Aerobic Plate Count, Gram Stain, and Isolation

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Aerobic Plate Count, Gram Stain, and Isolation

• Food Microbiology Laboratory

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Aerobic Plate Count

• Provides general estimate of live, aerobic,
  bacteria
• Excludes
• Obligate Anaerobes
• Microaerophiles

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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

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APC Results

• Evaluate Sanitation of Product
• Predict Shelf-life
• Safety Indicator
• Monitor Environment

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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
• Dont Use on Fermented Foods
• Colonies may be too small to see

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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

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Protocol for Plate Counts

• Prepare a Sample Homogenate
• 110 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
110 Dilution 10-1
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Formula

• 10 ml/g sample, want 1100 dilution
• 100 10 90 ml of diluent needed
• Start with Different Sample Sizes
• 50 g sample
• Must have 500 g total volume for 110
• 500 50 450 ml diluent needed
• 95 ml sample
• Must have 950 total volume for 110
• 950 95 855 ml of diluent

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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

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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
(1100)
(11000)
(1100000)
(110)
(110000)
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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
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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

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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
• Dont Put Lids on tightly

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Gram Stain

• Gram Positive or Gram Negative
• Based on Cell wall Structure
• Gram
• Very Thick Cell Wall due to Peptidoglycan Layer
• N-acetylglucosamine
• N-acetylmuramic acid
• Two amino sugars linked by beta 1,4, bonds
• Gram
• Thin Cell Wall with a Lipopolysaccharide layer

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Obtaining Isolated Colonies

• Goal is to get Isolated Colonies from Food and/or
  Cultures
• Colonies can be Identified and Further Evaluated

• Collect loopful of culture
• Streak in each area starting
• with area 1
• Flame Loop in between
• areas

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1
3
4
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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

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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

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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
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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
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Counting Examples
If Both Dilutions are outside Range, use the
Higher Dilution (LOWER COUNTS) 7.5 x 103 cfu/ml
or g
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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 lt10 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 gt10 colonies/cm2
• Count 4 squares, average and multiply by 56

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APC Variations

• Psychrotrophic
• Incubate at 5-7 C for 10 days
• Use Pre-poured Plates
• Thermoduric
• Hold 5 ml liquid sample or 110 diluent of solid
  sample in 60-80 C water bath for 30 min
• Cool on ice for 10 min
• Plate and incubate

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Dilution Variations
99 ml Dilution Blanks
1 ml
1 ml
1 ml
10-7
10-3
10-5
10-1
0.1 ml
1 ml
1 ml
0.1 ml
0.1 ml
1 ml
1 ml
0.1 ml
-7
-5
-3
-1
-8
-6
-4
-2
CAN NOT use with petri-film