5.0 MICRO-ORGANISMS
 

5.1 GRAM STAIN
Bacteria Acid Fast Bacilli Donovan Bodies Viral Inclusions Fungi and Pneumocystis carinii

5.1.1 CONTROL FOR GRAM STAIN
A method for making Gram Positive and Negative Controls in Tissue

PRINCIPLE
The probability of obtaining a well-fixed piece of tissue containing both Gram-positive and Gram-negative bacteria in the exponential growth phase, uncompromised by antimicrobial therapy and therefore, displaying typical Gram staining, is remote.
1. Gram controls can be made using placental tissue which has been incubated with a heavy suspension of bacteria in a nutrient broth.
2. Lung may also be used - obtain from autopsy. Culture 24 hours then process.

REFERENCES
p 209 Ms Masaye Tanaka, & Mr Colin Henderson, Foothills General Provincial Hospital, Calgary, Alberta, Canada. Histologic Technical Bulletin Vol xiii, No.3, Nov 1983. p182 Histopathology Note book, Roy C Ellis 1992, 1st Ed.

SPECIMEN
Fresh unfixed placenta or lung - for use on 2nd day of procedure. Gram - positive cocci (Staphylococcus)eg. Staph. aureus Gram - negative bacillus eg. E. coli, Proteus or Klebsiella

REAGENTS
(A) BLOOD AGAR PLATES (Microbiology Dept)
(B) BRAIN HEART INFUSION BROTH (Microbiology Dept)

PROCEDURE
Day 1 Blood agar plates are heavily inoculated with pure cultures of a Gram- positive cocci (Staphylococcus) and a Gram- negative bacillus (Proteus or Klebsiella) and incubated overnight at 37øC.
Day 2 The next day the entire inoculum from each plate is suspended in 15 mL of Brain Heart Infusion broth. Small 0.5cm clippings of placenta are suspended in the inoculated broth and incubated at 37øC for three hours. Placenta is chosen because it is porous and is available, fresh and unfixed, from the labour and delivery rooms of most hospitals. After incubation, the clippings of placenta are fixed in 10% neutral buffered formalin overnight and processed in the usual manner.

RESULTS
The stained slides show extracellular Gram-positive and Gram-negative bacteria throughout the tissue.
Note that the control may exhibit better Gram staining than the test slide as bacteria in the control are from an exponential growth phase population and fixed under ideal conditions.

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5.1.2 GRAM STAIN FOR BACTERIA
A method for Gram Positive and Gram Negative Bacteria (Brown and Brenn)

PRINCIPLE
Crystal Violet stains the nucleic acids of the bacteria (and background tissue) and after treatment with iodine, the sections are differentiated in acetone and counterstained with basic fuchsin. The tissue background and Gram-negative bacteria lose their blue staining and are subsequently stained with basic fuchsin. Gram-positive bacteria retain the crystal violet/iodine blue staining.

REFERENCES
p 222 Lee G Luna, Manual of Histological Staining Methods of the AFIP. p 121 Cook H C, Manual of Histological Demonstration Techniques p 280 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd Ed.

SPECIMEN
Standard paraffin section Fixative 10% neutral buffered formalin

CONTROL
Use control section containing both Gram positive and Gram negative bacteria. See method of preparing a Gram control - method 5 1.1

REAGENTS
(A) 1% CRYSTAL VIOLET SOLUTION
Crystal violet (C.I 42555) 1.0 g Distilled water 100 mL
(B) 5 % SODIUM BICARBONATE SOLUTION
Sodium bicarbonate 5.0 g Distilled water 100 mL
(C) GRAM'S IODINE SOLUTION
Iodine 1.0 g Potassium iodide 2.0 g Distilled water 300 mL
(D) ACETONE
Solution 1 & 3) Flammable liquid. Store in refrigerator.
(E) 0.25 % BASIC FUCHSIN SOLUTION (STOCK)
Basic Fuchsin (C.I. 42510) 0.25 g Distilled water 100 mL
(F) BASIC FUCHSIN SOLUTION (WORKING)
Basic fuchsin solution (Stock) 10 mL Distilled water 100 mL
(G) PICRIC - ACID ACETONE (Solution 2) (stored in fridge) Picric acid 0.1 g Acetone 100 mL
(H) ACETONE - HISTOLENE SOLUTION (Solution 4) Acetone 50 mL Histolene 50 mL

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Place slides on a staining rack. Pour on approx. 1 mL (or 20 drops) of crystal violet solution and add 5 drops of sodium bicarbonate solution for 1 minute. Agitate gently.
3. Rinse in tap water.
4. Flood slides with Gram's iodine solution for 1 minute.
5. Rinse in water and blot with filter paper to complete dryness.
6. Differentiate briefly with acetone (Solution 1 in coplin jar) until no more colour runs off. Do not overdifferentiate as Gram-positive bacteria will become negative. Rinse in water.
7. Repeat Steps 5 and 6 for further differentiation if section background is too blue.
8. Working basic fuchsin solution for 3 minutes. Wash in water.
9. Blot gently, but do not allow sections to dry completely.
10. Dip in acetone to start differentiation reaction.
11. Differentiate immediately with picric acid-acetone (Solution 2) until sections are yellowish pink.
12. Rinse quickly in acetone (Solution 3), then in acetone-histolene (Solution 4).
13. Clear in histolene, several changes.
14. Mount with Depex or Safe-t-mount.

NOTES:
1. This method is invaluable in the demonstration of the filaments of Nocardia and Actinomyces. It must be realised however, that these filaments are not completely or strongly Gram positive.
2. It is possible to obtain either Gram positive or Gram negative results depending on the degree of differentiation. It is suggested therefore, that at least two slides be run with varying degrees of differentiation in order to lessen the possibility of over-differentiation at Step 9 of the staining procedure.

RESULTS
Gram positive bacteria Blue
Gram negative bacteria Red
Filaments of Nocardia & Actinomyces Blue
Nuclei Red Other tissue elements Yellow

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5.2 DIFF-QUIK STAIN FOR CAMPYLOBACTER-LIKE ORGANISMS.

Diff-Quik Stain. Modified for the demonstration of Campylobacter-like organisms in gastric biopsies.

PRINCIPLE
The Diff-Quik stain is a modification of Wright's stain which is used mainly for blood films and bone marrow preparations. The original Diff-Quik method included a fixative stage for use with unfixed blood and bone marrow films. For use with paraffin sections we omitted this step.

REFERENCE
Walkeden, P.J. and B.E. Douglas. Rapid method for the demonstration of Campylobacter-like organisms (CLO's) in paraffin sections of gastric biopsies. J. Histotechnol. 13:113, 1990.

SPECIMEN
Fixation: Neutral buffered formalin Sections: Standard paraffin section

CONTROL SECTIONS
Tissue containing Helicobacter pylori.

REAGENTS
1. Commercial Diff-Quik Solution (Red) in a staining jar.
2. Commercial Diff-Quik Solution 3 (Blue) in a staining jar.

PROCEDURE
1. Deparaffinise sections and hydrate to water.
2. Dip slides into Diff-Quik Solution 2 (Red) for 3 seconds. (3x1 second dips) Allow excess stain to drain.
3. Dip slides into Diff-Quik Solution 3 (blue) for 6 seconds (6x1 second dips) Allow excess stain to drain.
4. Rinse with distilled water.
5. Blot sections carefully and air-dry using gentle warmth.
6. Dip in histolene and mount in Safety-Mount.

RESULT
CLOs Dark blue
Tissue elements Shades of blue, pink and red
 

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5.3 ZIEHL - NEELSEN STAIN FOR TUBERCLE BACILLI (A.F.B.)
(Ziehl, 1882, Neelsen, 1883)

PRINCIPLE
Mycobacteria tuberculosis (tubercle bacilli) have a lipid-rich cell wall which is capable of taking up strong phenol dye solutions (eg. carbol fuchsin solution) such that the dye is retained upon subsequent differentiation in acid or alcohol ie. they are said to be acid and alcohol fast (AAFB = acid and alcohol fast bacilli).

REFERENCES
p 123 Cook H C, Manual of Histological Demonstration Techniques p 283 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard paraffin section Fixative 10% neutral buffered formalin

CONTROL
Use a control positive for tubercle bacilli.

REAGENTS
(A) CARBOL FUCHSIN SOLUTION
Dissolve 1 g basic fuchsin (CI 42510) in 10 mL absolute alcohol Dissolve 5 g of phenol in 100 mL distilled water Mix the two solutions and filter before use Warning: Phenol is highly toxic/corrosive Wear gloves. Re-seal bottle with paraffin wax
(B) 1 % ACID ALCOHOL 70 % alcohol 99.0 mL Conc. HCl 1.0 mL
(C) STOCK ETHYLENE BLUE SOLUTION
Dissolve 1.4g methylene blue (CI 52015) in 100 mL 95 % alcohol
WORKING METHYLENE BLUE SOLUTION
Stock methylene blue 10 mL Tap water 90 mL

PROCEDURE
1. Deparaffinise sections and hydrate to distilled water.
2. Pre-heat filtered carbol fuchsin to 56øC in Coplin jar in water bath/oven for 15 mins, then add sections and heat in water bath/oven for 30 mins.
3. Wash well in tap water.
4. Differentiate in acid alcohol until faint pink (do NOT over differentiate).
5. Wash in water.
6. Counterstain lightly with Working methylene blue solution for 2 - 3 seconds.
7. Dehydrate in alcohol.
8. Clear and mount.

NOTE:
Blue counterstain should be pale to facilitate detection of the red staining organisms.

RESULTS
AFB (acid-fast bacilli) Magenta
Background Weak blue

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5.4 WADE - FITE FOR DEMONSTRATION OF MYCOBACTERIUM LEPRAE

PRINCIPLE
The Wade-Fite technique is recommended for the demonstration of Mycobacterium leprae. In this technique the more gentle dewaxing of the section, and the minimal exposure to organic solvents, protects the rather precarious acid-and alcohol fastness of the organism.

REFERENCES
p 126 Cook H C, Manual of Histological Demonstration Techniques p 284 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd Edition p 221 Luna (for Carbol-new fuchsin solution).

SPECIMEN
Standard paraffin section Fixative 10% neutral buffered formalin

CONTROL
Always use a control that is positive for Mycobacterium leprae.

REAGENTS
(A) DEPARAFFINISING SOLUTION
Prepare two coplin jars of the following mixture. Turpentine Oil 40 mL Xylene substitute (Histolene) 20 mL
(B) CARBOL NEW FUCHSIN SOLUTION
Dissolve 0.5 g new fuchsin (CI 42520) in 10 mL absolute alcohol Dissolve 5 g phenol in 90 mL distilled water Mix the two solutions and filter. Caution: Phenol is highly toxic/corrosive Wear gloves Re-seal bottle with paraffin wax.
(C) 10 % AQUEOUS SULPHURIC ACID
(D) WORKING METHYLENE BLUE SOLUTION Stock methylene blue (See 5.4) 10 mL Tap water 90 mL

PROCEDURE
1. Take section straight from hot-plate and de-paraffinise in two changes of Turpentine Oil/Histolene mixture - approximately 5 minutes each change.
2. Blot dry on paper hand towel.
3. Boil Carbol Fuchsin Solution and filter onto section - leave on for 10 minutes.
4. Wash in water 10 minutes.
5. Differentiate in 10% aq sulphuric acid approximately 3 seconds depending on tissue. Sections turn blue/black.
6. Wash in running water 15 minutes.
7. Counterstain with 2-3 dips in working Methylene Blue Solution.
8. Dehydrate rapidly.

RESULTS
Leprosy bacilli Magenta
Background Pale blue

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5.5 WARTHIN STARRY METHOD FOR SPIROCHAETES AND DONOVAN BODIES
 

(Warthin and Starry, 1920) Treponema pallidum is the causative organism of syphilis and can be demonstrated with this silver technique. Granuloma inguinale is a venereal disease caused by a gram-negative bacteria, Calymmatobacterium granulomatosis. These bean-shaped bacilli, are also called Donovan bodies and can be demonstrated with the Warthin-Starry silver technique.

PRINCIPLE
Organisms are demonstrated by silver impregnation technique.

REFERENCES
p 129 Cook H C, Manual of Histological Demonstration Techniques p 286 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd Edition Dept. of Pathology, Royal Perth Hospital, Perth WA.

SPECIMEN
Standard paraffin section - three per block Fixative 10% neutral buffered formalin

CONTROL
Use a Control that is positive for Treponema pallidum or Donovan bodies. 3 Control slides, developed from 5 to 10 minutes are used.

EQUIPMENT
Plastic forceps, 2 x water baths, one at 43øC and one at 54øC

REAGENTS
All glassware should be clean, rinsed in distilled water and dried.
(A) ACIDULATED WATER 1000 mLs (Distilled water brought to pH4 with a few granules of Citric Acid) All solutions are prepared using acidulated water. Prepare fresh solutions in acidulated water of the following:
(B) 2 % SILVER NITRATE 100 mL
(C) 5 % GELATIN 100 mL
(D) 0.15 % HYDROQUINONE 100 mL
(E) DEVELOPER SOLUTION 2 % silver nitrate (in acidulated water) 15 mL 5 % gelatin (in acidulated water) 37.5 mL 0.15 % hydroquinone (in acidulated water) 20.0 mL Heat separately to 54øC Mix well just before use.

PROCEDURE
1. Rinse all glassware with acidulated water.
2. Deparaffinise test sections x 3 + Control Sections x 3, hydrate to distilled water and then rinse in acidulated water.
3. Dilute 25 mLs of the 2% solution silver nitrate with 25 mLs of acidulated water. Heat the 1% silver solution in a water bath to 43øC for 15 mins. Impregnate the sections with 1% silver nitrate at 43øC for 30 mins.
4. Measure solutions for developer and keep separately at 54øC. Place a coplin jar of acidulated water into 54øC water bath for rinsing after development.
5. Mix developer in a coplin jar. Place slides in developer solution at 54øC (approx. 5 - 10 minutes).

NOTES:
a) Development is critical. Sections develop from pale yellow (under-developed) to golden brown (over-developed).
b) Process 3 control sections and remove the patient's slide and a control slide at 3 different time intervals e.g. 3, 5 and 8 minutes.
c) Developing time varies from approximately 3 to 10 mins and even up to 15 minutes.
d) Donovan bodies require longer development than spirochaetes.
e) Correct temperatures and impregnation times are very important.
f) Rinse in acidulated water at 54 øC
g) Rinse in distilled water.
h) Dehydrate, clear and mount.

RESULTS
Spirochaetes Black
Donovan bodies Black
Background Yellow - brown
 

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5.6 GIEMSA STAIN FOR DONOVAN BODIES AND PARASITES
(Giemsa, 1902)

PRINCIPLE
This method is a modified version of the original Giemsa technique used for haematological smears and gives good results for sections. Giemsa is a Romanowsky stain which contains azure B and eosin Y and is capable of making subtle distinction in shades of staining. The acidic groupings of the nucleic acids and proteins of the cell nuclei determine their uptake of the basic dye azure B and the presence of basic groupings result in an affinity for acidic dyes and their staining by eosin.

REFERENCES
p 61 Cook H C, Manual of Histological Demonstration Techniques p 295 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd Edition

SPECIMEN
Standard paraffin section fixed in 10% neutral buffered formalin. Air dried smears, post fixed in methanol or 95% alcohol - 20 mins. CONTROL Use a positive control section to demonstrate Donovan bodies.

REAGENTS
(A) STOCK COMMERCIAL GIEMSA (GURR'S IMPROVED R66 # 35086)
(B) PHOSPHATE BUFFER The pH 6.8 buffer can be prepared from buffer tablets. Dissolve one tablet in distilled water and make up to one litre. Alternative method. Stock A 0.2 M Sodium dihydrogen orthophosphate (M.W156) 3.12 g Sodium dihydrogen orthophosphate in 100 mLs distilled water Stock B 0.2 M disodium hydrogen orthophosphate (M.W142) 2.83 g disodium hydrogen orthophosphate in 100 mLs distilled water Composition of Buffer pH 6.8 25.5 mL Stock A + 24.5 mL Stock B made up to 100 mLs with distilled water.
(C) 1 % AQUEOUS ACETIC ACID. OVERNIGHT STAINING Dilute 1.0 mL of (A) with 50 mL of (B) 40 MINUTE STAINING Dilute 5 mL of (A) with 50 mL (B)

PROCEDURE
1. Deparaffinise and hydrate sections to water.
2. Hydrate post-fixed smears (see specimen requirements) in water for one minute.
3. Stain in Giemsa overnight or 40 mins in stronger solution. Wash in water.
4. Differentiate with acetic acid until sections are pale purple in colour (approximately 4 secs).
5. Wash in water.
6. Air dry sections.
7. Mount in Safety Mount

NOTES
To achieve a good colour balance it is necessary to initially overstain with the Giemsa stain, and then differentiate with 1% acetic acid.

RESULTS
Nuclei Blue - purple
Azurophilic granules Blue
Collagen Pale pink
Donovan bodies Pale blue
Protozoons Dark blue
Eosinophil granules, } Red blood cells & } Pink Other acidiphil structures }

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5.7 PHLOXINE-TARTRAZINE TECHNIQUE FOR VIRAL INCLUSIONS
(Lendrum, 1947)

PRINCIPLE
All tissues are stained red with phloxine, then the red stain is differentiated out with tartrazine in cellosolve which also acts as a yellow counterstain. This stain is used to demonstrate many structures according to the degree of differentiation.

REFERENCES
p 53 Cook H C, Manual of Histological Demonstration Techniques p 292 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd Edition

SPECIMEN
Standard paraffin section fixed in 10% neutral buffered formalin.

CONTROL
Viral inclusion bodies - viral warts Molluscum contagiosum, cytomegalic disease Paneth cell granules - small intestine

NOTES
1. The red colour is removed first from muscle and other connective tissues; the last structures to lose their red colour are viral inclusions, Paneth cell granules and red blood cells.
2. With less extensive differentiation this method can also be used to display pancreatic beta cells, Russell bodies (within plasma cell cytoplasm), keratin (e.g. keratin pearls and individual cell keratinisation in squamous carcinoma, and inhaled squames in the lungs of still-births and neonatal deaths) and fibrin.
3. The variable staining of keratin can be a problem when attempting to demonstrate viral inclusions in skin lesions.

REAGENTS
(A) PHLOXINE SOLUTION
Phloxine (Phloxin B CI 45410) 0.5 g Calcium chloride 0.5 g Distilled water 100 mL
(B) TARTRAZINE SOLUTION (CI 19140)
Saturated solution of tartrazine in 2-ethoxy-ethanol (`cellosolve') - store in flammables cupboard.

PROCEDURE
1. Dewax in histolene, then hydrate through graded alcohols to water.
2. Stain nuclei in alum haematoxylin for 2 minutes.
3. Wash in running tap water for 5 minutes.
4. Stain in phloxine solution for 20 minutes.
5. Rinse in tap water, then blot dry.
6. Stain in tartrazine solution under microscopic control until only the viral inclusions remain strongly red. An average time is 5 - 10 minutes.
7. Rinse in 95 % alcohol.
8. Dehydrate in absolute alcohol, clear in histoclear and mount in Safe-t-mount.

RESULTS
Viral inclusions Bright red
Red blood cells Variably orange-red
Nuclei Blue - grey
Background Yellow

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5.8 ORCEIN STAIN FOR HEPATITIS B ANTIGEN (HBSAg) IN LIVER TISSUE

(Australian Antigen) The Hepatitis B virus causes an acute inflammation of the liver. Hepatic cell necrosis is associated with leucocytic and histiocytic reaction and infiltration. HBSAg may be stained by orcein in liver of carriers and chronic hepatitis, not acute.

REFERENCES
p25- 36 Japan J. Exp. Med., Vol.44, I, 1974

SPECIMEN
Standard paraffin section Fixation Neutral buffered Formalin.

CONTROL
HBAg in liver tissue.

REAGENTS
(A) ACIDIFIED POTASSIUM PERMANGANATE SOLUTION Potassium permanganate 0.3g Distilled water 100mL Concentrated Sulphuric acid 0.3mL
(B) 1.5 % AQUEOUS OXALIC ACID
(C) ORCEIN STAIN Dissolve 1.0 g of synthetic orcein in small amount of 70 % alcohol with aid of gentle heat e.g. 56øC water bath. Make up final volume to 100 mL of 70% alcohol, then add 1.0 mL concentrated hydrochloric acid.
(D) 1 % ACID ALCOHOL

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Oxidise in permanganate for 5 mins.
3. Wash in water
4. Bleach in oxalic acid until white
5. Wash in 70% alcohol.
6. Stain in orcein for one and a half hours.
7. Wash in water.
8. Differentiate in acid alcohol until no colour runs off the slides.
9. Wash in water.
10. Dehydrate, clear and mount.

RESULTS
Australia antigen purplish (The distribution of Australian antigen varies: fine diffuse granules in the cytoplasm in sub- lobar clumps of hepatocytes to round or oval cytoplasmic clumps in single scattered hepatocytes.) Cytoplasm much lighter brown

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5.9 VICTORIA BLUE - NUCLEAR FAST RED FOR HBSAg

PRINCIPLE
To demonstrate copper associated protein, elastic and Hepatitis B surface antigen (HBSAg) in paraffin wax sections.

REFERENCE
Method obtained from Royal Free Hampstead NHS Trust Histopathology Department 1998. Their method date 20/5/97. AFIP laboratory methods in Histotechnology p210 Tanaka method Ref:Acta Pathol Jpn 1981;31:93

SPECIMEN
10% neutral buffered formalin. Standard paraffin wax sections.

CONTROL
Known case of primary biliary cirrhosis.

REAGENTS
(A) VICTORIA BLUE
Distilled water 200 mL Dextrine 0.5 g Victoria Blue 2 g Resorcinol 4 g Gradually warm up the mixed solution of all the above until it boils. Remove form heat then gradually add 25mL of boiling 29% ferric chloride solution (12mL commercial 60% ferric chloride solution plus 13mL distilled water) and boil the solution for a further 3 minutes. Cool. After cooling filter it. Dry the filtrate on the filter paper in a 56øC oven until completely dry. Dissolve the dried filtrate in 400mL of 70% alcohol. Finally add 4mL concentrated hydrochloric acid and 6g phenol. It is best to leave this solution to mature for at least two weeks prior to use.
(B) NUCLEAR FAST RED
Dissolve 0.5g nuclear fast red in 500mL of warmed 5% aluminium sulphate. Filter when cool.
(C) 4% AQUEOUS SODIUM METABISULPHITE

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Treat with acidified potassium permanganate (as for Gordon and Sweet's Reticular fibres method 2.4) for 5 minutes.
3. Treat with 4% sodium bisulphite for 1 minute.
4. Wash in running tap water.
5. Wash well with 70% alcohol.
6. Stain in the Victoria Blue solution, in a Coplin jar, for a minimum of 4 hours (preferably overnight).
7. Wash well with 70% alcohol for approx 1 minute. This is the differentiation step - ensure the background of the section is clear.
8. Wash in running tap water for 1 minute.
9. Stain with nuclear fast red solution for 5 minutes.
10. Wash in running water for 2 minutes.
11. Dehydrate, clear and mount.

RESULTS
HBsAg, elastic fibres, copper associated protein blue
All other tissue components pink/red

HEALTH AND SAFETY
Potassium permanganate Harmful by ingestion. Irritating to eyes and skin Ferric chloride Irritant. Hydrochloric acid Vesicant. Causes burns. Phenol Harmful by ingestion. Irritating to eyes and skin.
 

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5.10 GROCOTT - GOMORI METHENAMINE SILVER METHOD FOR FUNGI.
(Grocott, 1955, Gomori, 1946)

PRINCIPLE
This method depends upon the reduction of the silver by the aldehyde groups produced after oxidation of fungal wall components with chromic acid.

REFERENCES
p 288 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd ed.

SPECIMEN
Standard Paraffin section fixation Neutral buffered Formalin.

CONTROL
Use a control positive for Fungi.

REAGENTS
(A) 5 % AQUEOUS CHROMIC ACID (Chromium trioxide)
(B) 1 % AQUEOUS SODIUM METABISULPHITE
(C) STOCK HEXAMINE - SILVER SOLUTION To 100 mL 3% hexamine (methenamine) add 5 mL of 5% aqueous silver nitrate. (HIGHLY TOXIC - OXIDISER) A white precipitate will form which dissolves on shaking. This solution will keep for a few months at 4øC. WORKING HEXAMINE - SILVER SOLUTION Dilute 2 mL of 5 % aqueous sodium tetraborate (borax) with 25 mL distilled water, then add 25 mL stock solution. Filter and warm in 56øC oven.
(D) STOCK LIGHT GREEN (CI 42095) SOLUTION Dissolve 0.2 g of light green in 100 mL distilled water. Add 0.2 mL glacial acetic acid.
WORKING LIGHT GREEN SOLUTION Dilute 10 mL stock solution with 50 mL distilled water. Add 1 drop glacial acetic acid.
(E) 5 % SODIUM THIOSULPHATE
(F) 0.2 % GOLD CHLORIDE (SODIUM CHLORO-AURATE) IRRITANT Kept with reticulin fibre stains method 2.5.

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Oxidise in chromic acid for 1 hour.
3. Wash in water
4. Bleach in metabisulphite for 1 minute.
5. Wash in distilled water.
6. Treat sections in pre-heated Working hexamine-silver solution at 56øC water bath/oven until sections are yellowish/brown in colour (1/2 - 1 hour).
7. Wash in distilled water.
8. Tone in gold chloride for 2 minutes.
9. Wash in water.
10. Fix in 5 % thiosulphate for 5 minutes.
11. Wash in water.
12. Counterstain lightly with Working light green solution 5 - 10 seconds.
13. Dehydrate in alcohol.
14. Clear and mount.

RESULTS
Fungal elements Black
Background Pale green
Uric acid & Urates Black
Pneumocystis carinii Black
 

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5.11 GRAM WEIGERT STAIN FOR PNEUMOCYSTIS CARINII AND GRAM- POSITIVE ORGANISMS

Pneumocystis carinii is a protozoon which mainly infects the lungs and is often a fatal pneumonia in patients on immunosuppressive therapy or patients with AIDS.

PRINCIPLE
This is a modified Gram technique. The Gram stain is superimposed on a haematoxylin and eosin stain. Only Gram-positive organisms are demonstrated by this technique and not Gram-negative organisms. Pneumocystis carinii can also be demonstrated.

REFERENCES
p 392 Culling, Handbook of Histopathological & Histochemical Techniques p 122 Cook, H C, Manual of Histological Demonstration Techniques

SPECIMEN
Standard paraffin section Fixation Neutral buffered Formalin.

CONTROL
Use a control positive for Pneumocystis carinii.

REAGENTS
(A) 2.5 % AQUEOUS EOSIN
(B) ANILINE CRYSTAL VIOLET Crystal violet CI 42555 5 g Absolute alcohol 10 mL Aniline 2 mL Distilled water 88 mL This solution keeps well
(C) LUGOL'S IODINE Iodine 1 g Potassium iodide 2 g Distilled water to 100 mL Dissolve the potassium iodide in 4-5 mL of water; dissolve the iodine in this. Dilute to 100 mL to make Lugol's solution.
(D) 50 / 50 ANILINE / HISTOLENE

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Stain nuclei lightly (approx 1 minute) with alum haematoxylin.
3. Blue in tap water
4. Stain in 2.5 % aqueous phloxine or eosin for 10 minutes at 56øC
5. Wash in water.
6. Stain in aniline crystal violet for 1/2 - 1 hour.
7. Rinse in water.
8. Treat with Lugol's iodine for 1 minute.
9. Blot with fine filter paper.
10. Differentiate with equal part of histoclear and aniline until only bacteria and fibrin are blue-black (it is almost impossible to over-differentiate).
11. Rinse in xylol to remove aniline, and mount in Canada balsam or DPX.

RESULTS
Gram-positive organisms Blue -black
Nuclei Blue
Other tissue constituents Shades of red
Pneumocystis carinii Blue with one side denser Blue stain. Approx. round 4-5æ diam.

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