1. Principle
- Method used bacterial culture plates of dry medium and cold H2O – soluble gel. Diluted test suspension are added to plates at a rate of 1.0 mL per plate. Pressure, when applied to plastic spreader placed on overlay film, spreads test portion over ca 20 sq.cm growtn area. Gelling agent is allowed to solidify and plates are incubate and then counted.
2. Scope
- Analysis Coliform and E.coli in Non Dairy Creamer by Petrifilm Coliform/ E.coli Count Plate method.
3. Reference
- AOAC Official method 991.14 – Coliform and Escherichia coli Counts in Foods – Dry rehyratable Film Methods
4. Appartus and Reagent
- Petrifilm Coliform/ E.coli Count Plates
- Plastic spreader – designed to spread test portion evenly over plate growth area.
- Pipets – Calibrated for bacteriological use to deliver 1.0 mL.
- Dilution pepton (water) – aseptically. Autoclave 15 min at 1210C.
5. Preparation of test suspensions
- Aseptically prepare 1:10 dilution (10 g/ 90 mL dilution pepton). Mix well and plate. Prepare additional dilutions as required. Ordinarily, 1:10 and 1:100 dilutions are sufficient.
6. Analysis
- Use dry-film aerobic count plates. Place plate on flat surface. Lift top film and inoculate 1 mL test suspension onto ceter of film base.
- Carefully roll top film down onto inoculum. Distribute test suspension over prescibed growth area with downward pressure on center of plastic spreader device.
- Leave plate undisturbed 1 min to permit gel to solidify.
- Coliform count: Incubate plate 24 h ± 2 h at 350C ± 10C.
- E.Coli count: Incubate plate 48 h ± 4 h at 350C ± 10C.
- In incubator, place plate in horizontal postion, clear side up, in stacks not exceeding 20 units.
7. Result
- Magnifier – illumination may also be used to facilitate counting.
- Colifroms appear as red colonies that have one or more gas bubbles associated (within one colony diameter) with them. Red colonies without gas bubbles are not counted as coliform organisms.
- E.coli colonies appear as blue colonies associate with gas bubbles; other coliforms appear as red colonies with gas.
- Count all colonies in countable range.
- To calculate Coliform and E.Coli count, multiply total number of colonies per plate (or average number of colonies per plate if counting duplicate plates of same dilution) by reciprocal of dilution used.