TECHNIQUES FOR THE STUDY OF MICRORGANISMS IN FOOD
Microbiologic quality of food production is determined in order;
1. to estimate the shelf life of the food products
2. Used to determine the suitability of the food.
3. It can also be used to determine the cause of food spoilage
4. The presence of a pathogen where the food has been implicated in the food borne illness.
5. To monitor specific pathogens such as an indicator organism.
Microbiological quality can be determined to by direct examination of the food or by culturing the food on suitable agar medium.
A. Direct examination of food
It is sometime possible to detect the presence of microorganisms in a food sample by directly viewing the sample under the microscope. A small amount of the food can be mounted and teased out in a drop of water on a slide and covered with a cover slip. The slide can be examined at low magnification at X40. Yeasts and mold can be seen readily while bacteria and their spores can be seen after careful preparation.
PROBLEMS ASSOCIATED WITH DIRECT EXAMINATION OF FOOD.
1. Since only a small sample of the product is examined the microorganism may not be seen if they are present in large nos.
2. Some food constituents may interfere with the heat fixing thus allow the smear to be washed up during staining
3. The method does not identify live and dead cells
4. It has limited sensitivity
B. CULTURAL TECHNIQUES
1. The total plate count may be used to detect the presence of viable materials. The total plate count can also be said to be the total viable count.
2. It may be used to determine if the food meets established microbiological criteria
3. It can also be used to estimate if a particular component of the total flora may be determined.
4. Total viable count may be used to estimate the total number of organisms present.
The total place count (TPC) can be achieved by the pour plate method or the spread plate method.
LIMITATION OF VIABLE COUNT OR PLATE COUNT
1. The small volume of innoculum is of limitation
2. Thermal shock from molten agar used during pour plate.
C. SERIAL DILUTION TECHNIQUE
Serial dilution, as the name suggests, is a series of sequential dilutions that are performed to convert a dense solution into a more usable concentration.
• In simple words, serial dilution is the process of stepwise dilution of a solution with an associated dilution factor.
• In biology, serial dilution is often associated with reducing the concentration of cells in a culture to simplify the operation.
• Serial dilution involves the process of taking a sample and diluting it through a series of standard volumes of sterile diluents, which can either, be distilled water or 0.9 % saline.
• Then, a small measured volume of each dilution is used to make a series of pour or spread plates.
• Depending on the estimated concentration of cells/organisms in a sample, the extent of dilution is determined. For e.g., if a water sample is taken from an extremely polluted environment, the dilution factor is increased. In contrast, for a less contaminated sample, a low dilution factor might be sufficient.
• The dilution factor in a serial dilution can be determined either for an individual test tube or can be calculated as a total dilution factor in the entire series.
• The dilution factor of each tube in a set:
• For a ten-fold dilution, 1 ml of sample is added to 9 ml of diluent. In this case, the dilution factor for that test tube will be:
• After the first tube, each tube is the dilution of the previous dilution tube.
Now, for total dilution factor,
• Total dilution factor for the second tube = dilution of first tube × dilution of the second tube.
Example:
For the first tube, dilution factor = 10-1 (1 ml added to 9 ml)
For the second tube, dilution factor = 10-1 (1ml added to 9 ml)
Total dilution factor = previous dilution × dilution of next tube
= total dilution of 10-1 × 10-1 = 10
Applications/Uses
.
Serial dilution is used in microbiology to estimate the concentration or number of cells/organisms in a sample to obtain an incubated plate with an easily countable number of colonies.
Limitation/Problems
1. An error might occur during the propagation of the sample, and the transfer inaccuracies lead to less accurate and less precise transfer. This results in the highest dilution to have the most inaccuracies and the least accuracy.
2. Because serial dilution is performed in a stepwise manner, it requires a more extended period of time which limits the efficiency of the method.
3. Serial dilution only allows the reduction of bacteria/cells but not the separation of bacteria/cells like in other techniques like flow cytometry.
4. This technique also requires highly trained microbiologists and experts in aseptic techniques.
D. The pour-plate technique
1. The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette.
2. Molten agar cooled to 45°C, is poured into a Petri dish containing a specified amount of the diluted sample.
3. Following the addition of the molten-then cooled agar, the cover is replaced, and the plates gently rotated in a circular motion to achieve uniform distribution of microorganisms.
4. This procedure is repeated for all dilutions to be plated.
5. Dilutions should be plated in duplicate for greater accuracy, incubated overnight, and counted on a Quebec colony counter either by hand or by an electronically modified version of this instrument.
Significance of Pour Plate Technique
• This technique is used to perform viable plate counts, in which the total number of colony-forming units within the agar and on the surface of the agar on a single plate is enumerated.
• Viable plate counts provide scientists a standardized means to generate growth curves, to calculate the concentration of cells in the tube from which the sample was plated, and to investigate the effect of various environments or growth conditions on bacterial cell survival or growth rate.
Advantages of Pour Plate Technique
• Easy to undertake.
• Will detect lower concentrations than surface spread method because of the larger sample volume.
• It requires no pre-drying of the agar surface.
• The most common method for determining the total viable count is the pour-plate method.
• The pour plate technique can be used to determine the number of microbes/ mL in a specimen.
• It has the advantage of not requiring previously prepared plates and is often used to assay bacterial contamination of foodstuffs.
Disadvantages of Pour Plate Technique
• The use of relatively hot agar carries the risk of killing some sensitive contaminants, so giving a low result.
• Small colonies may be overlooked.
• In the case of solid sample dissolving in water, some species may suffer a degree of viability loss if diluted quickly in cold water; consequently, isotonic buffer (phosphate-buffered saline for example) or peptone water are used as solvents or diluents.
• Colonies of different species within the agar appear similar — so it is difficult to detect contaminants.
• The reduced growth rate of obligate aerobes in the depth of the agar.
• Preparation for the pour plate method is time consuming compared with streak plate/and or spread plate technique.
E. THE COLIFORM TEST.
Coliform is a facultative anaerobe that ferments lactose to produce gas and is a gram -ve, non spore forming rod capable of fermenting lactose in the presence of bile at 37oC. Eg E. coli, Citrobacter & Enterobater. Fecal coliform also can be determined and these are coliforms that can grow at higher temp than normal such as at 44oC or 44.5oC eg E. coli
ENTEROBACTERIEACE COUNT can also be used to determine the microbiological quality of food. The absence of total coliform and fecal coliform may give a false confidence of safety when lactose negative organisms predominate. Enterobacteria counts are used more generally as an indicator of hygienic quality rather than fecal contamination and can therefore say more about general microbiological quality than possible health risk posed by the food products e.g of non-lactose fermenter include Salmonella, Shigella, Erwinia, Serratia
E coli- feacal coliform intestinal pathogen
E coli & S. faecalis are classified as good sewage indicators, both are non spore formers.
The method for detecting coliform in water therefore includes:
• Presumptive test
• Confirmatory test
• Completed test
Lactose broth is prepared as single and double concentration and the test tubes are inoculated with sample of the water. Tiny tubes known as Durham tubes are inserted inside the lactose broth tube.
After incubation, the test tubes that show gas production evidence by displacement of lactose broth within the Durham tube are considered to be presumptive positive for coliform.
A confirmatory test follows by the streaking of the EMB plates with samples from the presumptive positive test-tubes. The characteristics colonies which develop on the EMB agar can be confirmed as E coli.
The completed test now consists of inoculating fresh lactose broth test-tube again with samples of the colony that has grown on the EMB plate. Gas production is the evidence that the coliform test is completed and that the water was definitely contaminated from a fecal source and potential pathogens may be present in such sample.