmacêuticas, 38(3), 315-322. http://dx.doi.org/10.1590/s1516-93322002000300007
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Giannella, R. (1996). Medical Microbiology. 4th edition (p. Chapter 21). Galveston: University of Texas Medical Branch at Galveston.
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Hendriksen, R. (2003). Global Salm-Surv (4th ed., p. 4). Retrieved from http://www.antimicrobialresistance.dk/data/images/salmonella1_pdf.pdf
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Microbiological quality guide for ready-to-eat foods. (2009) (1st ed., p. 5). NSW. Retrieved from https://www.foodstandards.gov.au/publications/documents/Guidelines%20for%20Micro%20exam.pdf
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rapidmicrobiology » Salmonella Detection and Identification Methods. Rapidmicrobiology.com. Retrieved 16 March 2016, from http://www.rapidmicrobiology.com/test-method/salmonella-detection-and-identification-methods/
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rapidmicrobiology » Salmonella Detection and Identification Methods. Rapidmicrobiology.com. Retrieved 16 March 2016, from http://www.rapidmicrobiology.com/test-method/salmonella-detection-and-identification-methods/
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