Useful Notes on Preparing Slides of Microbes and Selectively Staining for Microscopic Examination

Not only is the microbiologist able to determine the size and shape of microbe, but many chemical characteristics may be ascertained from the staining reactions of different microbes. The two types of microscopic slide preparations generally made in the laboratory are wet mounts and dry mounts (smear preparations).

A standard wet mount specimen enables the microseopist to examine large microbes suspended in a drop of water. The flat, glass microscope slide is cleaned thoroughly to remove any grease film, and a drop of water is placed on the slide.

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The organisms to be examined are transferred to the slide using aseptic technique, and a cover glass is placed on top. Living fungi, protozoans, and algae can be studied to determine whether or not they are motile, and what cell arrangements might be formed.

Because of the small size of microbes such as the bacteria, it is very difficult to bring the cells into sharp focus using a standard wet mount. To resolve this problem, an alternative is used called the hanging drop slide preparation.

To prepare this slide, small amounts of petroleum jelly are placed on the corners of clean cover glass, and an inoculating loop is used to aseptically transfer a sample of culture to the center of the glass.

A special microscope slide with a depression in its surface is then placed over the cover glass to center die specimen in the depression. The completed slide is then flipped right-side-up and placed on the microscope for examination.

Studies prepared in this manner will not dry out as rapidly, and they allow the microscopist to easily magnify the microbes one thousand times their normal size.

Dry slides may also be prepared in two ways. The negative smear preparation may be used to examine microbes that are difficult to dye with the usual laboratory stains.

This preparation technique provides little information about the chemical nature of the cells but is very useful in determining the accurate size and shape of the cells. A small amount of the chemical nigrosin or India ink is placed on the end of a clean, flat glass slide.

The microbes are mixed into this chemical with an inoculating loop, and a second glass slide is used to spread the suspended cells over the slide’s surface. As the suspension covers the slide, it quickly dries to a dark film that may be examined without a cover glass.

Nigrosin and Indian ink do not stick to the cell surface but only surround the microbes, which appear as shiny little rods, cocci, or spirals in a dark gray-purple background when viewed through the microscope.

The other dry slide technique is the smear preparation, which is a method of placing a sample of microbes on a clean glass slide in preparation for staining. Culture from liquid media is placed on the slide with an inoculating loop, while those from solid media (petri plates, or slants) are transferred by inoculating needle into a drop of sterile water.

The specimen is smeared over the surface of the slide and allowed to air dry. In order to prevent the cells from being washed off during the staining procedure, the slide is quickly passed through a Bunsen burner flame to heat-fix the cells to the slide.

Heat-fixed smear preparations may be kept for a long time, since the cells have been killed and fixed to the surface of the glass. However, since the cells are small and contain a high percentage of water, very little information may be gained by examining the killed, unstained bacteria.

To identify internal structures and learn something about the chemical nature of the microbes, stains may be added to the smear before microscope examination.

Two staining methods commonly used in the lab are the simple stain and the differential stain. Only a single dye is used in the simple staining procedure, while the differential technique makes use of more than one stain to highlight the differences between cell parts or between cell types.

The most commonly used dyes in microbiological work are known as cationic dyes, since the coloured portion of their ions has the positive charge. The stain methylene blue is methylene chloride (CH, C12).

The colour-containing portion of the molecule is called the chromatophore, while the nonpigment-containing ion is called the auxochrome. When dye is washed over the surface of bacterial cells, the positively charged methylene ion (chromatophore) will chemically combine with the negative electrical charge that normally occur on the cells’ surface.

More commonly, these dyes are called basic dyes. The acidic dyes (e.g. sodium eosinate) have a negatively charged chromatophore and will not attach to the surface. Simple staining is a very quick method.

After the smear preparation has been heat-fixed, the dye is poured onto the surface and allowed to remain therefore about 45 seconds. Then it is washed off with water, and the slide is gently blotted dry. Other simple stains commonly used for bacterial cells include crystal violet, safranin, and carbolfuchsin.

These same dyes may be used for differential staining methods when applied in combination. Each particular dye is chosen on the basis of its ability to selectively react with certain cell parts or certain cell types.

The Gram stain uses crystal violet, Gram’s iodine, alcohol, and safranin to show the differences between two major bacterial cell types, Gram positive and Gram-negative.

Keep in mind that there is no single dye called a “Gram stain”, but that this is differential staining technique requiring the use of several chemicals.

Three other useful methods are the acid-fast, endospore, and capsular staining. All of these methods are performed on smear preparations in a specified sequence.

The acid-fast stain method is similar to the Gram method in that it is based on the ability of cell types to be decolourized after staining and enables microbiologists to categorize microbes.

Acid-fast bacteria (AFB) are not easily decolourized with acid-alcohol (HC1 +95% ethyl alcohol) after staining with hot carbolfuchsin since these bacteria have a thick, waxy lipid material that prevents the dye from being removed through the cell membrane If these AFB cells are damaged after the dye is added, the acid-alcohol will remove the red dye and the cell will appear to be non-acid-fact.

This staining method is used in the clinical laboratory for the identification of bacteria such as Mycobacterium tuberculosis (the cause of tuberculosis) and Mycobacterium laprae (the cause of leprosy).

A counterstain is used in the acid-fast technique to colour those cells that are non-acid-fast and have been decolourized by the acid-alcohol. Since the acid-fast cells are carbolfuchsin red, the counterstain usually used in methylene blue.

If admixture of acid-fast (Mycobacterium) and non-acid-fast (e.g., Streptococcus) bacteria are stained on the same slide, mycobacteria will appear as red bacilli and the streptococci as blue cocci. This staining procedure demonstrates differences in cell types, whereas the endospore and capsular stains demonstrate differences in cell parts.

Endospores produced by the genera Bacillus and Clostridium are chemically resistant to most dyes. Therefore, a special staining must be used to highlight their presence inside vegetative cells.

The Schaeffer-Fulton method utilizes hot malachite green stain to colour the resistant endospores and cold safranin (red) to colour the surrounding vegetative cell. When this staining procedure has been completed, cells that have formed endospores will show pink cytoplasm and green endospores.

In order to display the capsule of bacteria, crystal violet is used to stain cells in a swear preparation. Copper sulphate is then washed over the slide and the slide is dried in air.