Aerobic plate count,

original sources
www.depts.ttu.edu/afs/.../Aerobic%20Plate%20Count,.ppt

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.
10 g/ml sample
90 ml of diluent
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 HomogenateDilution Blanks Containing 90 ml Diluent
10 ml 10 ml 10 ml10 ml
10
-1 10
-2
10
-3
10
-4
10
-5
(1:10)
(1:100)(1:1000)
(1:10000)
(1:100000)

11. Plating
10
-1
10
-2
10
-3 10
-4
10
-5
Put 1 ml of Each Dilution into Empty Petri-Dish
1 ml1 ml1 ml1 ml
1 ml1 ml
1 ml1 ml
1 ml1 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
1
2
3
4
•Collect loopful of culture
•Streak in each area starting
with area 1
•Flame Loop in between
areas
•Goal is to get Isolated Colonies from Food and/or Cultures
•Colonies can be Identified and Further Evaluated

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 1:100 1:10001:100001:100000
1 TNTC
1
TNTCTNTC 200 22
2
2 TNTCTNTCTNTC 150 10
Average- - - 175 -
1
Too Numerous to Count
2
Too Few to Count
•Average two countable plates and Multiply by Dilution Factor
•Count is 175 x 10
4
•Must Convert to TWO Significant Digits
•1.8 x 10
6
cfu/ml or g

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 10
2
– 2.5 x 10
4
125 x 10
3
– 1.3 x 10
5
AVERAGE – 7.8 x 10
4
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 10
4
– 3.5 x 10
6
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 10
3
cfu/ml or g*

21. Overloaded Plates
•Use Highest Dilution and Use Grid on Colony
Counter
–1 Grid = 1 cm
2
–A standard Plastic Plate has 56 cm
2
surface area
•If <10 colonies/cm
2
, 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/cm
2
–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
10
-1
10
-3
10
-5 10
-7
-8
-7
0.1 ml1 ml
-4
-3
0.1 ml1 ml
-6
-5
1 ml
-2
-1
1 ml
99 ml Dilution Blanks
0.1 ml
0.1 ml
1 ml 1 ml1 ml
CAN NOT use with petri-film