Counting bacterial colonies on agar plates is a simple and effective method for determining the number of viable bacteria in a sample.
This method relies on the growth of a bacterial cell in an agar plate to form a visible colony, only living or viable bacterial cells will be counted. If the total cell count is required, please use a counting chamber (Haemocytometer).
Aseptic technique is used for all steps.
There are four major steps in the procedure:
· preparation of serial dilutions
· mixing the serial dilutions into agar
· counting the resulting bacterial colonies
· calculation of total numbers of viable bacteria from these counts.
Preparation of Serial Dilutions
Take a liquid sample containing the bacteria to be counted and mix well.
Take 6 x 20 mL McCartney (or universal) bottles and label them 10-1, 10-2, 10-3,10-4, 10-5 and 10-6.
Pipette 9 mL of sterile PBS or sterile Saline (or other isotonic diluent) into each of the bottles.
Pipette 1 mL of the undiluted sample into the bottle marked 10-1. Discard the pipette and using a new pipette, mix the contents and pipette 1 mL from the 10-1 bottle into the 10-2 bottle.
Continue like this until transfers have been completed to the 10-6 bottle.
You now have the following dilutions of the original liquid sample:
Tube
Dilution Factor
Dilution
1
10-1
1/10
2
10-2
1/100
3
10-3
1/1,000
4
10-4
1/10,000
5
10-5
1/100,000
6
10-6
1/1,000,000
Mixing the dilutions into agar plates
Liquefy at least 100 mL of appropriate agar by autoclaving.
Place the bottle of molten agar in a 50°C water bath and allow the agar to cool to 50°C.
Mark six empty sterile agar plates (Petri dishes) 10-1 to 10-6 on the base of the plate NOT the lid. Add other required details such as sample type, HL number, date and culture conditions to the base of the plates.
Remove the agar bottle from the 50°C water bath and wipe the outside of the bottle with paper towelling to remove water. Working quickly to avoid cooling of the agar to 42°C (this is the temperature at which it sets) pour about 15 mL of molten agar into each of the six plates. The agar should be approximately 7 mm thick.
Pipette 1 mL of each of the dilutions into the base of correctly labelled plates using a separate pipette to avoid carryover errors.
Gently swirl each plate to mix the 1 mL of diluted sample into the 15 mL of agar.
Leave the plate without moving for at least 13 minutes to allow the agar to set.
When the agar is set, incubate the plate as appropriate.
Counting bacterial colonies
After an appropriate incubation period examine the plates for colonial growth.
Colonies will form on the top of the agar as well as in the agar. Those on top of the agar will be larger but all colonies must be counted.
Select plates that appear to have between 30 – 300 colonies in and on the agar as this gives the best statistical representation of the number of bacteria in the undiluted sample.
Using a light box or colony counter (if one is available) and marker pen (put a dot above each colony as you count it), count the number of colonies in each of the dilutions having between 30 – 300 colonies. This will become easier with practice.
Write the numbers and relevant dilutions down.
ASSUMPTIONS: There are two assumptions that are used:
· All viable cells form colonies.
· Each colony counted is formed from one bacterial cell.
The above assumptions do not allow the use of the term bacteria/mL as a unit. Instead the unit Colony Forming Units/mL or CFU/mL is used.
(a) The pour-plate method is employed for bacterial-cell enumeration and isolation
(b) In the pour-plate method of addition of cells to solid medium contained within a petri dish, cells are added to melted (but not too hot) solid medium
(c) The melted solid medium is then poured into a petri dish and allowed to harden
(d) Colonies appear both within, beneath, and on top of the agar
(e) See Figure 6.7, Calculation of the number of bacteria per milliliter of culture using serial dilution
(16) Spread plate
(a) The spread plate method is employed for bacterial-cell enumeration and isolation
(b) In the spread-plate method of addition of cells to solid medium, a small volume of culture is dropped onto the surface of agar that has already hardened in a petri dish
(c) The volume is then spread around the agar surface
(d) Colonies will grow solely on the surface of the agar
(e) This technique is advantageous particularly when cells are sensitive to exposure to relatively high temperatures plus the method does not require a prior melting of the solid medium
(f) See Figure 6.7, Calculation of the number of bacteria per milliliter of culture using serial dilution
