Total Plate Count Lab 1 TPC Rough estimate of total microbial content of food Limitation anisms may not grow Incubation temperature not ideal for anisms Oxygen content eliminates anaerobes, and may suppress microaerophiles Molds and yeast require longer incubation periods Fermented foods have a higher content of microbes than is preferred Overview Ground beef/cheese Blender/Stomacher 11g/99ml peptone water Meat -10-5 Cheese- 10-7 TSA not PCA 35ºC for 48 hr Slow anisms 2 min 2 min Spread plates Petrifilms Petrifilms Quick plating method Contains a dye to make it easier to observe live cells TETRAZOLIUM dye is reduced by growing cells to produce a red color You are counting red dots when using petrifilms Disadvantages- No colony morphology and expensive How to work with a Petrifilm 1 2 1 ml onto center 3 4 Place spreader with ridge side down onto center of Petrifilm Press center of device Let top flap down Results of TPC Petrifilm Release spreader and allow to dry Second and Third Period Count colonies by rules (p. 9-12) and follow flowchart (p. 32) Calculate the total plate count by dividing your value by the dilution factor X volume plate Morphological and microscopic evaluations CFU dilution factor X volume plated Evaluations 1. Colony shape and size: round, irregular, punctiform (tiny) 2. Margin (edge): entire (smooth), undulate (wavy), lobate (lobed) 3. Elevation: convex, umbonate, flat, raised 4. Color: color or pigment, plus opaque, translucent, shiny or dull 5. Texture: moist, mucoid, dry (or rough). Gram Stain Air dry specimen, heat fix Crystal Violet – 1 min Gram’s Iodine – 1 min Acetone Alcohol – quickly decolorize (3-5 sec) Safranin – 1 min Note cell shape and size Purple = +, Pink = – COLONY MORPHOLOGY CELL MORPHOLOGY
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