Gram’s staining

Gram Staining is use to decide whether the bacterium is

→Coccus or bacillus(rod)

→Gram positive or Gram negative.

On these two characters bacteria are classified and are used for identification in routine practice.

Principle:-

Gram Staining depends on cell wall structure and cytoplasmic membrane. When Bacteria are at first stained with crystal violet & then mordant with iodine, certain bacteria resist decolorization by alcohol whereas other do not. Bacteria that resist decolorization are ‘’Gram positive’’, and the bacteria that are decolorized are “Gram negative’’ bacteria are then stained with a red counter stain. Gram positive bacteria stain dark purple-blue & Gram negative bacteria red.

Materials Required:

1.     Clean glass slides

2.     Inoculating loop

3.     Bunsen burner

4.     Microscope

5.     Immersion oil

6.     Distilled water

7.     18 to 24 hour cultures of organisms

Gram staining consists of four components:

1.    Primary stain -       (Crystal violet, methyl violet or Gentian violet)

2.    Mordant        -        (Gram's Iodine)

3.    Decolourizer -        (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture)

4.    Counterstain-         (Dilute carbol fuchsin, safranin or neutral red)

***An easy way to remember the steps of the Gram stain is...

⇒COME= Crystal violet

⇒IN        = Iodine

⇒AND    = Alcohol

      ⇒STAIN= Safranin 

Preparation & fixation of the glass microscopic slide

Slide:  Clean & Grease or oil free slides are essential for the preparation of microbial smears.

***Grease or oil from the fingers on the slides is removed by washing the slides with soap and water. Wipe the slides with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until ready for use.

Labeling of the slides

Label the slide with the initials of the name of the organism on the edge of the slide. Care should be taken that the label should not be in contact with the staining reagents.

Specimen

Direct collection of swab

Slant cultures of Escherichia coli, Micrococcus roseus, or other bacteria.

Preparation of the smear

The first consideration is the correct preparation of the smear. Make a thin film of the material on a clean glass slide, using a sterile loop or swab for viscous specimens. Air dry, then heat fix the slide by passing it several times through

a flame (the slide should not become too hot to touch). Failure to follow these directions may cause staining artifacts and disrupt the normal morphology of bacteria and cells.

Please note: It is very important to prevent preparing thick, dense smears which contain an excess of the bacterial sample. A very thick smear diminishes the amount of light that can pass through

Staining procedure

3.   Cover the smear with Crystal Violet (primary stain) for 1 min.

4.   Gently wash off the slide with water.

5.   Add Gram’s Iodine (mordant) for 1 min.

6.   Wash with water.

7.  Carefully decolorize with 95% ethanol until thinnest parts of the smear are colorless. (Wash with water).

 ***This is the "tricky" step.  Stop decolorizing with alcohol as soon as the purple color has stopped leaching off the slide (time will vary depending on thickness of smear). Immediately wash with water.  Be sure to dispose of all ethanol waste in the appropriately labeled waste container.

8.   Cover the smear with Safranin or carbol fuchsin  for 30 seconds.

9.   Wash with water.

*** both the top & the bottom of the slide

10. Air dry, or blot with absorbent paper.

11. Observe first with low power (10X) to locate a good field.  Add a drop of oil and swing the oil immersion lens into the oil.  Use only the fine focus to bring the image into clear focus

***Appearance of the Gram positive coccus and Gram negative bacillus at different stages of the gram staining procedure are illustrated below

GRAM  STAIN

Procedure	Reagent	Cell Color
		Gram Positive	Gram Negative
Fixed cells on slide		COLORLESS	COLORLESS
Primary stain	Crystal Violet	PURPLE	PURPLE
Mordant	Iodine	PURPLE	PURPLE
Decolorizer	Alcohol	PURPLE	COLORLESS
Counterstain	Safranin	PURPLE	RED

Results

1.     Blue or Violet- Gram Positive

2.     Pink or Red color -Gram Negative

***As shown below, organisms that retain the violet-iodine complexes after washing in ethanol stain purple and are termed Gram-positive, those that lose this complex stain red from the safranin counter stain are termed Gram- negative.