Gram staining | History | Application |Principle | Procedure

Gram Stain

• In 1884, Hans Christian Gram discovered a staining technique that could help to visualise the infectious bacteria clearly and can easily study their morphology.

•  In this technique he used 

1. crystal violet (the primary dye), 
2. Gram’s iodine (IKI, the mordant), 
3. an alcohol rinse (decolorizer), 
4.  Safranin (counter stain ).

• A Gram stain is a test that checks for bacteria at the site of a suspected infection such as the throat, lungs, genitals, or in skin wounds. 

• Gram stains may also be used to check for bacteria in certain body fluids, such as blood or urine.

• This color choice provides differentiation between bacteria that stain purple, called gram-positive, and those that stain red, called gram-negative.

• The difference between gram-positive and gram-negative bacteria is due to the physical nature of their cell walls and how it reacts to the series of reagents applied to the cells.

• Gram positive bacteria has thick peptidoglycan layer in the cell wall and gram bacteria has thin peptidoglycan layer. 

Application :

Gram staining is a technique used in microbiology laboratory to stain a bacteria to differentiate them through their cell wall.

A Gram stain is most often used to find out if you have a bacterial infection. If you do, the test will show if your infection is Gram-positive or Gram-negative.

Principle of Gram staining :

Gram staining is based on the ability of bacterial cell wall to retain the primary stain crystal violet dye during alcohol treatment. 

The cell wall for Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than gram-negative bacteria. 

Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed easily.

 This step fixed the dye to the cell wall.

 However, subsequent treatment with a decolorizer, which is 95% ethanol, dissolves the lipid layer present in the gram-negative cells. 

The removal of the lipid layer enhances the leaching of the primary stain i.e. crystal violet from the cells into the surrounding.

 In contrast, the alcohol dehydrates the thicker Gram-positive cell walls, closing the pores as the cell wall shrinks during dehydration. 

As a result, the diffusion of the violet-iodine complex is blocked, and the bacteria remain stained. 

The length of the decolorization is critical in differentiating the gram-positive bacteria from the gram-negative bacteria.

 A prolonged exposure to the decolorizing agent will remove all the stain from both types of bacteria. 

Finally, a counterstain safranin is applied to the smear to give decolorized gram-negative bacteria a pink colour. 

Requirements

1. 24 hour bacterial culture

2. Gram staining reagents - Crystal violet, Grams’s iodine solution, 95% ethanol and safranin

3. Staining tray

4. Wash bottles

5. Droppers

6. Inoculating loops

7. Glass slides

8. Spirit lamp

9. Microscope

Procedure:

1. A fresh clean slide is taken and the smear area is marked on the underside of the slide with a marker.
2. The slide is flame and is placed on the working table.
3. The tube containing the culture is rocked for attaining uniform distribution of the microorganisms throughout the broth.
4. Inoculation loop is heated till red hot by placing the loop at 60ºC angle into the upper core of the spirit lamp flame by holding it in the right hand.
5. The loop is allowed to cool and one loopful of the bacterial culture is transferred from the tube to the slide.
6. The mouth of the culture tube is flame and the cotton plug is replaced.
7. The inoculation loop is heated again and when it is cool the bacterial suspension is spread thinly over an area with the help of the inoculation loop.
8. Finally the smear is allowed to air dry.

So, that was the preparation of smear of the liquid culture and now we’ll see for the solid cultures.

For solid cultures

i. A loopful of sterile distilled water is placed on a clean grease free glass slide.

ii. Then aseptically a small amout of bacterial colony is transferred into the distilled water droplet with the help of inoculating loop.

iii. The culture is emulsified with the water droplet with the loop.

iv. Gently the slide is heated by holding it in the right hand above the flame.

Fixing a smear

9. The smear is passed through the tip of the flame very rapidly for around four to five times.

10. Then the smear is allowed to cool and air dry.
Staining

11. Further the slide is place on the slide rack.

12. The smear is flooded with crystal violet stain and allowed to react for 1 minute.

13. Then the slide is wash with distilled water for few seconds using the wash bottle.

14. The smear is again flooded with gram’s iodine solution and allowed to react for one minute (the addition of Gram iodine solution to the smear forms the crystal violet-iodine complex and the complex so formed prevent the dye from washing away easily. This step fixed the dye to the cell wall).

15. The iodine solution from the slide is washed off with 95% ethanol. Ethyl alcohol should be added drop by drop until no more colour flows from the smear. (In this step the Gram positive bacteria are not affected while all the Gram negative bacteria are completely decolourized.)

16. Then the slide is wash with distilled water for few seconds using the wash bottle.

17. Finally the slide is flooded with the counter stain safranin and allowed to react for another 1 minute.

18. The slide is wash with distilled water for few seconds using the wash bottle.

19. The slide is blot dry by using absorbent paper and the stained slide is allowed to air dry.

OBSERVATION

 The slide is microscopically observed using the oil immersion objective.

RESULT

Under the microscope the given bacteria appear purple in colour and are arranged in cluster and hence it is the Gram positive bacteria.

 

This classification is based on chemical and physical properties of their cell wall constituents. 

The result of Gram staining reaction reveals whether the organism is positive or negative based on the colour the organism appears after staining. 

Reference:

1. https://medlineplus.gov/lab-tests/gram-stain/#:~:text=What%20is%20it%20used%20for,used%20to%20diagnose%20fungal%20infections.