CLINICAL BACTERIOLOGY

LABORATORY | MODULE 3 STAINING| 1ST SEMESTER

STAINING DYAR METHOD & VICTORIA BLUE DYE (BLUE)

❖ a procedure that applies colored chemicals called dyes to ➢ cell wall
the specimen in order to facilitate identification
❖ Artificially coloring the microorganisms. CAPSULAR STAINS
❖ Dye / Stain may be classified as: ➢ Capsule is a thick gelatinous or slimy layer excreted by some
✓ Basic bacteria outside the cells which is produced by the plasma
✓ Acidic membrane. This substance is also known as slimy layer or
glycocalyx. This substance also aid bacteria to attach
PURPOSE themselves to mucous membrane and tooth surface which
➢ To observe and appreciate the appearance also contribute to its virulence by inhibiting phagocytosis.
➢ To differentiate one organism from another ▪ Capsule → clear halos zone against
➢ It helps in identification of organisms and its special dark bg
structures ▪ No capsule → no clear halos zone
➢ Examples of Capsule:
Bacillus anthracis, Klebsiella pneumoniae,
STAINS Streptococcus pneumonia Neisseria
POSITIVE
❖ Are salts composed of positive and negative ion, one of meningitidis Clostridium spp, Rhizaobium
which is colored (chromophore – color bearing ion), which spp
imparts a color to a cell or cell parts and fixed them through NEGATIVE Neisseria gonorrhoreae
a chemical reaction.
STAINS:
▪ Hiss method
TYPES OF STAINING TECHNIQUES o Capsules appear faint blue halos
A. SIMPLE STAINING around purple colored organisms
❖ One (single) particular dye is used o Capsule is stained brown
❖ For visualization of morphological shape & arrangement ▪ Tyler’s Stain
o Capsule is stained light violet
EXAMPLES / STAINS ▪ Muir’s Stain
➢ Crystal violet → 30-45 secs → violet o Capsule is stained light blue
➢ Carbol Fuchsin → 15-30 secs → red ▪ Gin’s method
➢ Methylene Blue → 3-5 mins → blue o Bacteria will be stained but the capsule is
➢ Safranin → 60 secs → red or pink unstained w/ their margin delineated by the ink
▪ Welch’s Method
If methylene blue is used, some granules in the interior of the cells o Capsule stains pale violet
of some bacteria may appear more deeply stained than the rest of ▪ Wadsworth’s Method
the cell, which is due to presence of different chemical substances. o Bacteria → blue
o Capsule → pinkish

B. INDIRECT, RELIEF OR NEGATIVE STAIN

METACHROMATIC GRANULES
❖ In Negative staining technique, an acidic stain such as
Nigrosin, India ink, Eosin or Congo red is used. ➢ Also known as volutin granules, are composed of
o These stains do not penetrate and stains the polyphosphate which are stained reddish-purple with
bacterial cell due to repulsion between the negative methylene blue.
charge of the stain and the negatively charged ➢ These granules serves as a reserve source of phosphate
bacterial cell wall. that can be used by bacterial as a reserve energy source in
o Hence in Negative staining background is stained case there is decrease nutrient available for microbial survival
instead of bacterial cells. .
❖ Microorganisms appear as colorless (clear transparent STAINS:
bodies) ➢ Loeffler’s alkaline methylene blue stain (LAMB)
o Granules are dark blue
➢ Neisser’s stain
INDIA INK / BARRI’S MTD Cryptococcus neoformans
o Granules are dark blue
Capsule is unstained; bg is ➢ Albert’s stain
NIGROSIN MTD
black o Granules are blue-black ; cytoplasm
Capsule is unstained; bg is appears green
CONGO RED
blue ➢ Lindegran Stain
Capsule is unstained; bg is o Granules are reddish brown
ANTHONY’S STAIN
purple ➢ Burke’s technique
o Modified gram’s technique for ganules
C. SPECIAL STAINS ➢ Ljubinsky method
❖ seek to identify various bacterial structures of importance. ➢ Ponder
❖ It is used to examine bacterial spores, capsules, flagella, and
metachromatic granules. ENDOSPORES / SPORE STAIN
❖ For instance, a special stain technique highlights the flagella ➢ Bacterial spores (endospores) are thick-walled, highly
of bacteria by coating the flagella with dyes or metals to refractile bodies that are produced by Bacillus and
increase their width. Flagella so stained can then be observed. Clostridium.

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 CLINICAL BACTERIOLOGY
LABORATORY | MODULE 3 STAINING| 1ST SEMESTER

STAINS: GRAM STAIN

➢ Heat and Acetic method
❖ To differentiate between Gram + bacteria (large
➢ Dorner’s method → 10% nigrosin + carbol
peptidoglycan layer on the outer surface of the cell) and Gram
fuchsin
- bacteria.
o Spores are red
❖ Principle:
o Others are gray
o based on the differential structure of the cellular
➢ Wirtz and Conklin
membranes and cell walls of the two groups.
o Spores
o Gram-positive organisms contain a highly cross-
are red
linked layer of peptidoglycan
o Vegetative
o The Gram-negative organisms do not contain a
cell is red ; endospore is blue-green
thick cross-linked layer of peptidoglycan
➢ Schaeffer & Fulton
❖ Should use 24hr culture
o Spores are green due to malachite green

FLAGELLA STAIN
➢ Flagella are whip-like protein appendages which make
bacteria move. Not all bacteria have flagella, however, for Note: In
those that have, the number and the arrangement of the between wash
95% with water
flagella are a characteristic of the specie. The flagella of
bacteria arise from a basal body in the cell membrane and
Or blue To red
project outwardly through the cell wall and capsule.

STAINS:
➢ Gray’s stain
➢ Leifson stain → flagella are red
➢ Fisher & Conn method

MECHANISMS:
✓ First of all in this procedure thickness of flagella is increase
so it can be visible.
✓ The Leifson’s stain is made up of tannic acid ,basic fuschin
stain prepared in alcohol base.
✓ When we treat Leifson’s stain with cell the tannic acid get
attach to the flagella and alcohol get evaporated.
✓ After evaporation of alcohol the thickness of flagella is
increased due to deposition of tannic acid.
✓ Where as Basic fuschin stain the Flagella.
✓ After Leifson’s stain treatment cells are treated with
Methylene blue stain.
✓ This Methylene blue stains the cell.

RESULT:
➢ Flagella appear red and bacterial cell appear blue

D. DIFFERENTIAL STAIN
❖ Use of two contrasting stains (more than one chemical stain)
separated by a decolorizing agent
❖ used to detect abnormalities in the proportion of different
white blood cells in the blood.
o The process or results are called a WBC
differential.
❖ 4 solutions usually employed:
✓ Primary Stain
✓ Mordant
✓ Decolorizer
✓ Secondary Stain
❖ For identification
o Gram stain and Acid Fast stain
❖ For visualization of structure Gram + Staphylococcus aureus, Streptococcus
o Spore stain & Capsule stain bacteria pyogenes, Corynebacterium diphteriae
Gram – Neisseria meningitidis, Escherichia coli,
bacteria Pseudomonas aeruginosa

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 CLINICAL BACTERIOLOGY
LABORATORY | MODULE 3 STAINING| 1ST SEMESTER

RULES IN GRAM STAINING: ZIEHL NEELSEN (hot method)

(MCCBELL)

STAIN/REAGENT ACID FAST NON-ACID FAST

Carbon Fuchsin Red red
(primary stain)
Heat
WHY DOES GRAM + BECOMES GRAM - (mordant)
✓ Overdecolorization ; use of acidic gram’s iodine Acid alcohol 3% red Colorless
(decolorizer)
WHY DOES
WHY DOESGRAM
GRAM+- BECOMES
BECOMESGRAM GRAM-+ Methylene Blue red Blue
✓ Underdecolorization ; thick bacterial smear (counter stain)
❖ ACID FAST: Red on blue bg (Bright red to
LIMITATIONS intensive purple. Red, straight or slightly curved
✓ Over-decolorization may result in the identification of false rods, occurring singly or in small grps, may
gram-negative results, whereas under-decolorization may appear beaded)
result in the identification of false gram-positive results. ❖ NON-ACID FAST: Blue; bg is blue
✓ Smears that are too thick or viscous may retain too much ❖ Best method for DSSM
primary stain, making the identification of proper Gram stain
reactions difficult. Gram-negative organisms may not LIMITATIONS
decolorize properly. ✓ The filter paper must remain moist and in contact with the
✓ Cultures older than 16 to 18 hours will contain living and dead specimen during heating to allow for proper penetration of the
cells. Cells that are dead will be deteriorating and will not primary stain.
retain the stain properly. ✓ Organisms cultivated on blood agar may experience nutrient
✓ The stain may form a precipitate with aging. Filtering through deprivation, resulting in a lower lipid content in the outer
gauze will remove excess crystals. membrane resulting in poor staining
✓ Gram stains from patients on antibiotics or antimicrobial
therapy may have altered Gram stain reactivity due to the
successful treatment.
KINYOUN’S (cold method)
✓ Occasionally, pneumococci identified in the lower respiratory
tract on a direct smear will not grow in culture. Some strains STAIN/REAGENT ACID FAST NON-ACID FAST
are obligate anaerobes. Carbon Fuchsin Red red
✓ Toxin-producing organisms such as Clostridia, staphylococci, (primary stain)
and streptococci may destroy white blood cells within a Tergitol, Phenol
purulent specimen. (mordant)
✓ Faintly staining Gram-negative organisms, such as Acid alcohol 3% red Colorless
Campylobacter and Brucella, may be visualized using an (decolorizer)
alternative counterstain (e.g., basic fuchsin). Malachite Green red green
(counter stain)
ACID FAST STAIN ❖ ACID FAST: Red on green bg (Mycobacterium
❖ To distinguish acid fast from non-acid fast organisms spp., will appear pink.
❖ Acid Fast Organism ❖ NON-ACID FAST: Green or blue-green; bg is
o Organisms that are very hard to stain but once blue
stained they are difficult to decolorize due to the ❖ Best stain for AFO in tissues
presence of unsaponifiable wax called mycolic
LIMITATIONS
acid that envelopes the bacteria.
✓ May be less sensitive than the Ziehl-Neelsen method.
✓ Smears that are too thick may not properlystain.

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 CLINICAL BACTERIOLOGY
LABORATORY | MODULE 3 STAINING| 1ST SEMESTER

ADDITIONAL NOTES

SPECIAL STAINS FOR BACTERIA

❖ Brown and Brenn Technique
o Modified gram’s
o Stain for bacteria in tissue section
❖ Warthin-Starry Silver Stain
o Special Stain for Bartonella, Helicobacter, Borrelia
and Treponema
❖ Hucker’s Modification of Gram’s Stain
o For fungi and are all gram +

STAIN FOR BIPOLAR BODIES

➢ Wayson stain → Bipolar Bodies are red

STAINS FOR RICKETTSIA & CHLAMYDIA

AURAMINE-RHODAMINE (fluorochrome)
For Rickettsia:
❖ Can use LPO ➢ Castaneda → blue
❖ Alternative for AFS which is more sensitive but less specific
➢ Machiavello → red
STAIN/REAGENT ➢ Giemsa → blue
Auramine- Primary
Rhodamine Stain For Chlamydia:
None Mordant ➢ Gimenez → inclusion bodies are red
➢ Machiavello → red
0.5% acid alcohol Decolorizer
➢ Giemsa → purple
Quenching /
0.5% KMnO4
Counterstain
STAINS FOR SPIROCHETES
Result Yellow on back
bg ➢ Fuelgen’s → Nucleic Acid
❖ Following staining, microscopy is done ➢ Levaditi Silver Impregnation, Warthin Starry →
Spirochetes are black
➢ Fontana Tribondeau → spirochetes are dark brown or
black
➢ India Ink negative staining → spirochetes are unstained;
black bg

STAINS FOR ACID-FAST BACILLI

❖ Pappenheim’s Method – differentiates M. tuberculosis
(red/positive) from M. smegmatis (blue/negative)
STAIN FOR MYCOPLASMA
❖ Baumgarten’s Method – differentiates M. tuberculosis
(blue/negative) from M. leprae (red/positive) ➢ Dienes stain → blue
❖ Wadefite technique / Fite Faraco technique – staining
DNA – PROBE MEDIATED STAINING
intended for M. leprae in tissue section; uses hematoxylin
as secondary / counterstain ➢ It specifically identifies selected pathogens
❖ Ziehl Neelsen (Hot method) – AFB are red o Chlamydia trachomatis
❖ Kinyoun (cold method) – AFB are red o Bordetella pertussis
❖ Truant’s Fluorescence technique o Legionella pneumophilia
❖ Flickerson and Kammer’s Fluorescence technique o HSV and VZV
o Cytomegalovirus
o Adenovirus and respiratory viruses
OTHER STAINING TECHNIQUE
FLUORESCENT STAINING
➢ It utilizes special dyes (fluorophores) to visualize bacterial
cells through a fluorescent microscope
➢ TYPES:
o Fluorochrome staining
▪ Used a single fluorescent dye
(fluorophore) and is directed towards
staining the bacterial wall
a non specific dye which binds to the
Acridine
nucleic acid component of every host
Orange
cell and microorganism

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 CLINICAL BACTERIOLOGY
LABORATORY | MODULE 3 STAINING| 1ST SEMESTER

stains the cell wall of the mycobacteria

Auramine-
across species specially the mycolic
Rhodamine
acid
o Immunofluorescence
▪ It involves the use of a fluorescent dye
and a specific antibody reagent to
identify microorganisms

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