LUSAKA APEX MEDICAL UNIVERSITY

STAINING TECHNIQUES

Dr Simpokolwe K.
 STAINING

• Microbial Staining – This is the giving of colour

to microbes, and it is employed because
microbes are colourless and have highly
transparent structures.
 Fixation
• The process by which the internal and
external structures of microorganisms are
preserved and fixed in place. There are two
types of fixation:
1. Heat fixation – fixation by means of application
of heat. The prepared smear of microorganisms
is gently heated and air-dried.
2. Chemical fixation – involves the use of chemicals
such as ethanol and formaldehyde.
 Reasons for staining
1. Used to increase visibility of microorganisms
being studied.
2. Used to identify the shape of bacteria.
3. Used to determine the morphological
features of microorganisms.
4. Used to detect contamination.
5. Used to differentiate and classify
microorganisms (differential stains).
6. Used to detect bacterial parts such as
capsule, spores, flagella or inclusion bodies
(special stains).
 Simple Staining Technique
• Only one stain is used
• Crystal Violet, Carbol Fuchsin, and Methylene
Blue are examples of basic dyes used in simple
staining technique.
 Differential Staining Technique
• Used to classify bacteria into separate groups
based on their distinct staining properties.
• Makes use of a primary stain, a decolorizer,
and a counterstain.
• Gram Stain and Acid-Fast Stain are examples
• TYPES OF STAINS

1. Simple staining – only one dye is used-

differentiation among bacteria is impossible-
examples of dyes used are Crystal Violet, Carbol
Fuchsin, and Methylene Blue.

2. Differential staining- more than one dye is used-

Differentiation among bacteria is possible- Eg.
Gram’s staining, Acid-fast staining.

3. Special staining – more than one dye used -

Special structures are seen.
Eg. Capsule staining, Spore staining.
 Basic requirements for staining:

1. Clean grease-free slide.

2. Microbe to be stained.
3. Inoculating loops- to transfer Microbial
suspension to slide.
4. Bunsen burner – to sterilize inoculating loops
before and after smear preparation.
5. Pencil marker – to mark where bacterial
smear is applied, label the slide.
 Basic initial steps before staining:

1. Collect the specimen

2. Prepare Smear
• Putting of microbial suspension (bacteria in
liquid for example) to be stained on the central
portion of glass slide in a circular fashion, air-
dried, heat-fixed, the resultant preparation
called bacterial smear- appears dull white.
 SIMPLE STAINING:
This is simple to perform- only one basic stain used. Examples of
stains used are:
• Crystal violet
• Methylene blue
• Basic fuschin
• Malachite green

Principle:
- All bacteria in smear takes stain and appears in colour of stain.
- Basic stain more affinity towards bacterial surface & stains the
bacteria.

Uses:
To study morphology and arrangement of bacteria.
 Procedure:
• A bacterial smear is prepared, air-
dried and heat-fixed.
• A Heat-fixed smear is flooded with
either one of the basic stain and
allowed to react for 1-2 minutes and
then washed under running tap
water.
• Air dried and focused with 10x,45x &
100x on a microscope.

Results:
• Morphology – spherical / rod.
• Arrangement – cocci –clusters/chains.
 DIFFERENTIAL STAINING

• 1. GRAM STAINING

• DANISH BACTERIOLOGIST HANS CHRISTIAN GRAM

(1880)
• Based on this reaction, bacteria classified into Gram
positive and Gram negative bacteria.
• The cell wall composition differences makes
difference.
Differences between Gram -Ve and Gram +Ve Bacteria

Gram-Negative Bacteria Gram-Positive Bacteria

More complex cell wall. Simple cell wall.

Thin peptidoglycan cell wall layer. Thick peptidoglycan cell wall layer.

Outer lipopolysaccharide wall layer. No outer lipopolysaccharide wall layer.

Retain safranin. Retain crystal violet/iodine.

Appear pink/red. Appear blue/purple.

• Gram-positive cells have
– A thick outer layer of peptidoglycan
– A very narrow periplasmic space
– Teichoic acids in the peptidoglycan
• Gram-negative cells have
– A varying width periplasmic space containing a very
thin layer of peptidoglycan
– An outer membrane composed of lipopolysaccharide
(LPS)
REQUIREMENTS – STAINING REAGENTS:

1. Crystal violet – Primary stain

2. Gram’s iodine- mordant/fixative
3. Acetone (95%)- decoloriser
4. Safranine/dilute carbol fuchsin –counterstain
PRINCIPLE:
1. CRYSTAL VIOLET - all bacteria take crystal violet- so all
appears violet.
2. IODINE – Crystal Violet-iodine(CV-I) complex is formed.
3. ACETONE- bacteria with high lipid content loose CV-I
complex(Crystal violet Iodine complex). They appear
colourless but bacteria with less lipid content retains
CV-I complex , and thus appear violet.
4. SAFRANINE/ DILUTE CARBOL FUCHSIN – only colourless
bacteria takes in – appear pink.
RESULT:

Colour:
Purple/blue colored bacteria – Gram positive
Pink/red colored bacteria – Gram negative
Shape:
Spherical – cocci
Rod – bacilli
 PROCEDURE:
• Prepare slide.
• Add Crystal violet – 1 min – wash with water.
• Add Iodine – 1 min – wash with water.
• Add Acetone drop by drop and watch out colour
comes out – wash immediately.
• Add Safranine/dilute carbol fuchsin – 1 min- wash.
• Allow to dry – examine under microscope.

Note: Results should be confirmed only with 100x

magnification.
Examples of Gram Positive Bacteria

Staphylococcus aureus

Streptococcus pyogenes

Clostridium perfringens

Listeria monocytogenes
Examples of Gram Negative Bacteria

Escherichia coli

Haemophilus influenzae

Vibrio cholerae

Neisseria meningitidis
 2.ACID-FAST STAINING:
(Ziehl-Neelsen stain)
• To stain Mycobacterium species especially
M.tuberculosis.
• High lipid content – makes decolorisation very
difficult –extraordinary property.
• Principle:
• Acid fast(resist) – Property of Mycobacterium
species - once this bacteria stained with primary
dye – difficult to decolorise with acid.
• This property due to Mycolic acid in cell wall.
Staining reagents
1.Strong carbol fuchsin – primary stain
2.20% sulphuric acid/3% Hcl – decoloriser –
acid-fast property.
3. 95% alcohol- decoloriser- alcohol – fast
property
4. Methylene blue/ Malachite green-
counterstain.
Procedure:
1. Strong carbol fucshin-heat till steam rises – allow 5-10 min
to act– wash with water.

2. Decolorise with acid-alcohol mixture till get a faint pink

colour in the smear (take 3-5 min) – wash.

3. Methylene blue/Malachite green – 2 min – wash.

4. Allow to dry and focus under microscope.

Result:

• Pink bacilli – Acid fast bacteria/bacilli

Eg., M.tuberculosis – long slender bacilli.

• Blue colored bacteria – Non-acid fast

Eg. Epithelial cells, pus cells, other
bacteria.
Acid fast stain:
 Special Staining Techniques
• Negative Staining using India Ink or
Negrosine is used to reveal the presence of
capsule.

• Schaefer-Fulton using Malachite Green and

Safranin is used to stain endospores