Staining

Bacterial cells are so small that, when examined in hanging drop preparations, little of their finer structure can be made out to be studied more closely. They must be colored with some dye. This process is called staining. The dyes most often used are aniline dyes which are derivatives of the coal tar product aniline. It is good that dye-impregnated paper strips used for staining bacteria are now available commercially. The different types of bacteria when stained can be studied well in their different colors under the microscope.

For a stained preparation, place a small amount of the material to be examined on a clean glass slide and spread out into a thin film with a wire loop or swab. Subsequent flame sterilization of the inoculating loop is a must. The film is known as a smear. Let the film dry in the air; then slowly pass the slide, smear side up, through a flame two or three times. Flaming kills the bacteria in the smear and causes them to stick to the slide. The smear is fixed through the process called is fixing. Other methods of fixing, such as immersion in methyl alcohol or Zenker’s solution, are sometimes used, but heat fixing is the most suitable for routine work. Apply the desired stain to the fixed smear. Wash off excess stain with water. Blot the slide dry between sheets of absorbent paper. Observe stained smear with the microscope.

Three classes of stains are used in bacteriology today, namely the simple stains, the differential stains, and the special stains. In addition, there is the process known as negative staining.

SIMPLE STAINS

A simple stain is usually made up of an aqueous or alcoholic solution of a single dye. It is applied to the fixed smear from 1 to 5 minutes and washed off. The stained preparation is then ready for microscopic examination. Widely used simple stains include Loffler’s alkaline methylene blue, carbolfuchsin, gentian violet, and safranine. The length of time that the stain remains on the smear depends on the avidity with which it acts. Sometimes a chemical to make it stain more intensely is added to the solution. Such a chemical is called a mordant.

With simple stains most bacteria stain easily and quickly, some do not stain so readily, and a few do not stain at all. Capsules and spores are not stained with these simple stains but may give contrast as clear unstained structures. Flagella cannot be demonstrated at all in this way. Stains help in distinguishing one kind of bacteria from another and these can be viewed well with the aid of the microscope.

DIFFERENTIAL STAINS

More complex staining methods divide bacteria intro groups, depending on their reaction to the chemicals used for staining. Of these, the Gram stain and the acid fast stain are most often used.

The method of staining introduced by Hans Christian Gram divides bacteria into two great groups: those that are gram-positive and those that are gram-negative. This method depends on the fact that, when bacteria are stained with either crystal violet or gentian violet (Gram I) and the smear is then treated with a weak solution of iodine as a mordant (Gram II), some bacterial cells combine with the dye and iodine to produce a color that cannot be removed easily by alcohol, acetone, or aniline (Gram III), whereas the color is readily removed from certain other bacteria by these solvents. In the last step of the staining procedure a counterstain (Gram IV), such as safranine, is applied to give a contrasting color to the decolorized bacterial cells. Stains used to give contrast in color are counter stains. The ones most often used in the Gram stain are safranine and dilute carbolfuchsin, both of which give a red color, and Bismarck brown, which, as its name implies, gives a brown color. Many modifications have been devised for the original Gram’s Method.

Bacteria from which the blue color of the stain-iodine-bacterial cell complex cannot be removed are designated gram-positive, and those from which it can be removed and which are stained with the counterstain, as gram-negative. Gram-positive bacteria do not stain with the counterstain because they are already completely stained. A few bacteria that sometimes retain the stain and that at other times do not are referred to as gram-variable. The reactions of the bacteria to the Gram stain are very important in the study of pathogenic bacteria.

It must be noted that the chemical composition of the cell wall in gram-positive bacteria being different from that in gram-negative bacteria explains the alternate reactions of the two groups to gram staining. However the reason that the cell wall of gram-positive bacteria prevents their decolorization in the staining process is not clear. Certain physiologic differences are generally correlated with Gram staining. Gram-positive bacteria tend to be more resistant to the action of oxidizing agents, alkalis, and proteolytic enzymes than are gram-negative ones. They are more susceptible to the acids, detergents, sulfonamides, and antibiotics, such as penicillin, than are gram-negative ones.

Another method of differential staining is the acidfast or Ziehl-Neelsen stain, for which method several standard modifications exist. When most bacteria and related forms are stained with carbolfuchsin, they stain easily, but when the smear is treated with acid-alcohol, they are completely decolorized. It is relatively difficult to stain certain other microbes with carbolfuchsin, but once stained, they retain the dye even when treated with acid-alcohol. Those that retain the stain are spoken of as being acid-fast. That property of being acid-fast derives from the presence of many complex lipids, fatty acids, and waxes within the bacterial cell wall, not possessed by cell walls of non-acid fast bacteria.

Important examples of acid-fast organisms encountered in medicine, some of which are nonpathogenic include e Mycobacterium tuberculosis, Mycobacterium leprae, and other of the Mycobacterium species, and asteroides. These different species can be examined well with under the microscope.

SPECIAL STAINS

Important special stains are those for capsules, spores, flagella, and metachromatic granules. Stains primarily designed to demonstrate metachromatic granules are especially valuable in identifying Corynebacterium diphtheriae and in differentiating it from related bacteria. Important stains of this type are Albert’s stain, using toluidine blue and malachite green, and Neisser’s stain, using methylene blue.

Negative (relief) staining. Microorganisms such as Treponema pallidum, not stained by ordinary dyes, may be made visible by the process known as negative or relief staining, in which the background, but not the microorganisms, is stained. The microorganisms are mixed with India ink or 10% nigrosin both of which are black. The mixture is spread out into a thin smear and allowed to dry. The microbes appear as colorless objects against a gray-black background. Negative staining may be done with the dye Congo red and this has been used to display spiral organisms and Chlamydia (bedsoniae). By a technique of negative staining using an electron-dense material, such as phosphotungstic acid, viruses are prepared for visualization in the electron microscope.