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 Dont 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/Swab Collect 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.

90 ml of diluent 10 g/ml sample

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 (1:10)

10-2 10-3 (1:100) (1:1000)

10-4 (1:10000)

10-5 (1:100000)

Plating
Put 1 ml of Each Dilution into Empty Petri-Dish
10-1 10-2 10-3

10-4

10-5

1 ml 1 ml

1 ml 1 ml

1 ml 1 ml 1 ml 1 ml 1 ml 1 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 Dont 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

Goal is to get Isolated Colonies from Food and/or Cultures Colonies can be Identified and Further Evaluated 2 1 4 Collect loopful of culture Streak in each area starting with area 1 Flame Loop in between areas

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 2
Average
1

TNTC1

TNTC TNTC

200 150 175

222 10 -

TNTC TNTC TNTC -

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

Counting - Examples
Plate 1 2 Average 10-1 TNTC TNTC 10-2 300 200 250 10-3 150 100 125 10-4 10 20 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 1 2 Average 10-1 TNTC TNTC 10-2 TNTC TNTC 10-3 TNTC TNTC 10-4 300 400 350

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

Counting Examples
Plate 1 2 Average 10-1 TNTC TNTC 10-2 300 400 250 10-3 10 5 125

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
99 ml Dilution Blanks
1 ml 1 ml 1 ml

10-1

10-3

10-5

10-7

1 ml 0.1 ml 1 ml 0.1 ml 1 ml 0.1 ml 1 ml 0.1 ml

-1 -2

-3 -4

-5

-7

-6

-8

CAN NOT use with petri-film