WHAT IS STAIN?

Chemical compound used to enhance the visibility of a

microscopic object or organism.

SIMPLE STAINING AND DIFFERENTIAL STAINING

DIFFERENTIAL STAINING TECHNIQUE

it involves more than one dye

most used in Microbiology

Gram Staining and Acid Fast Staining

POSITIVE STAINING VS NEGATIVE STAINING

SIMPLE STAINING TECHNIQUE

it determines bacterial shape and morphologic arrangements

Methylene blue is a classic example of a simple stain

Positive and Negative Staining

Positive Staining
-where the actual cells are colored and appear in a clear
background.
Negative Staining
-where the cells remain clear(uncolored) and the background is
colored to create a contrast to aid in the better visualization of the
image.

PREPARATION OF SMEAR AND HEAT FIXING:

Using a sterilized inoculating loop, transfer loopful of liquid

suspension containing bacteria or fungi to a slide or transfer an
isolated colony from a culture plate to a slide with a water drop

Disperse the bacteria on the loop in the drop of water on the slide
and spread the drop over an area the size of a dime

Allow the smear to dry thoroughly.

Heat-fix the smear cautiously by passing the underside of the slide

through the burner flame

PROCEDURES:

Cover or flood the smear with either methylene blue (2 min) or

crystal violet (1 min) or Safranin (2 min) and allow the dye to
remain in the smear for approximately one minute

Using distilled water wash bottle, gently wash off the excess stain
from the slide by directing a gentle stream of water over the
surface of the slide

Wash off any stain that got on the bottom of the slide as well

Saturate the smear again but this time with Iodine. Iodine will set
the stain

Wash of any excess iodine with gently running tap water. Rinse
thoroughly.

Wipe the back of the slide and blot the stained surface with
bibulous paper or with a paper towel.

Place the stained smear on the microscope stage smear side up and
focus the smear using the 10X objective.

Choose an area of the smear in which the cells are well spread in a
monolayer. Center the area to be studied, apply immersion oil
directly to the smear, and focus the smear under oil with the 100X
objective.

Simple Staining Procedure

Simple Staining
The simple stain is a very simple staining procedure involving single
solution of stain.
Any basic dye such as methylene blue, safranin, or crystal violet can
be used to color bacterial cells.
Preparation of a smear and heat fixing
Using a sterilized inoculating loop, transfer loopful of liquid
suspension containing bacteria to a slide (clean grease free
microscopic slide) or transfer an isolated colony from a culture plate
to a slide with a water drop.
Disperse the bacteria on the loop in the drop of water on the slide and
spread the drop over an area the size of a dime. It should be a thin,
even smear.
Allow the smear to dry thoroughly.
Heat-fix the smear cautiously by passing the underside of the slide
through the burner flame two or three times. It fixes the cell in the
slide. Do not overheat the slide as it will distort the bacterial cells.
Staining
Cover the smear with methylene blue and allow the dye to remain in
the smear for approximately one minute (Staining time is not critical
here; somewhere between 30 seconds to 2 minutes should give you an
acceptable stain, the longer you leave the dye in it, the darker will be
the stain).
Using distilled water wash bottle, gently wash off the excess
methylene blue from the slide by directing a gentle stream of water
over the surface of the slide.
Wash off any stain that got on the bottom of the slide as well.
Saturate the smear again but this time with Iodine. Iodine will set the
stain.
Wash of any excess iodine with gently running tap water. Rinse
thoroughly.

 Wipe the back of the slide and blot the stained surface with
bibulous paper or with a paper towel.
Place the stained smear on the microscope stage smear side up and
focus the smear using the 10X objective.
Choose an area of the smear in which the cells are well spread in a
monolayer. Center the area to be studied, apply immersion oil
directly to the smear, and focus the smear under oil with the 100X
objective.
Gram Staining

Gram staining is a common technique used to differentiate two

large groups of bacteria based on their different cell wall
constituents.

Gram Staining

Prepare a Slide Smear

Transfer a drop of the suspended culture to be examined on a slide

with an inoculation loop. If the culture is to be taken from a Petri
dish or a slant culture tube, first add a drop or a few loopful of
water on the slide and aseptically transfer a minute amount of a
colony from the Petri dish. Note that only a very small amount of
culture is needed; a visual detection of the culture on an
inoculation loop already indicates that too much is taken.
Spread the culture with an inoculation loop to an even thin film
over a circle of 1.5 cm in diameter, approximately the size of a
dime. Thus, a typical slide can simultaneously accommodate 3 to 4
small smears if more than one culture is to be examined.

Prepare a Slide Smear

Air-dry the culture and fix it or over a gentle flame, while moving
the slide in a circular fashion to avoid localized overheating. The
applied heat helps the cell adhesion on the glass slide to make
possible the subsequent rinsing of the smear with water without a
significant loss of the culture. Heat can also be applied to facilitate
drying the the smear. However, ring patterns can form if heating is
not uniform, e.g. taking the slide in and out of the flame.

Add crystal violet stain over the fixed culture. Let stand for 10 to
60 seconds; for thinly prepared slides, it is usually acceptable to
pour the stain on and off immediately. Pour off the stain and gently
rinse the excess stain with a stream of water from a faucet or a
plastic water bottle. Note that the objective of this step is to wash
off the stain, not the fixed culture.
Flood the fixed culture with Grams Iodine. After 1 min, wash it
with water.
Apply alcohol three to five times on the slide. After 5 seconds,
wash it with water.
Counterstain with basic fuchsin solution for one minute. Wash off
the solution with water. Blot with fliter paper to remove the excess
water. Alternatively, the slide may shaken to remove most of the
water and air-dried.

Acid-Fast Stain

Is used to identify acid-fast organisms that are characterized by

nearly impermeable cell walls.
They contain mycolic acid and large amounts of fatty acids, waxes,
and complex lipids.

Preparation

Prepare bacterial smear on clean and grease free slide, using sterile
technique.
Allow smear to air dry and then heat fix.
Cover the smear with carbol fuchsin stain.
Heat the stain until vapour just begins to rise (i.e. about 60 C). Do
not overheat. Allow the heated stain to remain on the slide for
5 minutes.
Wash off the stain with clean water.
Cover the smear with 3% v/v acid alcohol for 5 minutes or until the
smear is sufciently decolorized, i.e. pale pink.
Wash well with clean water.

Cover the smear with malachite green stain for 12 minutes, using
the longer time when the smear is thin.
Wash off the stain with clean water.
Wipe the back of the slide clean, and place it in a draining rack for
the smear to air-dry (do not blot dry).
Examine the smear microscopically, using the 100 X oil immersion
objective.

Cell Membrane

Proteins

Chemical Composition and Function

Cell Membrane

The cell membrane (also known as the plasma membrane or

cytoplasmic membrane) is a biological membrane that separates
the interior of all cells from the outside environment.
The cell membrane is selectively permeable to ions and organic
molecules and controls the movement of substances in and out of
cells.

GRAM STAINING
Gram Staining

Chemical Composition and Function

The plasma membrane is composed of a bilayer . This bilayer

behaves very much like a fluid, that means the lipids in this bilayer
are in constant lateral motions. The phospholipids can laterally
exchange their places, however they can not diffuse transversely.
Glycolipid and phospholipid
Act as cell identity marker

Cholesterol

It is one of the most commonly used differential staining technique

Introduced by Hans Christian Gram
This staining technique helps in the classification and
differentiation of microorganisms
This test differentiates the bacteria into Gram Positive and Gram
Negative bacteria

Importance of Gram Stain reaction of Pathogens

Lipid

The other important components of the plasma membrane are

proteins. Proteins can be associated with the outside (peripheral)
of the membrane or they extend throughout the entire membrane
(transmembrane). Many of the plasma proteins are not fixed into a
certain position, but can freely move within the membrane.
Proteins in the plasma membrane serve as transporter molecules.
They also function as recognition sites for substances such as
hormones, transmitting chemical signal to the cell.
Some proteins on the periphery of the plasma membrane function
as enzymes, catalyzing chemical reactions

Cholesterol partially immobilizes the fatty acid tails making the

membrane less flexible and thus less permeable to smaller
molecules.

It can help determine what kind of pathogen is present

Enables the doctors to determine whether the pathogen is causing
an infection or not
It can help determine the appropriate treatment for an infection

Peptidoglycan

Consists of polysaccharide chain linked by small peptide or protein

chains
Only found in bacteria
Serves as the protective layer of most bacteria

Common Sources of Errors in Performing Gram Staining

Excessive washing between steps

Excessive heat during fixation
Old cultures of specimen used
Insufficient Iodine exposure
High or Low concentration of reagent used

ACID FAST STAIN (ZIEHL-NEELSEN)

differential stain
used to identify acid-fast organism such as genus Myobacterium
-Acid fast organisms are characterized by wax like, nearly
impermeable cell walls

Reagents:

Carbol Fuchsin- primary dye

Steam
Acid Alcohol- decolorizing agent
Methylene blue- counterstain

Procedure:

Prepare bacterial smear on clean and grease free slide.

Allow smear to air dry and then heat fix.
Cover the smear with carbol fuchsin stain.
Heat the stain until vapour just begins to rise (i.e. about 60 oC). Do
not overheat. Allow the heated stain to remain on the slide for
5 minutes.
Wash off the stain with clean water for 30 seconds.
Decolorize by adding acid-alcohol drop by drop until the slide
remains only slightly pink. This requires 10 to 30 seconds and
must be done carefully.
Rinse with clean water for 5 seconds.
Cover the smear with methylene blue stain for 12 minutes, using
the longer time when the smear is thin.

Blot dry with bibulous paper.

SOURCES OF ERROR:

Excessive heating during fixation

Excessive washing

INTERPRETATION
Acid fast: Bright red to intensive purple (B), Red, straight or slightly
curved rods, occurring singly or in small groups, may appear beaded
Non-acid fast: Blue color (A)
EXAMPLE
Acidfast: Mycobacterium tuberculosis, Mycobacterium smegmatis.
Non-Mycobacterial bacteria: Nocardia
Coccidian Parasites: Cryptosporidium

ACID FAST STAIN

ZIEHL-NEELSEN
o Hot Stain
o Methylene blue
KINYOUN
o Cold Stain
o Malachite green
PREPARAITON OF SPUTUM SPECIMEN

To prepare your patient, have him drink plenty of fluids on the

evening before the test, provided that he's not on a fluid restriction.
Obtain the sample first thing in the morning.
Instruct the patient to take at least three deep breaths, then force
out a deep cough.

Set up your equipment: a sterile specimen cup with a tight-fitting

cap, the appropriate label, and aerosol of 10% sodium chloride or
sterile water on hand.
Ask to remove his dentures.
Have the patient rinse his mouth with plain water but don't allow
him to brush his teeth or use mouthwash.
Avoid touching the inside to ensure that it is sterile.
Collect the specimen, securely cap the container and send the
sample to the lab immediately.

PROCEDURE (Grays Method)

Leave the preparation for about 5-10 minutes.

Wash the preparation with water.
Air dry the preparation. DO NOT HEAT-FIX!
Observe the preparation under OIO.

spirillum volutans
ENDOSPORE STAINING
ENDOSPORES are special resistant, dormant structure formed within a
cell that protects a bacterium from adverse environmental conditions.

SPECIAL STAINING

is used to color and isolate specific parts of microorganisms, such

as spores and flagella, and to reveal the presence of capsules.

FLAGELLA STAINING

FLAGELLA are structures of locomotion too small to be seen

with a light microscope without staining.

4 DIFFERENT FLAGELLAR ARRANGEMENTS

Monotrichous*
Lophotrichous*
4 DIFFERENT FLAGELLAR ARRANGEMENTS
Amphitrichous*
Peritrichous*
PROCEDURE (Grays Method)

Flood the smear with the mordant, tannic acid.

Incubate the preparation at room temperature for about 20
minutes.
Wash the preparation with water.
Apply a few drops of carbol fuchsin into the preparation.

ENDOSPORES cannot be stained by ordinary methods, such as simple

staining and Gram staining, because the dyes do not penetrate the wall of
the endospore.
PROCEDURE (Schaeffer-Fultons Method)

Heat-fix the smear.

Flood the slide with the primary stain, malachite green.*
Steam the preparation for about 5 minutes.*
Wash the preparation with water for about 30 seconds.*

PROCEDURE (Schaeffer-Fultons Method)

Counterstain the smear with safranin.*

Wash the preparation with water.
Dry the preparation then observe under OIO.

BACILLUS CEREUS
CAPSULE STAINING
CAPSULES are gelatinous coverings of microorganisms that are mostly
made up of polysaccharides.
CAPSULES are considered virulence factors since they contribute to the
pathogenicity of an organism.

CAPSULES are difficult to stain since capsular materials are soluble in

water and may be dislodged or removed during rigorous washing.
PROCEDURE (Negative Staining Method)*

Place a small of drop of the negative stain, India ink or nigrosin

on a slide.*
Smear the bacterial culture into the dye.
Air dry the preparation. DO NOT HEAT-FIX!*
Flood the smear with crystal violet or safranin for about 1
minute.*

PROCEDURE (Negative Staining Method)

Drain the crystal violet or safranin by tilting the slide at a 45

degree angle.
Let the stain run off until it air dries.
Observe the preparation under OIO.

klebsiella pneumoniae