Staining Techniques in Microbiology
By Semester V
What is Staining
Staining is the biochemical technique of adding a class-specific (DNA, proteins, lipids, CHO’s) dye to a substrate to qualify or quantify the presence of a specific compound. It is similar to fluorescent tagging Staining is also used in flow cytometry Staining of specimen can provide contrast between bacteria and surrounding media Types of staining:
Simple staining Differential Staining Special stains: capsules, endospores
Differential Stain – The Gram Stain
The Gram stain is based on the structure of the bacterial cell wall The Gram stain is an important rapid test In Gram-positive bacteria, the purple crystal violet stain is trapped by the layer of peptidoglycan which forms the outer layer of the cell. In Gram-negative bacteria, the outer membrane prevents the stain from reaching the peptidoglycan layer in the periplasm. The outer membrane is then permeabilized by acetone treatment, and the pink safranin counterstain is trapped by the peptidoglycan layer.
Simple Staining Methods – Negative Staining
Negative staining is a simple staining method used when methods like Gram staining fail First, smear the sample on to the slide, followed this by an application of indian ink. After drying, the microorganisms may be viewed in bright field microscopy as lighter inclusions wellcontrasted against the dark environment surrounding them. Negative staining is a mild technique which may not destroy the microorganisms therefore it is unsuitable for studying pathogens.
Which is Which?
Procedure for Gram Stain
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Place a slide with a bacterial smear on a staining rack. STAIN the slide with crystal violet for 1-2 min. Pour off the stain with forceps Flood slide with Gram's iodine for 1-2 min. Pour off the iodine. Decolorize by washing the slide briefly with acetone (23 seconds). Wash slide thoroughly with water to remove the acetone - do not delay with this step. Flood slide with safranin counterstain for 2 min. Wash with water. Blot excess water and dry in hand over bunsen flame
Gram Positive bacteria
Staphylococcus, Streptococcus and Clostridium
Gram Negative bacteria
Escherichia coli, Salmonella typhi, Vibrio, and Bordetella
Acid Fast Staining
The high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria, is responsible for the staining pattern of poor absorption followed by high retention. The most common staining technique used to identify acid-fast bacteria is the Ziehl-Neelsen stain, in which the bacteria are stained bright red and stand out clearly against a blue background. Acid-fast bacteria can also be visualized by fluorescence microscopy using specific fluorescent dyes (auramine-rhodamine stain)
Acid Fast Staining
Mycobacterium avium complex (MAC) with acid fast stain often has the characteristic appearance shown here with numerous mycobacteria filling macrophages. Such macrophages may be distributed diffusely or in clusters.
Capsule Staining
India ink staining – capsule appears as a clear halo around the bacteria. Capsulated organisms
Streptococcus pneumoniae, Klebsiella sp., Bacillus anthracis, Haemophilus influenzae
Endospore Staining
With Gram staining, pores will appear as unstained refractile body within cell Modified Ziehl-Neelson staining Malachite Green stain can also be directly used
Giemsa Staining
Used in staining chromosomes Used in staining blood smears as well It can be used to identify malaria and other parasites like trypanosomas
Giemsa Stain
Stains associated with the Giemsa stain are the Romanowsky stain, Wright, Jenner’s and Leishman stains
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