GRAM STAINING

Gram staining is a DIFFERENTIAL BACTERIAL STAINING technique

used to differentiate bacteria into Gram Positive and Gram
Negative types according to their cell wall composition.
History
The Gram stain was first used in 1884 by Hans Christian Gram (Gram,1884). Gram was
searching for a method that would allow visualization of cocci in tissue sections of lungs of
those who had died of pneumonia.
Already available was a staining method designed by Robert Koch for visualizing turbercle
bacilli. Gram devised a method that used Crystal Violet (also known as Gentian Violet) as the
primary stain, an iodine solution as a mordant, followed by treatment with ethanol as a
decolourizer.
This staining procedure left the nuclei of eukaryotic cells in tissue samples unstained while
the cocci found in the lungs of those who had succumbed to pneumonia were stained
blue/violet.
Gram found that his stain worked for visualising a series of bacteria associated with disease
such as the “cocci of suppurative arthritis following scarlet fever”.
He found however that Typhoid bacilli were easily decolorized after the treatment with
crystal violet and iodine, when ethanol was added.
We now know that those organisms that stained blue/violet with Gram’s stain are gram-
positive bacteria and include Streptococcus pneumoniae (found in the lungs of those with
pneumonia) and Streptococcus pyogenes (from patients with Scarlet fever), while those that
were decolorized are gram-negative bacteria such as Salmonella Typhi that is associated with
Typhoid fever.
.
PROCEDURE
1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent.
Please note that the quality of the smear (too heavy or too light cell concentration) will affect
the Gram Stain results.
2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
3. Flood slide with the mordant: Gram's iodine. Wait 1 minute.
4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
5. Add drop by drop to slide until decolorizing agent running from the slide runs clear.
6. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
7. Wash slide in a gentile and indirect stream of tap water until no colour appears in the
effluent and air dry or blot dry with absorbent paper.
 8. Observe the results of the staining procedure under oil immersion using a Brightfield
microscope. At the completion of the Gram Stain, gram-negative bacteria will stain pink/red
and gram-positive bacteria will stain blue/purple. Record your findings.
PRINCIPLE:
The principle of Gram staining is based on the differences in the chemical and physical
properties of bacterial cell walls, primarily the thickness of the peptidoglycan layer. Gram-
positive bacteria have a thick peptidoglycan layer that retains the primary stain (crystal
violet) through a decolorization step, appearing purple. Gram-negative bacteria have a thinner
peptidoglycan layer and an outer membrane, which allows the primary stain to be washed out
during decolorization, causing them to take up the pink or red counterstain.
Key steps and their role in the principle

 Primary Stain (Crystal Violet):

Stains all bacterial cells purple.

 Mordant (Iodine):

Forms a larger, insoluble crystal violet-iodine complex within the cell wall.

 Decolorizer (Alcohol or Acetone):

 For Gram-positive bacteria, it dehydrates the thick peptidoglycan layer, shrinking the pores
and trapping the crystal violet-iodine complex.

 For Gram-negative bacteria, it disrupts the outer membrane and the thin peptidoglycan layer,
allowing the complex to be washed out.
 Counterstain (Safranin or Basic Fuchsin):
Stains the decolorized Gram-negative bacteria pink/red. Gram-positive cells remain purple
and are not affected by the counterstain.

THEORIES OF GRAMS STAIN

1. Cell wall theory

 Gram-positive bacteria: Have a thick cell wall (50-90% peptidoglycan). During

decolorization with alcohol, the cell wall dehydrates, shrinking the pores and trapping the
crystal violet-iodine complex inside.

 Gram-negative bacteria:
Have a thin peptidoglycan layer and an outer membrane rich in lipids. Alcohol dissolves the
lipid-rich outer membrane, creating large pores through which the crystal violet-iodine
complex can easily escape.
[Link] content theory: This theory emphasizes the role of the outer membrane in Gram-
negative bacteria. The higher lipid content of this membrane is dissolved by the decolorizing
agent (alcohol or acetone), leading to increased permeability and the loss of the stain.

[Link] ribonucleate theory:

This theory suggests that Gram-positive bacteria have a compound of magnesium
ribonucleate and basic protein in their cell membrane that helps retain the primary dye, a
substance that is absent in Gram-negative bacteria.

4. PH Theory:

Gram-positive bacteria: The cytoplasm is considered more acidic, allowing the basic crystal
violet dye to bind with greater affinity.

Gram-negative bacteria: The cytoplasm is thought to be less acidic, meaning the crystal
violet is not retained as strongly and is more easily washed away during decolorization

MODIFICATIONS OF GRAMS STAINING:

[Link] and Beerman's: Used for staining tissue sections, this modification uses methyl
violet as the primary stain and basic fuchsin as the counterstain instead of safranin.

[Link]'s: Preferred for staining gonococci, this method uses an absolute alcohol
decolorizer.

[Link]'s: Adds ammonium oxalate to the crystal violet solution to help prevent
precipitation.
APPLICATIONS OF GRAMS STAIN:

1. Direct grams smear aids in starting empirical antibiotic.

2. We can choose the culture media by seeing the organism in grams smear.
3. We can assess the quality of the sample ex: sputum – presence of more number
of epithelial cells indicates poor quality.
4. We can classify the bacteria into gram positive and gram negative organism.

The ideal time to perform a Gram stain is during the log phase (or exponential
phase) of the bacterial growth curve, typically between 18 and 24 hours after
incubation, because this is when cells have a stable and consistent cell wall
structure. Cells in this phase are actively dividing, but they are not yet affected
by nutrient depletion
AIM: To stain the given smear by grams stain and interpret the observation
Observation: I

COLOUR SHAPE ARRANGEMENT

PURPLE SPHERICAL CLUSTERS

INFERENCE:
The given smear contains Gram positive cocci arranged in clusters
EXAMPLE: STAPHYLOCOCCUS AUREUS
Observation: II

COLOUR SHAPE ARRANGEMENT

PINK ROD SCATTERED
INFERENCE:
The given smear contains Gram-negative bacilli arranged scatteredly.
EXAMPLE: ESCHERICHIA COLI
Observation: III

[Link] COLOUR SHAPE ARRANGEMENT

1. PINK ROD SCATTERED
2. PURPLE SPHERICAL CLUSTERS

INFERENCE:
The given smear contains Gram-positive cocci arranged in clusters AND Gram-
negative bacilli in scattered arrangement.
Viva questions:

1. What type of stain is Gram stain? It is a differential stain which is used to differentiate
between Gram positive and Gram negative bacteria.
2. Who invented Gram stain? Gram stain was invented by Hans Christian Gram who is a
histologist.
3. Mention different types of primary stain. The Primary stains can be Crystal violet, Gentian
violet, Methyl violet
4. Mention the different counter stains that can be used?. The different counter stains include
Neutral red (1%), Safranin (0.5%) and Dilute carbol fuchsin and Basic fuchsin. Neutral red
for 2-4min, demonstrating Gonococci and other intracellular gram negative bacteria.
5. How do you prepare Dilute carbol fuchsin?. Dilute carbolfuchsin is prepared by 1:10
dilution of concentrated carbol fuchsin.
6. Mention the different decolorisers you know? Different decolorisers are absolute alcohol,
acetone, iodine acetone, acetone-alcohol.
7. What is the theory of Gram stain.?
8. What is the positive control and negative control used for Gram stain? Positive control
[Link] ATCC 25923 Negative control [Link] ATCC 25922
9. What is ATCC? American Type Culture Collection.
10. What is NCTC?. National Collection of Type Cultures.
11. What are all the different modification of Gram stain? Kopeloff and Beerman’s--- Methyl
violet for 5min, Gram’s iodine for 2 min, decoloriser is acetone and counter stain basic
fuchsinfor 30 sec Jensen’s method—Methyl violet for 30 sec, Gram’s iodine for 30 sec,
decoloriser absolute alcohol for 30 sec, neutral red (0.1%) for 2min. Preston and Morollel's
—Crystal violet for 30 sec, Gram ‘s iodine for 30 sec, Iodine acetone for 30 sec, dilute carbol
fuchsin for 30 sec. Quick gram stain—Crystal violet for 5 sec, Gram's iodine for 5 sec,
acetone, Basic fuchsin for 5 sec Hucker's modification--Crystal violet for 30sec, Gram's
iodine for 30 sec, Acetone alcohol decoloriser for 10 sec, Safranin for 30 sec
12. Mention different types of stains.

Simple stain-Methyl violet, Dilute carbol fuchsin.

Differential stain- Gram stain, Ziehl -Neelsen stain, Albert stain

Negative stain—India ink, Nigrosin stain for demonstration of capsule

Silver impregnation stain, Fontana stain for demonstration of spirochaetes

Ryu’s stain for flagella

Malachite green stain, modified Ziehl-Neelsen stain (0.25%) sulphuric acid as decoloriser for
spores

Periodic acid -Schiff (PAS ) stain for demonstration of tissues

Fluorescent stains ( Acridine orange, Auramine rhodamine)

Romanowsky stain for malaria (impart a reddish-purple colour to the chromatin of malaria and
other parasites)

Leishman’s stain, Giemsa stain for protozoa

 Levaditi stain for staining of spirochaetes in tissue

13. What is gram variability? Gram positive organisms may lose their ability to retain crystal
violet and stain Gram negatively for the following reasons: (i) Cell wall damage due to
antibiotic therapy or excessive heat-fixation of the smear, overdecolorization of the smear.
(ii) Use of an iodine solution which is too old, i.e. yellow instead of brown in colour (always
store in a brown glass or other light opaque container). (iii) Smear has been prepared from
an old culture
14. When do you use only dilute carbol fuschin to stain? The use of dilute carbol fuchsin (1 in
10) is recommended for staining Vincents’ organisms, Yersinia, Haemophilus,
Campylobacter and Vibrio species.
15. Which are the bacteria or bacterial component that can’t be stained by Gram stain?
• Extremely slender bacteria such as Treponema
• Cells containing waxy substances impermeable to stain such as Mycobacteria
• Minute intracellular bacteria such as Chlamydia and Rickettsia
• Cell organelles such as capsule, spore, flagella etc
16. What are the applications of Gram staining?

 Rapid presumptive diagnosis of diseases such as bacterial meningitis

 Selection of empirical antibiotics based on Gram stain finding
 Selection of suitable culture media based on Gram stain finding
 Screening of quality of clinical specimens, such as sputum that contain many
pus cells and few epithelial cells.
 Counting of bacteria
 Appreciation of morphology and types of bacteria in a clinical specimen
 Presence of inflammatory cells

17. List a few organisms

Gram positive cocci In clusters:Staphylococci sp
In chains:Streptococcus pyogenes
In pairs:Enterococcus [Link] pneumonia
Gram negative cocci Neisseria meningitides,Neisseria gonorrhoeae

Gram positive bacilli Without spores:Corynebacterium diphtheria

With spores:Bacillus anthracis-(aerobe)

With spores:Clostridium perferingens,Clostridium tetani(anaerobe)

Gram negative bacilli Escherichia coloi,Klebsiella pneumoniae,Proteus sp.,Salmonella

species,Pseudomonas,