SIMPLE STAINING

Name two simple stain and their uses.

 Methyl violet

 Basic fuchsin

Uses : To study the morphology of bacteria like shape and arrangement

Cocci in cluster

procedure:
keep the given slide on the staining rack and pour the stain (Dilute corbol fuchsin or Methyl
violet ) over the smear . keep it for 1 minute .wash the slide with water . Blot dry using
blotting paper . observe under oil immersion .
OBSERVATION : Cocci in clusters
Colour: Violet
Shape : spherical
Arrangement : clusters
INFERENCE: Given smear shows violet colored cocci arrangement in clusters.
 GRAM STAIN

PRINCIPLE: Gram staining is a differential staining technique that differentiates bacteria

into two groups gram – positive & gram negative .The procedure is based on the ability of
micro oraganisms to retain color of the stains used during the gram stain reaction .Those
bacteria that resitdecolourisation and retain the primary dye are called Gram positive
and those bacteria that get decolourisation and get counter stained are called Gram
negative

PROCEDURE: There are four compounds of Gram stain.

 Primary stain with pararosaniline dye such as crystal violet, methyl violet or gentian
violet
 Mordant –Gram `s iodine
 Decolorizer – organic solvents such as acetone , ethanol or aniline
 Counter stain – such as safranine ,neutral red or carbolfuchsin

When stained with a primary stain crystal violet and fixed by mordant (Gram`s iodine) .a dye
iodine complex is formed. On subjecting to decolourisation with solvents such as acetone.
Gram positive bacteria are able to retain the dye iodine complex while gram negative
bacteria get decolorized .The decolourised gram negative bacteria take up the
counterstainsafranine and appear pink .

STEPS:
Identify the correct side of the slide. Flood the smear with crystal violet and leave it for 1
minute. Wash the slide with tap water. Add Gram `s iodine and leave for 1 minute. Wash slide
with tap water. Decolorize with acetone for 2 seconds [hold the slide close to the running
water in staining position. add acetone and wash immediately]. Add safranine and leave for 30
seconds, Wash the slide with tap water. Drain the water from the slide and allow it to dry.
Place one drop of oil and observe under oil immersion objective lens.
THE MECHANISM OF GRAM STAIN:
• The peptidoglycan cell wall of gram positive bacteria is thick and thus resists
decolourisation andretains the dye – iodine complex
• Gram positive bacteria have more acidic protoplasm then gram negative
bacteria .Hence the basic primary dye bind with more affinity to the Gram positive
bacteria
• The cell envelope of Gram negative bacteria contains more lipids in the outer
membrane than gram positive bacteria. Acetone or alcohol dissolves the lipid thus
forming large pores in gram negative bacteria through which the dye- iodine
complex leaks out .

1. Define differential staining and give examples.

Two stains are used which impart different colors to different bacteria or bacterial
structures, which help in differentiating bacteria. The most commonly employed
differential stains are:
 Gram stain: It differentiates bacteria into gram positive and gram-negative
groups
 Acid-fast stain: It differentiates bacteria into acid fast and nonacid-fast groups
 Albert stain: It differentiates bacteria having metachromatic granules from other
bacteria that do not have them.

2. State the function of iodine and acetone in Gram stain.

Iodine - Mordant/fixator
Acetone- Decolorizer

3. Which part of the bacteria gets stained?

It is the cytoplasm that gets stained. Presence of intact cell wall in important for
retaining the stain.

4. What are the alternatives used in gram stain?

• Primary stain with crystal violet, methyl violet or gentian violet

• Mordant –Gram `s iodine
• Decolorizer – acetone, ethanol or aniline
• Counter stain – such as safranine ,neutral red or carbolfuchsin

5. What is the crucial step in gram staining?

 Decolourisation is the most crucial step of Gram staining. If the decolorizer is

timed more, even the gram-positive bacteria would lose the primarycolor (over
decolourisation) and would appear gram negative.
  If the decolorizer it’s timed less, the gram-negative bacteria will not lose the
primary color properly (under decolourisation) and would appear gram positive.

6. What are the conditions when gram positive bacteria appears gram negative?

 Over decolourisation
 Old cultures
 Exposure to cell wall acting antibiotics.

7. List the differences between Gram positive and Gram negative cell walls.

Gram positive cell walls Gram negative cell walls

Peptidoglycan layer -Thicker (16–80 Peptidoglycan layer -Thinner (2 nm)

nm)

Lipid content- Nil or scanty (2–5%) Lipid content - Present (15–20%)

Lipopolysaccharide- Absent Lipopolysaccharide - Present

(endotoxin)

Teichoic acid- Present Teichoic acid - Absent

8. Name some special stains.

 Albert’s stain- for granules of Corynebacterium diptheriae

 Fontana stain- for spirochetes
 Leif son stain- for flagella
 Giemsa stain -for Chlamydia and Rickettsia
9. What are the bacteria that cannot be stained by Gram stain?

 Mycobacteria
 Chlamydia
 Rickettsia
 Treponema
 Mycoplasma

10.List the possible explanations of why some bacteria

are grampositive and others are gram negative.

 The peptidoglycan layer in the cell wall of gram positive organism are thick and they
resist decolourisation by the acetone and retains the dye iodine complex.
 Gram positive organisms have more acidic protoplasm than gram negative organism,
hence the basic primary dye binds with more affinity to gram positive organisms.
 The cell envelope of Gram negative bacteria contains more lipids in the outer
membrane than gram positive bacteria. Acetone or alcohol dissolves the lipid thus
forming large pores in gram negative bacteria through which the dye- iodine
complex leaks out .

11.Give examples for each of the following:

 Gram positive cocci

o Arranged in groups: Staphylococcus aureus
o Arranged in chains: Streptococcus pyogenes
o Arranged in pairs: Streptococcus pneumoniae

 Gram positive bacilli: Corynebacterium diptheriae

Bacillus anthracis

 Gram negative cocci: Neisseria meningitidis

Neisseria gonorrhoeae

 Gram negative bacilli : Escherichia coli

 Klebsiella pneumoniae

12.What is the clinical significance of performing gram stain?

 To start empirical antibiotics based on the gram stain report.

 To select appropriate culture media in the laboratory.
 To make presumptive diagnosis of meningitis, UTI & sepsis.
 Anaerobic organisms, such as Clostridium do not grow in routine culture.
Therefore, organisms detected in Gram stain, but aerobic culture-negative gives a
preliminary clue to perform an anaerobic culture of the specimen.
 In addition to stain the bacteria, Gram stain is useful for staining certain fungi
such as Candida and Cryptococcus (appear gram-positive)
 Gram stain helps in screening the quality of the sputum specimen before
processing it for culture. Presence of more pus cells and less epithelial cells
indicates good quality specimen.
GRAM STAINING:

Exercise 1:

Aim: To perform Gram staining on a heat fixed culture smear, focus under the
microscope, record the observations and interpret the result of Gram staining

OBSERVATION:

Grampositivecocciin groups

Organism 1

Colour Purple / Violet

Shape Cocci / Spherical

Arrangement Clusters

INFERENCE: The given smear shows Gram Positive cocci arranged

in clusters Example: Staphylococcus aureu

Exercise 2:

AIM: Aim: To perform Gram staining on a heat fixed

culture smear, focus under the microscope, record the
observations and interpret the result of Gram staining

Gramnegativebacilliinscattt

er ed

OBSERVATION:

Organism 1
Colour Pink
Shape Rod
Arrangement Scattered

INFERENCE: The given smear shows Gram Negative

Bacilli which shows scattered arrangement

Example: Escherichia coli

 Exercise 3:

AIM: Aim: To perform Gram staining on a heat fixed

culture smear, focus under the microscope, record the
observations and interpret the result of Gram staining

OBSERVATION:

Gramnegative bacilli in scattered

Grampositivecocciing roups

Organism 1 Organism 2

Colour Pink Violet

Shape Rod Spherical

Arrangement Scattered Cluster

INFERENCE: The given smear shows Gram Negative Bacilli which shows
scattered arrangement and Gram Positive Cocci arranged in clusters

Example: Gram negative bacilli:-Escherichia coli

Gram positive cocci:-Staphylococcus aureus