Measurement of bacterial growth

Contents

• Determination of total count

– Coulter counter

– Counting chamber

– Cell mass

– Turbidometry

– Membrane filtration

• Determination of viable count

– Plate count

– Membrane filtration

Intended
Learning objectives

At the end of this
lecture, student will be able to:

• Differentiate between total and viable count

• Explain the different methods for total and viable
counting

Measurement of Microbial Growth

Total count

Measurement of cell
numbers by direct counting using a counting chamber

– Easy, inexpensive, and relatively quick

– Gives information about the size and morphology of
microorganisms

– Petroff-Hausser counting chamber – counting procaryotes

– Hemocytometers can be used for both procaryotes and
eucaryotes

– Procaryotes are more easily counted in these chambers if
they are stained, or when a phase-contrast or a fluorescence microscope

These specially designed slides have chambers of known depth
with an etched grid on the chamber bottom. The number of microorganisms in a
sample can be calculated by taking into account the chamber’s volume and any
sample dilutions required

Disadvantages of
counting chamber

• The microbial population must be fairly large for accuracy
because such a small volume is sampled

• It is also difficult to distinguish between living and
dead cells in counting chambers without special techniques.

Coulter Counter

• Larger microorganisms such as protozoa, algae, and
non-filamentous yeasts

• The microbial suspension is forced through a small hole or
orifice.

• An electrical current flows through the hole, and
electrodes placed on both sides of the orifice measure its electrical
resistance

• Every time a microbial cell passes through the orifice,
electrical resistance increases (or the conductivity drops) and the cell is
counted.

Coulter counter

Coulter Counter –
Advantages and disadvantages

• The Coulter Counter gives accurate results with larger
cells and is extensively used in hospital laboratories to count red and white
blood cells.

• It is not as useful in counting bacteria because of
interference by small debris particles, the formation of filaments, and other
problems.

Membrane filtration

• The sample is filtered through a black polycarbonate
membrane filter to provide a good background for observing fluorescent objects

• The bacteria then are stained with a fluorescent dye such
as acridine orange or DAPI and observed microscopically

• Acridine orange–stained microorganisms glow orange or
green and are easily counted with an epifluorescence microscope

Counting bacteria by
membrane filtration

Viable
count

Colony counting using
streak plate or spread plate technique

• A diluted sample of bacteria or other microorganisms is
dispersed over a solid agar surface.

• Each microorganism or group of microorganisms develops
into a distinct colony.

• The original number of viable microorganisms in the sample
can be calculated from the number of colonies formed and the sample dilution.

• Since it is not possible to be absolutely certain that
each colony arose from an individual cell, the results are often expressed in
terms of colony forming units (CFU) rather than the number of microorganisms.

Limitations of colony
counting

• Low counts will result if clumps of cells are not broken
up and the microorganisms well dispersed.

• Counts will also be low if the agar medium employed cannot
support growth of all the viable microorganisms present

• The hot agar used in the pour-plate technique may injure
or kill sensitive cells

Membrane filtration
technique

• Counts of colonies growing on special membrane filters
having pores small enough to trap bacteria

• Sample is drawn through a special membrane filter

• The filter is then placed on an agar medium or on a pad
soaked with liquid media

Incubated until each cell forms a separate colony colony
count gives the number of microorganisms in the filtered • sample

Membrane filtration
for total or viable count

Measurement
of cell mass

• Increases in the total cell mass, as well as in cell
numbers, accompany population growth

• Therefore techniques for measuring changes in cell mass
can be used in following growth

Determination of
microbial dry weight

• Cells growing in liquid medium are collected by
centrifugation,

• washed, dried in an oven, and weighed.

• This is an especially useful technique for measuring the
growth of fungi.

• Time consuming, however, and not very sensitive

Turbidity and
Microbial Mass Measurement

• Determination of microbial mass by measurement of light
absorption.

• As the population and turbidity increase, more light is
scattered and the absorbance reading given by the spectrophotometer increases.

Turbidimetric method

Total protein or
nitrogen content

• If the amount of a substance in each cell is constant, the
total quantity of that cell constituent is directly related to the total
microbial cell mass.

• For example, a sample of washed cells collected from a
known volume of medium can be analyzed for total protein or nitrogen.

• An increase in the microbial population will be reflected
in higher total protein levels

Summary

• Total count – counting the living and dead cells

• Viable count – counting only the living cells that are
capable of multiplication

• Total count is determined by counting chambers, coulter
counter, cell mass determination, turbidity measurement, membrane filtration

• Viable count can be determined by plate count and membrane
filtration