Types of Staining

1. Gram staining:-

                                                   The most widely used staining procedure in microbiology is the Gram stain which was discovered by the 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. 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, 93% 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 bacteria. At the end, the smear is counterstained with the basic dye,different in colour than crystal violet. This counterstain is 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.

Gram Staining

   2. 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 cell surface is negatively charged, they repel the above 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.

3. Acid-fast staining:-

                              

                                                           Some bacteria of genera Mycobacterium and Nocardia, do not get stained easily with simple stains. But if this microorganisms are heated with carbolfuchsin, they can be stained. As this staining can not 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 this microorganisms, Acid-fast microorganism that retain this dye and appear red and microorganism non-acid fast micro, appear blue or brown due to the counter staining with methylene blue after they have been decolourized by the acid-alcohol.

4. Endospore staining:-

                                                                  

                                                                  Endospore produced by Bacillus and Clostridium do not stain easily. Endospore 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 decolourized and counter stained a light red with safranin.

Endospore staining

5. 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 colour. Further, 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. 

                                       

6. 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 (Grey method), pararosaniline (Leifson method), silver nitrate (West method), or crystal violet (Difco's method).