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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

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aerobic plate count
Aerobic Plate Count
  • Provides general estimate of live, aerobic, bacteria
  • Excludes
    • Obligate Anaerobes
    • Microaerophiles
plate counts
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
APC Results
  • Evaluate Sanitation of Product
  • Predict Shelf-life
  • “Safety” Indicator
  • Monitor Environment
limitations of apc
Limitations of APC
  • Only aerobic organisms 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
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
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.

90 ml of diluent

10 g/ml sample

1:10 Dilution – 10-1

formula
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
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
Dilutions

Dilution Blanks Containing 90 ml Diluent

Sample Homogenate

10 ml

10 ml

10 ml

10 ml

10-2

10-3

10-4

10-5

10-1

(1:100)

(1:1000)

(1:100000)

(1:10)

(1:10000)

plating
Plating

Put 1 ml of Each Dilution into Empty Petri-Dish

10-4

10-5

10-2

10-3

10-1

1 ml

1 ml

1 ml

1 ml

1 ml

1 ml

1 ml

1 ml

1 ml

1 ml

apc protocol
APC – Protocol
  • Add 18-20 ml of tempered (45-50 F), molten plate count agar to the petri dish.
    • Agar MUST be tempered or the bacteria will be killed by heat
  • Standard Methods or Plate Count Agar
  • Swirl 10 times in each direction
  • Allow to Solidify
  • Incubate inverted at 35-37 C for 48 hours
sterilization
Sterilization
  • Equipment and Media MUST be Sterile
  • Hot Air Sterilization
    • 170 C for 1 hour
      • Equipment Temperature
      • Put in oven for 2 hours
      • Wrap in paper, foil, etc.
  • Steam Sterilization
    • 121 C for 15 min. MUST have 15 psi pressure
      • Liquid Media or Equipment
      • Don’t Put Lids on tightly
counting plates
Counting Plates
  • Only count plates with 25-250 colonies
  • More than 250
    • Too Numerous To Count – TNTC
  • Less than 25
    • Too Few to Count - TFTC
counting plates1
Counting Plates

1 Too Numerous to Count

2 Too Few to Count

  • Average two countable plates and Multiply by Dilution Factor
    • Count is 175 x 104
  • Must Convert to TWO Significant Digits
    • 1.8 x 106 cfu/ml or g
counting examples
Counting - Examples

Use ALL FOUR even though 300 is outside range. If ONE

PLATE is in RANGE, use BOTH for Average.

250 x 102 – 2.5 x 104

125 x 103 – 1.3 x 105

AVERAGE – 7.8 x 104 cfu/g or ml

counting examples1
Counting Examples

All Dilutions are outside Range so we MUST use counts

Outside range

350 x 104 – 3.5 x 106 cfu/ml or g*

Use an “*” when using dilutions outside countable ranges

This means it is an ESTIMATED count

counting examples2
Counting Examples

If Both Dilutions are outside Range, use the Higher Dilution

(LOWER COUNTS)

7.5 x 103 cfu/ml or g*

overloaded plates
Overloaded Plates
  • Use Highest Dilution and Use Grid on Colony Counter
    • 1 Grid = 1 cm2
    • A standard Plastic Plate has 56 cm2 surface area
  • If <10 colonies/cm2, count 12 squares (6 consecutive horizontally and 6 consecutive vertically)
    • Total and Divide by 12 (average). Multiply by 56 to get total colonies on plate. Report as Estimate
  • If >10 colonies/cm2
    • Count 4 squares, average and multiply by 56
apc variations
APC Variations
  • Psychrotrophic
    • Incubate at 5-7 C for 10 days
    • Use Pre-poured Plates
  • Thermoduric
    • Hold 5 ml liquid sample or 1:10 diluent of solid sample in 60-80 C water bath for 30 min
    • Cool on ice for 10 min
    • Plate and incubate
dilution variations
Dilution Variations

99 ml Dilution Blanks

1 ml

1 ml

1 ml

10-7

10-3

10-5

10-1

1 ml

1 ml

0.1 ml

0.1 ml

1 ml

1 ml

0.1 ml

0.1 ml

-5

-7

-1

-3

-6

-8

-2

-4

CAN NOT use with petri-film