Classic Culture Procedures

MAJ (First Mi. Last)

Chief, Food Analysis Department

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.

                 

Procedures

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

                 

Terminology Quiz

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

• SPC = ??• HPC = ??• PPC = ??• APC = ??• TPC = ??

                 

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.

                 

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?

                 

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 ± 1˚C for 48 ± 3 hours– Should be <15 CFU/15 minutes

                 

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 ± 1˚C for 48 ± 3 hours, weigh after incubation

– Agar loss should be <15%

                 

Other QC Parameters

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

                 

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

                 

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

                 

Standard Plate Count

• Incubate plates at 32 ± 1˚C for 48 ± 3 hours

• Count plates promptly after incubation– If impossible to count promptly after

incubation, may store plates at 0 – 4.4˚C for <24 hours

• When computing the SPC, report only the first two significant digits– i.e., 12,700 reported as 13,000

                 

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

                 

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 ± 1˚C for 24 ± 2

hours

                 

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?

                 

Yeast and Mold

• Surface (spread) plating technique is preferable to the pour plate method

• Incubate plates at 20 – 25˚C for 5-7 days

• Countable plates are 15-150 colonies

                 

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?

                 

Water Testing

• Heterotrophic plate count (HPC)– Direct and -1 dilution– Incubate at 32 ± 1˚C for 48 ± 3 hours

• Coliform count– Incubate at 32 ± 1˚C for 24 ± 2 hours

• Fecal streptococci– Incubate at 32 ± 1˚C for 24 ± 2 hours

                 

Water Testing

• Pseudomonas aeruginosa– Incubate at 32 ± 1˚C for 24 ± 2 hours

• Sulfite-reducing anaerobes– Incubate anaerobically at 32 ± 1˚C

for 48 ± 3 hours

                 

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