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 Report as 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 Solid Sponge/Swab 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 10-2 10-3 10-4 10-5 10-1 (1:100) (1:1000) (1:100000) (1:10) Sample Homogenate Dilution Blanks Containing 90 ml Diluent 10 ml 10 ml 10 ml 10 ml 10-2 10-3 10-4 10-5 10-1 (1:100) (1:1000) (1:100000) (1:10) (1:10000)

Plating Put 1 ml of Each Dilution into Empty Petri-Dish 10-2 10-3 10-4 10-5 10-1 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 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 1 TNTC 200 222 2 150 10 - 175 TNTC1 Average Plate 1:10 1:100 1:1000 1:10000 1:100000 1 TNTC1 TNTC 200 222 2 150 10 Average - 175 1 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 10-1 10-2 10-3 10-4 1 TNTC 300 150 10 2 200 100 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 300 2 400 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 ranges This means it is an ESTIMATED count

Counting Examples Plate 10-1 10-2 10-3 1 TNTC 300 10 2 400 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 Thermoduric 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 -5 -7 -1 -3 -6 -8 -2 -4 Characteristics of major bacterial groups. Dilution Variations 99 ml Dilution Blanks 1 ml 1 ml 1 ml 10-3 10-5 10-7 10-1 1 ml 1 ml 0.1 ml 1 ml 0.1 ml 1 ml 0.1 ml 0.1 ml -5 -7 -1 -3 -6 -8 -2 -4 CAN NOT use with petri-film

direct microscopic count: 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

viable 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)

protocol for serial dilution and viable count of a sample:

measuring culture turbidity: