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Staining techniques
• Staining is coloring with dye to make some structures more visible.

• Staining is used to increase visibility as most bacterial are transparent

and difficult to see under light microscope.
Staining techniques • Before microorganisms can be stained, they must be fixed or attached to
the microscopic slide; otherwise they might be washed a way by the dye.

• For fixing smear preparation is needed.

• Smear: A thin film of a solution of microbes on a slide.

• A smear is usually fixed to attach the microbes to the slide and to kill the
microbes.

Wet mount versus stained smear

• The specimen is fixed ether by heat or chemicals (ethanol,
v Wet mount
formaldehyde, etc) after the smear is air dries.
• Cell suspended in fluid a drop or two of the culture is then placed
• The stain is applied and then washed off with water .
on a slide and overlaid with a cover glass
• The slide is then blotted with absorbent paper.
• Cove glass can damage larger cells and might dry or contaminate
the observers fingers • The stained microorganisms are now ready for microscopic
examination.
v Smear preparation
• A smear is a thin film of bacterial suspension prepared on a slide
for microscopic examination
• It is prepared by spreading the material (suspension) over the
surface of a slide and allowing it to air dry.

Stains (dyes)
• In acidic dye (negatively charged), the color is in the negative ion.
• Chemically, dyes are usually salt, although a few are bases or acids
composed of a positive and a negative ion, one of which is colored and • They are not attached by most bacteria because the negative ions
is called the chromophore (the part of molecule that is responding for are repelled by the negatively charged bacterial surface.
color).
• Negatively charged dyes stain the background instead of the cell.
• Dyes can be basic, acidic or neutral.
• The color of basic dyes is in the positive ion e.g. methylene blue , This is called negative staining(preparation of colorless bacteria
crystal violet, basic fuchsine, malachite green and safranin are positive against a colored background) .
charged.
• They are important in the observation of overall cell shapes, size
• Positive charged dyes react with /bind to the negatively charge nucleic
acid and proteins inside cell and to polysaccharides on the bacterial cell and capsule. Example of acidic dyes include eosin, nigrosin,
surface. indian ink, rose Bengal and acid fuchsin.

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• Neutral dyes are made by combining acidic and basic dyes. v Types of staining techniques

• The colored part is contained in both positive and negative ions • There are two categories of staining techniques: simple and
(e.g. Giemsa stain). differential staining.

• They are most useful for staining complex cells of higher forms A) simple staining
because they permit differentiation of interior structures, some • Simple staining employs a single dye, most commonly methylene
of which are basic and some are acidic. blue, crystal violet, basic fuchsin and safranin.
• They are used to stain nucleic acids and cytoplasm. • It generally stains the bacterial cell the same color.

• Simple staining is used to reveal size, shape and arrangement of

bacteria .

B) differential staining Staining Principles

• Differential staining used more than one dye and is used to

• Acidic (-ve) / Basic (+ve)
distinguish between structure within a cell or between types of

bacteria by staining them different colors.

• Simple Stains
• Gram staining, acid-fast staining, flagella, capsule and endospore

staining are examples of differential staining. However, the first • Differential Stains

two are the most frequently used.

• Special Stains

C) Gram Stain • Decolorized Alcohol wash

• Cannot wash out of the Gram + ve cell wall
• Differential Stain • Gram –ve wall is easily disrupted by alcohol wash
• Saffranin counterstain
– 2 colors for Gram + ve and Gram –ve
• Gram +ve cells stay purple
– Developed by Hans Gram • Gram –ve cells stain pink
• Diagnostic importance
– Distinguishes microbes based on peptidoglycan content in cell wall – Peptidoglycan is a target for many antibiotics when present in a
• In the first step the smear is stained with basic dye crystal violet
thick layer
• Penicillins
(Primary stain) followed by treatment with iodine solution functioning • Monobactams
as mordant. • Carbapenems

• CV-I complex

– Crystal violet + iodine forms a large molecular complex

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Appearance After Each Step

• Iodine increases the interaction between cell & dye so that cell

stains strongly. 1. Crystal violet

• The smear is next decolorized by washing with ethanol or acetone.

2. Addition of Iodine
• Finally smear is counter-stained with a simple basic dye different in

color from Crystal violet. 3. Alcohol rinse step

• Safranin is the most common counter stain which colours Gram

4. Safranin Counterstain
negative bacteria pink to red and leaves Gram positive bacteria dark

purple.
14

D)Ziehl-Neelsen Acid Fast Stain • They can be stained by Ziehl-Nulsen method, which uses heat and

• Acid Fast Bacteria phenol to derive basic fuchsin into the cells.
• Mycobacterium genus
identification – Mycrobacterium spp. were penetrated with basic fuchsin, not
• These bacteria have cell wall with
high lipid content such as mycolic easily decolourized by acidified alcohol ( acid alcohol) and thus
acid . are said to be acid fast.
• A group of branched chain hydroxy
lipids, – Diagnosis
• which prevent dyes from readily
• Tuberculosis
binding to cells.
• Leprosy

E) Negative (Capsule) Stain

• Visualizes the Capsule F) Endospore Stain (Schaefer-Fulton)
– Thick glycocalyx around • Endospores are hard to see
cell with Gram stain
– Negatively charged dyes – Waxy dipicolinic acid
• Repelled by –ve charge • Visualizes pathogens
on glycocalyx – Clostridium tetani
• Halo effect is seen – C. botulinum
– Cells are counterstained – C. perfringens
with positively charged
dye – Bacillus anthracis
• Virulence factor • Vegetative cells vs.
Endospores
– Repels phagocytic WBC
– Sterilization challenge
– May prevent antibiotic
entry into cells – Heat and dessication-
resisitant

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• Spore formation takes place in some bacterial genera to

• In the Schaffer-Fulton procedure, endospores are first stained by
withstand unfavourable conditions.
heating bacteria with malachite green,
• All bacteria cannot form spores, only few bacterial genera
including Bacillus and Clostridium, ü which is very strong stain that can penetrate endospores.

ü It produce sporulating structure inside vegetative cells called endospore. • After malachite green treatment, the rest of the cell is washed free
• Endospore morphology and location vary with species and are of dye with water and is counter-stained with safranin.
valuable for identification.
• This technique yields a green endospore with red vegetative cell.
• Endospores are not stained well by most dyes, but once stained,
they strongly resist decolorization.

Special Stains See Fig 3.14 Review of different staining techniques

Important Staining Reactions in Microbiology

• Endospore stain: Heat is required to drive a

stain into the endospore.

• Flagella staining: requires a mordant to make

the flagella wide enough to see.
• Capsule stain uses basic stain and negative
stain

For Gram stain

technique compare to
Fig 3-12

CHAPTER THREE

Taxonomy Of Microbes(Major groups

of microorganisms)