Aerobic plate count,

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Aerobic plate count,

  1. 1. Aerobic Plate Count, Gram Stain, and Isolation Food Microbiology Laboratory
  2. 2. Aerobic Plate Count • Provides general estimate of live, aerobic, bacteria • Excludes – Obligate Anaerobes – Microaerophiles
  3. 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. 4. APC Results • • • • Evaluate Sanitation of Product Predict Shelf-life “Safety” Indicator Monitor Environment
  5. 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. 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. 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. 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. 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. 10. Dilutions Sample Homogenate Dilution Blanks Containing 90 ml Diluent 10 ml 10-1 (1:10) 10 ml 10 ml 10-2 10-3 (1:100) (1:1000) 10 ml 10-4 (1:10000) 10-5 (1:100000)
  11. 11. Plating Put 1 ml of Each Dilution into Empty Petri-Dish 10 -1 1 ml 1 ml 10-2 1 ml 1 ml 10-3 10-4 10-5 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml
  12. 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. 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. 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. 15. Obtaining Isolated Colonies •Goal is to get Isolated Colonies from Food and/or Cultures •Colonies can be Identified and Further Evaluated 2 1 4 3 •Collect loopful of culture •Streak in each area starting with area 1 •Flame Loop in between areas
  16. 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. 17. Counting Plates Plate 1:10 1 TNTC1 2 Average 1:10000 1:100000 TNTC TNTC 200 222 TNTC TNTC TNTC 150 10 175 - - 1:100 - 1:1000 - 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 1
  18. 18. 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
  19. 19. 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 ranges This means it is an ESTIMATED count
  20. 20. 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*
  21. 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. 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. 23. Dilution Variations 99 ml Dilution Blanks 1 ml 1 ml 10-1 10-3 1 ml 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 -3 -5 -7 -2 -4 -6 -8 CAN NOT use with petri-film

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