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Aerobic Plate Count, Gram Stain, and Isolation

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

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Aerobic Plate Count, Gram Stain, and Isolation

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

  2. Aerobic Plate Count • Provides general estimate of live, aerobic, bacteria • Excludes • Obligate Anaerobes • Microaerophiles

  3. 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

  4. APC Results • Evaluate Sanitation of Product • Predict Shelf-life • “Safety” Indicator • Monitor Environment

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Dilutions Dilution Blanks Containing 90 ml Diluent Sample Homogenate 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)

  11. Plating Put 1 ml of Each Dilution into Empty Petri-Dish 10-4 10-5 10-2 10-3 10-1 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml

  12. 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

  13. 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

  14. 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

  15. Obtaining Isolated Colonies • Goal is to get Isolated Colonies from Food and/or Cultures • Colonies can be Identified and Further Evaluated • Collect loopful of culture • Streak in each area starting • with area 1 • Flame Loop in between • areas 2 1 3 4

  16. 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

  17. Counting Plates 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

  18. Counting - Examples 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

  19. Counting Examples 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

  20. Counting Examples If Both Dilutions are outside Range, use the Higher Dilution (LOWER COUNTS) 7.5 x 103 cfu/ml or g*

  21. 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

  22. 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

  23. Dilution Variations 99 ml Dilution Blanks 1 ml 1 ml 1 ml 10-7 10-3 10-5 10-1 1 ml 1 ml 0.1 ml 0.1 ml 1 ml 1 ml 0.1 ml 0.1 ml -5 -7 -1 -3 -6 -8 -2 -4 CAN NOT use with petri-film

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