Microbial CountMicrobial Count

Aim: Count the number of bacterial cells in a provided sample

Methods: Total count, Viable count

I. Total count This technique involves the count of all bacteria in a

given suspension either viable or dead. This can be

performed either:

1) Turbidimetry

Total number of bacteria is calculated from a standard calibration curve which is obtained by plotting log bacterial count of a suspension against absorbance of this suspension.

2) Counting chamber The most common method of enumerating the total

microbial cells is the direct counting of cell suspension in

a counting chamber of known volume using a

microscope. There are many types of counting chambers

from which the most common type is called

hemocytometer (originally designed for performing

blood cell counts). It consists of a thick glass microscopic

slide with a rectangular groove that creates a chamber

of known dimensions.

This chamber is marked with a counting grid (network)

of perpendicular lines divided into:

- Nine large squares (1mm x 1mm)

- The center square is divided into 25 smaller squares

(0.2mm x 0.2mm).

- Depth of the counting chamber : 0.1 mm.

Therefore it is possible to count the number of cells in

a specific volume of fluid (10-20 ul in each slide notch),

and thereby calculate the concentration of cells in the

original fluid volume.

Loading the counting chamber

II. Viable count1) Pour plate (seed) methodPrinciple High numbers of bacteria are often present in samples.Some samples may contain up to 1010 bacteria /ml. Atthis concentration, a 0.1 ml volume plated onto anutrient agar plate could potentially yield one billion colonies which is impossible to count in a Petri dish. To overcome this problem, serial dilutions of the sampleprior to plating reduce the number of organisms suchthat when a small volume of the diluted sample is plated,the colonies are countable. For statistical accuracy, onlyplates containing between 30 and 300 colonies arecounted.

Procedure: 1. Make a serial dilution of the supplied bacterial

culture by transferring aseptically exactly 1 ml amounts (using a sterile 1 ml pipette) into the sterile test tubes containing 9 ml saline solutions.

2. Each of the tubes must be thoroughly mixed (by means of a new 1 ml pipette) to insure even distribution of the organism.

3. With a sterile 1 ml pipette, aseptically transfer 1 ml of each dilution into each of 3 sterile Petri dishes. Mark each plate with the dilution present.

4. Melt the nutrient agar, pour into Petri dishes, and mix the bacterial suspension with the nutrient agar.

5. Incubate; count-the number of colonies choosing plates that have between 30 and 300 colonies.

1/10 1/100 1/1000

Precautions for a succesful experiment: 1) Good mixing of the m.o. both in: a) the 9-ml saline tubes b) the Petri dishes together with melted nutrient agar. 2) Melted agar tubes must be poured at a temp. not too

high (to avoid killing of the m.o.) & not too low (to allow for a sufficient mixing time that will insure an even distribution of the m.o. throughout the plate).

3) Petri dishes must be inverted upside-down immediately after solidification of poured nutrient agar to prevent condensed water vapor droplets in the inner side of the plate lid from falling-down over the agar surface, otherwise, wet agar surface will cause overlapping of the microbial colonies appearing as an uncountable patch over agar surface.

Expected result

2) Membrane filtration method

In this method cells are filtered onto membrane filter which is then placed over nutrient agar surface plates. Number of c.f.u/ml is determined after incubation at suitable temp. & time.