STAINS USED IN MEDICAL

MICROBIOLOGY

BY DR NUBWA MEDUGU
 INTRODUCTION

• Most bacteria are difficult to see under the bright field

microscope.

• Bacteria are almost colorless

• To visualize bacteria, either dyes or stains, or an

alternative source of illumination (phase contrast or
differential interferrence constrast) are used.
• Being relatively fast, inexpensive, and simple, staining is the
most commonly used technique to visualize bacterial cells.

• Staining not only makes bacteria more easily seen, but it

allows their morphology (e.g. size and shape) to be
visualized more easily.

• In some cases, specific stains can be used to visualize

certain structures (flagella, capsules, endospores, etc) of
bacterial cells.
 WHAT ARE STAINS

• Stains (dyes) are chemicals containing chromophores (groups that

impart color).

• Their specificity is determined by their chemical structure.

• Stains are generally salts in which one of the ions is colored.

• For example, the dye methylene blue is actually the salt methylene
blue chloride which will dissociate in water into a positively charged
methylene blue ion which is blue in color and a negatively charged
chloride ion which is colorless.
 CLASSIFICATION OF STAINS

• Based on method : may be classified as

• simple (nonspecific)
• differential (specific)

• Simple stains will react with all microbes in an identical

fashion.

• Differential stains give varying results depending on the

organism being treated.
• Simple stains consist of one dye
• They are useful solely for increasing contrast so that
morphology, size, and arrangement of organisms can be
determined.

• Methylene blue, carbol fuchsin, safranin, india ink, and

crystal violet are some of the most commonly used stains.
• Differential stains on the other hand, use two or more
dyes and help us to distinguish between two or more
organisms or two or different parts of the organism.

• Types of differential stains are gram, Ziehl-Neelsen acid

fast, negative, flagella, and endospore stains.
• Based on chemical nature : Commonly used
microbiological stains generally fall into one of two
categories
- basic stains
- acidic stains

• however there are a few stains such as India Ink which are
neutral.
 CAUTION

Note: The dyes used for bacteriological staining are

generally aniline dyes, derived from coal tar, which means
they are POTENTIALLY CARCINOGENIC and should be
handled carefully. Avoid contact with them by keeping them
off skin, clothing and benches.
GENERAL STAINING
METHODOLOGY
 FIXING

• A simple method is that of air drying and heat fixing.

• The organisms are heat fixed by passing an air-dried smear
of the organisms through the flame of a gas burner.
• The heat coagulates the organisms' proteins causing the
bacteria to stick to the slide.
• Be very careful not to over heat the organisms when fixing
them to a slide. This distorts the sample of the organisms.
• When you heat fix a slide, you want to apply enough heat to
precipitate the proteins to allow the cells to stick to the slide
but not to drastically change the shape of the cells (or reduce
them to charred remains).
 PREPARATION OF A BACTERIAL
SMEAR AND HEAT FIXATION

• Clean the slide

• Place a very small drop of normal saline on the surface of the slide.
• Aseptically remove a small amount of the culture from the agar surface
and just touch it several times to the drop of water until it just turns
cloudy.
• Using the loop, spread the suspension over the entire slide to form a thin
film.
• Allow this thin suspension to completely air dry.
• Pass the slide (film-side up) through the flame of the bunsen burner 3 or
4 times to heat-fix. Caution: Too much heat might distort the organism
and, in the case of the gram stain, may cause gram-positive organisms to
stain gram-negatively.
SPECIFIC STAINS AND
METHODOLOGY
 ROMANOWSKY STAINS

The Romanowsky stains are all based on a combination of

eosinate and methylene blue (sometimes with its oxidation
products azure A and azure B).

Common variants include Wright's stain, Jenner's stain, May-

Grunwald stain, Leishman stain, Fields stain and Giemsa
stain.

All are also suited to examination of blood to detect blood-borne

parasites like malaria.
 IODINE

Iodine is one component in the staining technique known

as Gram staining.
Lugol's solution or Lugol's iodine (IKI) is a brown solution
that turns black in the presence of starches and can be used
as a cell stain’
Iodine is also used as a mordant in Gram's staining, it
enhances dye to enter through the pore present in the cell
wall/membrane.
 MALACHITE GREEN

Malachite green (also known as diamond green B or victoria

green B) can be used as a blue-green counterstain in ZN stain
It also can be used to directly stain spores.
 CRYSTAL VIOLET

Crystal violet, when combined with a suitable mordant,

stains cell walls purple.
Crystal violet is an important component in Gram staining.
 AURAMINE AND RHODAMINE

Auramine O can be used to stain acid-

fast bacteria (e.g. Mycobacterium, where it binds to
the mycolic acid in its cell wall) in a way similar
to Ziehl-Neelsen stain.

Rhodamine is a protein specific fluorescent stain commonly

used in fluorescence microscopy.
 STAINING TECHNIQUES

Some of the commonly-used staining techniques are:

Methylene blue staining
Gram staining
Albert staining
Ziehl Neelsen staining (Acid fast staining)
India ink staining
Iodine staining for ova and cysts in faeces
 QUALITY CONTROL OF STAINS

Control organism/
Stain ATCC No* Expected result
material

Ziehl-Neelsen Mycobacterium spp. 25177 Pink red bacilli

E. coli 25922 Blue bacilli

Gram E. coli 25922 Gram -ve bacilli

S. aureus 25923 Gram +ve cocci

Iodine solution Formalin treated stool Visible cyst nuclei

specimen with cysts

* If no standard strains are available, known laboratory

strains should be used as controls.
 GRAM STAINING

• The most widely used staining procedure in

microbiology is the Gram stain, discovered by the Danish
scientist and physician Hans Christian Joachim Gram in
1884.

• Gram staining is used to determine gram status to classify

bacteria broadly. It is based on the composition of
their cell wall. Gram staining uses crystal violet to stain
cell walls, iodine as a mordant, and
a fuchsin or safranin counterstain to mark all bacteria.
Solutions
Methyl violet stain
Iodine solution (sodium or potassium iodide)
Basic fuchsin stain
Decoloriser – acetone or acid alchohol
This is basically Kopeloff and Beermans 1922
modification
 The Basic Method

1. First, a loopful of a pure culture is smeared on a slide and allowed to air dry.

2. Fix the cells to the slide by heat or by exposure to methanol.

3. Crystal violet (a basic dye) is then added for approximately 1 minute.

4. Briefly rinse the slide with water.

5. Add iodine (Gram’s iodine) solution for 1 minute. This acts as a mordant
and fixes the dye, making it more difficult to decolorize and reducing some
of the variability of the test.
6. Briefly rinse with water.
7. Decolorize the sample by applying 95% ethanol or a mixture of
acetone and alcohol. This can be done in a steady stream, or a series of
washes.
 This step washes away unbound crystal violet, leaving Gram-positive organisms
stained purple with Gram-negative organisms colorless.
 The decolorization of the cells is the most “operator-dependent” step of the process and
the one that is most likely to be performed incorrectly.
8. Rinse with water to stop decolorization.

9. Rinse the slide with a counterstain (safranin or carbol fuchsin).

10. Blot gently and allow the slide to dry. Do not smear.
 STAIN REACTION:

The four basic steps of the Gram Stain are:

1) Application of the primary stain Crystal Violet (CV) to
a heat-fixed smear of bacterial culture.
CV dissociates in aqueous solutions into CV+ and Cl –
ions.
These two ions then penetrate through the cell wall and cell
membrane of both Gram-positive and Gram-negative cells.
The CV+ ions later interacts with negatively charged
bacterial components and stains the bacterial cells purple.
2) Addition of Gram’s Iodine.
Iodine (I – or I3 –) acts as a mordant and as a trapping
agent.
A mordant is a substance that increases the affinity of the cell
wall for a stain by binding to the primary stain, thus forming
an insoluble complex which gets trapped in the cell wall.
In the Gram stain reaction, the crystal violet and iodine
form an insoluble complex (CV-I) which serves to turn the
smear a dark purple color.
At this stage, all cells will turn purple.
3) Decolorization with 95% ethyl alcohol.
Alcohol or acetone dissolves the lipid outer membrane of
Gram negative bacteria, thus leaving the peptidoglycan layer
exposed and increases the porosity of the cell wall.
The CV-I complex is then washed away from the thin
peptidoglycan layer, leaving Gram negative bacteria
colorless.
On the other hand, alcohol has a dehydrating effect on the
cell walls of Gram positive bacteria which causes the pores
of the cell wall to shrink.
The CV-I complex gets tightly bound into the multi-layered,
highly cross-linked Gram positive cell wall thus staining the
4) Counterstain with Safranin
The decolorized Gram negative cells can be rendered visible
with a suitable counterstain, which is usually positively
charged safranin, which stains them pink.
Pink colour which adheres to the Gram positive bacteria is
masked by the purple of the crystal violet (Basic fuschin is
sometimes used instead of safranin in rare situations).
It is a prudent practice to always include a positive and negative control on the
staining procedure to confirm the accuracy of the results (Murray et al 1994)
and to perform proficiency testing on the ability of the technicians to correctly
interpret the stains (Andserson, et al. 2005).
 ZIEHL-NEELSEN STAINING

• Ingredients and preparations

• Carbol fuchsin 3% (strong)
• Sulphuric acid 20-25%
• 95% alchohol
• Methylene blue 0.1% (malachite green is an alternative)

• Uses
• Distinguishes acid fast bacilli such as Mycobacterium
tuberculosis and M.leprae from other non-acid fast bacilli.

•
Smearing and fixation :
1. Make a smear from yellow purulent portion of the sputum
using applicator stick.

2. Let the smear air-dry for 15-30 minutes.

3. Fix the smear by passing the slide over the flame 3-5 times
for 3-4 seconds each time.
 STAINING :

1. Place the fixed slide on the staining rack with the smeared side facing
upwards.

2. Pour filtered 1% carbol fuchsin over the slide so as to cover the entire slide.

3. Heat the slide underneath until vapours start rising. Do not let boil or dry.
Continue the process up to five minutes.

4. Gently rinse the slide with tap water to remove the

excess carbol fuchsin stain. At this point, the smear on the slide looks red
in colour.

5. Decolor the stained slide by pouring 25% sulphuric acid on the slide and
leaving the acid for 2-4 minutes.
7. Counter stain the slide by pouring 0.1% methylene blue

solution onto the slide and let it stand for one minute.

8. Gently rinse the slide with tap water and tip the slide to drain

off the water.

9. Place the slide in the slide tray and allow it to dry.

10. Examine the slide under a microscope

 INDIA INK STAINING

Staining procedure :
Place a loopful of India ink on the side of a clean slide.
A small portion of the solid culture is suspended in saline on
the slide near the ink and then emulsified in the drop of ink,
or else, mix a loopful of liquid culture of specimens like CSF
with the ink.
Place a clean cover slip over the preparation avoiding air
bubbles.
Press down, or blot gently with a filter paper strip to get a
thin, even film.
IN CLOSING