HISTOLOGY

LECTURE # 31

INTRODUCTION TO SPECIAL STAINS TECHNIQUES:

MICROOORGANISMS STAINING

Rationale: Special stain techniques are one of the major processes done in the histology laboratory.
These techniques are performed to be evaluated with the diagnostics slides from H&E’s. These stains are
used as an aid and a diagnostic tool for a final diagnosis.

Objective:
Once completed this lecture, the student should be able to:

a) Describe the different types of microorganisms demonstration staining.

b) Learn the various methods of stain demonstration.

c) Learn the classification for gram (+/-), Acid fast bacilli, rod-S shape organisms.

d) Learn the procedures and diagnostic tools for each stain.

MICROORGANISMS
DEFINITION: A living organism too small to be seen with the naked eye; includes bacteria, fungi,
protozoa’s, and microscopic algae, also includes viruses.
CLASSIFICATION OF MICROORGANISMS

It is important to classify living organisms, establish the relationship between one group of organism and
another, and to differentiate between them. A method known as taxonomy is used for the classification of
living forms and provides a method of identifying organisms already classified.
Example: Kingdom
Division or phylum
Class
Order
Family
Genus
Species
SCIENTIFIC NOMENCLATURE

In a world inhabited by millions of living organisms, biologists must be sure they known exactly which
organisms is being discussed; to avoid a misleading name of organisms, a system of scientific
nomenclature was developed by Carolus Linnaeus, where organisms are given two names derived from
Latin or Greek using the genus name and the scientific epithet (species) printed underlined or italicized.
Genus is always capitalized and is always a noun and the species is written in lowercase and is usually an
adjective. This nomenclature is known as binomial nomenclature because it involves two names.

Example: Klebsiella pneumoniae

Klebsiella pneumoniae

Once the organism is mentioned for at least one time, then abbreviation can be use for the rest of article,
presentation, etc.

Example: K. pneumoniae
K. pneumoniae

I. BACTERIA

A. Definition:

1. Tiny, small-celled organisms that are widely distributed in nature. Each bacteria cell is a complete
organism that is able to metabolize, grow and reproduce. The cell wall is composed of peptidoglycan, a
mucopolysaccharide, size range from 0.2 to 10 um.

2. All bacteria share the property of prokaryotic cellular organization. The most widely accepted
taxonomic classification for bacteria is Bergey's Manual of systematic Bacteriology dividing bacteria into
four divisions according to the characteristics of cell walls.

3. Bacteria’s are classified in three groups according to their shapes.

a) Spherical or ovoid - classified as cocci, these cocci can occur in different ways and be termed
according to their arrangement.

1. Diplococci - cocci occurring in pairs.

2. Staphylococci - cocci occurring grape-like clusters. They are gram negative (-), non-spore forming
cocci. Their antibiotic resistance poses major problems. Many stains produce Β-lactamase (penicillinase),
an enzyme that destroys penicillin by opening the lactam ring. Staphylococcus food poisoning symptoms
range from 1 to 8 hours causing, nausea’s, vomiting (often projectile) and diarrhea due to the ingestion of
a performed enterotoxin and not to infection

a) Staphylococci aureus is the agent causative of toxic shock syndrome and the cause
of many life-threatening hospital infections.

3. Streptococci - cocci that occur in chains. Their classification are based on the presence of
carbohydrates (C) in the cell wall. The method of classification used is called the Lancefield
Classification.
b) Rod-shaped - classified as bacilli. Gram Positive(+) and Gram Negative (-) bacteria have cell
envelopes that consist of the cell wall and the cell membrane that acts as a diffusion barrier to large
charged molecules.

1. Gram negative bacilli red color staining. Their cell envelopes contains molecules of lipoprotein,
lipopolysaccharide, and peptidoglycan.

a) Salmonella organisms-food poisoning, it doesn’t appear until 1 to 3 days after the

ingestion of the organism and fever is noted.

b) Pasteurella multocida - coccobacillary gram-negative rod, is part of the normal

mouth flora of dogs and cats. Many animals’ bites become infected with this
microorganism.

c) Donovan Bodies - are intracellular gram (-) negative

coccobacillary organisms taught to be the cause of granuloma
inguinale=> causes a painless, nonhealing ulceration of the
genitals.

2. Gram positive bacilli - blue to blue-violet color staining. They contain large amounts of teichoic
acids, which are important surface antigens for these bacteria’s. The cell wall of gram (+) positive
bacteria acts as a barrier to the extraction of Crystal Violet-Iodine Complex by alcohol; this
property is the basis of the Gram stain.

a) Clostridium botulinum- (Endospore forming) infection of this organism in human is rare.

c) Spiral or corkscrew shaped - are classified as spirochetes.

1) Organisms

a) Treponema pallidum - causative organism of syphilis.

b) Lyme disease - caused by a spirochete.

c) Borrelia burgdorferi

d) Leptospira interrogams

e) Helicobacter pylori - Found in gastric mucosa and believe to be the causative

organism of gastritis.

Other structures used in classification of bacteria include spores, flagella, and capsules.

1. Spores - round or oval structures located single or at one area of the bacilli and termed according
to their location.

a) Terminal spore - spore located at one end of the bacilli.

b) Central spore - spore is located in the middle of the bacilli.

c) Subterminal spore - spore located just before the end of the bacilli.
All these spores are different from their parental cell in physiological functions, enzymatic contents, and
chemical compositions. They are able to withstand various environments than bacterium and are resistant
to being destroyed by heat, drying and disinfectants. They are difficult to stain and once stained they are
difficult to decolorize.

2. Flagella - thin appendages that arise from one or more locations on the surface of a cell and are
used for cellular locomotion. A bacterium may possess one or more flagellum or various
flagella’s
and are termed according to the location of their flagella’s.

a) Monotrichous flagella - only one flagella present.

b) Lophotrichous flagella - two flagella present one at each side of the bacteria.

c) Peritrichous flagella - various flagella’s present, located all around the bacteria.

Flagella’s cannot be seen under light microscopy unless they are thickened by deposition of stain on the
surface.

3. Capsule - an outer, viscous covering composed of polysaccharide or polypeptide. They are not
stained with regular staining but a negative staining will demonstrate the bacterial bodies, which
will not stain and the spaces between bacteria will be filled with some opaque material such as
India ink. The capsules are seen as unstained haloes around the bacteria.

Non-Endospore forming bacteria’s

1. Irregular Nonsporing Gram Positive Organisms

The organisms in this group are often grouped under the general term Corynebacteria. They tend to be
pleomorphic, which often varies with the age of the cells.

The genus Actinomyces consist of anaerobes that are opportunistic members of normal oral microbiota
(found in the mouth and throat) in humans and animals.

a) Actinomyces israelii - is a pathogenic gram positive, branched, funguslike bacterium that can
cause osteomyelitis in the craniofacial region. It also causes actinomycosis, a tissue-destroying
disease affecting the head, neck, or lungs.

2. Mycobacteria

Aerobic, non-motile, rod-shaped organisms that contains large amounts of lipids in the cell walls resisting
decolorization, they appear to be related to the presence of lipids in the walls of organisms. Their name
(myco means fungus), was suggested by their occasional exhibition of filamentous growth. Most of
the pathogenic species are acid-fast bacteria’s.

a) Mycobacterium tuberculosis - Causes tuberculosis

b) Mycobacterium leprae - Causes leprosy. This organisms can persist in a host for many years
3. Nocardioforms

Aerobic organism that reproduces by forming rudimentary filaments, which fragment into short rods. The
structure of their cell wall resembles that of the mycobacteria; therefore, they are often acid-fast.
Nocardia is the best known genus in this group and are common in soil. Nocardia is an aerobic, branched,
gram (+) positive rod shaped bacterium that is only capnophilic.

a) Nocardia asteroides - occasionally cause a chronic, pulmonary infection (Nocardiosis) that

resembles tuberculosis.

Other bacteria’s that do not possess the bacterial attributes are Rickettsias, Chlamydias, and
Mycoplasmas.

1. Rickettsias and Chlamydias

They are similar to viruses, and even smaller than some larger viruses. They resemble bacteria and are
therefore classified as such, containing DNA and RNA.

Rickettsias - rod shaped bacteria or coccobacilli, gram negative and non-motile that divides by binary
fission, and is detected with light microscopic observation, appearing as small pleomorphic coccobacilli.
Ranging from 1 to 2 um, Most rickettsial diseases are transmitted to humans by ways of arthropods
(insects and ticks) vectors. The only exception is Coxiella burnetii which causes Q Fever which can be
transmitted by inhalation of contaminated dust and aerosols or by ingestion of contaminated milk.

Examples of rickettsias subgroups include typhus (mites), and spotted fever (Rocky mountain spotted
fever) which has been found in the saliva of wood ticks. Even that is called the Rocky mountain spotted
fever this disease has been found in large communities in the Eastern and Southeastern regions of the
United States.

Chlamydias - are coccoid bacteria that have a complex growth cycle, which is obligated intracellular.
They are gram-negative and non-motile and, unlike most rickettsias, they do not require insects for
transmission. They are transmitted by interpersonal contact or by airborne respiratory routes. There are
only two species of chlamydias known at the present time.

a) Chlamydia trachomatis - is the causative agent of trachoma, the most common cause of blindness
in humans.

b) Chlamydia psittaci - is the causative agent of psittacosis. Human usually contact the disease from
infected birds kept as pets, infected poultry or during employment in poultry dressing plants.

2. Mycoplasma

They are free living bacteria’s that do not actively penetrate cells and do not form cell walls; their growth
is not inhibited by penicillin (which inhibits synthesis of the cell wall), but sensitive to tetracycline and
sulfonamides. Mycoplasma cells are extremely small, highly pleomorphic organisms. They stain poorly
with Gram stain but well with Giemsa stain.

a) Mycoplasma pneumoniae - the causative agent of primary atypical pneumonia (PAP), commonly
called "walking" pneumonia. Sometimes these symptoms may be confused clinically with
influenza or legionellosis.
Staining Reactions:

In the histology field the histopathology laboratory most likely is involved with surgical or autopsy tissue
specimens. The performance of histochemicals will allow us to identify the organism based on
characteristics they show and the tissue reaction seen in response to the organisms’ presence.

The staining techniques used in identifying bacteria may be divided into three classifications: simple
stains, differential stains, and special stains.

Simple stains are used as screening device to identify the presence of bacteria. Example of simple stains
includes Giemsa, Methylene blue, and crystal violet.

Differential stains are performed in order to divide bacteria into groups depending on their reaction to the
chemicals used in the staining techniques. Gram stains and acid-fast stains are classified under this
category since gram stains divide bacteria’s into gram-positive and gram-negative bacteria’s, while acid-
fast techniques divide bacteria’s into acid-fast and non acid-fast groups.

Special stains are techniques used to demonstrate special bacterial structures such as spores, flagella, and
capsules and are performed normally in the bacteriology laboratory.

I. Techniques for Differential Demonstration

A. Gram Reactions

These techniques besides differentiating the bacteria into gram-positive and gram-negative they are also
useful in the determination of whether an abscess or necrosis is bacterial in origin. Gram-positive fungal
filaments of Nocardia and Actinomyces may also been shown.

Gram stains reacts differently to some kinds of bacteria, this could be due to the difference in their cell
walls affecting the retention or escape of the crystal violet and iodine, called the crystal violet-iodine (CV-
I) complex. Gram positive bacteria have thicker cell wall composed of peptidoglycan whereas gram
negative organisms have thinner cell walls also composed of peptidoglycan in addition they contain a
layer of lipopolysaccharide external to their cell wall. When the crystal violet is applied to the cells of
gram positive and gram negative and then the iodine this will readily enter the cells. The crystal violet
and iodine will form the CV-I complex which is larger than the molecules of the crystal violet that entered
the cells, and because of their size, it cannot be washed out of the intact peptidoglycan layer of the gram
positive cells by alcohol. In the other hand the alcohol wash disrupt the outer polysaccharide layer, and
the CV-I complex is washed out through the thin layer of the peptidoglycan. As a result, gram negative is
rendered colorless until counterstained with safranin, after which the gram-negative organisms will stain
pink.

1. Principles of Gram stains

To explain gram stain is necessary to examine the steps in the technique.

1. Application of the Crystal Violet (Purple dye)

2. Application of the Iodine solution (Mordant)
3. Alcohol wash (Decolorizer)
4. Application of Safranin (Counterstain)
B. Acid-Fast Reactions
1. Acid-fast methods are used to demonstrate the presence of acid-fast bacteria in limited group of
microorganisms in tissue sections. This includes bacteria’s from the genus Mycobacterium, of which
several species is pathogenic to man. These stains are based on the chemical composition of the cell
walls; therefore they are not useful in identifying either the wall-less bacteria or the bacteria with unusual
walls. The cell walls of acid-fast bacilli such as mycobacterium, consist of peptidoglycan and as much as
60% of this are lipids. They are resistant to normal staining procedures when stained with carbol-fuchsin
dye, they cannot be decolorized with a mixture of acid and alcohol due to the composition of the cell wall,
which contains large amount of lipids. They contain a waxy material in their cell walls. The acid-fast
microorganisms retain the red color since carbol-fuchsin is more soluble in the waxes in the cell wall than
in acid-alcohol. The decolorizer will remove the stain from bacteria’s that are not acid-fast.
1. Principles of acid-Fast Stains

a) Steps in the acid-fast techniques;

1. Application of a phenylmethane dye (Carbol-fuchsin)

2. Application of an acid alcohol decolorizer
3. Application of a counterstain
C. Silver Impregnation Reactions
Silver stains are used frequently to demonstrate spirochetes, legionella, and helicobacter organisms; these
organisms are argyrophilic; that is, they will adsorb silver from a silver solution, but need a separate
solution of a reducing agent to reduce the adsorbed silver to the visible metallic state. The most effective
reducing agent is hydroquinone, a phenolic compound that becomes oxidized to a quinone
as the silver is reduced to the metallic state.
1. Principles of Silver Impregnation Stains
a) Steps in the Silver Impregnation

1) Application of a sensitizer.
2) Application of a Silver solution.
3) Application of a reducer.

The procedure involves the impregnation of silver to tissue sections, and since these organisms are
argyrophilic after a silver impregnation then the procedure is followed by a reducer solution known as
Hydroquinone which will reduce the adsorbed silver to the visible metallic state. This technique is used
also for demonstration of Afipia felis the causative agent of Cat-Scratch disease (CSD).
D. Giemsa Reactions
The Giemsa technique is the technique most used for demonstration of rickettsias, differentiated in an
alcoholic rosin solution.
1. Principle of Giemsa technique.
a) Steps in the Giemsa staining.
1. Application of Giemsa working solution.
2. Application of a working buffer solution.
The procedure involves the application of a Giemsa stain solution, followed by a differentiator solution
until rickettsias organisms are visible.

II. FUNGI

A. Definition:

1. Primitive plants of one cell or multiple cells that do not possess root stems, leaves, or
chlorophyll, but have a distinct membrane-nucleus that contains genetic material.

2. Mycology is the study of fungi, and diseases produced by fungi are called mycosis.

3. Fungi are identified according to their appearance and microscopic morphology due to the
large and varied group they belong to.

B. All fungus considered medically important are classified in four groups.

1. Filamentous fungus - also known as molds, have a structured filament called hypha, which with more
growth produces more hyphae, which is then known as mycelium - a collection of hyphae.

a) Hyphae may be divided by partitions called septa.

The mycelia can be classified as vegetative or reproductive, the reproductive mycelium gives rise to
spores which are characteristic for each type of fungus.

1. Chlamydospore formation

2. Blastospore formation

3. Arthrospore formation

Yeast -Unicellular fungus round or oval that reproduces by forming "Buds" that enlarge and develop into
new yeast cells.

Budding - a protuberance formed in the outer surface of the parent cell and the nucleus of the parent cell
divides.

One nucleus migrate to the bud, cell-wall material is laid down between the parent cell and the bud breaks
away from the parent cell.

Ex. Cryptococcus neoformans

Yeast-Like fungus => like yeast they reproduce by budding, but the buds tend to elongate into
filamentous structure called "Pseudohyphae".

Pseudohyphae - link together in chains that somewhat resemble the mycelia of the filamentous fungi.
They do not result in spore formation and no true branching occurs.

Ex. Candida albicans

Dimorphic fungi - Possess two different forms of growth morphology depending on temperature.
Yeast morphology => is observed when is grown in the body or on artificial media at 37oC.

Ex. Blastomyces dermatitides

Filamentous appearance => is observed when is grown in soil or when cultured on artificial media at
25oC.

Ex. Blastomyces dermatitides

C. Pathogenic fungi can be divided in three groups.

Superficial fungi (mycoses) or dermatophytoses (cutaneous mycoses)=> fungi that affects the superficial
keratinized layers of skin, hair and nails.

Ex. Ringworm and athlete's foot

Systemic fungi or deep => fungi that affect deeper tissues or organs.

Ex. Blastomyces dermatitides

Coccidioides immitis
Histoplasma capsulatum

Fungi capable of producing either deep or systemic disease.

Ex. Candida albicans

D. Methods of demonstration of fungus

There are various staining techniques used in the demonstration of fungus. These techniques start with
the oxidation of the tissue using oxidizing agents such as Chromic acid or Periodic acid to change fungal
polysaccharides to aldehyde groups.

Chromic Acid

R-CH-CH-R CrO3 H-C-R

| | H5IO6 ||
OH OH O

1,2 Glycol Group Aldehyde Group

Periodic Acid

1. Principle of Silver fungus stains.

a) Application of an oxidizing agent

1. Application of a Silver solution

2. Application of a toner
3. Application of Sodium thiosulfate (Hypo)
 4. Application of a counterstain

The procedure involves the application of an oxidizing agent that will change the fungal polysaccharides
to an aldehyde group followed by a solution of sodium bisulfite to remove any traces of oxidizing agent.
The next step involves the impregnation of the tissue with an alkaline silver reagent followed by a
solution of Gold chloride to tone the tissue sections and eliminates yellow tones from the section. a
solution of sodium thiosulfate (Hypo) is applied to remove any unreacted silver, and then is
counterstained with a light green solution.

1. Principle of Fungus stains.

a) Application of an oxidizing agent.

b) Application of a Schiff's reagent.
c) Application of Aldehyde Fuchsin solution for the Gridley's fungus demonstration.
d) Application of a counterstain

The procedure involves the application of an oxidizing agent that will change the fungal polysaccharides
to an aldehyde group followed by a solution of Schiff's reagent to stain the polysaccharides cell walls. The
next step involves good washing in tap water to obtain a full development. For the Gridley's fungus
demonstration a solution of Aldehyde fuchsin is applied, and a counterstain of light green solution is used
for background.

III. PROTOZOANS AND MISCELLANEOUS PARASITES

A. Definition

Protozoa are considered single celled animals that appear to be simple structurally, but are complex
functionally. They vary in shape, but they possess a nucleus and cytoplasm surrounded by a cell
membrane.

The protozoa in their majority possess special structures like cilia or flagella for their movement. The
cytoplasm of the trophozoite or vegetative stage, may contain food reserves in the form of glycogen or
chromatoid bodies.

According to the protozoa type, there will be a cyst stage which is more resistant than the trophozoite
stage to unfavorable conditions because of the tough cyst membrane that protects the organism; in some
cases the cyst stage provides a better opportunity for transfer from one host to another like in the case of
parasitic amebas.

Giardia lamblia is a protozoon that can infest the human duodenum and jejunum. Giardia cysts may be
found in formed or liquid stool.

Protozoa are responsible for various diseases such as:

a. Malaria (Plasmodium spp.)

b. Amebiasis (Entamoeba histolytica)
c. Sleeping Sickness (Trypanosoma spp.)
d. Leishmaniasis (Leishmania donovani)⇒ causative agent of Kala-azar, multiplies in
reticulo-endothelial cells, especially in macrophages of the spleen, lymph nodes and bone
marrow.
 e. Toxoplasmosis (Toxoplasma gondii)⇒is transmitted by contact with raw meat or cat feces
infected with oocysts.

B. Demonstration Techniques.

Various methods are used to demonstrate some protozoans like the Giemsa method which is great for
demonstrating malarial parasites, Trypanosoma, and Leishmania, as well as Toxoplasma and
Pneumocystis. Amebas in tissue sections may be demonstrated, by virtue of their glycogen content with
Best's Carmine or Periodic acid Schiff’s - Hematoxylin, nuclei, chromatoid bars and fibrils are colored
blue.

IV. VIRUSES

A. Definition:

A submicroscopic, parasitic, filterable agent ranging in size from 16 µm to 300 µm, consisting of a
nucleic acid core surrounded by a protein coat called capsid that sometimes is enclosed by an envelope
composed of lipids, protein and carbohydrates. Each capsid is composed of protein subunits called
capsomeres The nucleic acid core could either be DNA or RNA, subdividing the viruses in two
groups known as:
a) DNA-containing viruses
b) RNA-containing viruses
In order for the virus to survive free in the environment the protein coat is necessary, once the virus
particle penetrates and infects a host cell, this protein coat is no longer needed for their survival. The
nucleic acid is still needed once the virus particle is inside a host because it stimulates the host cells to
form new virus particles.
Viruses are obligate intracellular parasites since they can reproduce only inside host cells. They cannot
grow on artificial media. Host cells supply enzyme and other materials for the infecting virus to
synthesize new viruses. They affect any cells including plants, animals or bacterial cells. A virion is a
complete, fully developed viral particle composed of nucleic acid surrounded by a coat.
There is a spectrum of host cells in which a virus can multiply, depending on the host range, a virus is
generally classified as an animal virus, bacterial virus (bacteriophage), or a plant virus.
B. General Morphology of Virus
1. Helical - resemble long rods, and their capsids are hollow cylinders surrounding the nucleic acid.

Example: Tobacco Mosaic virus

2. Polyhedral - they are many sided.

3. Enveloped - covered by an envelope, they are roughly spherical but highly pleomorphic. There are
enveloped helical viruses (Ex. Influenza) and enveloped polyhedral virus (Ex. Herpes simplex).

Example: Spherical => Polio virus

4. Complex - have complex structures, many bacteriophages have a polyhedral capsid with a helical tail
attached.
 Example: Large loaves=> Vaccinia virus
Tadpoles => Bacterial viruses or bacteriophages.

C. Viral Inclusion Bodies

1. They can be as large as 30 µm in diameter when inside the host cell, and can be visible under light
microscopy. They are normally named after their discoverer.

Examples:

a) Rabies infection are transmitted through a bite wound from an infected animal either sensitive
RNA virus that forms Negri bodies in the cytoplasm of infected nerve cells.

b) Smallpox and Vaccinia infections are called Guarnieri bodies.

2. These inclusion bodies may be found in:

a) Cytoplasm (Guarnieri bodies; Negri bodies)

b) Nucleus (Inclusion bodies of Herpes simplex & Chickenpox)
c) Nucleus & Cytoplasm (Inclusion bodies of smallpox)

3. Rotaviruses ⇒ they were initially identified by direct electron microscopy (EM) of duodenal mucosa
in infants with gastroenteritis, studies shown them to be the cause of 30 to 40 percent of acute diarrhea
in infants.

4. Herpesviruses ⇒ contain a double stranded DNA genome surrounded by a protein coat that is in turns
enclosed by a lipid envelope.

Ex. Herpes Simplex

a) Cytomegalovirus (CMV) - is a herpes virus that induces a cellular swelling characterized as

cytomegaly. This virus is transmitted by saliva, urine, semen, cervical secretions, and human milk. CMV
inclusions can be asymptomatic, a mild disease, or progressive and fatal. Once a person is infected this
infection persists for life.

5. Influenza virus ⇒ is a myxovirus causing influenza and similar illnesses in humans.

Chemical composition and staining reactions of the inclusions. They are in their majority acidophilic in
staining, but cytoplasmic inclusions of the psittacosis group are basophilic.

D. Viral hepatitis is a term that refers to an inflammation of the liver caused by any of the three hepatitis
viruses (Hepatitis A, B & C).

A hepatitis-associated antigen associated with hepatitis B virus can be described, it is called "Hepatitis B
surface Antigen (HBsAg), may be stained in paraffin sections by use of either Orcein or the aldehyde
fuchsin methods.
 Special Staining Techniques
Kinyoun Acid-Fast Stain
Purpose: To detect the presence of acid-fast mycobacteria in tissue section.

Principle: The organisms known as the Mycobacteria tuberculosis are difficult to demonstrate because of
the lipid capsule which surrounds them. It takes considerable effort to force a stain through this capsule
into the organism, but once staining has been achieved it is resistant to removal by acid and alcohol, This
is the basis of the best known technique for demonstrating the most important organism of the group,
Mycobacterium tuberculosis.

This method uses the carbo-fuchsin stain in which the red dye carbol fuchsin is forced into the bacteria
and other structures with heat, and is then removed from the other structures with a dilute acid alcohol.
The acid fast bacillus, with its lipid capsule, withstands the effects of the dilute acid alcohol and remains
red-stained whereas, the stain is removed from almost all other structures (except red cells); a blue
counterstain is used for contrast.

Most acid-fast stains involve the application of a Phenylmethane Dye (eg. Pararosaniline, rosaniline, or
new fuchsin) in a phenol solution. The phenol enhances the staining and appears to combine with the
dyes above within the acid fast bacilli. It also functions to dissolve the fuchsin dye generally used.
Alcohol is added to the carbol-fuchsin solution (see below) as a solvent and because it too enhances the
staining.

The carbol-fuchsin dye stains all structures red. The section is then treated with a dilute acid-alcohol
which removes the carbol-fuchsin dye from all except "acid-fast" structures.

Fixative: 10% Neutral buffered formalin is the preferred fixative although others fixatives except Carnoy
solution may be used.

Technique: 4 to 5 µm paraffin sections.

Control: a section containing acid fast bacilli must be used.

Reagents:
a) Kinyoun Carbol-Fuchsin Solution:
1. Basic Fuchsin
2. Phenol crystals
3. 95% Alcohol
4. Distilled water.
b) 1% Acid Alcohol:Hydrochloric acid in 70% alcohol
c) Methylene Blue Solution (Stock): Methylene Blue in 95% Alcohol
d) Methylene Blue Solution (Working): Stock Methylene Blue
Procedure:
1. Deparaffinize and hydrate to deionized water.
2. Stain in Kinyoun carbol-fuchsin solution for 1 hour minutes at room temperature. Filter before use.
3. Rinse slides in deionized water for 5 minutes.
4. Differentiate slides in two changes of 1% acid alcohol until tissue is pale pink.
5. Rinse slides in deionized water for 2 minutes. Carry slides through the remainder of the procedure one
slide at a time.
6. Counterstain in Working Methylene blue solution for a few dips. DO NOT OVERSTAIN; the
sections should be sky-blue.
7. Rinse sections in deionized water.
8. Rinse slides quickly in 95% and 100% ethanol.
9. Clear in two to three changes of xylene, and mount with synthetic resin.

Results:
Acid-fast bacteria………………………….…………………………………………………...…Bright Red
Background………………………………………………………………………………….…….Light blue

Mycobacterium tuberculosis
Ziehl-Neelsen Method for Acid-Fast Bacteria
Purpose: To detect the presence of acid-fast mycobacteria in tissue section.

Principle: The organisms known as the Mycobacteria tuberculosis are difficult to demonstrate because of
the lipid capsule which surrounds them. It takes considerable effort to force a stain through this capsule
into the organism, but once staining has been achieved it is resistant to removal by acid and alcohol, This
is the basis of the best known technique for demonstrating the most important organism of the group,
Mycobacterium tuberculosis.

This method uses the carbol-fuchsin stain in which the red dye carbol fuchsin is forced into the bacteria
and other structures with heat, and is then removed from the other structures with a dilute acid alcohol.
The acid fast bacillus, with its lipid capsule, withstands the effects of the dilute acid alcohol and remains
red-stained whereas, the stain is removed from almost all other structures (except red cells); a blue
counterstain is used for contrast.

Most acid-fast stains involve the application of a Phenylmethane Dye (eg. Pararosaniline, rosaniline, or
new fuchsin) in a phenol solution. The phenol enhances the staining and appears to combine with the
dyes above within the acid fast bacilli. It also functions to dissolve the fuchsin dye generally used.
Alcohol is added to the carbol-fuchsin solution (see below) as a solvent and because it too enhances the
staining.

The carbol-fuchsin dye stains all structures red. The section is then treated with a dilute acid-alcohol
which removes the carbol-fuchsin dye from all except "acid-fast" structures.

Fixative: 10% Neutral buffered formalin is the preferred fixative although others fixatives except Carnoy
solution may be used.

Technique: 4 to 5 µm paraffin sections.

Control: a section containing acid fast bacilli must be used.

Reagents:
a) Ziehl-Neelsen Carbol-Fuchsin Solution:
a) Basic Fuchsin
b) Absolute Ethyl alcohol
c) Phenol crystals
d) Distilled water.
b) 1% Acid Alcohol: Hydrochloric acid in 70% alcohol
c) Methylene Blue Solution (Stock): Methylene Blue in 95% Alcohol
d) Methylene Blue Solution (Working): Stock Methylene Blue

Procedure:
1. Deparaffinize and hydrate to deionized water.
2. Stain in Ziehl-Neelsen carbol-fuchsin solution for 30 minutes at room temperature. Filter before use.
3. Rinse slides in deionized water for 5 minutes.
4. Decolorize slides in 1% acid alcohol until tissue is pale pink.
5. Rinse slides in deionized water for 8 minutes. Carry slides through the remainder of the procedure one
slide at a time.
6. Counterstain in Working Methylene blue solution for a few dips. DO NOT OVERSTAIN; the
sections should be pale blue.
7. Rinse sections in deionized water.
8. Rinse slides quickly in 95% and 100% ethanol.
9. Clear in two to three changes of xylene, and mount with synthetic resin.

Results:
Acid-fast bacteria………………………….…………………………………………………...…Bright Red
Background………………………………………………………………………………….…….Light blue

Mycobacterium tuberculosis
Fite Acid-Fast Stain for Leprosy Organisms
Purpose: To detect the presence of Mycobacterium leprae (causative agent of leprosy) in tissue section.

Principle: The lipoid capsule of the organism takes up carbol-fuchsin and resists decolorization with
dilute mineral acid.

Fixative: 10% Neutral buffered formalin is the preferred fixative although others fixatives except Carnoy
solution may be used.

Technique: 4 to 5 µm paraffin sections.

Control: a section containing acid fast bacilli must be used.

Reagents:
1. Xylene Peanut oil solution: 1 part Peanut oil & 2 parts Xylene
2. 1% Acid Alcohol: Hydrochloric acid in 70% alcohol
3. Ziehl-Neelsen Carbol-Fuchsin Solution:
a) Basic Fuchsin
b) Absolute Ethyl alcohol
c) Phenol crystals
d) Distilled water.
4. Methylene Blue Solution: Methylene Blue, glacial acetic acid & distilled water.

Procedure:
1. Deparaffinize sections in two changes of the Xylene-Peanut oil mixture for 12 minutes each change.
2. Drain sections, wipe off excess oil, and blot to opacity. The residual oil helps to prevent shrinkage and
injury of the section.
3. Stain in Ziehl-Neelsen carbol-fuchsin solution for 20 to 30 minutes at room temperature. Filter before
use.
4. Rinse slides in deionized water for 5 minutes.
5. Decolorize slides individually in 1% acid alcohol until tissue is faint pink.
6. Rinse slides in tap water.
7. Counterstain in Working Methylene blue solution for a few dips. DO NOT OVERSTAIN; the
sections should be pale blue.
8. Rinse off excess Methylene blue in tap water.
9. Blot sections and let stand for a few minutes to air dry completely.
10. Mount air-dried sections with synthetic resin. Do not use alcohol and xylene.

Results:

M. leprae …………………..………...Bright Red

and other acid-fast bacteria……....…..Bright Red
Background……………………..…...Light blue
Auramine-Rhodamine Fluorescence Method
Purpose: To detect the presence of Mycobacterium tuberculosis or other acid-fast organism.

Principle: The exact mechanism of this stain is unknown. Both of the dyes used are basic dyes that
fluoresce at short wavelengths. Both dyes used in combination yield better staining than either dye alone.

Fixative: 10% Neutral buffered formalin is preferred.

Technique: 4 to 5 µm paraffin sections.

Control: a section containing acid fast bacilli must be used.

Reagents:
Auramine O-Rhodamine B Solution:
Auramine O
Rhodamine B
Glycerol
Phenol
0.5% Acid Alcohol: Hydrochloric acid, 70% alcohol
0.3% Eriochrome Black T Solution: Eriochrome black T & Distilled water.

Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in Auramine O-Rhodamine B solution in a glass Coplin jar and microwave for 4
minutes on high.
3. Rinse sections in three changes of distilled water.
4. Differentiate sections in two changes of acid alcohol for 1 ½ minutes each.
5. Rinse slides in four changes of distilled water.
6. Stain slides in 0.3% Eriochrome black T solution for 15 seconds.
7. Rinse slides in three changes of distilled water.
8. Stand slides on end and thoroughly air dry.
9. Dip slides in xylene and mount with synthetic resin.
10. Examine sections with a high-dry objective, a UG1 or UG2 exciter filter, and a colorless UV
barrier filter.

Results:
Acid-fast organisms…….……………………….............................…Reddish-yellow fluorescence
Background.……………………………………………..……………Black
Brown-Hopps Gram Stain
Purpose: Demonstration of Gram-negative and Gram-positive bacteria in tissue.

Principle: Crystal violet is applied first and then followed by an iodine mordant forming a dye lake. At
this point, both Gram-negative and Gram-positive organisms are stained Although both types of bacteria
have a cell wall composed of peptidoglycan, the wall of Gram-positive bacteria is thicker than that of
Gram-negative organisms, and Gram-negative bacteria also contain a layer of lipopolysaccharide external
to the cell wall. These differences in the cell wall account for differences in the way that bacteria are
decolorized in the next procedural step. The large crystal violet-iodine molecular complex cannot easily
be washed out of the intact peptidoglycan layer of Gram-positive cells; however, it is easily removed from
Gram-negative bacteria because alcohol or acetone disrupt the outer lipopolysaccharide layer, and the
remaining thin peptidoglycan cell wall cannot retain the complex. Undamaged Gram-positive cell walls
will retain the crystal violet-iodine complex, unless the walls have been damaged or disrupted for some
other reason (eg, old or dead organisms). If the cell wall of a normally Gram-positive organism is
damaged, the organism will then stain Gram-negative. The decolorization step is a relative one, and
sections can be over decolorized, removing stain from both Gram-negative and Gram-positive organisms.
After decolorization, a counterstain is applied to color the Gram-negative organisms.

Fixative: 10% Neutral buffered formalin.

Technique: 4 to 5 µm paraffin sections

Control: Sections containing both Gram-positive and Gram-negative organisms should be used.

Reagents:
1% Crystal Violet: Crystal violet & Distilled water.
Gram Iodine: Iodine, potassium iodide & distilled water.
Basic fuchsin solution: Basic fuchsin & distilled water.
Gallegos Solution: Formalin, 37-40%, Distilled water & Glacial acetic acid.
Picric-Acid Acetone: Picric acid & Acetone.

Procedure:
1. Deparaffinize and hydrate slides to water.
2. Stain sections in crystal violet for 2 minutes.
3. Rinse slides in distilled water.
4. Stain slides in Grams iodine solution for 5 minutes.
5. Rinse slides I distilled water to remove excess iodine.
6. Blot one slide at a time with slightly damp filter paper and decolorize quickly in acetone.
7. Rinse slides quickly in distilled water.
8. Stain slides in working Basic fuchsin solution for 5 minutes.
9. Rinse slides in distilled water.
10. Differentiate slides in Gallego’s solution for 5 minutes.
11. Rinse slides in distilled water and blot sections, but not to dryness.
12. Dip slides quickly in acetone three times.
13. Dip slides in picric acid-acetone solution quickly three times.
14. Dip slides quickly in acetone three times.
15. Pass slides through a mixture of acetone-xylene (1:2) in five quick dips, then clear in two changes of
xylene.
16. Mount with synthetic mounting media.
Results:

Gram-positive bacteria…………………..........................................................…Blue
Gram-negative bacteria……………………………………………………….…Red
Nuclei……………………………...............…………………………………… Light Red
Background……………………………………………………...………………Yellow
Hotchkiss-McManus PAS Reaction for Fungi
Purpose: Demonstration of fungi in tissue.

Principle: The principle is similar to that described in the PAS procedure. Polysaccharides present in the
fungal cell walls are oxidized by the periodic acid to aldehydes. The aldehydes react with Schiff’s reagent
to yield rose-colored fungi.

Fixative: 10% Neutral buffered formalin, Bouin’s or Zenker’s solution.

Technique: 4 to 5 µm paraffin sections

Control: Sections containing fungi must be used as a control.

Reagents:
1% Periodic acid: Periodic acid & distilled water
Schiff’s Reagent: See PAS Procedure
1N Hydrochloric Acid: Hydrochloric acid & distilled water
10% Sodium Metabisulfite: Sodium Metabisulfite & distilled water
Fast Green Solution 1:500- fast Green FCF, Distilled water & Glacial Acetic acid

Sulfurous Rinse:
Distilled water, 1N Hydrochloric acid, 10% Sodium Metabisulfite. Prepare fresh just before use.

Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place sections in 1% periodic acid solution for 5 minutes.
3. Wash slides in three changes of distilled water.
4. Place sections in Schiff’s reagent for 15 minutes.
5. Place sections in three changes of sulfurous rinse for 2 minutes each.
6. Wash slides in running tap water for 10 minutes to develop full color.
7. Counterstain slides in fast green solution for 1 minute.
8. Rinse slides in distilled water.
9. Dehydrate slides in 95% alcohol, followed by 100% alcohol, clear with xylene.
10. Mount with synthetic mounting media.

Results:

Fungi………………….....................................................................................…Rose
Background……………………………………………………...………………Green
Gridley’s Fungus Stain
Purpose: Demonstration of fungi in tissue.

Principle: This procedure uses chromic acid to oxidize adjacent glycol groups to aldehydes. The
aldehydes are then reacted with Schiff’s reagent. Since Chromic acid is a stronger oxidizing agent than
periodic acid, it further attacks and destroys aldehydes, so fewer reactive groups are left to react with the
Schiff reagent. A less intense reaction is obtained than with the PAS technique, but background staining
is also decreased.

Fixative: 10% Neutral buffered formalin.

Technique: 4 to 5 µm paraffin sections

Control: Sections containing fungi must be used as a control.

Reagents:
4% Chromic acid: Chromium trioxide & distilled water
Schiff’s Reagent: See PAS Procedure
Aldehyde Fuchsin Solution: Basic fuchsin, 70% alcohol, Hydrochloric acid, Paraldehyde. Let
solution stand for 2 to 3 days at room temperature or until it turns deep purple. Filter and store in
the refrigerator.
0.25% Metanil Yellow: Metanil yellow, distilled water & glacial acetic acid

Procedure:
1. Deparaffinize and hydrate slides to water.
2. Oxidize the sections in 4% chromic acid for 1 hour.
3. Wash slide in running water for 5 minutes.
4. Place sections in Schiff’s reagent for 15 minutes.
5. Wash slides in running water for 15 minutes.
6. Rinse slides in several changes of 70% alcohol.
7. Stain slides in the Aldehyde fuchsin solution for 30 minutes.
8. Rinse off the excess stain with 95% alcohol.
9. Rinse slides in distilled water.
10. Counterstain slides in Metanil yellow solution for 30 seconds to 1 minute. Do not overstain.
11. Dehydrate slides in 95% alcohol, followed by 100% alcohol, clear with xylene.
12. Mount with synthetic mounting media.

Results:
Mycelia…………………......................................................................................Deep purple
Conidia…………………………………………………………………………..Deep rose to purple
Background……………………………………………………...………………Yellow
Elastic fibers and mucin………………................................................................Deep purple
Grocott’s Methenamine Silver (GMS) Nitrate Fungus Stain
Purpose: Demonstration of fungal organisms in tissue.

Principle: Polysaccharides in the fungal cell wall are oxidized to aldehydes by chromic acid. Chromic
acid is a strong oxidant, further oxidizing many of the newly released aldehyde groups to breakdown
products that will not react; this helps suppress the weaker background reactions of collagen fibers and
basement membranes. Only substances that possess large quantities of polysaccharides, such as fungal
cell walls, glycogen, and mucins, will remain reactive with the methenamine silver, reducing it to metallic
silver. Methenamine gives the solution the alkaline properties necessary for proper reaction and sodium
borate acts as a buffer. Gold chloride is a toning solution and the sodium thiosulfate removes any
unreduced silver.

Fixative: 10% Neutral buffered formalin is preferred.

Technique: 4 to 5 µm paraffin sections

Control: Sections containing fungi must be used as a control. If staining for Pneumocystis carinii use a
control with this type of organism since the timing in the impregnation process is different.

Reagents:
5% Chromic acid: Chromium trioxide & distilled water
5% Silver nitrate: Silver nitrate & distilled water
3% Methenamine Solution: Methenamine & distilled water
5% Borax: Sodium borate & distilled water
Stock Methenamine Silver
3% Methenamine Solution & 5% Silver nitrate solution
Working Methenamine-Silver Solution
5% Borax solution & distilled water, then add the Stock Methenamine-Silver Nitrate
solution.
1% Sodium bisulfite: Sodium bisulfite & distilled water
0.1% Gold Chloride: 1% Gold chloride solution & distilled water
2% Sodium thiosulfate (Hypo): Sodium thiosulfate & distilled water
Stock light green solution: Light green SF (yellowish), distilled water and glacial acetic acid
Working light green solution: Stock light green solution & distilled water

Procedure:
1. Deparaffinize sections and hydrate to distilled water.
2. Oxidize sections in chromic acid solution for 1 hour. After 40 minutes, begin preheating the silver.
The chromic acid solution may be reused until it turns dark.
3. Wash slides in running tap for a few seconds.
4. Rinse in 1% sodium bisulfite for 1 minute to remove any residual chromic acid.
5. Wash in tap water for 5 to 10 minutes.
6. Wash with three to four changes of distilled water. I
7. Using nonmetallic forceps, place slides in preheated working methenamine solution in the water
bath at 56 to 58°C for 15 minutes or until sections yellowish brown (paper-bag brown). Remove
the control, rinse in distilled water, and check microscopically for adequate silver impregnation.
Fungi should be dark brown at this stage. If impregnation is not sufficient, return the slide to the
methenamine silver and check every 3 to 5 minutes.
8. Rinse slides in six changes of distilled water.
9. Tone in 0.1 % gold chloride solution for 2 to 5 minutes. This solution may be used until brown
precipitate appears and the solution is cloudy.
10. Rinse sections in distilled water.
11. Remove unreduced silver by placing the slides in 2% sodium thiosulfate solution for 2 to 5
minutes.
12. Wash thoroughly in tap water.
13. Counterstain with working light green solution for 1 to 2 minutes.
14. Dehydrate with two changes each of 95% and absolute alcohol.
15. Clear with two to three changes of xylene and mount with a synthetic resin.

Results:
Fungi…………………......................................................................................Black
Pneumocystis carinii…......................................................................................Black
Mucin………………………………………………………………………….Taupe to dark gray
Background……………………………………………………...…………….Green
Warthin-Starry Technique for Spirochetes
Purpose: Demonstration of spirochetes in tissue.

Principle: This is an argyrophil method; that is, the spirochetes have the ability to bind silver ions from a
solution, but they do not have the ability to reduce the silver to a visible metallic form. A chemical
reducer, hydroquinone, is used for that purpose.

Fixative: 10% Neutral buffered formalin is preferred.

Technique: 4 to 5 µm paraffin sections

Control: The tissue must contain spirochetes.

Reagents:
1% Citric acid: Citric acid & Triple distilled water
Acidulated water: Triple distilled water and 1% Citric acid to bring the water to a pH of 4.0
2% Silver nitrate: Silver nitrate & distilled water
1% Silver nitrate: Silver nitrate & distilled water
5% Gelatin solution: Gelatin high grade & acidulated water
0.15% Hydroquinone solution: hydroquinone & acidulated water
Developer Solution Prepare immediately before use and in the order given.
2% Silver nitrate
5% Gelatin solution
0.15% Hydroquinone solution
Procedure:
1. Place the 2% silver nitrate, 5% gelatin, and hydroquinone solutions in separate 50 mL plastic centrifuge
tubes. Heat in a water bath at 54°C for at least 1 hour.
2. Place a 100-mL graduated cylinder and a chemically cleaned Coplin jar in the oven for at least 1 hour
(for developer).
3. Deparaffinize and hydrate sections to acidulated water.
4. Place slides in the 1 % silver nitrate impregnating solution in a water bath at 43°C for 30 minutes. Do
not preheat the solution.
5. Just before the slides are due out of the impregnating solution, prepare the developer (place in the
warm Coplin jar) and place in the 54°C water bath.
6. Put slides in developer for 3 or 4 minutes. Check after 2 minutes and continue checking frequently until
they are ready.
7. Wash slides quickly and thoroughly in distilled water.
8. Dehydrate sections in 95% and absolute alcohols, and clear in xylene (two changes of each).
9. Mount sections with synthetic resin.

Results:
Spirochetes………………….........................................................................Black
Background…………………………………………………...…………….Pale yellow to light brown

Steiner-Spirochetes
Dieterle Method for Spirochetes and Legionella Organisms
Purpose: Demonstration of spirochetes or the causative organism of legionellosis.

Principle: This is an argyrophil method; that is, the spirochetes have the ability to bind silver ions from a
solution, but they do not have the ability to reduce the silver to a visible metallic form. A chemical
reducer, hydroquinone, is used for that purpose.

Fixative: 10% Neutral buffered formalin is preferred.

Technique: 4 to 5 µm paraffin sections

Control: The tissue must contain spirochetes or Legionella organisms.

Reagents:
5% Alcoholic Uranyl Nitrate: Uranyl nitrate & 70% ethyl alcohol
10% Alcoholic Gum Mastic: Gum mastic & Absolute alcohol
It takes 2 to 3 days for gum mastic to dissolve, then filter and store in the refrigerator.
1% Silver nitrate: Silver nitrate & distilled water
10% Formic acid
Developer Solution Prepare immediately before use and in the order given.
Hydroquinone
Sodium sulfite
Distilled water
Acetone
Formaldehyde, 37-40%
Pyridine
10% alcoholic gum mastic

Procedure:
1. Preheat the 5% alcoholic uranyl nitrate solution and 1% silver nitrate solution in a 55 to 58oC oven for
at least 30 minutes.
2. Deparaffinize and hydrate sections to distilled water. Use three control slides.
3. Place sections in preheated 5% alcoholic uranyl nitrate in a 55 to 58oC oven for 30 minutes to 1 hour.
4. Dip sections once in distilled water.
5. Dip slides once in 95% alcohol.
6. Place slides in 10% alcoholic gum mastic for 3 minutes.
7. Dip sections once quickly in 95% alcohol.
8. Place sections in distilled water for 1 minute, then allow slides to drain for 15 to 20 minutes until
almost dry. Slides may be left overnight at this point.
9. Place slides in the 1 % silver nitrate impregnating solution in a 55 to 58oC oven, in the dark for 5 hours.
sections from old blocks may require longer incubation time.
10. Quickly dip slides twice in distilled water.
11. Place sections in developer and dip until the sections are tan to gold. Check a control at 4, 8 and 12
minutes. Finish all sections when the control is ready.
12. Quickly dip slides twice in distilled water.
13. Place slides in 10% formic acid for 45 seconds.
14. Dip slides twice in distilled water.
15. Dip slides twice in 95% alcohol.
16. Dip slides twice in acetone.
17. Clear slides in two changes of xylene and mount with synthetic resin.
Results:
Spirochetes, bacteria ………………….........................................................................Brown to black
Background…………………………………………………...……………………….Pale yellow to tan
Other structures that may stain are melanin granules, chromatin, formalin pigments, and some foreign
materials present in macrophages.

Legionella organisms Spirochetes: Treponema pallidum

Steiner & Steiner Technique for Spirochetes, Helicobacter &
Legionella organisms
Purpose: Demonstration of spirochetes, Helicobacter pylori, or the causative organism of legionellosis.

Principle: This is an argyrophil method; that is, the spirochetes have the ability to bind silver ions from a
solution, but they do not have the ability to reduce the silver to a visible metallic form. A chemical
reducer, hydroquinone, is used for that purpose.

Fixative: 10% Neutral buffered formalin is preferred. Mercuric and chromium fixative must be avoided.

Technique: 4 to 5 µm paraffin sections

Control: The tissue must contain spirochetes, helicobacter or legionella.

Reagents:
1% Alcoholic Uranyl Nitrate: Uranyl nitrate & 70% ethyl alcohol
2.5% Alcoholic Gum Mastic: Gum mastic & Absolute alcohol
It takes 2 to 3 days for gum mastic to dissolve, then filter and store in the refrigerator.
1% Silver nitrate: Silver nitrate & distilled water
0.04% Silver nitrate: Silver nitrate & distilled water
2% Hydroquinone solution: Hydroquinone & distilled water
Reducing Solution
2.5% Gum mastic
2% Hydroquinone
Absolute alcohol
Prepare immediately before us, filter and add 2.5 mL of 0.04% Silver nitrate solution.

Procedure:
Before staining, place a plastic Coplin jar in a 45°C to 50°C water bath to heat. Prepare the Reducing
solution and place in the preheated Coplin jar.
1. Deparaffinize and hydrate sections to distilled water.
2. Sensitize sections in 1% uranyl nitrate and microwave for 42 seconds, remove sections from oven and
place slides in distilled water.
3. rinse slides in distilled water until possible contamination is eliminated.
4. Place slides in the 1 % silver nitrate impregnating solution and heat in the microwave for 42 seconds,
remove slides from the oven and let stand on counter for 10 minutes.
5. Rinse slides in three changes of distilled water.
6. Rinse slides in two changes of 95% alcohol.
7. Rinse slides in two changes of 100% alcohol.
8. Place slides in gum mastic for 5 minutes.
9. Let the slides air dry for 1 minute.
10. Rinse slides in two changes of distilled water.
11. Reduce slides in the reducing solution in a 45°C water bath for 10 to 25 minutes or until sections have
developed satisfactorily.
12. Rinse sections in distilled water to stop the reduction.
13. Dehydrate sections in 95% and absolute alcohols, and clear in xylene (two changes of each).
14. Mount sections with synthetic resin.
Results:
Spirochetes, Helicobacter and legionella…………………......................................Dark brown to black
Background…………………………………………………………..…………….Light yellow

Helicobacter pylori Helicobacter pylori:

The Genta Stain
A combination of Steiner, H&E
and Alcian Blue pH 2.5