Aerobic Plate Count,.ppt

7/29/2019 Aerobic Plate Count,.ppt

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Aerobic Plate Count, Gram

Stain, and Isolation

Food Microbiology Laboratory


2/23

Aerobic Plate Count

Provides general estimate of live, aerobic,

bacteria

ExcludesObligate Anaerobes

Microaerophiles


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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


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APC Results

Evaluate Sanitation of Product

Predict Shelf-life

Safety Indicator Monitor Environment


5/23

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

Dont Use on Fermented Foods

Colonies may be too small to see


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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


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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 Dilution10-1


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Formula

10 ml/g sample, want 1:100 dilution

10010 = 90 ml of diluent needed

Start with Different Sample Sizes50 g sample Must have 500 g total volume for 1:10

50050 = 450 ml diluent needed

95 ml sample Must have 950 total volume for 1:10

95095 = 855 ml of diluent


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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


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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)


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Plating

10-110-2 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


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APCProtocol

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


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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

Dont Put Lids on tightly


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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


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Obtaining Isolated Colonies

12

34

Collect loopful of culture

Streak in each area startingwith 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


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Counting Plates

Only count plates with 25-250 colonies

More than 250

Too Numerous To CountTNTC Less than 25

Too Few to Count - TFTC


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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 CountAverage 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


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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 ONEPLATE is in RANGE, use BOTH for Average.

250 x 1022.5 x 104

125 x 1031.3 x 105

AVERAGE7.8 x 104 cfu/g or ml


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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 range350 x 1043.5 x 106 cfu/ml or g*

Use an * when using dilutions outside countable ranges

This means it is an ESTIMATED count


20/23

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*


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Overloaded Plates

Use Highest Dilution and Use Grid on ColonyCounter

1 Grid = 1 cm2

A standard Plastic Plate has 56 cm2 surface area If 10 colonies/cm2

Count 4 squares, average and multiply by 56


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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 ofsolid sample in 60-80 C water bath for 30 min

Cool on ice for 10 minPlate and incubate


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Dilution Variations

10-110-3 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

Aerobic Plate Count,.ppt

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