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
• Don’t 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
– 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

8. 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

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
Sample Homogenate Dilution Blanks Containing 90 ml Diluent
10 ml
10-1
(1:10)
10 ml
10 ml
10-2
10-3
(1:100) (1:1000)
10 ml
10-4
(1:10000)
10-5
(1:100000)

11. Plating
Put 1 ml of Each Dilution into Empty Petri-Dish
10
-1
1 ml 1 ml
10-2
1 ml 1 ml
10-3
10-4
10-5
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
• Don’t 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
2
1
4
3
•Collect loopful of culture
•Streak in each area starting
with area 1
•Flame Loop in between
areas

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
Plate
1:10
1
TNTC1
2
Average
1:10000
1:100000
TNTC TNTC
200
222
TNTC TNTC TNTC
150
10
175
-
-
1:100
-
1:1000
-
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
1

18. Counting - Examples
Plate
10-1
10-2
10-3
10-4
1
TNTC
300
150
10
2
TNTC
200
100
20
Average
-
250
125
TFTC
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
Plate
10-1
10-2
10-3
10-4
1
TNTC
TNTC
TNTC
300
2
TNTC
TNTC
TNTC
400
Average
-
-
-
350
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
Plate
10-1
10-2
10-3
1
TNTC
300
10
2
TNTC
400
5
Average
-
250
125
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 <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

22. 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

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