GRAM’S STAIN

GRAM STAIN
INTRODUCTION :

• It was originally developed by Hans Christian

Gram in 1884 to differentiate the bacteria's
into Gram positive and Gram negative.

• even after 130 years its used widely in

bacteriology
PRINCIPLE:
• Gram positive cell wall have a more acidic protoplasm,
• Which retaining the basic dye more strongly than Gram
negative bacteria

• Iodine makes the protoplasm more acidic and serves as

mordant, i.e. iodine combines with dye to form a dye-iodine
complex and fixes the dye in bacterial cell

• Gram positive cell wall or cytoplasmic membrane being less

permeable, the dye-iodine complex gets trapped within the
cell

• The Gram negative cell wall has increased permeability to

acetone or alcohol, permitting the outflow of complex during
decolourization
 Gram stain theories
1. PH theory: Gram positive bacteria cell wall have more
acidic, hence, can retain basic dye for longer time.
Iodine acts as fixative

2. Magnesium ribonucleate theory: magnesium

ribonucleate and basic protiens are concentrated at the
cell membrane of Gram positive bacteria

Magnesium ribonucleate retain the basic dye (C.V)

hence Gram positive bacteria appear violet in colour.
 Conti…..
Cell wall theory:
• Gram positive cell wall have a thick peptidoglycan layer
• The stain gets trapped into this layer and the bacteria turned
violet.
• Gram negative cell wall its more permeable, thus allowing the
outflow of crystal violet, attributed to thin peptidoglycan layer
• Presence of lipopolysaccharides layers in the distrupted by
the action of acetone or alcohol
• Mordanting with Gram’s iodine complexes are formed in the
cytoplasm
• Following decolorization, as a more lipid content in Gram
negative bacterial cell wall gets dissolved leading to formation
of larger spores.
 MODIFICATIONS OF GRAM’S STAINING :
• Kopeloff’s & beermans modification: Primary stain methyl
violet and counter stain is basic fuchsin respectively

• Jensens modification: here absolute alcohol used as

decolorize and neutral red as counter stain. (useful in
meningococci and gonococci)

• Weigerts modification: Aniline-xylol used as decolorize

(useful in tissue staining )

• Preston & morrells modification: iodine-acetone is used as

decolorizer.

• Brown and Brenn modification: This is used for

 SMEAR PREPARATION
Before staining is necessary to make smears of
either patients specimen or by the bacterial cultures.

1. DIRECT SMEAR:
A direct smear is a preparation of the primary
clinical sample received in the laboratory for
processing.
A direct smear provides a mechanism to identify
the number and type of cells present in a specimen,
including white blood cells, epithelial cells, and
predominant organism type
2. Culture smear:
preparation from solid or semisolid
media or broth.

HOW TO MAKE A SMEAR?

- Take a clean glass slide and loop
- Labeling
- Take a loop full of sample and smear it on clean
glass slide.
- Air dried and heat fixed.
 FIXATION(PRELIMNARY STEP) :
• It’s a process by which the internal & external structures
of cells are preserved and fixed in position.

• It toughens the cell structure so that they do not change

during staining.

There are two types of fixation

• Heat fixation

• Chemical fixation (Ethanol and methanol)

Smear made on a clean glass slide, is air dried or heat fixed
 PROCEDURE (STEPS FOR GRAM STAINING ):
• Primary stain: the smear is stained with dyes such as crystal
violet or methyl violet for a minute. Then rinse with water.
Crystal violet stains all bacteria's to violet color
• Mordant : Here Grams iodine is poured on slide for one
minute & rinsed with water
• Decolorization : a few drops of decolorize such as acetone
(1-2 sec) or ethyl alcohol(20-30sec) is flowed on the slide
and immediately rinsed with water. It helps to remove
primary stain from gram negative bacteria's but gram
positive bacteria resist.
• Counter stain/ secondary stain : such as safranin or dilute
carbol fuchsin is poured for 30 sec.it gives pink or red color
to gram negative bacteria.
• Finally the slide is dried and examined under oil immersion
objective.
Interpretation
• Gram-positive bacteria • Gram-negative bacteria
observed under oil observed under oil
immersion appear violet. immersion appear pink.
 LIMITATIONS :
• If decolorizer is poured for more time , even
gram positive bacteria loose color and appear
as gram negative bacteria

• If decolorizer is poured for less time gram

negative bacteria do not loose color of
primary stain properly.
GRAM POSITIVE COCCI
 Examples
• Gram positive cocci: Staphylococci aureus, Staph.
epidermidis, Staph. saprophyticus, Microcci,
Streptococcus pyogenes, Strept. agalactiae,
Strept.pneumoniae, Enterococcus faecalis, Viridans
streptococci etc.
• Gram negative cocci: Neisseria meningitidis, Neisseria
gonorrhoeae, Veillonella sp.
• Gram positive bacilli: Bacillus, corynebacterium sp.,
Diphtheroids, Listeria monocytogenes, Eubacterium sp.
Etc.
• Gram negative bacilli: Esch.coli, Salmonella sp., Proteus
sp., Morganella morganii, Pseudomonas aeruginosa,
Burkholderia sp., Enterobacter sp., Citrobacter sp., Vibrio
cholerae, Serratia sp., Klebsiella sp., Shigella sp., etc.
APPLICATIONS :
• To differentiate bacteria into gram positive and gram negative.

• To start empirical treatment by direct smear from specimen.

• Gram staining from bacterial culture gives an idea to put required biochemical tests for
further identification of bacteria.

• For fastidious organisms, such as Haemophilus which takes time to grow in culture,
gram staining helps in early presumptive identification based on their morphology
(pleomorphic gram negative bacilli)

• Anaerobic organisms, (Clostridium ) do not grow in routine culture. Therefore

organisms detect in Gram stain.

• To know the structure of the bacteria's

• Its useful for stain certain fungi such as candida & Cryptococcus.

• Gram staining helps in screening the quality of the specimen (Sputum) before
 DIFFERENCES BETWEEN GRAM-POSITIVE
AND GRAM-NEGATIVE CELL WALL
CHARACTERS GRAM-POSITIVE CELL GRAM-NEGATIVE CELL
WALL WALL
Peptidoglycan layer Thicker (15-80nm) Thinner (2nm)
Lipid content Nil or Scanty (2-5%) Present (15%-20%)
Lipopolysaccharide Absent Present
Teichoic acid Present Absent
Aromatic amino acids Absent Present
THANK YOU