GRAM STAINING

GRAM STAINING 

AIM

            To differentiate two important groups of bacteria such as Gram positive and Gram negative.

PRINCIPLE

            Gram staining technique is a Differential staining method used to differentiate bacterial species into two large groups (Gram positive and Gram negative) based on its cell wall composition Peptidoglycan. The name comes from the Danish Bacteriologist Hans Christian Gram, who developed the technique in 1884 (originally devised in 1882 but published in 1884). The Crystal violet act as a Primary stain, Iodine act as a Mordant, 95 % Ethyl alcohol or Acetone act as a Decolourizing agent and Safranine act as a Counter stain. The Mordant forms the link between the cell wall of the bacteria and the Primary stain Crystal violet.

            Gram staining is still the cornerstone of bacterial identification and taxonomic division. Gram positive bacteria resist decolourization (due to thick Cell wall peptidoglycan) and appear violet in colour due to the Primary stain Crystal Violet. Gram negative bacteria undergo decolourization (due to thin Cell wall peptidoglycan) and takes the Counter stain Safranine and appear pink in colour. Decolourization step is an important step of Gram staining process because the Decolourization step distinguishes Gram positive from Gram negative cells. If the decolourizer 95 % Ethyl alcohol or Acetone remains on the sample for too long, it may also decolorize Gram positive cells.

Based on shape, bacteria are classified into two types. They are Cocci and Bacilli.

a)  Gram positive cocci – Violet colour and Spherical shape. Example, Staphylococcus sp. (arranged in clusters) and Streptococcus sp. (arranged in chains).

b)     Gram positive bacilli - Violet colour and Rod shape. Example, Bacillus sp. & Lactobacillus sp.

c)     Gram negative cocci – Pink colour and Spherical shape. Example, Neisseria sp.

d)     Gram negative bacilli - Pink colour and Rod shape. Example, E. coli and Salmonella typhi.

MATERIALS REQUIRED

i.       Bacterial culture

ii.     Microscopic slide

iii.   Inoculation loop

iv.   Crystal violet

v.     Gram’s Iodine

vi.   Gram’s Decolourizer (95 % Ethyl Alcohol/ Acetone)

vii. Safranine

viii.  Bunsen Burner

ix.   Microscope

PROCEDURE

a)     Clean and dry the microscopic slide thoroughly.

b)     Prepare thin smear of given bacteria on a clean glass slide.

c)     Allow the smear to air dry and fixed with heat.

d)     Flood the smear with Crystal violet and allow it for one minute.

e)     Wash the smear with distilled water for few seconds using running water.

f)      Stain the smear with Iodine solution for one minute.

g)     Wash Iodine solution with running water.

h)   Add Decolourizer (95 % Ethyl alcohol/Acetone) drop wise for 10 - 15 seconds, until no more color flows from the smear.

i)      Wash the slide with distilled water and drained properly.

j)      Stain the smear finally with counter stain Safranin for 2 minutes.

k)     Wash the slide with running water and dried properly.

l)     Observe the slide under Low power objective (10 x or 40 x) and High power objectives (100 x Oil immersion) of the microscope.

OBSERVATION AND RESULTS

a)     Gram positive cocci – Violet colour and Spherical shape.

b)     Gram positive bacilli - Violet colour and Rod shape.

c)     Gram negative cocci – Pink colour and Spherical shape.

d)     Gram negative bacilli - Pink colour and Rod shape.

 

Figure – 1: Steps involved in Gram staining (Source: globalindoorhealthnetwork.com)

Figure – 2: Gram positive cocci

Figure – 3: Gram positive bacilli

Figure – 4: Gram negative cocci

Figure – 5: Gram negative bacilli