Aerobic Plate Count, Gram Stain, and Isolation.ppt
Aerobic Plate Count, Gram Stain, and Isolation Food Microbiology Laboratory Aerobic Plate Count Provides general estimate of live, aerobic, bacteria Excludes Obligate Anaerobes Microaerophiles Plate Counts Assumption Each colonies arises from a single bacterial cell Bacteria like to “clump” together so some colonies may arise from more than one cell Report as Colony Forming Unit (CFU)/gram or ml NOT at total bacteria APC Results Evaluate Sanitation of Product Predict Shelf-life “Safety” Indicator Monitor Environment Limitations of APC Only anisms are counted Bacteria Type not known Media may not support growth of certain bacteria Eye strain/Human Error Hard to Distinguish Between food particles and bacteria Don’t Use on Fermented Foods Colonies may be too small to see Types of Samples Liquid Non-viscous Liquids can be measured with pipet Viscous liquids should be weighed Solid Aseptically weigh Sample Sponge/Swab Collect sample by swabbing a defined area Environmental and Container Rinse inside of Containers Open Plate to Collect Air Samples RODAC Plates Protocol for Plate Counts Prepare a Sample Homogenate 1:10 dilution 1 part sample to 10 parts total volume Blend in Blender or Stomacher for 2 min. 10 g/ml sample 90 ml of diluent 1:10 Dilution – 10-1 Formula 10 ml/g sample, want 1:100 dilution 100 – 10 = 90 ml of diluent needed Start with Different Sample Sizes 50 g sample Must have 500 g total volume for 1:10 500 – 50 = 450 ml diluent needed 95 ml sample Must have 950 total volume for 1:10 950 – 95 = 855 ml of diluent Plate Count Protocol Prepare Serial Dilutions Dilute to a level where you will get countable colonies on plates Use a NEW STERILE PIPET between each dilution Place pipet tip down in pipet tanks Shake each dilution bottle 25 times in a 90 degree arc within 7 seconds. Phosphate Buffer or Peptone Buffer to Dilute Dilutions Sample Homogenate Dilution Blanks Containing 90 ml Diluent 10 ml 10 ml 10 ml 10 ml 10-1 10-2 10-3 1
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