Introductory Microbiology

Magnification

• Magnification beyond the resolving power is of no value since the larger image will be less distinct in detail and fuzzy in appearance. The situation is analogous to that of a movie screen. If we more closer to the screen the image is larger but is also less sharp than when viewed at a distance.
• Most laboratory microscopes are equipped with three objectives. Each capable of a different degree of magnification. These are referred to as the oil immersion, high dry, and low-power objectives. The primary magnification provided by each objective is engraved on its barrel. The total magnification of the system is determined by multiplying the magnifying power of the objective by that of the eyepiece. Generally, an eyepiece having a magnification of X10 is used, although eyepieces of higher or lower magnifications are available.

Preparations for light microscope examinations:
Two general techniques are used to prepare specimens for light microscope examinations. One is to suspend organisms in a liquid (the wet mount or the hanging drop techniques) and the other is to dry, fix, and stain films or smears of the specimen.

The wet mount and hanging drop technique:
Wet preparations permit examination of organisms in a normal living condition. A wet mount is made by placing a drop of fluid containing the organisms on to a glass slide and covering the drop with a cover slip. To reduce the rate of evaporation and exclude the effect of air currents, the drop may be ringed with petroleum jelly or a jelly or a similar material to provide a seal between the slide and cover slip. A special slide with a circular concave depression is sometimes used for examination of wet preparations. A suspension of microbial specimen is placed on a cover slip, then inverted over the concave depression to produce a ‘hanging drop’ of the specimen. Examination of microorganisms in wet preparation is desirable in the following instances.
1) The morphology of spiral bacteria is greatly distorted when these bacteria are dried and stained; they should be examined in living condition.
2) The observations of bacteria to determine whether or not they are motile obviously requires that they be suspended in a liquid medium, free to move about.
3) To observe cytological changes occurring during cell division and to determine the rate at which the division occurs, the organisms must be examined in the living state (i.e wet mount). Spore formation and germination must also be observed in living cells.
4) Some cell inclusion bodies, e.g. vacuoles and lipid material, can be observed readily by this method.
When wet preparations are examined by bright field microscopy, it is extremely important to control the light source. The reason is that the lack of a stain makes the cells less distinctly visible; adjustment of the intensity of the light source can enhance their visibility.
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