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Differential Stains
Staining procedure which differentiates or distinguishes between types of bacteria is termed as differential staining technique. Methods for simple staining impart same colour to all bacteria and other biological material, may be slight variation in shade. On the other hand, differential staining methods impart distinctive colour only to certain types of bacteria.
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The basic principle underlying this differentiation is due to the different chemical and physical properties of cell and as a result, they react differently with the staining reagents. Differential staining procedure utilizes more than one stain. In some techniques the stains are applied separately, while in other as combination. There are two most important differential stains, namely, (A) Gram stain and (B) Acid-fast stain.
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(A) Gram Stain:
Christian Gram in 1883, while working on the etiology of respiratory disease in the municipal hospital in Berlin, accidently discovered a new staining technique. He applied crystal violet and Lugol's iodine to the biopsy specimen which resulted into heavy precipitation. To remove these precipitates he tried 95% ethanol and it was found that tissues were decolourized faster than bacteria.
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This led him to conclude that he had developed a new differential stain for bacteria in tissue. In 1884 he demonstrated that some bacteria are loosing primary stain within the tissues and accepting the counter stain (I). Thus, first differential staining procedure came into use, which divided bacteria in two groups the Gram negative and the Gram positive. Many modifications of this method exist, but in general, they are all based on the same principle.
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Gram stain is one of the most important and widely used differential stains. It has great taxonomic significance and is often the first step in the identification of an unknown prokaryotic organism. For eukaryotic cells this stain is not significant, since most of them are Gram negative, except yeasts and few molds which are Gram positive. In general, Gram negativity is wide spreaded than Gram positivity. This technique divides bacteria into two groups (i) Gram positive those which retain primary dye like crystal violet and appear deep violet in colour and (ii) Gram negative, which lose the primary dye on application of decolourizer and take the colour of counterstain like safranin or basic fuchsin.
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They appear red in colour. In routine diagnostic work a Gram stained smear is often the only preparation examined microscopically. It should be noted that Gram positive species sometime may show Gram negativity. On the other hand, Gram negative species do not produce cells which show Gram positivity. Gram positive bacteria are more sensitive to the antibacteriaI action of penicillins, acids, iodine, basic dyes, detergents and lysozyme as compared to Gram negative bacteria. They are also less susceptible, to alkalies, azides, tellurite, proteases and lysis by antibodies and complement.
Several theories have been offered in the literature to explain the mechanism of Gram reaction by various investigators. However, the basic mechanism is still not completely under stood. The most accepted hypothesis is based on the difference in the structure and composition of cell wall between these two groups of cells, proposed by Salton (2). The cell wall of Gram negative cells contains higher percentage of lipid as compared to that in the cell wall of Gram positive cells. Moreover, cell wall of Gram negative cell is found to be thinner as compared to that of Gram positive cell. Major differences between Gram positive and Gram negative bacteria are listed in Table
When we apply crystal violet, it reacts with cell and stains it. Subsequently, on application of mordant, Gram’s iodine, it reacts with dye and forms Crystal Violet-Iodine (CVI) complex in the cell. This complex is not extracted out in the decolourizing solution from Gram positive cells. The reason
TABLE
Some characteristics differences between Gram-positive and Gram-negative
bacteria.
Characteristic |
Gram-positive |
Gram-negative |
Cell wall composition |
Low in lipid (1-4%) |
High in lipid (11-20%) |
Sensitivity to penicillin |
More |
Less |
Susceptibility to basic dyes, e.g. crystal violet |
Marked |
Less |
Nutritional requirements |
Generally complex, only few species autotrophic |
Relatively simple, many species autotrophic |
Resistance to physical disruption |
More resistant |
Less resistant |
for this is as follows. Cell wall of Gram positive cell contains less lipid. On application of decolourizing agent like alcohol or acetone, due to dehydration, shrinkage of cell wall takes place which in turn, decreases the permeability for CVI complex. Thus, the complex is retained in the cell and hence cell is stained deep violet in colour. On the other hand the treatment of decolourizing agent extracts lipid from cell wall of Gram negative cell and with the result, there is increase in permeabiity property of cell wall. Due to this, CVI complex is extracted out and cells get decolourized (lose violet colour). On application of counterstain, cells take colour of counter stain. If the decolourizing step is omitted from Gram stain technique all bacteria will appear as Gram positive
The Gram reaction has been found to be affected by several factors and to obtain satisfactory results they should be kept in mind. They are discussed below:
(i) The age of bacterial culture should not be more than 24 h.
At older age cell loses Gram positivity and will appear as Gram negative.
(ii) Application of heat during the fixation of smears is another important step. Too much heating during this step will lead to loss in, Gram positiveness.
(iii) Overcrowding of cells in smear also affects the result, due to improper decolourization.
(iv) Staining reagents should be freshly prepared.
(v) In Gram staining decolourizing step is very important. To obtain satisfactory differentiation, the nature and the exposure time of decolourizing agent should be standardized with the material to be stained. Acetone alone is more powerful decolourizing agent than ethanol.
(vi) It is also important not to allow a .bacterial smear to dry.
There are many variations of original Gram staining procedure. Here we describe only few of them.
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