Differential

staining
⦿The Gram stain, developed in 1884 by the
Danish physician Christian Gram, is the most
widely employed staining method in
bacteriology.
⦿It is an example of differential staining—
procedures that are used to distinguish
organisms based on their staining properties.
⦿Use of the Gram stain divides Bacteria into two
classes— gram negative and gram positive.
⦿The Gram stain is a differential stain
commonly used in the microbiology
laboratory that differentiates bacteria on
the basis of their cell wall structure. Most
bacteria can be divided into two groups
based on the composition of their cell wall:
⦿1) Gram-positive cell walls have a thick
peptidoglycan layer beyond the plasma
membrane.
⦿Characteristic polymers called teichoic and
lipoteichoic acids stick out above the
peptidoglycan and it is because of their
negative charge that the cell wall is overall
negative. These acids are also very important in
the body’s ability to recognize foreign bacteria.
⦿Gram-positive cell walls stain blue/purple with
the Gram stain.
⦿Gram-negative cell walls are more complex. They have a
thin peptidoglycan layer and an outer membrane beyond
the plasma membrane. The space between the plasma
membrane and the outer membrane is called the
periplasmic space.
⦿The outer leaflet of the outer membrane is composed
largely of a molecule called lipopolysaccharide (LPS).
LPS is an endotoxin that is important in triggering the
body’s immune response and contributing to the overall
negative charge of the cell.
⦿Spanning the outer membrane are porin proteins that
enable the passage of small molecules.
⦿Lipoproteins join the outer membrane and the thin
peptidoglycan layer.
⦿Gram-negative cells will stain pink with the Gram stain.
⦿1. A heat -fixed smear is covered with a basic
purple dye, usually crystal violet. Because the
purple stain imparts its color to all cells, it is
referred to as a primary stain.
⦿2. After a short time, the purple dye is washed
off, and the smear is covered with iodine, a
mordant. When the iodine is washed off, both
gram- positive and gram-negative bacteria
appear dark violet or purple.
⦿3. Next, the slide is washed with alcohol or
an alcohol-acetone solution. This solution is
a decoloruing agent. Which removes the
purple from the cells of some species but not
from others.
⦿4. The alcohol is rinsed off, and the slide is
then stained with safranin, a basic red dye.
The smear is washed again, blotted dry, and
examined microscopically.
 Preparing Specimens for Viewing – Differential Stains

Gram Stain
Distinguishes between two large groups of microorganisms:
- purple staining, Gram-positive cells
- pink staining, Gram-negative cells

These two types of cells differ significantly in the chemical and physical structure of their cell wall.

Mordant is a
chemical used to fix
the staining reaction

The structure of the thinner cell walls of Gram negative bacteria cannot hold the dyes previously used, once the
decolorizer is applied.

Crystal violet (1 min) : rinse : Iodine (1 min) : rinse : Acetone Alcohol (10–15 sec) : rinse : Safrinin (1 min)
⦿PROCEDURE:
⦿1. Using a sterile inoculating loop, add 1 drop of sterile water to
the slide. Prepare a mixed smear of Escherichia coli (G- rod) and
Staphylococcus epidermidis (G+ coccus).
⦿2. Air dry and Heat fix.
⦿3. Cover the smear with Crystal Violet (primary stain) for 1 min.
⦿4. Gently wash off the slide with water.
⦿5. Add Gram’s Iodine (mordant) for 1 min.
⦿6. Wash with water.
⦿7. Decolorize with 95% ethanol. (time will vary depending on
thickness of smear). Immediately wash with water.
⦿8. Cover the smear with Safranin for 30 seconds.
⦿9. Wash both the top & the bottom of the slide with water.
⦿10. Blot the slide with bibulous paper.
⦿11. Using the 10X objective lens, focus first on the line and then on
the smear. Then using the 100X (oil immersion lens).
 Acid-Fast Stain
⦿ Another important differential stain (one that differentiates
bacteria into distinctive groups) is the acid-fast stain, which
binds strongly only to bacteria that have a waxy material in their
cell walls.
⦿ Microbiologists use this stain to identify all bacteria in the genus
Mycobacterium, including the two important pathogens
Mycobacterium tubercuIlosis, the causative agent of tuberculosis,
and Mycobacterium leprae (the causative agent of leprosy.
⦿ This stain is also used to identify the pathogenic strains of the
genus Nocardia (no-kar'de-a). Bacteria in the genera
Mycobacterium and Nocardia are acid-fast.
⦿Mycobacterium and many Nocardia species are called
acid-fast because during an acid-fast staining
procedure they retain the primary dye carbol
fuchsin despite decolorization with the powerful
solvent acid-alcohol (95% ethanol with 3% HCl).
⦿Nearly all other genera of bacteria are nonacid-fast.
⦿The acidfast genera have the waxy hydroxy-lipid
called mycolic acid in their cell walls. It is assumed
that mycolic acid prevents acid-alcohol from
decolorizing protoplasm.
⦿The acid-fast stain is a differential stain.
⦿ [n the acid-fast staining procedure, the red dye carbolfuchsin is applied
to a fixed smear, and the slide is gently heated for several minutes.
(Heating enhances penetration and retention of the dye.) Then the slide
is cooled and washed with water. The smear is next treated with acid-
alcohol, a decolorizer, which removes the red stain from bacteria that
are not acid -fast.
⦿ The acid-fast microorganisms retain the red color because the
carbolfuchsin is more soluble in the cell wall lipids than in the acid-
alcohol.
⦿ In non- acid-fast bacteria, whose cell walls lack the lipid components,
the carbolfuchsin is rapidly removed during decolorization, leaving the
cells colorless.
⦿ The smear is then stained with a methylene blue counterstain.
⦿ Non-acid-fast cells appear blue after application of the counterstain.
 Preparing Specimens for Viewing – Differential Stains

Acid-Fast Stain
This stains the cells of the genera
Mycobacterium and Nocardia, which cause
many diseases in humans, including
tuberculosis, leprosy, and other lung and skin Create a smear of the organism
infections. that you are testing. Cover the
smear with a strip of blotting
paper.

Cells of these bacteria have large amounts of Saturate paper with Ziehl’s
carbolfuchsin and heat for 3 – 5
waxy lipid in their cells walls, so the Gram minutes. Remove blotting paper.
stain and other water-based stains don’t work
well on them. Wash slide with tap water and then
decolorize the smear for 10 - 15 seconds
with acid alcohol. Rinse again with tap
water.
Cells are determined to be acid-fast or not, based
on whether they retain their primary stain
after decolorization. Apply crystal violet for 1 minute,
wash, blot dry.

Blotting paper : Ziehls carbolfuchsin (3 – 5 min heat) : rinse: Acid Alcohol (10 – 15 sec) : rinse : crystal violet (1 min)
OBSERVATION OF MICROORGANISMS
⦿ PROCEDURE
⦿ 1. Add one loopful of sterile water to a microscope slide.
⦿ 2. Make a heavy smear of Mycobacterium smegmatis. Mix thoroughly with
your loop. Then transfer a small amount of Staphylococcus epidermidis to the
same drop of water. You will now have a mixture of M. smegmatis and S.
epidermidis.
⦿ 3. Air dry and heat fix well.
⦿ 4. Cover the smear with carbolfuchsin dye. Place a piece of paper towel on
top of the dye. Be sure the paper towel is saturated with the dye.
Carbolfuchsin is a potential carcinogen. Please wear gloves when working
with this dye.
⦿ 5. Place the slide on the rack over dry heat for 2 minutes.
⦿ 6. Cool and rinse with water.
⦿ 7. Decolorize by placing a drop of acid alcohol on the slide and allowing it to
sit for 15 seconds.
⦿ 8. Wash the top and bottom of slide with water and clean the slide bottom
well.
⦿ 9. Counterstain with Methylene Blue for 30 seconds to 1 minute.
⦿ 10. Wash and blot the slide with bibulous paper.
⦿ 11. Focus 10X - then use oil immersion.
Acid-fast bacteria. The Mycobacterium leprae bacteria that have infected this tissue
have been stained red with an acid-fast stain. Non- acid-fast cells are stained with the
methylene blue counterstain.
Note: The acid-fast Mycobacterium retains carbolfuchsin and stains hot pink. The
Staphylococcus epidermidis is decolorized and the counterstain colors them blue.
 Endospore staining
⦿Endospore staining, like acid-fast staining, also requires
harsh treatment to drive dye into a target, in this case an
endospore.
⦿An endospore is an exceptionally resistant structure
produced by some bacterial genera (e.g., Bacillus and
Clostridium). It is capable of surviving for long periods in
an unfavorable environment and is called an endospore
because it develops within the parent bacterial cell.
⦿Endospore morphology and location vary with species and
often are valuable in identification; endospores may be
spherical to elliptical and either smaller or larger than the
diameter of the parent bacterium.
⦿Endospores are not stained well by most dyes, but
once stained, they strongly resist decolorization. This
property is the basis of most endospore staining
methods. In the Schaeffer-Fulton procedure,
endospores are first stained by heating bacteria with
malachite green, which is a very strong stain that
can penetrate endospores.
⦿After malachite green treatment, the rest of the cell
is washed free of dye with water and is
counterstained with safranin. This technique yields a
green endospore resting in a pink to red cell.
 Preparing Specimens for Viewing – Differential Stains

Endospore Stain
Some bacteria produce endospores, dormant, highly-resistant cells
inside the cytoplasm of the bacteria, that can survive
environmental extremes (desiccation, heat, harmful chemicals)

Most notable genera: Bacillus and Clostridium

Endospores cannot be stained by normal staining procedures

Endospore stain uses heat to drive the primary stain, malachite

After cooling, the sample is decolorized with water and counter

Results in green stained endospores and red-colored vegetative

Malachite Green (5 min heat) : rinse : safrinin (20 sec)