Introduction: Bacteria are microscopic organisms that can not be seen with unaided
eye. They can be seen even in unstained preparations such as a wet mount
or hanging drop preparation but the morphology is not clear. Bacteria
are colorless and when suspended in saline they dont offer any
contrast. Besides, bacterial motility makes it difficult to observe the
morphology clearly. Hence, bacteria have to be stained to observe them.
The dyes often used are toxic chemicals that kill the bacteria. The
process of smearing, fixing and drying often kill the bacteria. This
process fixes the bacteria to the slide and their position on slide
Preparation of smear:
A grease free glass slide is taken and a circle is marked one side of
the slide using a wax pencil or a glass marker pen. Marking the slide
makes it convenient to identify the surface of the slide that contains
the smear. On the opposite side, a small drop of sterile saline is
taken. Saline may also be transferred using a bacteriological loop. A
small portion of the colony is picked up using a sterile straight wire
and emulsified well in the saline on the slide. The colony is spread
evenly within the circled are to produce a smear. Presence of grease on
the slide would prevent uniform distribution resulting in an uneven
smear. Taking too much of the colony would result in excessively thick
smear. Similarly, taking a very miniscule part of the colony make it
very difficult to look for bacteria. Once a uniform smear is prepared,
it has to be air dried. The smear must be held above the Bunsen flame at
comfortable height. The smear must be not be forcibly dried by applying
heat. After the smear dries, it is fixed. Smears can be fixed physically
or chemically. Chemical fixatives are not useful for regular
bacteriological studies. It is convenient to heat-fix the smear by
gently passing the slide through the Bunsen flame once or twice.
Excessive heating of the slide must be strictly avoided.
You don't have to prepare smears, slides containing smear would be
provided to you in the practical class.
Smears on glass slide, staining rack,
staining solutions, blotting paper, immersion oil and microscope
There are several dyes that can be used to stain the bacteria. Simple
staining technique utilizes single basic dye such as crystal violet,
methylene blue, basic fuchsin etc. All bacteria take up the basic dye
uniformly and appear in the same colour. Only the morphology of the
bacteria can be appreciated upon staining.
We use two simple staining solutions, namely crystal violet solution and
dilute carbol fuchsin solution. We use the former to demonstrate cocci
and the latter to demonstrate bacilli. This choice is not compulsory but
is used by convention. The slide with cocci in the smear would be market
C and the one with bacilli would be marked B only to avoid mixing up
the two slides.
The slide marked C is placed on the staining rack above the sink. Few
drops of crystal violet solution is poured over the smear enough to
cover the circled area. Care must be taken not to flood the slide, which
would be a wastage of staining solution. The stain must be allowed to
remain on the smear for a minute. The slide is then lifted from the
rack, tilted to run the stain down to the sink and then washed under
gentle stream of running tap water. A washing bottle may also be used
for the same purpose. The slide must be held at inclined position and a
thin stream of water must fall on the slide a little above the smear.
Water must not be allowed to fall directly on the smear as there are
chances of the entire smear getting washed away. Once the smear is
sufficiently washed, it is placed on a piece of blotting paper. The
sides of the paper are then folded over the slide and gentle pressure is
applied to dry the slide. Excessive pressure or wiping the paper over
the slide may completely wipe off the smear.
The procedure for staining the slide marked B is similar to the
procedure mentioned above except that the staining solution to be used
is dilute carbol fuchsin and the time of contact is only 30 seconds.
After the slide is dry, a drop of cedar wood oil (or liquid paraffin) is
placed on the smear. The slide is placed on the mechanical stage and
smear portion is centered above the condenser. The microscope is
adjusted for viewing under oil immersion objective (100x) by raising the
condenser fully, opening the diaphragm fully and using the plane mirror.
This ensures that maximum light is available. The stage is raised (or optical body is lowered; depending on
the type of microscope) using coarse adjustment till the oil immersion
objective touches the oil. It is subsequently focused using fine
adjustment until a clear image appears. The small circular area of smear
that is seen through the eyepiece is called a field.
Observation for slide marked C stained by crystal violet solution:
Violet coloured, spherical shaped bacteria that are arranged in singles,
pairs, tetrads, short chains and irregular grape like clusters are seen. Inference: The given slide contains one of the cocci.
Observation for slide marked B stained by dilute carbol fuchsin
Pink coloured, rod shaped bacteria that are present haphazardly (without
any characteristic arrangement) are seen. Inference: The given slide contains one of the bacilli.
Simple staining with crystal violet
Simple staining with dilute carbol fuchsin
Note: a. Observation must be made in the following order: colour > shape >
b. Spherical shaped bacteria
are referred as cocci (coccus is singular) and rod shaped bacteria as
bacilli (bacillus is singular)
b. This exercise is not
included in the practical examination, it is only for your learning.
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