STAINING

METHODS TO ASSESS MORPHOLOGY

o Assessed in the living state (unstained- organism appears colorless)

o Wet smear – inoculum or specimen is mixed with normal saline solution (NSS)
o Hanging Drop Preparation – uses concave slide; can be used to demonstrate the motility of the
organism
o Assessed in fixed state (stained)
STEPS
o Bacterial Smear Preparation
▪ For staining microorganisms, directly from the specimen, smear can be prepared through
imprint (touch prep), or by rolling swab in slide, or by placing a drop or pellet or sediment
from the fluid
▪ For staining microorganisms grown in a solid culture medium, a sterile needle may be used to
transfer a small amount of growth from the medium to the slide, then emulsified using a drop
of sterile water or saline
▪ For staining microorganisms grown in liquid culture medium, an aspirated sample of the
broth culture is applied to the slide, dried and fixed before staining
o Fixation
▪ Using heat; or 70 – 95% alcohol; 95% methanol for 1 minute; fixation kills the organism and
forces it to stick to the slide; following fixation, slide must be dried before applying the stains
o Staining
▪ Application of dyes or stain to better demonstrate the organism

Staining Techniques

a. Simple Staining
• Application of single staining solution
• Basic dyes have greater affinity for nuclei; acid dyes have greater affinity for cytoplasm
• Example: methylene blue, iodine
b. Differential Staining
• Application of 2 or more stains; aids in differentiation of one group to another
• Makes use of a primary stain, a mordant, decolorizer and counterstain
• Mordant increases affinity or binding of dye
• Examples:
i. Gram Staining to differentiate gram positive from gram negative
ii. Acid Fast Staining to differentiate acid-fast from non-acid-fast organisms

GRAM STAINING

o Developed by Danish bacteriologist Hans Christian Gram in 1884

o Stains the teichoic acid present in cell walls of Gram-positive bacteria
o Not applicable to organisms that exists almost exclusively within host cells (e.g. Chlamydia), those that lack
cell wall (e.g, Mycoplasma and Ureaplasma), and those of insufficient dimension to be resolved by light
microscopy (e.g. spirochetes)
o When is Gram staining done?
o direct from the specimen
o direct from the culture
 Steps:

V Violet ▪ Crystal violet/ Gentian violet; primary stain

I Iodine ▪ Gram’s iodine; mordant; connects the stain to the tissue
A Acetone ▪ Decolorizer
Alcohol ▪ Mixture of equal parts of 95% ethyl alcohol in acetone
▪ The most critical step in the procedure
S Safranin ▪ Secondary stain or counter stain
▪ 1% aqueous safranin or basic fuchsin

Steps Gram Positive Gram Negative

Crystal violet – 1 min (10-30 seconds) Purple – blue Purple – blue
Gram’s Iodine – 1 min (20-60 seconds) Purple – blue Purple – blue
Alcohol (10 seconds) Purple – blue Colorless
Safranin – 10 seconds (30 seconds) Purple – blue Pink - red

GRAM POSITIVE APPEARS: PURPLE BLUE

GRAM NEGATIVE APPEARS: PINK RED

o Gram variable is the description used if some bacterial cells stain pink and other cells stain purple
o Gram's iodine should always be alkaline since acid pH causes gram positive bacteria to become gram
negative. It should be stored in amber bottles to prevent deterioration
o Gram positive bacteria may become gram negative as a result of loss of cell wall integrity caused by antibiotic
treatment, autolysis, aging, media or temperature of incubation
o During the examination of a Gram-stained film, one must note the presence of other cells, such as the
inflammatory cells like phagocytes, which might indicate an infectious process; or the presence of squamous
epithelial cells in respiratory specimen to identify possible contamination with organisms and cells from the
mouth
o Stained smear must be viewed using oil immersion objective

MODIFICATIONS OF GRAM STAINING:

• Gram Stain for Metachromatic Granules – methyl violet-sodium bicarbonate as primary stain, Gram's
iodine as mordant, acetone as decolorizer, safranin O (or safranin Y) as counterstain
• Other modifications
HUCKER’S MODIFICATION BURKE’S MODIFICATION
Primary stain Crystal violet Crystal violet
Mordant Gram’s iodine Gram’s iodine
Decolorizer Acetone-alcohol Sodium bicarbonate + Ether
Acetone
Counterstain Safranin Safranin

ACID-FAST STAINING (AFB Stain)

o Stains the mycolic acid in the cell wall of bacteria

o Organisms with mycolic acid in cell wall are difficult to Gram-stain
o Acid fast organism: Mycobacterium, Nocardia, Cryptosporidium, Isospora, Cyclospora
o Mycobacterium - appears as Gram ghost after gram staining
o Nocardia - appears as partially acid fast
o Steps:
o Primary stain = Carbol Fuchsin (basic fuchsin + phenol)
▪ Phenol acts as an accentuator
 o Mordant = heating/steaming or Water with detergent to speed up staining
o Decolorizer = 0.5 or 1.0% Acid Alcohol (3% HCI in 95% ethanol) *
o Counterstain = methylene blue or malachite green

Steps Acid-Fast Non-Acid-Fast

Primary stain (Carbol Fuchsin) – 5 min Red Red
Acid Alcohol – 1 min Red Colorless
Counterstain (methylene blue) – 1 min Red Blue

ACID FAST ORGANISMS = RED; NON-ACID-FAST ORGANISMS = BLUE

MODIFICATIONS:

▪ Ziehl-Neelsen Stain - hot method; uses steaming as mordant

▪ Kinyoun Stain - cold method; for acid-fast organisms in tissue; no heat application necessary due to higher
concentration of phenol on the carbol fuchsin: tergitol (a detergent, union carbide) may also be used
▪ Modified Kinyoun - uses HNO3-alcohol or H2SO4-alcohol as decolorizer instead of HCl alcohol combination
▪ Pappenheim's method
▪ used to differentiate Mycobacterium smegmatis (neg) from M. tuberculosis (pos)
▪ Decolorizer: rosolic acid and absolute alcohol
▪ Baumgarten's method
▪ used to differentiate Mycobacterium leprae (red) from M. tuberculosis (blue)
▪ reagent: dilute alcoholic fuchsin and nitric acid-alcohol

Special Stains

▪ Stains that are used for the demonstration of a specific part of the cell or specific microorganisms
▪ Example:
o CAPSULE = Hiss stain, Tyler capsule staining, Welch's capsule staining, Grin's stain, Anthony's stain
o FLAGELA = Leifson stain, Gray's method, Caesares Gil method, Loeffler's method, Van Ermenger
method, Fisher & Cohn method
o METACHROMATIC GRANULES = Albert, Neisser, Ponder, Ljubensky method, and Loefflers’ Alkaline
Methylene Blue (LAMB)
o SPORES = Schaeffer-Fulton; Heat and acetic acid method; Dorner's spore stain; Wirtz method
o GLYCOGEN VACUOLES = lodine or Periodic Acid Schiff (PAS)
o MITOCHONDRIA = Janus green B
o FUNGI = Lactophenol cotton blue
▪ cotton blue stains chitin
▪ phenol kills other microorganisms
▪ lactic acid preserves fungal structure
o SPIROCHETES = Fontana-Tribondeau
▪ Contains ammonical silver nitrate as stain, tannic acid as mordant and glacial acetic acid as
fixative
o Fluorescent Stains
▪ Use of fluorochrome dyes with the aid of fluorescent microscope
▪ Example:
• Calcofluor White
o Stains the chitin found in cell wall
o Positive result is bright apple green or blue-white fluorescence
o Truant's Rhodamine-Auramine stain
▪ Used for acid-fast organisms; examination is done using low power objective
▪ Positive result: fluorescent yellow (or orange) organisms on black background
▪ Potassium permanganate acts as counterstain and quenching agent. It helps prevent non-
specific fluorescence
 ▪ Acid-alcohol serves as decolorizer
o Acridine Orange - bacteria will fluoresce bright orange against a green-fluorescing or dark
background
▪ Antibody-conjugated Stains
o Use of antibody with fluorochrome or enzyme for more specific and targeted staining
o Example:
▪ FITC (fluorescein isothiocyanate) - conjugated stains used in Direct Fluorescence Assay (DFA)
test for Legionella and Bordetella
▪ Negative Staining
o is a technique where the organisms are not stained but the background; uses special stain to color
the background such as the organism may appear colorless
o Example:
▪ Nigrosin method
▪ India ink
• the background appears black or very dark while bacterial capsules, spirochetes, or
the thick gelatinous capsule of Cryptococcus neoformans remain colorless
• can be used to count the lateral uterine branches of the proglottids of the adult
Taenia parasites

GENERAL RULES IN STAINING AND MORPHOLOGIC EVALUATION

- Bacillus and Clostridium are the only spore-formers

- Mycobacterium sp. are difficult to stain with Gram stain. They are gram positive but they most often
described as "gram ghost"
- Legionella and Spirochetes are difficult to Gram stain; silver impregnation techniques are most useful.
- Mycoplasma and Ureaplasma do not have cell walls
- All cocci are Gram positive except
o Veilonella
o Branhamella (Moraxella)
o Neisseria
- All bacilli are Gram negative except: (not a complete list)
o Listeria o Lactobacillus
o Mycobacterium o Erysiphelothrix
o Nocardia o Corynebacterium
o Clostridium o Actinomyces
o Bacillus