Obtaining Pure Culture of Microorganisms: 6 Methods

The methods are: 1. Streak Plate Method 2. Pour Plate Method 3. Spread Plate Method 4. Serial
Dilution Method 5. Single Cell Isolation Methods 6. Enrichment Culture Method.

1. Streak Plate Method:

This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed
culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar
medium (Fig. 16.13). The successive streaks “thin out” the inoculum sufficiently and the micro-
organisms are separated from each other.

It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. These
plates are incubated to allow the growth of colonies. The key principle of this method is that, by
streaking, a dilution gradient is established across the face of the Petri plate as bacterial cells are
deposited on the agar surface.

Because of this dilution gradient, confluent growth does not take place on that part of the medium where
few bacterial cells are deposited. Presumably, each colony is the progeny of a single microbial cell thus
representing a clone of pure culture. Such isolated colonies are picked up separately using sterile
inoculating loop/needle and re-streaked onto fresh media to ensure purity.

2. Pour Plate Method:

This method involves plating of diluted samples mixed with melted agar medium (Fig. 16.14). The
main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to
permit a thorough distribution of bacterial cells within the medium.

Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium
maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C). The bacteria
and the melted medium are mixed well.
The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated.
When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium
(subsurface colonies) and on the medium (surface colonies). These isolated colonies are then picked up
by inoculation loop and streaked onto another Petri plate to insure purity.

Pour plate method has certain disadvantages as follows:

(i) The picking up of subsurface colonies needs digging them out of the agar medium thus interfering
with other colonies, and

(ii) The microbes being isolated must be able to withstand temporary exposure to the 42-45°
temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of
psychrophilic microorganisms.

However, the pour plate method, in addition to its use in isolating pure cultures, is also used for
determining the number of viable bacterial cells present in a culture.

3. Spread Plate Method:

In this method (Fig. 16.15), the mixed culture or microorganisms is not diluted in the melted agar
medium (unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid,
usually, water or physiological saline.
A drop of so diluted liquid from each tube is placed on the center of an agar plate and spread evenly
over the surface by means of a sterilized bent-glass-rod. The medium is now incubated.

When the colonies develop on the agar medium plates, it is found that there are some plates in which
well-isolated colonies grow. This happens as a result of separation of individual microorganisms by
spreading over the drop of diluted liquid on the medium of the plate.

The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In contrast to
pour plate method, only surface colonies develop in this method and the microorganisms are not
required to withstand the temperature of the melted agar medium.

4. Serial Dilution Method:

As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that
have not yet been successfully cultivated on solid media and grow only in liquid media.

A microorganism that predominates in a mixed culture can be isolated in pure form by a series of
dilutions. The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of
tubes of sterile liquid medium are inoculated with aliquots of each successive dilution.

The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there
are some tubes showing growth of only one individual microbe. For convenience, suppose we have a
culture containing 10 ml of liquid medium, containing 1,000 microorganisms (Fig. 16.16.), i.e., 100
microorganisms/ml of the liquid medium.

If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then
have 100 microorganisms in 10 ml or 10 microorganisms/ml. If we add 1 ml of this suspension to
another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism.
If this tube shows any microbial growth, there is a very high probability that this growth has resulted
from the introduction of a single microorganism in the medium and represents the pure culture of that
microorganism.

5. Single Cell Isolation Methods:

An individual cell of the required kind is picked out by this method from the mixed culture and is
permitted to grow.

i. Capillary pipette method:

Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile
pipette drawn to a capillary. One then examines each drop under the microscope until one finds such a
drop, which contains only one microorganism. This drop is removed with a sterile capillars pipette to
fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure
culture (Fig. 16.17).
ii. Micromanipulator method:
Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture.
This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial
cell) from a hanging drop preparation. The micro-manipulator has micrometer adjustments by means
of which its micropipette can be moved right and left, forward, and backward, and up and down.

A series of hanging drops of a diluted culture are placed on a special sterile coverslip by a micropipette.
Now a hanging drop is searched, which contains only a single microorganism cell.

This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile
medium on another sterile coverslip. When the number of cells increases in that drop as a result of
multiplication, the drop is transferred to a culture tube having suitable medium. This yields a pure
culture of the required microorganism.

The advantages of this method are that one can be reasonably sure that the cultures come from a single
cell and one can obtain strains with in the species. The disadvantages are that the equipment is
expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason why
this method is reserved for use in highly specialised studies.

6. Enrichment Culture Method:

Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or
that have slow growth rates compared to the other species present in the mixed culture.

The enrichment culture strategy provides a specially designed cultural environment by incorporating a
specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium
of known composition and, specific condition of incubation favours the growth of desired
microorganisms but, is unsuitable for the growth of other types of microorganisms.
Methods of measuring bacterial growth

Estimating the growth of bacteria is extremely important. Environmental health officers regularly
inspect food premises and take sample for analysis. Water boards check water supplies daily. Many
products are produced using bacteria grown in fermenters. Measuring their growth is an important
part of the process.

There are several different methods of measuring growth:

• Rough estimates of growth rates can be made by regularly measuring the diameter of a bacterial or
fungal colony as it spreads from a central point to cover the surface of a solid growth medium (such
as an agar plate)

• The size of a population of microorganisms in liquid culture may be measured by counting cells
directly or by first diluting the original sample and then counting cell numbers (see below), or
by taking some indirect method such as the turbidity (cloudiness) of the culture.

Direct cell counts may be divided into:

1. Total counts = which include both living cells and dead cells
2. Viable counts = which count living cells only

In practice, it is never possible to count whole populations of microorganisms. Instead, the cells in
a very small sample of culture are counted, and the result multiplied up to give a population
density in organisms per cm3 of culture. Even then, the population density is likely to be so high
that cell counts are usually made in known dilutions of the culture, usually in 10-fold steps. This is
known as serial dilution.

The dilution plating technique is as follows:

A culture medium, such as milk or a water sample, is made into a series of dilutions using the serial
dilution technique.
1cm3 (the same as saying 1ml) of each of the diluted samples (see diagram above), are
individually streaked onto sterile agar plates, which are then placed in an incubator at 25oC for two
days to allow time for the bacteria to grow. After all the streaks have been allowed to grow, the
dilution at which the colonies are distinct and separate is noted down (this might be the 1/1000
dilution from the example above). If the dilution is insufficient then colonies will merge, referred to as
‘clumping’, and counting is inaccurate (the example above would be the 1/10 and the 1/100 dilution).
The distinct and separate colonies of bacteria from the agar plate you have chosen are counted with
the assumption being made that each colony has arisen from a single cell, which has divided
asexually, from the original culture medium. To find the total viable cell count the number of
colonies you count is multiplied by the appropriate dilution factor. So in the example above this
would be:

159 colonies counted on the 1/1000 dilution agar plate

So 159 x 1000 = 1.59 x 10 to the power 5 bacteria in the original sample.

However, if the dilution is insufficient then colonies will merge (see 1/10 and 1/100 dilution agar
plates above), referred to as ‘clumping’, and counting is inaccurate, resulting in an
underestimate of numbers
Using a Haemocytometer

A more accurate method involves using a haemocytometer. This is a specialised microscope

slide originally used to count red blood cells. Using the haemocytometer gives total cell counts as it
is not possible to distinguish between living and dead cells (you are not required to describe or use
a haemocytometer.)

Using Turbidimetry

A third method, known as turbidimetry, involves using a colorimeter to measure the cloudiness or
turbidity of the culture as cell numbers increase. Results are derived by comparison with a standard
graph of light absorbance plotted against known cell numbers (You are not required to describe or
use a calorimeter).