Classic Culture Procedures

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Classic Culture Procedures
MAJ John Beach Chief, Food Analysis Department
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Purpose

• Familiarize personnel with classic culture
  procedures for microbiological testing of dairy
  and water products.
• Identify specific requirements for standards
  relating to classic culture procedures.

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Procedures

• General information
• Standard plate count
• Coliform count
• Yeast and mold count
• Water testing

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Terminology Quiz

• What is the difference between an SPC, HPC, PPC,
  APC, TPC?
• SPC ??
• HPC ??
• PPC ??
• APC ??
• TPC ??

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Controls

• Steps or procedures performed to ensure
  reliability of laboratory data
• Controls and record keeping are a vital part of
  overall laboratory quality assurance
• Examples include diluent suitability, air
  quality, balance sensitivity, etc.

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Media and Dilution Blanks

• Verify sterility of dilution water and medium by
  pouring control plates for each lot/batch
• Plate dilution water with no sample added
• Pour plate with medium only
• Other dilution blank and media QC parameters?

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Air Quality

• What is air quality?
• Prepare air quality plates for both morning and
  afternoon platings
• Pour plate with medium only
• Leave lid off the plate for 15 minutes
• Incubate at 32 1C for 48 3 hours
• Should be lt15 CFU/15 minutes

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Incubation Parameters

• Put plates in incubator so there is at least 2.54
  cm between adjacent stacks of plates and between
  walls and stacks
• Why?
• Determine agar weight loss quarterly
• Weigh 5 empty plates, add SPC medium, weigh with
  medium, incubate at 32 1C for 48 3 hours,
  weigh after incubation
• Agar loss should be lt15

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Other QC Parameters

• Thermometers?
• pH meter?
• Autoclave?
• Water bath?
• Pipettes?
• Incubator?
• Balance?

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Standard Plate Count

• Suitable for estimating bacterial populations in
  most types of dairy products
• Ensure sample to be plated is homogeneous
• Why? How?
• Plate 2 or more dilutions per sample

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Standard Plate Count

• Select dilutions so that the total number of
  colonies will be between 25-250
• Mouth pipetting?
• Prepare medium
• QC requirements?
• Do not allow more than 20 minutes to elapse
  between diluting the first sample and pouring the
  last plate in the series

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Standard Plate Count

• Incubate plates at 32 1C for 48 3 hours
• Count plates promptly after incubation
• If impossible to count promptly after incubation,
  may store plates at 0 4.4C for lt24 hours
• When computing the SPC, report only the first two
  significant digits
• i.e., 12,700 reported as 13,000

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Coliform Count

• The coliform group comprises all aerobic and
  facultatively anaerboic, gram negative,
  nonspore-forming rods able to ferment lactose
  with production of acid and gas
• Typical coliforms include Escherichia,
  Enterobacter, and Klebsiella
• Presence is suggestive of unsanitary conditions
  or practices during production, processing, or
  storage

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Coliform Count

• QC procedures and techniques for the coliform
  count are similar to the SPC (Violet Red Bile
  Agar VRBA)
• Additional requirements are as follows
• After medium hardens, overlay with 3-4 mL of VRBA
• Incubate plates at 32 1C for 24 2 hours

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Yeast and Mold

• Standard Methods Agar (SPC) supplemented with
  chloramphenicol and chlortetracycline is the
  medium of choice
• Oxytetracycline-glucose-yeast extract (OGY) agar
• Dichloran-rose bengal-chloramphenicol (DRBC) agar
  
• Media and reagent QC requirements?

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Yeast and Mold

• Surface (spread) plating technique is preferable
  to the pour plate method
• Incubate plates at 20 25C for 5-7 days
• Countable plates are 15-150 colonies

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Water Testing

• All QC measures previously discussed also apply
  to microbiological testing of water (i.e., media,
  balance, autoclave, pH meter, etc.)
• Membrane filtration is very common
• Why?
• Are dilution blanks commonly used for water
  testing?

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Water Testing

• Heterotrophic plate count (HPC)
• Direct and -1 dilution
• Incubate at 32 1C for 48 3 hours
• Coliform count
• Incubate at 32 1C for 24 2 hours
• Fecal streptococci
• Incubate at 32 1C for 24 2 hours

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Water Testing

• Pseudomonas aeruginosa
• Incubate at 32 1C for 24 2 hours
• Sulfite-reducing anaerobes
• Incubate anaerobically at 32 1C for 48 3
  hours

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Questions

• THE BEST EXECUTIVE IS THE ONE WHO HAS SENSE
  ENOUGH TO PICK GOOD MEN TO DO WHAT HE WANTS DONE,
  AND SELF RESTRAINT TO KEEP FROM MEDDLING WITH
  THEM WHILE THEY DO IT.
• Theodore Roosevelt