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 don’t 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 remains unaltered.

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.

Note: You don't have to prepare smears, slides containing smear would be provided to you in the practical class.

Requirements:
Smears on glass slide, staining rack, staining solutions, blotting paper, immersion oil and microscope.

Staining:
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 solution:
Pink coloured, rod shaped bacteria that are present haphazardly (without any characteristic arrangement) are seen.
Inference: The given slide contains one of the bacilli.