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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 Anaerobes– Microaerophiles
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
APC Results
• Evaluate Sanitation of Product
• Predict Shelf-life
• “Safety” Indicator
• Monitor Environment
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
Types of Samples
• Liquid– Non-viscous Liquids can be measured with pipet– Viscous liquids should be weighed
• Solid– Aseptically weigh Sample
• Sponge/SwabCollect sample by swabbing a defined area
• Environmental and Container– Rinse inside of Containers– Open Plate to Collect Air Samples– RODAC Plates
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 sample90 ml of diluent
1:10 Dilution – 10-1
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
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
Dilutions
Sample Homogenate Dilution Blanks Containing 90 ml Diluent
10 ml 10 ml 10 ml 10 ml
10-1 10-2 10-3 10-4 10-5
(1:10) (1:100) (1:1000)(1:10000)
(1:100000)
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 ml1 ml1 ml 1 ml1 ml1 ml1 ml
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
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
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
Counting Plates
Plate 1:10 1:100 1:1000 1:10000 1:100000
1 TNTC1 TNTC TNTC 200 222
2 TNTC TNTC TNTC 150 10
Average - - - 175 -1 Too Numerous to Count2 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
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
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 rangesThis means it is an ESTIMATED count
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*
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
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
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
Characteristics of major•bacterial groups.
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
direct microscopic 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)
viable count:
protocol for serial dilution and viable count of a sample:
measuring culture turbidity: