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 Report as
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 Solid Sponge/Swab
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 10-2 10-3 10-4 10-5 10-1 (1:100) (1:1000) (1:100000) (1:10)
Sample Homogenate
Dilution Blanks Containing 90 ml Diluent
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)

11. Plating Put 1 ml of Each Dilution into Empty Petri-Dish 10-2 10-3 10-4
10-5
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 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 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. 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

15. Counting Plates 1 TNTC 200 222 2 150 10 - 175 TNTC1 Average Plate 1:10
1:100
1:1000
1:10000
1:100000 1
TNTC1
TNTC
200
222 2
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 104
Must Convert to TWO Significant Digits
1.8 x 106 cfu/ml or g

16. Counting - Examples Plate 10-1 10-2 10-3 10-4 1 TNTC 300 150 10 2 200
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

17. Counting Examples Plate 10-1 10-2 10-3 10-4 1 TNTC 300 2 400 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

18. Counting Examples Plate 10-1 10-2 10-3 1 TNTC 300 10 2 400 5 Average -
250
125
If Both Dilutions are outside Range, use the Higher Dilution
(LOWER COUNTS)
7.5 x 103 cfu/ml or g*

19. 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

20. APC Variations Psychrotrophic Thermoduric
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 C water bath for 30 min
Cool on ice for 10 min
Plate and incubate

21. Dilution Variations 99 ml Dilution Blanks -5 -7 -1 -3 -6 -8 -2 -4
Characteristics of major
bacterial groups.
Dilution Variations
99 ml Dilution Blanks
1 ml
1 ml
1 ml
10-3
10-5
10-7
10-1
1 ml
1 ml
0.1 ml
1 ml
0.1 ml
1 ml
0.1 ml
0.1 ml -5 -7 -1 -3 -6 -8 -2 -4
CAN NOT use with petri-film

22. direct microscopic count:
Petroff-Hauser counting chamber
requires at least 106 cells/mL
counts all cells (a total count)
without special stains, cannot distinguish between live and dead cells

23. viable count:
only those cells that are viable and able to grow and form colonies under the provided conditions are counted
results reported as colony forming units/mL (CFU/mL)

24. protocol for serial dilution and viable count of a sample:

25. measuring culture turbidity: