Limitations of electron microscopy and Preparations for light-microscope examinations

Limitations of electron microscopy:

The laying down of the great benefit of tremendous resolution and magnification, we can observe several types of limitations to electron microscopy. For example, the specimen being observed is in a chamber which is under a very high vacuum. Therefore the are not able to examine in a living state. In addition, the drying process may change some morphological characteristics. The next limitation of the technique is the low incursion power of the beam, necessitating the use of thin sections to bring out the internal structures of the cell.

The main problem confronting the investigator who effort to unravel the fine intracellular structure of the microbial cell is the designation of intracellular material. Many times at short interval is necessary to correlate results produced by the same organism examined by different microscopic techniques, like. Phase-contrast, bright-field (stained preparations) and electron microscopy. Each of the methods contributes different types of the idea. Reading of this information, especially comparison of what is revealed by each technique, makes it possible to identify cellular structures. But considerable experience in microscopy is needed before a investigator can correctly interpret the results.

Preparations for light-microscope examinations:

Two general techniques are used to prepare specimens for light microscopy examination. One is to suspend organism 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 techniques:

Wet preparation permits examination of organisms in a normal living condition. A wet mount is made by placing a drop of fluid containing the organisms onto 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 similar material to provide a seal between the slide and coverslip. A special slide with a circular concave depression is sometimes used for examination of wet preparations. A suspension of the microbial specimen is placed on a coverslip, 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. For example, in the examination of serious excludes suspected of containing the spirochete that causes syphilis, the wet preparations are examined by dark-field microscopy. This provides a sharp contrast between the organisms and the dark background. The normal arrangement of cells can also be better determined in a wet preparation.
2. The observation of bacteria to determine whether or not they are motile obviously requires that they are suspended in a liquid medium, free to move about.
3. To observe cytological changes occurring during cell division and occurs, the organisms must be examined in the living state (i.e. wet mount). Spore formation and generation 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. Partially closing the substage condenser diaphragm helps to increase contrast; however, some resolving power is lost. Dark-field and phase-contrast microscopy offer the distinct advantage of providing both high contrast and high resolving power for examination of unstained preparations.

Fixed, stained smears:

Fixed, stained preparations are most frequently used for the observation of the morphological characteristics of bacteria. The advantages of this procedure are that (1) the cells are made more clearly visible after they are colored, and (2) differences between cells of different species and within the same species can be demonstrated by use of appropriate staining solutions (differential or selective staining).

The essential steps in the preparation of a fixed, stained smear are (1) preparation of the film or smear, (2) fixation, and (3) application of one or more staining solutions.

Microbiological stains:

A large number of colored organic compounds (dyes) are available for staining microorganisms. These compounds are generally rather complex in terms of molecular structure. On this basis, they may be classified into groups such as triphenylmethane dyes, oxazine dyes, and thiazine dyes. A more practical classification for the cytologist is one based on the chemical behavior of the dye; namely acid, basic, or neutral. An acid (or anionic) dye is one in which the charge carried by the dye ion is negative; a basic (or cationic) dye is one in which the charge carried by the dye ion is positive. A neutral dye is a complex salt of a dye acid with a dye base, e.g., eliminate of methylene blue. Acid dyes generally stain basic cell components, and basic dyes generally stain acidic cell components.

The process of staining may involve ion-exchange reactions between the stain and active sites at the surface of or within the cell. For example, the colored ions of the dye may replace other ions on cellular components. Certain chemical groupings of cell proteins or nucleic acids may be involved in salt formation with positively charged ions such as Na⁺ or K⁺. thus we might view these peripheral areas of the cell as carrying a negative charge in combination with positively charged ions; for example,

(Bacterial cells) (Na⁺)

In a basic dye like methylene blue, the colored ion is positively charged (a cation), and if we represent this ion by the symbol MB, the dye, which is actually methylene blue chloride, may be represented as

MB⁺Cl^-

The ionic exchange which takes place during staining can be represented by the following equation, in which the MB⁺ cation replaces the Na⁺ cation in the cell.

(Bacterial cell^-) (Na⁺) + (MB⁺) (Cl^-) → (Bacterial cell^-) (MB⁺) ⁺ (Na⁺ Cl^-)

References:

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.