Food and Dairy Microbiology Lab 05 - Media Preparation PDF

FOOD & DAIRY MICROBIOLOGY LAB # 05 : Microbiological Media Preparation Dr. Muhammad Naseem Khan MSc, MBA, PhD PURCHASE, INSPECTION, AND STORAGE OF MEDIA Detailed specifications shall be provided to the supplier of media/chemicals No item shall be accepted if it does not conform...

FOOD & DAIRY MICROBIOLOGY LAB # 05 : Microbiological Media Preparation Dr. Muhammad Naseem Khan MSc, MBA, PhD PURCHASE, INSPECTION, AND STORAGE OF MEDIA Detailed specifications shall be provided to the supplier of media/chemicals No item shall be accepted if it does not conform to the specifications. On arrival, the items shall be checked for their general condition of packaging, expiry dates, and lot numbers. Receiving of any media, chemical, and reagent shall be recorded in a log book. PURCHASE, INSPECTION, AND STORAGE OF MEDIA The media, chemical, and reagents shall be stored as per instructions of the manufacturers. The date of opening is marked on each and every bottle/pack. Record of usage is maintained. Each lot of media is tested for its growth promotion and selectivity using quality control organisms. Chemicals are also tested against their positive and negative controls.  GROWTH PROMOTION TEST AND MEDIA SUITABILITY TEST ON NEW MEDIA USE FOR TESTING IN MICROBIOLOGY Each lot must be within optimum limits of pH and weight/volume ratio for a given media type. With the help of serial dilution and pour plate method prepare < 100cfu/ml working dilution of each challenge organism. Inoculate your challenge organism in the respective media and incubate at specified temperature for specified time mentioned with the organism. After incubation check the growth of challenge organism and record in their respective log book. If test medium failed to demonstrate growth against challenge organism, re test your medium for confirmation. Retesting proceeding should be treated in the same manner as initial testing. If, after retest any batch fails to meet the specifications claimed, the relevant agency must be informed, and that particular media may not be used for routine testing.  Challenge organism S.# Organism ATCC Incubation Incubation Temperature Time 1 Aspergillus niger 16404 20-25 °C 5 days 2 E coli 8739 30-35 °C 48-72 hours 3 Pseudomonas aeruginosa 9027 30-35 °C 48-72 hours 4 Staphylococcous aureus 6538 30-35 °C 48-72 hours 5 Salmonella typhimurium 14028 30-35 °C 48-72 hours  SELECTIVE AND DIFFERENTIAL MEDIA SUITABILITY TEST: Selective and Differential Media used in Microbiological laboratory should be check for negative control along with positive controlled testing / growth promotion testing Challenge organisms (negative control) for selective media are those groups of organisms which are suppressed by the inhibitory substances added to stop its growth on that particular medium. Challenge organisms (negative control) for differential media are those groups of organisms which show different biochemical reactions when inoculated on that particular medium.  examples for negative and positive controls are as follows: Challenge Organisms S.# Selective/Differential Positive Control Negative Control Media Lactose TTC Agar with Salmonella typhi, 1 Tergitol 7 E.coli Pseudomonas aeruginosa 2 Tryptone Broth E.coli Salmonella typhi, Pseudomonas aeruginosa 3 Bismuth Sulphite Agar Salmonella typhi E.coli, Pseudomonas aeruginosa  STERILIZATION OF APPARATUS AND MEDIA USED IN MICROBIOLOGICAL TESTING Glass bottles (with caps). Test tubes. Pipettes. Petri plates. Filtration assembly. Forceps. Wire loop. Measuring cylinders.  Cleaning: Wash the apparatus with plain water then use detergent or chromic acid (whichever is required) i.e., pipettes and test tubes are dipped in chromic acid for about 2-4 hours and then washed with tap water ten times and finally rinsed with distilled water. Glass bottles, filtration assembly, forceps and measuring cylinders are washed with detergent solution and then wash ten times with tap water and finally rinsed with distilled water. Wash thoroughly the bottom and the cover of the Petri plate with the detergent solution then rinse with water and dry in the basket.  Sterilization Pipettes, Petri plates are kept in their S.S. boxes and stand for sterilization through hot air sterilizer at 180˚c for 2 hours or 200˚c for 1.5 hours. Glass bottles, filtration assembly, forceps and measuring cylinders are wrapped in a paper sealed with an autoclave tape and sterilize through autoclave at 121˚c for 15 minutes. Wire loops are the basic tools of Microbiology and are sterilized through incineration.  Media Preparation: Follow the Manufacturer’s instructions for the preparation and sterilization of all media and other preparations used in microbiological analysis.  RECORD FOR STERILIZATION The boxes for pipettes and Petri plates are numbered and sterilization record is maintained in a log book. Record of media preparation and sterilization is maintained in a separate log book.  DISPOSAL OF WASTE MEDIA All the materials used for analysis shall be autoclaved at 1210C and 15 lbs pressure for 30 minutes before disposal. Cooled and discarded as follows: Liquids: drained off in the sink with excess amount of water Solids: collected in polythene bags After autoclaving the materials to be disposed off may either be incinerated or buried underground. The records of the disposal of samples shall be maintained.