9.1.1. Plate count
The plate count method means diluting bacteria with a diluent solution (e.g. sterile saline) until the bacteria are dilute enough to count accurately when spread on a plate. The assumption is that each viable bacterial cell will develop into a single colony. Bacterial cell numbers need to be reduced by dilution, because more than 200 colonies on a standard 9 cm plate are likely to produce colonies too close to each other to be distinguished as distinct colony-forming units (CFUs).
The materials needed to perform a plate count are:
- Sterile 0.9% NaCl (sterile saline)
- Sterile tubes, tips and spreaders
- Agar plates (three per sample)
- Make a ten-fold dilution serial dilution of your bacterial culture (broth). Dilute the suspension to a dilution factor of 10-6 (a million-fold dilution).
- Spread out aliquots using a sterile bacterial spreader (0.1 ml) of each dilution onto 3 agar plates.
- Incubate the plates for 4-7 days as previously described in section 5.3.
- Count the number of bacterial colonies
that appear on each of the plates that has between 30 and 200 colonies.
Any plate which has more than 200 colonies is designated as "too numerous to count”. Plates with fewer than 30 colonies do not have enough individuals to be statistically acceptable.
- To compute the estimated number of
bacteria on the surface that you tested, use the following formula:
B = N/d
where: B = number of bacteria; N = average number of colonies counted on three plates; d = dilution factor.
Example: Plate 1: 56 CFU; Plate 2: 75 CFU; Plate 3: 63 CFU; Average (N) = 64.7; Dilution (d ) = 1/1,000; B = (64.7x1,000) = 64,700 bacteria in 0.1 ml, 647,000 bacteria per ml.