Staining of bacteria

Staining of bacteria

 In 1958, W. Garlack and Carl Weigert in 1965 were the first to use stains for studying bacteria. Theoretically ,two theories have been put forward to understand the staining process of microbial cells

• Physical theory

• Chemical theory

In physical theory, the microbial cells are stained because of physical process such as osmosis ,absorption and capillary action.

 In chemical theory, the stains or the dyes combine with the chemicals present in the biological materials. This results in formation of any compound which gives the microbial cells the differential color i.e why the chemical does not get easily washed off

 Stain or staining material

 A stain is defined as an organic compound containing a benzene ring plus a chromophore and an auxochrome group.

 Chemically a stain can be

• Acidic stain anionic stains

Picric acid ,acid fuchsin or eosin are examples of acidic stains. These stain the cytoplasm component of the cells which are more alkaline in nature.

• Basic stains cationic stains

Methylene blue (crystal Violet), safranin are examples of basic stains.  These stain those cellular elements which are acidic in nature. Example nucleic acid

• Neutral stains

These are formed by mixing together both,the aqueous solution of acidic and basic stains . These stains are neutral and stain both negatively as well as positively charged cellular components.

Need of staining

 We have to stain the microbes because of the following reasons

1. Most of the microbes are colourless and cannot be seen distinctly without staining.

2. Sometimes to study particular structure in a microbe, a particular area is to be darkened.

3. Differentiate various structures, when a detailed study is required. Example for differential staining of nuclear material or the cell wall or endospore or flagella.

4. Some staining are aimed to classify bacteria into various groups example gram staining, acid fast staining etc.

Types of staining procedure

1. Simple staining

In this staining procedure ,a single stain is added to microorganism onto the slide. Now it is allowed to act for sometimes and then washed with water to remove the excess stain.

2. Differential staining

In this staining required more than one dye and is used to distinguish between structures within a cell.

It is well known fact that simple staining is used for examining shape of bacterial cells ,however differential staining is used for separation into groups like gram staining and acid fast staining.  Or visualization of the structures like flagella staining, nuclear staining, capsule staining and spore staining

Gram staining

 The gram staining which is differential staining was developed by Dr. Han Christian Gram, a Danish physician in 1884 that is why it is called gram staining. It is a very useful stain and is always the first step  to identify and classify bacteria. Gram staining is used to classify the bacteria into two major groups

 The gram positive and the gram negative bacteria.

• Crystal Violet (primary stain)is added to the smear

• Now iodine solution is added drop wise to this smear and this solution acts as a mordent.

• Smear is washed with water and alcohol acts as a decolorizing agent

• Smear is stained with safranin, it is a counter stain

Results of gram staining

 The bacteria which retains the primary stain appear dark blue or violet are called gram positive and the bacteria that lose the crystal violet stain and take the counterstain, appears red are called gram negative.

 Mechanism of gram staining

 The difference in the staining responses of bacteria to gram stain is because of chemical and physical difference in their cell walls. In gram negative bacteria the cell wall is the complex and multilayer structures and contain relatively high lipid content in addition to protein and mucopeptide . When crystal Violet is added to the cell it goes inside the cell wall and stains it . But as alcohol is added ,it readily dissolves the liquid present in the cell wall which results in the formation of large pores in the cell wall. Therefore the crystal Violet iodine complex leached out of the cell wall. In this way bacteria are discolorized . These decolourized gram negative bacteria later take the counter stain and appears red.

 In contrast gram positive, cell walls are thick and chemically simple. These cell walls are composed mainly of proteins and cross linked muco peptides when treated with alcohol causes dehydration and closure of the cell wall pores. therefore not allowing the loose of complex crystal Violet iodine complex and cells remain blue.

 Acid fast staining

 It is a differential stain, it was developed by Paul Ehrlich in 1882, this process was later modified by Ziehl Nelson and is being used by the present day microbiologist.

 Procedure

• Bacterial smear is stained with Ziehl carbol fuchsin for 3 to 5 minutes

• Then rinsed with tap water. decolourised with ethyl alcohol (containing 3% by volume of concentrated hydrochloric acid) until no stain comes out.

• Washed with tap water and counterstain with methylene blue solution.

 Results

 The bacteria which retain the primary stain and appear red in colour are called acid fast bacteria while the bacteria which lose their red colour on washing with acid alcohol and  counterstain with methylene blue are called non acid fast bacteria.

 Mechanism

 The acid fast bacteria contain very high contents of lipid called mycholic  acid in their cell wall.  This acid makes the penetration of strains extremely difficult. In fact the acid fastenes the relative solubilities . The red dye fuchsin is more soluble in phenol than in water or acid alcohol.  Phenol in turn is more soluble in waxy substances like mycholic acid present in acid fast bacteria than in water.  In the acid fast staining procedure red fuchsine enters the cell lipids (mycholic acid) and remain there because it is more soluble there than in the decolorizing agent. The intact cell coating prevent the red stain lipid from leaving the cells

 In nonacid fast bacteria wax substances are absent . So the red stain comes out with the decolorizing agent.  The fact that acid fastenes is due to waxy mycholic acid is proved by the fact that washing with hot water removes wax and so acidfastenes is also last.

 Acid fast staining is useful for the identification of the members of Mycobacterium, especially in staining of sputum for Mycobacterium , the cause of tuberculosis , This is because this bacillus (the acid fast microbe), commonly found in the sputum.

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