Hanging Drop Method

Hanging drop preparation is a special type of wet mount (in which a drop of
medium containing the organisms is placed on a microscope slide), often is used in
dark illumination to observe the motility of bacteria.

Figure 1. Hanging Drop Method Preparation

In this method a drop of culture is placed on a coverslip that is encircled with

petroleum jelly (or any other sticky material). The coverslip and drop are then
inverted over the well of a depression slide. The drop hangs from the coverslip, and
the petroleum jelly forms a seal that prevents evaporation. This preparation gives
good views of microbial motility.

Motile Bacteria

A bacteria, which has the intrinsic ability of movement in the surrounding

medium, in which it remains suspended, is a motile bacteria.

Non-motile Bacteria

A bacteria, which does not have the intrinsic ability of movement in the
surrounding medium, in which it remains suspended, is a non-motile bacteria. Non-
motile bacteria may show apparent motility, resulting from their brownian movement
caused by the bombardment of the water molecules in the surrounding medium, on
the bacteria cells.

Advantages and Disadvantages of Hanging Drop Method

Advantages: Like the wet mount, the hanging drop method preserves cell
shape and arrangement. The Vaseline-sealed depression also slows down the
drying-out process, so the organisms can be observed for longer periods.

Disadvantages: The hanging drop method is also far too risky to use with highly
pathogenic organisms.

Colony Morphology: Describing Bacterial Colonies

 When bacteria are growing on a solid medium like nutrient agar, they will form
a visible mass of growth within 24 hours. This mass is a colony.
A colony is a visible mass or clump of bacteria that tend to cling together
growing in one place on a solid medium.

The following outline will be helpful for verbally communicating the appearance
of observed colonial growth.

1. Form – The form refers to the shape of the colony. These forms represent the
most common colony shapes you are likely to encounter

a. Size – The size of the colony can be a useful characteristic for identification.
The diameter of a representative colony may be measured. Tiny colonies are
referred to as punctiform .

b. Surface – Bacterial colonies are frequently shiny and smooth in appearance.

Other surface descriptions might be veined, rough, dull, wrinkled (or
shriveled), glistening.

c. Texture – Several terms that may be appropriate for describing the texture or
consistency of bacterial growth are: dry, moist, mucoid , brittle, viscous,
butyrous (butterfly).

d. Color – It is important to describe the color or pigment of the colony. Also

include descriptive terms for any other relevant optical characteristics
such as: opaque, cloudy, translucent, irisdescent.

2. Elevation – This describes the “side view” of a colony. These are the most
common.

3. Margin – The margin or edge of colony (any growth) may be an important

characteristic in identifying an organisms. Several examples are shown below.
Obtaining Pure Culture of Microorganisms

The following points highlight the top six methods used for obtaining pure
culture of microorganisms. 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 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. 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:

a. The picking up of subsurface colonies needs digging them out of the agar
medium thus interfering with other colonies, and

b. 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, 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 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.

The following two methods are in use:

a. 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.

b. 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 specialized 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 favors the growth of desired microorganisms but, is
unsuitable for the growth of other types of microorganisms.