Gram Staining Laboratory

GRAM STAINING ( outlined by Algerico F. Baiño, Jr.
, RN )
I. HISTORY
 Bacteria are colorless, transparent and difficult to see that is why various staining methods have been
devised to enable scientist to examine bacteria
 Before staining, bacteria is smeared onto a glass microscope slide, air dried and then fixed
 Two most common methods of fixation are heat and methanol fixation. Heat fixation is accomplished by
placing the slide on a slide warmer and methanol fixation is flooding the smear with absolute methanol for
30 seconds.
 Methanol fixation id a more satisfactory technique and better preserves the morpholoogy of cells and
microorganisms
 Fixation serves 3 purposes :
1. It kills the organisms
2. It preserves the morphology ( shape )
3. It anchors the smear to the slide
 In 1883, Dr. Hans Christian Gram, a Danish physician, developed a staining technique that would enable him
to see bacteria in the lung tissues of patients who had died of pneumonia while working in a laboratory in
the morgue of Berlin Hospital
 His staining technique demonstrated that two general categories of bacteria causes pneumonia. The blue
stained bacteria are the Gram Positive and the Red stained bacteria are the Gram negative bacteria
 Since then, the Gram Staining has become the most important staining procedure in the bacteriology
laboratory because it differentiates between Gram (+ ) from Gram (- ), thus, it is a differential staining
procedure
 The organism’s gram reaction is serves as an extremely important clue when attempting to learn the identity
( species ) of a particular bacterium
 Application of Gram Staining Includes :
1. Used in research to classify the bacteria into Gram-positive and Gram-negative
2. Used in diagnostic labs for identification of the pathogen
3. Used in hospital for choosing spectrum of antibiotic for treatment before complete identification of
bacteria
4. Used to study the morphology of bacteria
II. PRINCIPLES OF GRAM STAINING
 When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the
bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram
positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is
low. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell
wall and prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal Violet-Iodine
complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or
purple in color.
 In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of
peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. When
they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-
iodine complex to leach out of the cells. Then when again stained with safranin, they take the stain and
appears red in color.
III. REAGENTS USED IN GRAM STAINING
1. Crystal Violet, the primary stain

 It is an intensely purple-colored organic compound chemically called triphenylmethane dye.
 It is also known as hexamethyl pararosaniline chloride or methyl violet 10B or gentian violet.
 In microbiology and molecular biology, it is used for staining bacteria, histological slide staining, DNA
staining, etc. It also shows antibacterial and antifungal properties, hence used in sterilization and
disinfection.
 In Gram Staining, it is used as a basic dye in the ionized form of CV+ and Cl-. It provides violet color
to Gram-Positive bacteria.
2. Iodine, the mordant

 It is an aqueous solution of iodine and potassium iodide used as mordant in Gram staining. It interacts
with CV+ and forms a CVI complex which gets trapped in the dehydrated peptidoglycan layer of the
Gram-Positive cell wall.
3. A decolorizer made of acetone and alcohol (95%)

 It is either acetone or ethanol (95%) or a mixture of acetone and ethanol in ratio 1:1 by volume. The
decolorizing solution dissolves the lipid content in the outer membrane of the Gram-Negative cell wall
and increases its permeability. Whereas, in the Gram-Positive cell wall the decolorizer dehydrates the
peptidoglycan layer and traps the CVI complex within the cell.
4. Safranin, the counterstain

 It is a red-colored counterstain used to stain decolorized Gram-Negative cells in the Gram Staining
technique. It is a basic dye that interacts with negatively charged components of the cell wall and
membrane.Besides safranin, dilute carbol fuchsin solution is also used as a counterstain.
IV. PROCEDURE
1. Take a clean, grease free slide.

2. Prepare the smear of suspension on the clean slide with a loopful of sample.
3. Air dry and heat fix
4. Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.
5. Flood the gram’s iodine for 1 minute and wash with water.
6. Then ,wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.
7. Add safranin for about 1 minute and wash with water.
8. Air dry, Blot dry and Observe under Microscope.
V. RESULTS AND INTERPRETATION OF GRAM STAINING
Gram-Positive bacteria appear violet or purple.

Gram-Negative bacteria appear pink or red.
Examples of Gram-positive bacteria:
1. Gram-positive cocci– Staphylococcus spp., Streptococcus spp., Enterococcus spp., etc.
2. Gram-positive bacilli– Bacillus spp., Clostridium spp., Lactobacillus spp., Streptomyces spp. and other
Actinobacteria, Listeria spp., Corynebacterium spp., etc.
Examples of Gram-Negative bacteria:
1. Gram negative cocci– Neisseria spp., Moraxella spp., Acinetobacter spp. Etc
2. Gram negative bacilli- E. coli, Klebsiella spp., Salmonella spp., Shigella spp., Pseudomonas spp., Proteus
spp., etc.

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GRAM STAINING ( outlined by Algerico F. Baiño, Jr.

II. PRINCIPLES OF GRAM STAINING

III. REAGENTS USED IN GRAM STAINING

1. Crystal Violet, the primary stain

2. Iodine, the mordant

3. A decolorizer made of acetone and alcohol (95%)

4. Safranin, the counterstain

1. Take a clean, grease free slide.

V. RESULTS AND INTERPRETATION OF GRAM STAINING

Gram-Positive bacteria appear violet or purple.

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