PARASITOLOGY (LAB)
SAINT PAUL
1A
Staining techniques
MRS. MALLARE |
STAINING TECHNIQUES
 Stains and staining techniques are necessary tools
for the microbiology. Like the protoplasm of all
cells, the cellular constituents of bacteria, yeasts,
moulds and protozoa are colorless to gray in color.
Because of the minute character of these micro-
organisms, they are difficult to see unless given an
artificial color. Stains are used for this purpose.
Dyes will also reveal certain structures, show
specific chemical affinities and influence the
growth and the development of micro-organisms.
 The dyes used in staining micro-organisms are
solutions of aniline compounds. The group most
commonly used are the basic dyes, because of
their affinity for nuclear material. It is generally
believed that bacteria have their nuclear material
scattered throughout the cell instead of being
concentrated in one mass, as is true of other cells.
Koch, Weigert and Erlich worked on this principle
and found that basic dyes stain bacterial cells
uniformly.
 The theories behind the staining process are
controversial. Some authorities believed that the
phenomenon is a chemical reaction in which the
protoplasm of the cells combine with the dye to
form a salt. Others link that the process of staining
is a physical one that the retention of color is due
to such physical forces as capillarity, osmosis,
adsorption and absorption. The general attitude
now assumed is that both theories play a part in
the staining reaction.
 The staining power of any solution may be
strengthened by heating, leaving the stain on
longer, by addition of alkalis, acids or phenol.
Mordants are also used to bind the stain.
TYPES OF STAINING PROCESS
1. Simple staining – uses methylene blue, eosin,
safranin, basic fuchsin, or crystal violet.
2. Differential staining
a. Gram stain – uses crystal violet, Gram’s
iodine, 95% alcohol and safranin; divides
bacteria into gram-positive (those which retain
the crystal violet) and gram-negative (those
which are decolorized by 95% alcohol and
take the counterstain).
 On the microscopic examination of a Gram-
stained smear containing mixed flora, the
differential features of the method will become
apparent; many bacteria will have retained crystal
violet-iodine complex and stained purple; other
will be stained red or pink. The Gram staining will
also show form, size and structural details of a
certain bacterial sample.
 The differential uptake is due to the difference in
concentration of protein and lipid in the bacterial
cell wall. Although evidence is quite incomplete
that it is now noticeable that a crystal violet-iodine
complex is formed within the Gram positive and
Gram negative cells. The Gram positive bacterial
cell, this crystal violet-iodine complex is trapped
within the cell because of the decrease in the cell
wall permeability caused by alcohol dehydration
of protein, while Gram negative bacterial cell, the
complex passes out because of the increase cell
wall permeability resulting from dissolution of
lipids in alcohol.
Following is a list of the more important bacteria and
their reaction to the Gram stain.
GRAM-POSITIVE GRAM-NEGATIVE
Streptococci Neisseria intracellularis
ILO
Staphylococci Neisseria gonorrhea
Diplococcus pneumoniae Neisseria catarrhalis
Corynebacterium diptheriae Escherichia coli
Mycobacterium tuberculosis (AFB) Klebsiella
pneumoniae
Mycobacterium leprae(AFB) Salmonella paratyphi
Bacillus anthracis Shigella dysenteriae
Clostridium tetani Salmonella typhosa
Clostridium welchii Hemophilus influenzae
Clostridium botulinum Hemophilus pertussis
Pasteurella pestis
Pasteurella tularensis
Brucella abortus
Vibrio comma
3. Acid-fast stain – uses basic fuchsin, acid alcohol and
methylene blue; divides bacteria into acid-fast and
nonacid-fast organisms. Acid-fast organisms resist
decolorization with acid alcohol, thereby retaining the
red color. The nonacid-fast organisms are decelorized
and take the counterstain. Acid-fast stain is used
chiefly to distinguish the Mycobacterium group.
4. Special stains:
a. Spore stain (Moeller’s method) – use carbolfuchsin
(steamed on smear) decolorizing agent and methylene
blue. Spores stain red and vegetative cells blue.
 PARASITOLOGY (LAB)
SAINT PAUL
1A
ILO
Staining techniques
MRS. MALLARE |

b. Capsule stain (Hiss method) – use crystal violet
(steamed) and cooper sulfate. Capsules appear as pale
blue halos around darkly stained cell bodies.
c. Flagella stain (Leifon’s) – use tannic acid, sodium
chloride, p-rosaniline acetate, alcohol and borax
methylene blue. Stains flagella black and vegetative
cells blue.
d. Metachromatic granules – use Löffler’s alkaline
methyne blue. Shows polar metachromatic granules. It
is used chiefly for the identification of C. diphtheriae.
e. Spirochete stain (Fontana-Tribondeau) – use
formalin tannic acid and silver nitrate. Spirochetes
stain black against maroon field.
I. Learning outcomes
At the end of this activity, the students will be able to:
1. prepare bacterial smears using stain(s).
2. apply different methods of staining
(simple/differential)
3. identify bacterial morphology, arrangement and
staining affinity
4. classify bacteria according to reaction with
differential stain
II. Materials
a. glass slides g. Stains i. gloves
b. inoculating loops malachite green j. forceps
c. alcohol lamp safranin k. basin
d. dropper methylene blue
e. Gram iodine solution crystal violet
f. isopropyl alcohol 95% h. bacterial culture (B.
subtilis, E. coli, K. pneumonia)
III. Procedures
Note: You may be used the cultured bacteria from
Activity 8 or may be provided by your instructor
A. Preparation of bacterial smear
a. Secure a clean glass slide. Using the masking tape
label one of the far ends of the slide with your group
number, date and staining method to be used.
b. Place a drop of NSS at the center of the slide (for
solid bacterial colony sample)
c. Using a sterilized inoculating loop, get a loopful of
bacteria from the culture.
d. Mix and emulsify to make a thin smear.
e. Sterilize the loop using the flame of an alcohol lamp
f. Air dry the smear (do not blow or dry it using a fan).
g. Fix the smear by passing the smear over the flame
(should be done in a quick manner to prevent charring
of the bacterial cells).
h. Prepare samples in triplicate (to be used in three
staining procedures).
B. Simple staining
a. Using the prepared bacterial smear, perform simple
staining by flooding the smear with methylene blue.
b. After one minute, gently wash off the smear with
water.
c. Blot dry
gently using a soft tissue paper.
d. Examine the stained smear under LPO, HPO, and
OIO.
e. Under OIO, draw the observed bacteria.
C. Gram’s staining
a. Using the prepared bacterial smear, perform
Gram staining by flooding the smear with
crystal violet.
b. After one minute, gently wash off the excess
stain with water.
c. Flood the smear again with iodine solution and
let it stand for 1 minute.
d. Gently wash the iodine with water.
e. Decolorize the smear by washing it with
alcohol, a drop at a time while holding the
slide at an angle. Drain.
f. Watch carefully. Stop applying alcohol when
no more color/stain is seen in the rinse.
g. Rinse with water immediately and blot dry.
h. Counter stain with safranin for 30 seconds.
i. Rinse the counter stain with water. Blot dry.
j. Examine the stained smear under LPO, HPO,
and OIO.
k. Under OIO, draw the observed bacteria.
D. Endospore staining
a. Obtain a smear of Bacillus subtilis from your
instructor and add malachite green stain.
b. Cover the smear and steam it over flame for five
minutes.
c. Do not boil. Make sure that the stain should not dry
out. Add more stain if needed.
 PARASITOLOGY (LAB)
SAINT PAUL
1A
ILO
Staining techniques
MRS. MALLARE |

d. Add safranin (counter stain) and leave for one

minute.

e. Drain excess stain and rinse with water.

f. Blot dry with tissue paper.

g. Examine the stained smear under LPO, HPO, and

OIO.

h. Under OIO, draw the observed bacteria. (endospore

appear green while the other parts of the cell are red)