Staining

Binita Adhikari
Lecturer
Microbiology
NAIHS-COM
 Staining
• Difficult to observe microorganisms by naked eyes
(minute and transparent and colourless)
• Colouring microbial cells with different stains
=staining
Purpose of staining
• Staining + microscopy aids to
- Differentiate microorganisms from their
surrounding environment,
- study properties of microorganisms, and
- divide microorganisms into specific groups for
diagnostic purposes.
Types of dyes
Classification based on Charge:
a) Acidic dye (stain)
• Anionic (chromogen portion exhibits negative charge)
• Provide background staining
eg. India ink, nigrosin,eosin
b) Basic dye (stain)
• Cationic (chromogen portion exhibits positive charge)
• Most commonly used for staining for bacteria
eg. Crystal violet, methylene blue
c) Neutral dye (stain)
• Formed from the precipitate when aqueous acidic and basic
stains combine
• Colouring matter is present both in anionic and cationic groups
• Stain nucleic acids and cytoplasm
eg. Neutral red, Giemsa stain
 Steps of Staining
1.Preparation of glass slides
2. Smear Preparation
3.Heat Fixation
4. Staining
1. Preparation of glass slides
• Dry, Clean, Oil or grease free
2. Smear Preparation
• Smear = thin preparation of microbial cells
• Should cover an area of about 15-20 mm diameter
on a slide
• Thin whitish layer/film=good smear
a) Broth cultures: 1 or 2 loopfuls of suspended cells at
the center of slide with sterile loop spreaded evenly
over an area about dime size
b) Cultures from a solid medium:
few colonies are emulsified with sterile water or
saline and should appear as semitransparent,
confluent, whitish film after drying.
3. Heat fixation
•Performed by rapid passage of air dried smear two
or three times over the flame of Bunsen burner.
-kills the bacteria
-firmly attaches the smear to the microscope slide
-allows the sample to more readily take up the stain

•Alcohol fixation for bacterial smear containing M.

tuberculosis, intracellular Gonococcus and
Meningococcus

4.Staining
 Types of staining techniques

Simple Staining Differential staining

(use of single stain) (use of two contrasting dyes)

For the visualisation of Visualisation of

Identification
morphological shape structure
and arrangement

Gram’s stain Acid Fast stain Spore stain Capsule

stain
 Staining Methods
1. Simple staining
-use of basic dye for visualising bacterial morphology and
arrangement
Negative staining
-Use of Acidic dyes , colourless organism against coloured
background
 Staining Methods
1. Simple staining
2. Negative staining
3. Differential staining
• Use of two or more contrasting dyes
• Useful for the separation of microorganisms into
groups (Gram’s stain and acid-fast stain) and for
the visualisation of structures (capsule stain,
spore stain, flagellar stain)
4. Special staining
 Simple staining
• Use of basic dyes like methylene blue, safranin, or
crystal violet to color the bacterial cells.
• bacterial cell morphology (shape and arrangement)
can be observed.
• As the surface of most bacterial cells and cytoplasm is
negatively charged, positively charged stains adhere
readily to the cell surface.
Negative staining (background staining)
• to demonstrate the capsule of Cryptococcus
neoformans, Streptococcus pneumoniae
• also to stain cells that are too delicate to be
heat fixed
• Acidic dye with negative charge on chromogen
will not penetrate the cells
• So unstained cells can be observed against
coloured backround
• NO HEAT FIXATION
Differential staining technique
Gram’s staining
Acid fast staining
 Gram’s Staining
• developed by Danish physician Hans Christian
Gram in 1884
• Differential staining technique dividing bacteria
into two groups: gram positive (violet colour) or
gram negative (red colour)
• Shape and arrangement can be seen
• Important preliminary step in initial
characterisation and classification of bacteria
Gram positive bacteria
Cocci: Staphylococcus spp., Streptococcus spp.,
Enterococcus spp., Peptostreptococcus spp.
Bacilli: Corynebacterium spp., Listeria spp., Lactobacillus
spp., Actinomyces, Clostridium spp

Gram negative bacteria

Bacilli: Pseudomonas aeruginosa, Salmonella
spp.,Shigella spp.,Vibrio spp.,Bacteroides,
Campylobacter spp
Spirochaetes: Leptospira, Treponema spp
Cocci: Neisseria meningitidis
Procedure
Primary stain: Crystal Violet
Mordant : Gram’s Iodine
Decolouriser: Alcohol, Alochol:Acetone or acetone only
Counterstain: Safranin
Principle of Gram’s Stain
1) Acid Protoplasm theory
2) Cell wall permeability theory
3) Magnesium Ribonucleate theory
• CV, basic dye, penetrate both in the cell wall of Gram
positive and Gram negative bacteria.(Violet colour)
• iodine (I- or I3-) interacts with CV+ to form large crystal
violet-iodine (CV-I) complexes within the cytoplasm and
outer layers of the cell.
-Gram positive bacteria (pH2-3) have more acidic
protoplasm, Gram negative bacteria (pH 4-5).
-iodine makes the cytoplasm more acidic ,so CVI complex
will be fixed more firmly in the bacterial cell(Acid-
Protoplasm theory)
Cell wall permeability theory
• decolorizing agent, (ethanol or an ethanol and acetone
solution), interacts with the lipids of the membranes of
both gram-positive and gram-negative bacteria.
Gram positive bacteria
• highly cross-linked and multi-layered peptidoglycan of
the gram-positive cell is dehydrated by the addition of
ethanol.
• traps the large CV-I complexes within the cell.
• After decolorization, the gram-positive cell remains
purple in color.
Gram negative bacteria
• outer membrane (lipopolysaccharide layer) is
lost from the cell, leaving the peptidoglycan layer
exposed.
• have thin layers of peptidoglycan, so with
ethanol treatment, cell walls become leaky and
allow the large CV-I complexes to be washed
from the cell.
• After decolourisation, gram negative cells will be
colourless
Mg-ribonucleate theory
• magnesium-ribonucleic acid (present only in
gram positive cells)
• CVI complex binds and form magnesium-
ribonucleic acid CVI complex which is more
difficult to remove
Procedure of Gram Staining; note the color change after each step
Reporting Gram smears
• Numbers of bacteria present, whether
many, moderate, few, or scanty
• Gram reaction of the bacteria, whether Gram positive
or Gram negative
• Morphology of the bacteria, whether cocci,
diplococci, streptococci, rods, or coccobacilli.
Also, whether the organisms are intracellular.
• Presence and number of pus cells
• Presence of yeast cells and epithelial cells.
 Acid Fast staining
• Differential staining technique
• differentiates species of Mycobacterium from other
bacteria
• stain is of diagnostic value (as it helps to identify M.
tuberculosis and M. leprae, which are pathogenic to
human)
• Acid fastness of bacteria is associated with the lipid
or waxy (mycolic acid) present in the outer covering
of Mycobacteria sp. (M. tuberculosis, M. bovis, M.
leprae, M. avium, M. smegmatis)
• other AFB : Nocardia spp.,Cryptosporidium
spp.
• Mycobacterium has lipid rich hydrophobic cell
walls which are relatively impermeable to
various basic dyes unless the dyes are
combined with phenol. Once the stain has
penetrated, it cannot be readily removed even
with the vigorous use of acid as a decolorizing
agent. So it’s called acid fast bacilli
3 common acid-fast staining methods:
a) Ziehl-Neelsen (hot),
b) Kinyoun (cold), and
c) Auramine-Rhodamine Fluorochrome (Truant method).
• acid fast stain uses three different reagents:
-Carbol fuchsin: 1°dye
- H2SO4 or acid alcohol (3% HCl, 95% ethanol):decolouriser
(5% for M. leprae,1% for Actinomyces in tissue, 0.5% for
cultures of Nocardia and 0.25-0.5% for spores of oocysts of
Cryptosporidium spp. and Isospora spp.)
- Methylene blue/malachite green: counterstain
-Colour of AFB is based on primary stain, counterstain
provide contrast background.
Red bacilli against blue
background= AFB
Mechanism/Principle
• The waxy covering of the cell wall are hydrophobic and are
impermeable to stain easily and does not allow the stain to
enter but when heated, the stain with phenol mordant
(Strong carbol fuschin solution) enters into the bacterial
cytoplasm.
• Heat treatment enhances the penetration of the primary stain
through the cell wall and into the cytoplasm. The stain once
entered, does not come out even during strong acid
treatment hence the name AFB.
• To make contrast in visualization counter stain can be done
by using methylene blue or malachite green. Note:The basic
dye carbol fuschin contains triethyl methane dye and phenol
which reacts with mycolic acid and increases the penetration
power of the dyes.
• Finally red coloured bacilli will be seen against blue/green
background.
 Recording and Reporting by WHO/IUATLD recommended method

Examination Result Grading No. of fields to be

observed
More than 10 AFB per Positive 3+ 20
oil immersion fields

1-10 AFB per oil Positive 2+ 50

immersion field

10-99 AFB per 100 oil Positive 1+ 100

immersion field

1-9 AFB per 100 oil Scanty Record exact number 100
immersion field seen and ask the
patient for repeating
sample

No AFB found per 100 Negative 0 100

oil immersion fields

Never report as “Mycobacterium tuberculosis” by smear report only.

 Special staining
• Used to distinguish parts of cells
a) Capsule stain
b) Spore stain
c) Flagella stain
d) Nuclear stain
 Capsule staining
• distinguish capsular material from the bacterial cells
• more difficult differential staining procedure
(capsular materials are water-soluble which may be dislodged
and removed with vigorous washing).
• NO HEATING OF SMEAR (resultant cell shrinkage may
create a clear zone around the organism, an artifact which may
be mistaken for the capsule).
• Few examples : Bacillus spp, Clostridium spp,
Klebsiella pneumonia, Streptococcus pneumonia,
several groups of streptococci, Neisseria
meningitides, Haemophilus influenza, Yersinia,
Rhizobium spp.
Mechanism/principle
• capsule is non-ionic unlike bacterial cells,
therefore the primary stain i.e. India ink is
unable to bind with the capsule, it only adhere
to the capsule.
• The primary stain is washed with crystal violet
rather than water.
• CV being basic dye will bind to the bacterial cell
and at the same time the primary stain is
removed from the capsule.
• So under microscope, capsule is seen
transparent surrounding the bacterial cell.
 Spore staining
• to differentiate bacterial spores from other
vegetative cells and to differentiate spore former
from non spore former bacteria.
• spores are classified as : central, subterminal or
terminal.
• eg. Bacillus anthracis, Bacillus subtilis, Clostridium
tetani, Clostridium welchii, Clostridium botulinum
common methods of spore staining :
1. Modified Z-N staining
• Carbol fuschin is mixed with heat fixed bacterial smear and given heat.
• Due to heating the strong stain enters the hard coat of bacterial spore
and stains it.
• Drops of 0.25 - 5% sulphuric acid over the smear acts as decolourizer.
• Methylene blue can be used as counter stain.

2.Dorner’s method
• Uses hot carbol fusion to penetrate the resistant endospore.
• Nigrosine serves as both a decolorizing agent and negative stain for the
smear.
• Finally, the endospore will have a red colour and vegetative portions of
the cells will be seen unstained in a dark background.

3. Schaeffer and Fulton method

• This method utilizes malachite green (5%) to stain the endospore and
0.25% Safranin to stain the vegetative portion of cells as a result spore
stains green where as vegetative part stains red.
red colored vegetative bacilli with green colored
endospores (intracellular spores).
 Flagella staining
• Flagella are cytoplasmic appendages, thread like
structure protruding through cell wall
• can be found both on Gram positive and gram
negative bacilli and most of the cocci are non motile.
• are about 0.02µm in thickness and hence beyond the
resolution limit of the light microscope.
• must be thickened by mordanting like tannic acid
• Two common procedure:
- A wet-mount procedure (Ryu method)
-Dried-smear preparation (Leifson staining technique)
 • Presence or absence of flagella
• Number of flagella per cell
• Location of flagella per cell
Observe for
-Presence or absence of flagella
-Number of flagella per cell
-Location of flagella per cell
Peritrichous: Escherichia coli
Polar: Pseudomonas aeruginosa
Negative: Klebsiella pneumoniae
Albert staining for Corynebacterium diphtheriae
• distinctly identifies metachromatic granules of
Corynebacterium diphtheriae.
• Metachromatic granules are also found in Yersinia pestis,
and Mycobacterium species.
• named as metachromatic because of its property of
changing color i.e when stained with blue stain they appear
red in color
• helps to distinguish Corneybacterium diphtheriae from
other nonpathogenic diphtheroid that lack the
metachromatic granules.
• stain the metachromatic granular bodies only but not any
other inclusions in the cytoplasmic membrane.
 Principle of Albert’s staining
• basically made up of two stains that are toluidine
blue’ O’ and malachite green (basic dye)
• pH of volutin granule is highly acidic
• So, on applying Albert’s stain to the smear,
toluidine blue’ O’ stains volutin granules i.e. the
most acidic part of cell and malachite green stains
the cytoplasm blue-green.
• On adding Albert’s iodine (mordant) due to effect of
iodine, the metachromatic property is not
observed and granules appear blue in colour.
green colored rod-shaped bacteria arranged at an angle to
each other, resembling English letter ‘L’, ‘V’, or Chinese letter
pattern along with bluish-black metachromatic granules at the
poles.