Microbial Staining Technique - Short Information


Most of living bacteria are colourless and studying them under microscope becomes difficult because of their lack of contrast with the water in which they may reside. Thus, staining makes them visible for observation of their intracellular structures as well as overall morphology


Gram staining

The most widely used staining procedure in microbiology is the Gram stain which was discovered by the Danish scientist and physician Hans Christian Joachim Gram. Gram staining depends on the fact that bacterial cell differ from one another chemically and physically and may react differently to a given staining procedure. The Gram stain is the most useful and widely employed differential stain used to differentiate bacteria. Based on gram staining, bacteria are divided into two groups—Gram negative and Gram positive.

Gram staining process involves staining with the basic dye crystal violet which acts as primary stain. It is followed by treatment with an iodine solution, which increases the interaction between the bacterial cell and the dye so that the cell is more strongly stained. Further, 95% ethanol or isopropanol-acetone is used as washing agent. The bacterial cell which retain the crystal violet-iodine complex when washed with the decolourizer is classified as Gram-positive bacteria, and those lose the stain is classified as Gram-negative. At the end, the smear is counterstained with a basic dye, different in colour than crystal violet. This counterstain is usually safranin. The safranin stains the colourless, Gram-negative bacteria pink but does not alter the dark purple colour of the Gram-positive bacteria. The end result is that Gram-positive bacteria are deep purple in colour and Gram-negative bacteria are pinkish to red in colour


(Picture 1: Gram-positive anthrax bacteria (purple rods) and white blood cells (round) in cerebrospinal fluid Gram-negative bacteria would have appeared pink)
Source :http://en.wikipedia.org/wiki/Gram_staining



Negative staining

Sometimes overall bacterial morphology can be determined without the use of harsh staining or heat-fixing techniques which otherwise change the shape of cells. When the bacterium does not stain well or when it becomes necessary to study shape and size of bacteria, negative staining helps in studying of this.  This kind of staining is done by mixing bacteria with an acidic stain such as nigrosin, India ink, or eosin, and then spreading out the mixture on a slide to form a film. As the bacterial cells surface is negatively charged, they repel the above mentioned negatively charged stain. So, these stains do not penetrate and stain the bacterial cells, instead, these stains either produce a deposit around the bacteria or produce a dark background so that the bacteria appear as unstained cells with a clear area around them


(Picture 2: Negative stain of Klebsiella pneumonia)

Source http://faculty.ntcc.edu/mhearron/staining_techniques.htm



Acid-fast staining

Some bacteria of genera Mycobacterium and Nocardia, do not get stained easily with simple stains. But, if these microorganisms are heated with carbolfuchsin, they can be stained. As this staining cannot be washed away even by acid-alcohol, this staining is called as acid-fast. This acid-fastness is due to the high lipid content (mycolic acid) in the cell wall of these microorganisms.  Acid-fast microorganisms that retain this dye and appear red and microorganisms non- acid fast micro, appear blue or brown due to the counterstaining with methylene blue after they have been decolorized by the acid-alcohol.


(Picture 3: Using a modified cold Kinyoun acid-fast staining technique, and under an oil immersion lens the Cryptosporidium sp. oocysts, which are acid-fast stain red, and the yeast cells, which are not acid-fast stain green)
Source :



Endospore staining

Endospores produced by Bacillus and Clostridium do not stain easily.  Endospores are stained by Schaeffer-Fulton or Wirtz-Conklin method where malachite green is used for staining and heat is used to penetrate stain.  The rest of the cell is then decolorized and counterstained a light red with safranin.


(Picture 4: Endospore stain of Bacillus megaterium . Note green endospores within pink bacilli.
Source: http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab7/endobmmg.html


Capsule staining

Capsules are slimy layer surrounding bacteria and are composed of polysaccharides, polypeptides, and glycoproteins. Simple staining process is not useful in determining the capsule. Presence of a capsule can be determined by Anthony’s capsule staining method and the Graham and

Evans procedure. In Anthony’s method, primary stain is crystal violet, which gives the bacterial cell and its capsular material a dark purple color. Furhter, copper sulphate removes excess primary stain as well as colour from the capsule. Copper sulphate also acts as counterstain as it is being absorbed into the capsule and turning it a light blue or pink.


(Picture 5: India Ink Capsule Stain of Klebsiella pneumoniae showing white capsules (Glycocalyx) surrounding purple cells)

Source : http://biosci.sierracollege.edu/materials/4/illustrations/capsule_s...


Flagella staining

Bacterial flagella are fine, threadlike organelles of locomotion and can be seen directly using only the electron microscope. Light microscope can also be used for the study of flagella, but, the thickness of the flagella needs to be increased by coating them with mordants such as tannic acid and potassium alum, and then they needs to be stained with basic stains. Different stains are used for this purpose – fuchsin (Gray method), pararosaniline (Leifson method), silver nitrate (West method), or crystal violet (Difco’s method). 


(Figure 6. Flagellar stain of a Salmonella Typhi. Like E. coli, Salmonella are motile by means of peritrichous flagella. A close relative that causes enteric infections is the bacterium Shigella. Shigella is not motile, and therefore it can be differentiated from Salmonella on the bais of a motility test or a flagellar stain. (CDC) )

Source: http://textbookofbacteriology.net/themicrobialworld/Salmonella.html


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Comment by pragya dwivedi on June 27, 2012 at 9:02pm

Thank you sir.............

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