Counting Micro-organisms

Counting elephants is easy – counting bacteria is not quite as easy!

Counting Micro-organisms

A single tiny drop of nutrient broth incubated overnight may contain 5 000 000 cells – this is a lot to count.1cm3 may contain 108 cells.In order to estimate numbers it is necessary to dilute the sample.

SERIAL DILUTION1ml into 9ml = 1:10 dilution

conc. 10-1 10-2 10-3 10-4 10-5 10-6

Counting Micro-organisms

Counting Micro-organisms

It is possible to dilute a cell suspension in known steps in order to plate a specific range of cells. This set of plates contain a 1 to 10 dilution series.

TOTAL COUNTS

Direct counts

The haemocytometer is a specialized microscope slide used to count cells. The centre portion of the slide has etched grids with precisely spaced lines.

TOTAL COUNTS

To get an accurate count there should be between 40 and 70 cells in a 1 mm square. If not you dilute or concentrate the cell suspension as necessary .

TOTAL COUNTS

TOTAL COUNTS

Indirect counts

These depend on the effects of the organisms to estimate their numbers.As organisms grow they make the nutrient broth turbid.

This turbidity can be measured with a colorimeter

TOTAL COUNTS

The more organisms the greater the optical density of the solution.

TOTAL COUNTS

Indirect count

The Coulter counter is a probe which measures the change in conductivity of a solution as a bacteria passes through a narrow gap.

TOTAL COUNTS

Problem with INDIRECT COUNTS and DIRECT COUNTS is that they can not tell living cells apart from dead cellsAdvantage of INDIRECT COUNT is that the process can be automated

VIABLE COUNTS

Count only those cells capable of growingViable counts can be accomplished by such techniques as pour plating.Assumption each viable cell gives rise to a colony.

VIABLE COUNTS

Advantages:The method can be made to be very sensitive. One can count subsets of the population

DisadvantagesColony-forming units may underestimate cell numbers because of clumping or chains of cells. Counts require at least a few hours, usually overnight, for incubation.