Role of Special Histochemical Stains in Staining ... - Dako

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Figure 1. Photomicrograph of ulcerated skin stained with Gram’s stain. The purple stain represents gram-positive bacteria which are seen as clumps (arrowhead) or as separate clusters of cocci (arrows). Everything other than gram-positive bacteria is stained pink by the carbol fuschin counterstain. The underlying structure of the skin cannot be seen. Figure 2. Giemsa stained section showing a gastric pit containing Helicobacter pylori which appear as delicate, slightly curved rod-shaped purple organisms (arrowheads). The stomach is inflamed and shows many neutrophils (arrows). The background is stained light pink by the eosin counterstain. 86 | Connection 2010
“ Silver stains are very sensitive for the staining of bacteria and therefore most useful for bacteria which do not stain or stain weakly with the Grams and Giemsa stains. ” components being azure A and B. Although the polychromatic stain was first used by Romanowsky to stain malarial parasites, the property of polychromasia is most useful in staining blood smears and bone marrow specimens to differentiate between the various hemopoeitic elements. Nowadays, the Giemsa stain is made up of weighted amounts of the azures to maintain consistency of staining which cannot be attained if methylene blue is allowed to “mature” naturally. Principles of Staining: The method consists of initial staining of the bacterial slide with crystal violet or methyl violet which stain everything blue. This is followed by Gram’s or Lugol’s iodine made up of iodine and potassium iodide, which act by allowing the crystal violet to adhere to the walls of gram-positive bacteria. Decolorization with an acetonealcohol mixture washes away the methyl violet which is not adherent to bacterial cell walls. At this stage, Gram-positive bacteria stain blue while the Gram-negative bacteria are colorless. A carbol fuschin counter-stain is then applied which stains the Gram-negative bacteria pink. Modifications: The Brown-Hopps and Brown-Brenn stains are modifications of the Gram stain and are used for demonstration of gram negative bacteria and rickettsia. Modifications: The Diff-Quick and Wright’s stains are modifications of the Giemsa stain. Carbol Fuschin Acid-Alcohol Stain Utility of the Stain: The carbol fuschin stain helps to identify mycobacteria which are bacilli containing thick waxy cell walls (Latin, myco=wax). Several mycobacteria can cause human disease; the two most significant ones are M. tuberculosis and M.leprae causing tuberculosis and leprosy respectively. Mycobacteria have large amounts of a lipid called mycolic acid in their cell walls which resists both staining as well as decolorization by acid-alcohol once staining has been achieved. The latter property is responsible for the commonly used term “acid-fast bacilli”. Mycobacteria cannot be stained by the Gram stain because it is an aqueous stain that cannot penetrate the lipid-rich mycobacterial cell walls. Giemsa Stain Utility of the Stain: The Giemsa is used to stain a variety of microorganisms including bacteria and several protozoans. Like the Gram stain, the Giemsa stain allows identification of the morphological characteristics of bacteria. However, it does not help further classification into Gram-negative or Gram-positive bacteria. The Giemsa stain is also useful to visualize H. pylori (Fig. 2, 3; See also Fig. 4 for a high resolution H&E stain showing Giardia). The Giemsa also stains atypical bacteria like rickettsia and chlamydiae which do not have the peptidoglycan walls typical of other bacteria and which therefore do not take up the Gram stain. The Giemsa stain is used to visualize several protozonas such as toxoplasma, leishmania, plasmodium, trichomonas, cryptosporidia and giardia. Principles of Staining: The Giemsa stain belongs to the class of polychromatic stains which consist of a mixture of dyes of different hues which provide subtle differences in staining. When methylene blue is prepared at an alkaline pH, it spontaneously forms other dyes, the major Principles of Staining: The mycobacteral cell walls are stained by carbol fuschin which is made up of basic fuschin dissolved in alcohol and phenol. Staining is aided by the application of heat. The organisms stain pink with the basic fuschin. Staining is followed by decolorisation in acid-alcohol; mycobacteria retain the carbol fuschin in their cell wall whereas other bacteria do not retain carbol fuschin, which is extracted into the acid-alcohol. Counterstaining is carried out by methylene blue. Mycobacteria stain bright pink with basic fuschin and the background stains a faint blue. Care has to be taken to not over-counter stain as this may mask the acid-fast bacilli. Modifications: The 2 commonly used methods for staining of M. tuberculosis are the Ziehl-Neelsen and Kinyoun’s acid fast stains. The Fite stain is used for staining of M.leprae which has cell walls that are more susceptible to damage in the deparaffinization process. The Fite procedure thus includes peanut oil in the deparaffinization solvent to protect the bacterial cell wall. The acid used for decolorization in the Fite procedure is also weaker (Fig. 5). Connection 2010 | 87
Page 1: Technical Articles Role of Special
Page 5 and 6: Figure 5. Ziehl-Neelsen stained sec
Page 7: Silver Stains (Warthin Starry Stain

Role of Special Histochemical Stains in Staining ... - Dako

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