aerobic plate count,

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

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

Obtaining Isolated Colonies

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•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

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