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-SpirochetesSteiner-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