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Aerobic Plate Count, Gram Stain, and Isolation Food

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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 ... – PowerPoint PPT presentation

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Title: Aerobic Plate Count, Gram Stain, and Isolation Food


1
Aerobic Plate Count, Gram Stain, and Isolation
  • Food Microbiology Laboratory

2
Aerobic Plate Count
  • Provides general estimate of live, aerobic,
    bacteria
  • Excludes
  • Obligate Anaerobes
  • Microaerophiles

3
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

4
APC Results
  • Evaluate Sanitation of Product
  • Predict Shelf-life
  • Safety Indicator
  • Monitor Environment

5
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

6
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

7
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
8
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

9
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

10
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)
11
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
12
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

13
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

14
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

15
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

2
1
3
4
16
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

17
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

18
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
19
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
20
Counting Examples
If Both Dilutions are outside Range, use the
Higher Dilution (LOWER COUNTS) 7.5 x 103 cfu/ml
or g
21
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

22
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

23
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
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