Salmonella can be passed from animal products to human, from animals to animals, and by oral route. This bacterium causes enteritis, enteric fever and systemic infection. The diagram 1 shows the cycle of Salmonella infection.
The cycle of Salmonella infection
Environmental Products eaten
Pollution by humans
Wildlife Farm livestock MAN
reservoirs
Meat/bone PETS
meal Products eaten
by animals
OFFAL Diagram 1
(BIO3525 Handaouts: week5)
There are many ways that the food could become infected. Taking the hypothesised micro-organism as the cause of the outbreak, the food could become contemned if the served meats were not cooked properly.
Salmonella inhabit in human and animal intestinal track. This bacterium could survive during the cooking. Also it could be possible if the row meats were cut in the cutting board, which wasn’t washed after this procedure, and the following products that were cut on this board have got contaminated.
There is another hypothesis where the food handler did not wash his/her hands.
In order to identify Salmonella, a blood or stool sample should be taken. The first step for the identification is to carry out selective medium, enrichment medium cultures, and blood cultures in differential medium. After this procedure the catalase and oxidase tests could perform. It would be also helpful to see if the micro-organism is glucose fermenter. After this, a number of biochemical tests can be applied.
Biochemical tests show the chemical properties of wanted bacterium and these characteristics then are compared to the known micro-organisms with the same chemical properties. The selection of the suitable biochemical test is very important for the identification of micro-organisms.
The identification could be made according to the results of these tests. These tests measure the presence or absence of the enzymes involved in the catabolism of the micro-organisms tested in differential media. According to these biochemical tests, the typical reaction patterns for a large number of various pathogens have been published. From the results, the characteristics of the unknown bacteria are compared to those reaction patterns and the wanted bacterium is identified.
Six different biochemical tests need to be applied for the identification of the Salmonella.
The biochemical tests that should be applied are Indole test, Methyl Red (MR), Voges-Proskauer Test (VP), Urase, Citrate test, Hydrogen sulphide test (H2S). The typical reaction patterns for Salmonella are shown in the table 2.
Table 2. Biochemical characteristics of Salmonella.
Taxonomy is a science of classification consisting of identification and nomenclature. The tests that would be taken in this case are closely related to the taxonomy of the wanted micro-organism. The most Gram –ve rods are associated with the intestinal infections and they are oxidase negative and they do ferment glucose. These micro-organisms belong to the family of Enterobacteriaceae. From the catalase test it could be assumed that the micro-organism could be Salmonella, Klebsiella, Eschirichia or Enterobacter. These genera appeared to be catalase positive. With the help of catalase and biochemical tests the genus from the wanted micro-organism can be identified.
Table 3 was given in order to calculate the generation time of organism Y at 24 ºC and to describe an appropriate method for determining the number of aerobic organisms/g of food, explaining why the results are reported as colony forming units (CFU).
Table 3 Log 10 number of colony forming units of aerobic organisms/g and the log 10 number of colony forming units Y organisms/g chicken in chicken held at room temperature (24 ºC) for 6 hours.
The generation time or doubling time is the time taken for the bacteria to double. The following formula is used in order to calculate the generation time:
g = t x 0.301
Log10Bf-Log10Bi where:
t = period of the log phase
Bf = numbers of organisms at the end of exponential phase
Bi = numbers of organisms at the start of the exponential phase
Calculations:
At time 0, the Log10 total number of colony forming units Y/g chicken was 2.83 Y/g.
After 6 hours the Log10 total number of colony forming units Y/g chicken was 5.98 Y/g.
Thus Bi = 2.83 Y/g and Bf = 5.98 Y/g
t = 6
g = 6x0.301 = 1.806 = 0.57 hours
5.98-2.83 3.15
The generation time of organism Y at 24 ºC is 0.57 hours or 57 minutes.
The appropriate method to determine the number of aerobic organisms/g of food is the viable count.
Agar plates are inoculated with the micro-organism, so as to obtain separate colonies after incubation. Each viable cell initially present in the sample multiplies to produce a visible colony during incubation. Therefore, by counting the number of colonies, the number of viable cells in the sample can be calculated.
As most microbial cultures are used at high concentrations considerable dilution of the sample is necessary in order to get a countable number of colonies per plate. This can be achieved by a series of small dilutions, usually one in ten (10-1) or one in a hundred (10-2). This is known as serial dilution. A one in ten dilution can be prepared by adding 1 ml of culture to 9 ml of diluent. A one in hundred dilution can be prepared by adding 0.1 ml of culture to 9.9 ml diluent or by carrying out two sequential one in ten dilutions. Each dilution is carried out by transferring the required amount of sample using a sterile pipette to the diluent. After swirling, or using a vortex mixer, to mix, a fresh pipette is used for the next dilution. When serial dilutions are prepared, a small quantity of each dilution is taken and plated onto agar plate. The plates are incubated at the appropriate temperature for the micro-organism. After incubation the number of colonies could be count and the number of CFU/g could be calculated.
CFU calculates the viable cells in a sample.
The results of viable count are reported as CFU/g or CFU/ml, because there is a theory that one colony comes from one viable cell during the cells growth. However not all cells grow separately. For example does not separate during its growth and the colony that will be formed could be developed from more than one cell.
The best number for viable count method would be 30-300 colonies.
There are problems if micro-organism spread on the surface of the plate. If there are too many colonies they merge into each other. Also some cells are alive, but not culturable in the lab.
In order to make the food safe, the caterer should treat the food carefully. The caterer has to make sure that the kitchen environment is clean and safe, in order to proceed to the preparation of the food. The use of sterile gloves could minimize the contamination of food.
The cooking and storing procedures must be done precisely. The products have to be cook at the appropriate temperature were bacteria can not survive, and the row products should be kept separately from cooked food. The equipments that have been used for a product have to be cleaned after they use. If a row meat was cut, the cutting board and the knife that have been used, have to be cleaned with an appropriate detergent. After this procedure, it would be safe to use the same equipments for other products. In this way cross contamination could be avoid. The hands of the caterer must be washed regularly in case He/She does not wear sterile gloves. Cross contamination could be occurred if the caterer erthei se epafi with row products and then with cooked food, He/She can transmit the bacteria with the hands.
The products have to be well cooked. The time of exposure of the food to the room environment have to be not very long before serving. If food is prepared and not served after a short period, the long time exposure of the food to the environment can attract organisms like flies. Flies will transmit faecal bacteria, which results to the contamination of the food.
Cooked products have to be cooled and reheat quickly. This will prevent the formation of vegetative cells.
Spores are found in many products such as vegetables, meats, chicken etc. They are released from vegetative cells and can survive very high and low temperatures. When food is left for a long time at room temperature, this would lead the spores to revert to a vegetative cell. Vegetative cells are bacteria which can lead to an outbreak.
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BIO 3525. Microbiology. Handouts.
