Total Plate Count

1
Total Plate Count

• Lab 1

2
TPC

• Rough estimate of total microbial content of food
• Limitation
• Fastidious organisms may not grow
• Incubation temperature not ideal for all
  organisms
• 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

3
Overview
Ground beef/cheese
11g/99ml peptone water
Blender/Stomacher
Cheese- 10-7
Meat -10-5
TSA not PCA
35ºC for 48 hr
Slow growing organisms
4
2 min
2 min
Spread plates
Petrifilms
5
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

6
How to work with a Petrifilm
1 ml onto center
7
Let top flap down
8
Release spreader and allow to dry
Results of TPC Petrifilm
9
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
10
Evaluations
COLONY MORPHOLOGY
CELL MORPHOLOGY

• Gram Stain
• Air dry specimen, heat fix
• Crystal Violet 1 min
• Grams Iodine 1 min
• Acetone Alcohol
• quickly decolorize
• (3-5 sec)
• Safranin 1 min
• Note cell shape and size
• Purple , Pink

• 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).

11
Lab Setup

• Each group will have either cheese or ground meat
• Within each group, one student blends the sample
  and the other will stomach a similar sample
• Lab Report due Friday, April 8th
• If you have any questions contact me at
  gramp.1_at_osu.edu