Materials
- Lab coat
- Eye protection
- Benchkote if necessary
- Disinfectant and paper towels
- Discard jar with disinfectant
- Bunsen burner and mat
- Dilution series of organism
- Sterile pipettes or syringes (0.1 cm3)
- Sterile Petri dishes
- 20 cm3 volumes of sterile molten nutrient agar at 45°C
- Glass spreader
- Alcohol
Instructions
- Wear a lab coat and use eye protection.
- Label the underside of the plates with initials, date, sample and dilution. For greatest accuracy, each dilution should be plated in triplicate and the average of the three counts used.
- Remove a sterile 0.1 cm3 syringe or pipette tip from its pack/container. Do not touch the parts which will come in contact with organism. If using a pipette tip, carefully attach to the dispenser.
- Start with the highest dilution (i.e. 10-7 ).
- Using aseptic technique (i.e. flaming the neck of the tube or bottle after removal and before replacement of its lid), remove exactly 0.1 cm3 of the sample and transfer to the base of a sterile Petri dish.
- Using aseptic technique, pour 20 cm3 sterile nutrient agar over the sample and mix gently.
- Place syringe or pipette tip into discard jar.
- Repeat steps 5 – 7 for dilutions 10-6 , 10-5 and 10-4.
- When the plates are solidified and dry, incubate upside down at the appropriate temperature for the appropriate time.
- After incubation, select plates for counting that contain 30 – 300 colonies (samples which contain <30 colonies/0.1 cm3 diluent are subject to large fluctuations in numbers or sampling errors, plates which contain >300 colonies are likely to have overlapping colonies).
- Count accurately and record the number of colonies on each plate.
- Calculate the concentration of viable cells or colony forming units (cfu) in the original suspension.
