Methods for Detecting Microbes in Food PDF

Methods for Detecting Microbes in Food Methods for Detecting Microbes in Food A.Conventional methods B.Rapid detection methods C.Nucleic acid-based methods D.Biosensors E.Immunological methods A. Conventional Methods 1. Direct Counting Methods 2. Culture Based...

Methods for Detecting Microbes in Food Methods for Detecting Microbes in Food A.Conventional methods B.Rapid detection methods C.Nucleic acid-based methods D.Biosensors E.Immunological methods A. Conventional Methods 1. Direct Counting Methods 2. Culture Based Methods I. DIRECT COUNTING METHODS I. DIRECT COUNTING METHODS Counted by observing the food sample directly or retaining the microorganisms on a filter paper by filtering the sample and then observing under microscope. A. Direct microscopic count (DMC) B. Direct counting on membrane filters  DIRECT MICROSCOPIC COUNT (DMC)  A. DIRECT MICROSCOPIC COUNT (DMC) DMC involves detecting the presence of microorganisms in food by microscopic observation; simple and easy Performed by making a smear of food specimen/cultures on to microscopic slides, staining with appropriate dye and viewing and counting all cells using microscope under oil immersion or different Magnifications. Commonly used in dairy industry for assessing microbial quality of raw milk and other dairy products. Enumeration (determine the numbers of bacteria in a sample) Direct Measurement of Microbial Growth Microscopic count - the microbes in a measured volume of a bacterial suspension are counted with the use of a specially designed slide (Petroff-Houser chamber).  Enumeration Direct Measurement of Microbial Growth Membrane filter method – used to test large volumes of sample In filtration, known volumes are filtered through membrane filter with pores 0.45 μm in diameter, bacteria are retained on the surface of a membrane filter and then transferred to a culture medium to grow and subsequently be counted.  Preparation of slide 1. Take 0.01 ml milk with the help of a sterile pipette and spread it evenly on a grease free slide of 1 cm2 marked area on a Breed slide. 2. Dry the smear on a warm surface at 40 - 45◦C. 3. Do not heat-fix the slide on direct flame. Rapid drying results in cracked surfaces on the film or peels off during further processing. 4. Immerse the slide in Newman's stain for to 1 minute. 5. Newman's stain removes the milk fat, fixes the smear and stains the bacteria in a single operation. 6. The tetrachloroethane of the stain helps to dissolve the milk fat globules, ethyl alcohol fixes the smear and methylene blue stains the smear  MICROSCOPIC EXAMINATION Examine under the oil immersion objective and count the number of micro-organisms (individual or clumps of cells) in a number of fields of the film. The fields for counting the bacterial cells are selected at random. The number of microscopic fields occurring in one square centimetre area of the smear is very high. Thus, a representative number of fields depending on the concentration of bacterial cells in a microscopic field are chosen for counting the bacterial cells as follows Calculate the average number of clumps per field and multiply by the microscopic factor to get the DMC per millilitre of milk. Interpret the results by comparing with the standards and assess the quality of milk samples as per Below Table GRADING OF MILK The quality of raw milk is adjusted using the following details ADVANTAGES DMC is widely used to screen incoming raw milk supplies as a platform test to determine whether the milk has an acceptable or legal bacterial load, as per BIS standards. Easy to perform. Less time is required to perform the test. Large number of samples can be screened in a given period of time Useful in providing the estimated counts, types of bacteria and somatic cells in milk. DMC can be used as a guide in identifying the types of bacteria present in a milk sample. Possible to find out the source of contamination of milk. DISADVANTAGES Not considered as a fool-proof/ legal method. Strain on the eyes of the operator is too much. Test is not reliable as both viable and non-viable cells are counted. Method is not suitable for pasteurized milk. Experienced person can carry out the test. Results are not reproducible because microbes are unevenly distributed in the smear. B. DIRECT COUNTING ON MEMBRANE FILTERS B. Direct Counting on Membrane Filters Membrane fillers with pore size smaller (0.45 um) than bacteria retain bacteria and the retained bacteria can be counted using microscope. Procedure involved : Concentrating/collecting bacteria on polycarbonate filters by filtering known volume of homogenized sample Staining and counting of retained bacteria Placing the membrane on a nutrient agar media or absorbent pad saturated with culture media of choice, and incubating Following growth , colonies are counted STEP-BY-STEP PROCEDURES 1. Collect the sample and make any necessary dilutions. 2. Select the appropriate nutrient or culture medium. Dispense the broth into a sterile Petri dish, evenly saturating the absorbent pad. 3. Flame the forceps, and remove the membrane from the sterile package. 4. Place the membrane filter into the funnel assembly. 5. Flame the pouring lip of the sample container and pour the sample into the funnel. STEP-BY-STEP PROCEDURE 6. Turn on the vacuum and allow the sample to draw completely through the filter. 7. Rinse funnel with sterile buffered water. Turn on the vacuum and allow the liquid to draw completely through the filter. 8. Flame the forceps and remove the membrane filter from the funnel. 9. Place the membrane filter into the prepared Petri dish. 10. Incubate at the proper temperature and for the appropriate time period. 11. Count and confirm the colonies and report the results.  Uses of Membrane Filters Membrane filters are used extensively in the laboratory and in the industry to sterilize materials likely to be damaged by heat sterilization. These materials include nutritional supplements of culture media, and pharmaceutical products such as drugs, hormones, sera, and vitamins. Effective and acceptable technique to monitor drinking water, air quality, etc. Useful for bacterial monitoring in the pharmaceutical, cosmetics, electronics, and food and beverage industries. Allows for removal of bacteriostatic or bactericidal agents that would not be removed in pour plate, spread plate, or MPN techniques. Can be used to allow selective passage of the organism of interest by selecting a membrane filter of appropriate porosity. Such practices are done in the pharmaceutical industry to allow the passage of a particular virus strain while preparing vaccines ADVANTAGES Well suited for samples containing low numbers of bacteria Facilities concertinaing bacteria by filtering large volume of sample Only small volume of food samples can be used for a single membrane Efficiency of membrane filter method can be increased by staining with florescent dyes (Ex. acridine orange) and observing under epiflorescence microscope (DEFT: Direct Epiflorescence Filter technique) Viable cells appear fluoresce green and Non viable cells appear orange and then counted counted. Can be used to enumerate microorganisms from a variety of foods (fresh fish, meat, fish/ meat products, water samples etc).  DIRECT EPIFLUORESCENT FILTER TECHNIQUE (DEFT) DEFT is a method originally developed for the rapid enumeration of microorganisms in raw milk samples. The method is based on a membrane filtration that captures the microorganisms, followed by a staining with a fluorochrome, acridine orange. After staining, the membrane is rinsed and mounted on a microscope slide, which can be easily visualized and counted with an epifluorescent microscope. The complete procedure can take as little as 30 min. II. Culture Based Methods II. Culture based methods Involve examination of microorganisms in food by encouraging them to multiply in a liquid or solid media On solid agar media bacteria develop as colonies and counting such viable colonies gives microbial load in foods Enumerating microorganisms by culture based methods can be done by using Plate Count Methods or Most Probable Number (MPN) technique Plate count method: Referred to as total plate count (TPC), standard plate count (SPC) or aerobic plate count (APC) Most widely used conventional method for determining viable cells or colony forming units (CFU) in foods. SPC involves blending/ homogenizing the sample, serially diluting in appropriate diluent, plating in or on suitable agar media, incubating at appropriate temperature for a given time, and counting visible colonies as CFU. Principle involved : SPC is based on the principle that each viable bacterial cells multiples and grows in to a visible colony Thus, counting number of colonies gives an idea about bacterial cells present in a sample Counts determined by taking average of replicate plates showing 30-300 colonies If there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. (too few to count (TFTC). Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together. (too numerous to count (TNTC). Factors affecting SPC: - Sampling method employed - Distribution of microorganisms in food - Nature of food biota - Nutritional adequecy of plating media - Incubation temperature and time - Type of diluents used - Presence of other competing organisms etc. - Plating on selective media for specific organisms is limited by degree of inhibition and effectiveness of selective/ differential agents employed. SPC can be performed by pour plate method or spread plate method (surface plating method) a) Pour plate method: Appropriate dilution of the sample (1 ml) is mixed with agar medium, allowed to set, incubated at appropriate temperature and colonies developed are counted. Here colonies develop both on surface and subsurface of agar plate Proper mixing of sample with agar medium is necessary so as to get isolated colonies which can be done by 2 ways One ml of appropriate sample dilution is added to Petri plate and about 15 ml of agar medium is added and mixed by rotating the plate in clockwise and anticlockwise direction One ml of appropriate sample dilution is added to testtube containing about 15 ml of molten agar medium, mixed by rolling the tube between the palm and poured to petriplates, allowed to set and incubated b). Spread plate method: Diluted sample (0.1 ml) is spread on the surface of pre poured, hardened agar plates using glass rod, incubated at appropriate temperature and colony developing on surface counted. Advantages: Suitable for heat sensitive psychrotrophs in food as they do not come in contact with molten agar. Enables providing colony features useful in presumptive identification especially on selective media. Favors strict aerobes on surface, but micro aerophils grow slowly Disadvantage: Problem of spreaders and colony crowding makes the enumeration difficult. 1. Collect the Food samples at random. 2. Weigh 1 g of the solid samples (vegetables/ fruits) and homogenise the samples in pestle and mortar. Thoroughly mix the sample into 9 ml of sterile diluents to make 10-1 dilution. 3. Transfer 1 ml of suspension from 10-1 dilution to a 9 ml sterile blank with a sterile pipette to make 10-2 dilution. Similarly prepare dilution up to 10-6. NOTE:- LIQUID SAMPLES ( CAN BE DIRECTLY USED FOR DILUTION)