Preparation and Staining of Specimens

Preparation and Staining of Specimens

Preparation and Staining of Specimens

Although living microorganisms can be directly examined with the light microscope, they often must be fixed and stained to increase visibility, accentuate specific morphological features, and preserve them for future study.


The stained cells seen in a microscope should resemble living cells as closely as possible. Fixation is the process by which the internal and external structures of cells and microorganisms are preserved and fixed in position. It inactivates enzymes that might disrupt cell morphology and toughens cell structures so that they do not change during staining and observation. A microorganism usually is killed and attached firmly to the microscope slide during fixation.

There are two fundamentally different types of fixation. Heat fixation is routinely used to observe prokaryotes. Typically, a film of cells (a smear) is gently heated as a slide is passed through a flame. Heat fixation preserves overall morphology but not structures within cells. Chemical fixation is used to protect fine cellular substructure and the morphology of larger, more delicate microorganisms. Chemical fixatives penetrate cells and react with cellular components, usually proteins and lipids, to render them inactive, insoluble, and immobile. Common fixative mixtures contain such components as ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.

Dyes and Simple staining

The many types of dyes used to, stain microorganisms have two features in common: they have Chromophore groups, groups with conjugated double bonds that give the dye its color, and then can bind with cells by ionic, covalent, or hydrophobic bonding. Most dyes are used to directly stain the cell or object or interest, but some dyes (e.g., India ink and nigrosin) are used in negative staining, where the background but not the cell is stained; the unstained cells appear as bright objects against a dark background.

Dyes that bind cells by ionic interactions are probably the most commonly used dyes. These ionisable dyes may be divided into two general classes based on the nature of their charged group.

  1. Basic dyes –

    methylene blue, basic fuchsin, drystal violet, safranin, malachite green – have positively charged groups (usually some form of pentavalent nitrogen) and are generally sold as chloride salts. Basic dyes bind to negatively charged molecules like nucleic acids, many proteins, and the surface of prokaryotic cells.

  2. Acidic dyes-

    eosin, rose Bengal, and acid fuchsin – possess negatively charged groups such as carboxyl (-COOH) and phenolic hydroxyls (-OH). Acidic dyes, because of their negative charge, bind to positively charged cell structures.

The staining effectiveness of ionizable dyes may be altered by pH, since the nature and degree of the charge on cell components change with pH. Thus acidic dyes stain best under acidic conditions when proteins and many other molecules carry a positive charge; basic dyes are most effective at higher pHs.

Dyes that bind through covalent bonds or because of their solubility characteristics are also useful. For instance, DNA can be stained by the Feulgen procedure in which the staining compound (Schiff’s reagent) is covalently attached to its deoxyribose sugars. Sudan III (Sudan Black) selectively stains lipids because it is, soluble but will not dissolve in aqueous portions of the cell.

Microorganisms often can be stained very satisfactorily by simple staining, in which a single dye is used. Simple staining’s value lies in its simplicity and ease of use. One covers the fixed smear with stain for a short period of time, washes the excess stain off with water, and blots the slide dry. Basic dyes like crystal violet, methylene blue, and carbolfuchsin are frequently used in simple staining to determine the size, shape, and arrangement of prokaryotic cells.

Differential Staining

The Gram stain, developed in 1884 by the Danish physician Christian Gram, is the most widely employed staining method in bacteriology. It is an example of differential staining-procedures that are used to distinguish organisms based on their staining properties. Use of the Gram stain divides Bacteria into classes – gram negative and gram positive.

The Gram-staining procedure is illustrated in. In the first step, the smear is stained with the basic dye crystal violet, the primary stain. This is followed by treatment with an iodine solution functioning as a mordant. The iodine increases the interaction between the cell and the dye so that the cell is stained more strongly. The smear is next decolorized by washing with ethanol or acetone. This step generates the differential aspect of the Gram stain; gram-positive bacteria retain the crystal violet, whereas gram-negative bacteria lose their crystal violet and become colorless. Finally, the smear is counterstained with a simple, basic dye different in color from crystal violet. Safranin, the most common counterstain, colors gram-negative bacteria pink to red and leaves gram-positive bacteria dark purple.

Acid-fast staining is another important differential staining procedure. It is most commonly used to identify Mycobacterium tuberculosis and M. Leprae, the pathogens responsible for tuberculosis and leprosy, respectively. These bacteria have cell walls with high lipid content; in particular, mycolic acids – a group of branched-chain hydroxyl lipids, which prevent dyes from readily binding to the cells. However, M. Tuberculosis and M. Leprae can be stained by harsh procedures such as the Ziehl-Neelsen method, which used heat and phenol to drive basic fuchsin into the cells. Once basic fuchsin has penetrated, M. Tuberculosis and M. Leprae are not easily decolorized by acidified alcohol (acid-alcohol), and thus are said to be acid-fast. Non-acid-fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain.

Staining Specific Structures

Many special staining procedures have been developed to study specific structures with the light microscope. One of the simplest is capsule staining, a technique that reveals the presence of capsules, a network usually made of polysaccharides that surrounds many bacteria and some fungi. Cells are mixed with India ink or nigrosin dye and spread out in a thin film on a side. After air-drying, the cells appear as lighter bodies in the midst of a blue-black background because ink and dye particles cannot penetrate either the cell or its capsule.

  1. Capsule staining- 

    It is an example of negative staining. The extent of the light region is determined by the size of the capsule and of the cell itself. There is little distortion of cell shape, and the cell can be counterstained for even greater visibility.

  2. Endospore staining-

    like acid-fast staining, also requires harsh treatment to drive dye into a target, in this case an endospore. An endospore is an exceptionally resistant structure produced by some bacterial genera (e.g. Bacillus and Clostridium.) It is capable of surviving for long periods in an unfavourable environment and is called an endospore because it develops within the parent bacterial cell. Endospore morphology and location vary with species and often are valuable in identification; endospores may be spherical to elliptical and either smaller or larger than the diameter of the parent bacterium. Endospores are not stained well by most dyes, but once stained, they strongly resist decolorization. This property is the basis of most endospore staining methods. In the Schaeffer-Fulton procedure, endospores are first stained by heating bacteria with malachite green, which is a very strong stain that can penetrate endospores. After malachite green treatment, the rest of the cell is washed free of dye with water and is counterstained with safranin. This technique yields a green endospore resting in a pink to red cell.

  3. Flagella staining-

    It provides taxonomically valuable information about the presence and distribution pattern of flagella on prokaryotic cells. Prokaryotic flagella are fine, thread-like organelles of locomotion that are so slender (about 10 to 30 nm in diameter) they can only be seen directly using the electron microscope. To observe them with the light microscope, the thickness of flagella in increased by coating them with mordants like tannic acid and potassium alum, and then staining with pararosani-line (Leifson method)or basic fuchsin (Gray method).

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