Frequently asked questions in staining exercises
|How are staining techniques classified?
• Simple stain: where only one stain is used and all bacteria are
stained similarly. Eg: Methylene blue, dilute carbol fuchsin
• Differential staining: where different bacteria stain differently to
a common staining technique depending on their physiological
properties. Eg: Gram’s stain and Acid fast staining
• Special stain: where structures of bacteria like spores, granules,
capsule etc are demonstrated. Eg: silver impregnation technique for
demonstration of spirochetes, Feulgen stain for demonstration of
nucleus, Sudan black stain for demonstration of lipid vacuoles, Ryu’s
stain for demonstration of flagella, Albert’s stain for demonstration
of metachromatic granules.
• Negative staining: where the background is stained with an acidic
dye such as India ink or Nigrosin. Used for demonstration of capsules.
How are stains classified?
• Stains are classified based on the pH of their chromophore (color
bearing ion) into acidic, basic and neutral. Acidic dyes have anionic
chromophore eg., sodium+ eosinate-. Basic dyes have cationic
chromophore eg., methylene blue+ chloride-. Acidic dyes combine more
strongly with cytoplasmic components of bacteria, especially the
nucleus that is basic in nature. Neutral dyes have both acidic and
basic component that nullify each other. They are Romanowsky’s stain
and are used in staining parasitic forms.
• Stains can be either natural (eg: carmine and hematoxylin) or
coal-tar derivatives /aniline stains (eg: methylene blue, crystal
• Supravital (cells removed from the body) and intravital (cells still
a part of the body).
What is polychrome methylene blue?
Loeffler’s methylene blue solution treated with Potassium hydroxide
turns into Polychrome methylene blue after prolonged storage with
shaking. Used in McFadyean’s reaction for Bacillus anthracis in blood
films and demonstration of metachromatic granules of Corynebacterium
Who invented Gram stain?
Hans Christian Gram invented this stain in 1884. The original
formulation was Aniline Gentian violet, Lugol’s iodine, absolute
alcohol and Bismark brown.
Which are the theories of Gram staining?
• Cell wall theory: Cell wall of Gram positive bacteria are 40 times
thicker than those of Gram negative cells, hence they are thought to
help retain the dye-iodine complex.
• Lipid Content Theory: Cell envelope of Gram negative bacteria
contains an additional membrane (outer membrane), hence containing
more lipids 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. Alcohol/acetone dehydrates
Gram positive bacteria shrinking the cell wall and the closing the
• Magnesium Ribonucleate Theory: A compound of magnesium ribonucleate
and basic protein concentrated at the cell membrane helps Gram
positive bacteria retain the primary dye. Gram negative bacteria do
not possess this substance.
• Cytoplasmic pH Theory: The cytoplasm of Gram positive bacteria are
said to be more acidic (2) than those of Gram negative ones (3). Hence
the dye is said to bind with more affinity to Gram positive cells.
Which part of the bacteria actually gets stained?
It is the cytoplasm (especially the nucleic acid) that gets stained
and not the cell wall. Presence of an intact cell wall is important for
retaining Gram positivity. Cell wall deficient forms such as
Mycoplasma and L forms are Gram negative.
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
• Minute intracellular bacteria such as Chlamydia and Rickettsia
• Cell organelles such as capsule, spore, flagella etc
Which are the alternatives used in Gram stain?
Primary stain: Crystal violet, Methyl violet and Gentian violet
Mordant: Gram’s iodine, rarely Lugol’s iodine
Decolorizer: Alcohol, acetone, acteone-alcohol mixture (1:1)
Counterstain: Dilute carbol fuchsin, safranin, neutral red, (Sandiford
stain for Gonococci)
Which are the positive and negative controls for Gram stain?
Postive control: Staphylococci
Negative control: E.coli, pus cells
What are the conditions when Gram positive bacteria can appear Gram
• When over-decolourized by either prolonged exposure to decolourizer
or using acetone alone.
• When cell wall gets damaged by exposure to lysozyme or cell wall
acting antibiotics such as Penicillin.
• Old cultures, where cell wall is weakened or action of autolytic
• Those bacteria that are phagocytosed, where cell wall is acted upon
by lysosomal contents
Which is the more important step in Gram stain?
Decolourization is the most important step as this step differentiates
between Gram positive and Gram negative bacteria. Over-decolourization
can result in Gram positive bacteria appearing Gram negative and
under-decolourization can result in Gram negative bacteria appearing
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
should contain many pus cells and few epithelial cells
• Counting of bacteria
• Appreciation of morphology and types of bacteria in a clinical
Name a fungus that is Gram positive?
What are the various modifications of Gram stain?
• Kopeloff and Beerman’s (Primary stain: Methyl violet, decolourizer:
acetone or alcohol-acetone mixture 1:1)
• Jensen’s (Primary stain: Methyl violet, decolourizer: absolute
alcohol, counterstain: Neutral red)
• Preston and Morrell’s (Primary stain: crystal violet, decolourizer:
• Weigert’s (Primary stain: Carbol gentian violet, decolourizer:
Aniline-xylol). This is used to stain tissue sections.
What is acid fast staining?
Certain bacteria or their structures have the ability to retain the
primary dye (strong carbol fuchsin) and resist decolourization by weak
mineral acids such as H2SO4, HCl. Such bacteria or their structure are
termed acid fast and this property is termed acid fastness. There are
two types of acid fast staining, the hot method and the cold method.
The hot method (Ziehl-Neelsen) involves heating the slide while the
cold methods such as Kinyoun’s and Gabbett’s do not involve heating
Who introduced Acid fast staining?
Ehrlich in 1882 discovered acid fastness. The original method involved
staining with aniline-gentian violet and decolourization with strong
nitric acid. It was later improved by Ziehl and Neelsen.
Why are Mycobacteria acid fast?
The cell walls of Mycobacteria are made up of waxy substance, Mycolic
acid that is relatively impermeable to ordinary staining techniques.
But, by application of heat and a mordant (phenol), the cell can be
stained. The purpose of heating is to soften the waxy material of the
cell wall and allow the stain to enter the cell. Basic fuchsin is more
soluble in phenol and phenol is a better solvent for lipids and waxes.
What are the components of Ziehl-Neelsen stain?
Primary stain: Strong Carbol Fuchsin (contain Basic fuchsin and
Decolourizer: 20% sulphuric acid
Counterstain: Loeffler’s Methylene blue or 1% Malachite green, Picric
acid for color-blind workers
What is acid-alcohol decolourizer?
3% HCl in 95% alcohol (methylated spirit). This is useful in
differentiating saprophytic Mycobacteria from pathogenic Mycobacteria.
Pathogenic Mycobacteria are both acid and alcohol fast but saprophytic
Mycobacteria are only acid-fast. Saprophytic Mycobacteria can get
declourized by alcohol. 95% alcohol can be used as a secondary
decolorizer after decolourizing with acid. Especially used in staining
smears prepared from urine that may contain Mycobacterium smegmatis.
Which are the various dilutions of sulfuric acid used?
Mycobacterium leprae - 5% H2SO4
Oocysts of Cryptosporidium, Isospora - 1% H2SO4
Tissue sections containing Actinomyctes, Nocardia - 1% H2SO4
Cultures of Nocardia - 0.5% H2SO4
Bacterial spores - 0.25-0.5% H2SO4
What are cold methods of acid fast staining?
The two methods namely Kinyoun’s and Gabbett’s don’t involve heating
of slides, hence called cold methods. Heating is substituted by
increased concentration of phenol and prolonging the duration of
staining. Kinyoun's method is favoured for detection of
Cryptosporidium oocysts in fecal samples. Gabbett’s method has decolourizer and counterstain in one
Why should the slide be flooded with strong carbol fuchsin?
For uniform distribution of heat, or else the slide may break.
What are the precautions to be taken while preparing or observing
smears for AFB?
• A new slide must be used for every specimen, because scratch marks
may give false positive.
• A uniform smear from thick portion of the sputum must be made.
• Staining jars should not be used to staining smear as there is risk
to cross contamination.
• Fresh blotting paper must be used for each smear for drying the
slide to prevent transfer from one slide to another.
How to interpret the smear?
At least 100 oil immersion fields must be viewed before declaring the
smear as negative. The sensitivity of smear is low because it requires
the presence of 104 bacilli/ml to be smear positive. If the number of
bacilli is less than this, the chances of detecting them are less. In
such a case, the sample should be subjected to concentration
techniques such as Petroff’s method. If the smear is positive for AFB,
it should be counted/graded. Failure to detect any AFB does not rule
tuberculosis. Grading of smears has prognostic value.
How is the smear graded?
Smears are graded depending on the number of bacilli seen.
3-9 bacilli/entire smear: +
≥10 bacilli/entire smear: ++
≥10 bacilli/in most oil immersion fields: +++
What other methods are available for staining Mycobacteria?
Sputum smears for Mycobacteria can be stained by fluorescent dyes such
as Auramine and Rhodamine as they have affinity for mycolic acid in
their cell walls. The fluorescent microscopy is useful in screening
large number of specimens. Large area of smear can be quickly observed
that too under high power dry objective.
What is beaded appearance of Mycobacteria?
Beaded appearance is used to describe the appearance of Mycobacteria
when the cell doesn’t stain uniformly, showing stained and unstained
regions. These forms are common in Mycobacterium tuberculosis while
Mycobacterium bovis stains uniformly. Most saprophytic Mycobacteria
What are metachromatic granules?
Metachromatic granules are polymetaphosphate reserves produced by
Corynebacterium diphtheriae in nutritious medium. These granules are
also known as Babes Ernst granules, Volutin granules, Polar bodies
etc. They are called metachromatic granules because of they exhibit
metachromasia, a property where the granules appear in a colour
different from that of the dye used. When stained with polychrome
methylene blue, they appear purple. They are produced in abundance in
serum containing medium such as Loeffler’s serum slope.
Which are the ways to demonstrate these granules?
Albert’s stain, Neisser’s stain, Ponder’s stain and Pugh’s stain. They
can be demonstrated as refractile bodies in wet mount or slightly more
gram positive structures in Gram stain.
Why are the bacilli arranged at angles to each other?
The bacilli are arranged at angles to each other resembling English
letter V or L or Chinese letter (cuneiform) pattern because the
daughter cells don’t separate completely after cell division (binary
What do Albert A(1) and B(2) solution contain?
Solution A(1) contains Toluidine blue, Malachite green, Glacial acetic
acid and Alcohol while solution B(2) contains iodine and potassium
iodide in distilled water.