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

Aerobic plate count,

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Page 1: Aerobic plate count,

Aerobic Plate Count, Gram Stain, and Isolation

Food Microbiology Laboratory

Page 2: Aerobic plate count,

Aerobic Plate Count

• Provides general estimate of live, aerobic, bacteria

• Excludes– Obligate Anaerobes– Microaerophiles

Page 3: Aerobic plate count,

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

Page 4: Aerobic plate count,

APC Results

• Evaluate Sanitation of Product

• Predict Shelf-life

• “Safety” Indicator

• Monitor Environment

Page 5: Aerobic plate count,

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

Page 6: Aerobic plate count,

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

Page 7: Aerobic plate count,

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

Page 8: Aerobic plate count,

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

Page 9: Aerobic plate count,

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

Page 10: Aerobic plate count,

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)

Page 11: Aerobic plate count,

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

Page 12: Aerobic plate count,

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

Page 13: Aerobic plate count,

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

Page 14: Aerobic plate count,

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

Page 15: Aerobic plate count,

Obtaining Isolated Colonies

12

34

•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

Page 16: Aerobic plate count,

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

Page 17: Aerobic plate count,

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

Page 18: Aerobic plate count,

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

Page 19: Aerobic plate count,

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

Page 20: Aerobic plate 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*

Page 21: Aerobic plate count,

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

Page 22: Aerobic plate count,

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

Page 23: Aerobic plate count,

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