3. Place the slide on the rack and flood with the crystal violet or gentian violet stain—stain for 1 minute. 4. Wash off the stain with Gram’s or Lugol’s iodine and leave the slide covered with iodine for 1 minute.

 

Microtatobiotes (The smallest living things)

Rickettsiales: Most of these are intracellular pathogens,

and filtrable forms and need special methods of culture.

Virales

Thallophyta

These are the Molds and Yeasts.

Bacterial Cell Constituents

Like other living cells, all bacteria possess the cell membrane,

cytoplasm and a nucleus. Special characteristics are seen

in certain strains.

Capsule

This is a protective outer covering layer possessed by some

bacteria.

Flagella

These assist in locomotion, their arrangement may vary.

Spores

Under unfavorable conditions for growth sporing occurs.

Spores are non-reproductive. Upon return of favorable

environment they are transformed into the reproducible vegetative form. Spores are spherical and have a

distinctive placement within the cell. They may be central

subterminal or terminal. Knowing their location assists in

identification of species.

Inclusion Granules

Some of the bacteria show inclusion granules. Volutin

granules are metachromatic granules and may appear as

aggregates of substances concerned with cell metabolism;

820 Concise Book of Medical Laboratory Technology: Methods and Interpretations

when stained with toluidine blue, they stain a red violet

color in contrast to blue staining of the cytoplasm. These

are considered to be made of polymerized inorganic phosphate. Lipid granules may be seen in bacteria and stained

with Sudan black. Polysaccharide granules stainable by

iodine (like glycogen or starch) can be seen in cytoplasm

of some bacteria.

Shape of Bacteria

1. Cocci

Spherical

a. Cocci in cluster—Staphylococci

b. Cocci in chain—Streptococci

c. Cocci in pair—Diplococci

d. Cocci in groups of four—Tetrad

e. Cocci in groups of eight—Sarcino.

2. Bacilli

These are cylindrical or rod-shaped organisms. They can

be of the following types:

a. Length of the cell equalling its breadth, called

coccobacilli, e.g. Brucella

b. Chinese letter arrangement as seen in corynebacteria

c. Vibrio are comma shaped, curved, rods and are named

so on account of their vibratory movement

d. Spirochetes are relatively longer, thinner, flexible and

coil shaped

e. Actinomycetes are the branching filamentous bacteria

f. Mycoplasma lack cell wall and hence have no definite

morphology. They may be round or oval bodies with

interconnecting filaments.

Bacterial Reproduction

Bacterial reproduction occurs by a simple process of

binary fission.

Bacterial Physiology

Bacterial physiology and biochemistry are studied by

observing cultures grown in the laboratory on artificially

prepared nutrient media. Various external factors influencing bacterial growth are—food, moisture, hydrogen ion

concentration, oxygen, carbon dioxide, temperature and

light.

1. Food

Bacterial growth is to large extent dependent on an

adequate supply of suitable food material, the specific

nutrient requirements vary from species to species. The

important nutrient requirements are carbon, nitrogen,

inorganic salts and for certain species, accessory growth

factors of bacterial vitamins.

2. Moisture

For bacterial growth moisture is essential. Drying in the air

damages bacteria.

3. Hydrogen-ion-concentration or pH

Most of the microbes growth better at a slightly alkaline pH

(pH 7.2–7.6). Some acidophilic bacteria flourish in acidic

pH. Those needing strong alkaline medium are termed

basophilic.

4. Oxygen needs

Most bacteria can grow in the presence of oxygen and air

and also in its absence. Those which grow in the presence

of oxygen are called aerobes, while those which grow in

its absence are termed anaerobes. Those which can grow

under both the conditions are called facultative anaerobes,

whereas bacteria that can grow in complete absence of

oxygen are named obligatory anaerobes.

5. Carbon dioxide

All bacteria need the presence of small amounts of CO2

for growth, an amount provided by atmosphere or by the

metabolic reactions occurring in the bacteria itself. However,

some bacteria need a higher concentration of CO2 (5–10%).

6. Temperature

For bacteria, there is a range of temperature at which growth

can occur. So there is a maximum, a minimum and the

intermediate optimum temperature (at which the growth

is most rapid). In the laboratory, this optimum temperature

is maintained in an incubator thermostatically controlled.

Majority of bacteria grow between 25 and 40°C and are

termed mesophilic. 30°C is optimal for free living and 37°C

is optimal for parasites in man or animals. Bacteria that

grow best between 60 and 70°C are called thermophilic,

while those growing best between 15 and 20°C are labeled

as psychrophilic.

7. Light

Darkness is a favorable condition for growth and viability

of bacteria. Direct sunlight is injurious to bacterial

growth. Some bacteria can produce pigmentation on

exposure to light and are called as photochromogens.

8. Symbiosis or mutual beneficial coexistence

A living organism multiplying in a human body is called as

a parasite and the person harboring is the host. When both

the parasite and the host derive benefit from each other—

it is termed symbiosis. Certain intestinal bacteria provide

Microbiology and Bacteriology 821

vitamins to their host without causing any pathogenic

effects—a symbiotic relationship.

Products of Bacterial Growth

While thriving in a host or on an artificial culture medium,

some bacteria produce substances that exert injurious effects

in the host—these are called ‘toxins’. In addition, certain

enzymes may be harmful to the host. Some bacteria produce

pigments (harmless, help in bacterial identification).

1. Bacterial toxins

These injurious products of bacteria are of two types:

(i) exotoxins (extracellular) and (ii) endotoxins (intracellular). Toxins diffuse readily from the living bacteria into

the surrounding medium. They can be obtained from the

medium after removal of the bacteria. This can be done

by centrifugation or by filtering through a Seitz filter.

The toxins remain in the supernatant fluid in the case of

centrifugation and in the filtrate in the case of filtration.

Certain gram-positive bacteria secrete exotoxins, for

example, Corynebacterium diphtheriae. Exotoxins are

antigenic and are rapidly destroyed by heat.

Endotoxins: These are toxins intimately associated with

the cell wall of the most gram-negative bacteria. They are

released after death and disintegration of the bacteria. The

majority of pathogenic bacteria produce endotoxins only.

As mentioned in the previous paragraph for exotoxins—the

endotoxins would be present in the residues and not in the

supernatant (centrifugation) or in the filtrate (filtration).

2. Bacterial enzymes

a. Proteolytic enzymes: An enzyme responsible for

decomposition of dead animal and vegetable matter

in nature.

b. Coagulase: This is often demonstrated during the

study of biochemical properties of some pathogenic

bacteria.

c. Amylase: This enzyme is capable of splitting starch and

is not much used in the study of bacteria.

d. Lactic acid fermentation.

3. Bacterial pigments

Many bacteria have the capacity to produce pigments,

e.g. Staphylococcus aureus—golden yellow pigment and

Pseudomonas pyocyaneus—green pigment. Certain

pigments are restricted to the bacterial colonies while

others can diffuse to surrounding medium.

Koch’s Postulates

The etiologic relationship between pathogen and a disease

is established by fulfilling Koch’s postulates, viz.

1. The pathogen must be constantly found in the body of

host either alive or dead.

2. It must regularly be isolated and it must be grown in

pure culture in vitro.

3. When such a pure culture is inoculated into a susceptible

animal species, the typical disease must result.

4. From such experimentally induced disease, the

pathogen must be again isolated.

Morphology and Staining Reactions

Bacterial identification is aided by their staining reactions.

Simple stains are used to show the presence of organisms

and the nature of the cellular contents in exudates.

1. Loeffler’s Methylene Blue

Saturated solution of methylene blue in alcohol 30 mL.

Potassium hydroxide 0.01% in distilled water—100 mL.

Method

Stain for 3 minutes after making and fixing the smear. This

stain does not readily overstain.

2. Dilute Carbol fuchsin

This is made by diluting Ziehl-Neelsen’s carbol fuchsin

stain ten times its volume in water. The smears are

stained for 10–25 seconds and are washed well with water

(Overstaining must be avoided here).

The two most frequently used differential stains are the

Gram and Ziehl-Neelsen techniques.

Gram’s Stain

This is the most widely used but not a fully understood

technique. Various theories put forward are:

a. It has been shown that gram-positive organisms

contain a substance known as magnesium ribonucleate, which gram-negative organisms lack. If this

substance is removed from gram-positive bacteria,

they will react as gram-negative organisms.

b. When iodine is applied for staining with crystal violet

or another stain of that group a compound is formed

which is insoluble in water, but soluble in alcohol

or acetone. It is said that the more permeable the

organism (i.e. the more easily water and other fluids

can pass through the cell wall), the more likely it is to

be gram-negative, since the acetone or alcohol has

easier access to the compound which it will dissolve.

c. It is also thought that the pH of the organism has at

least some influence of the reaction. Gram-positive

bacteria have a more acid cytoplasm and this is

increased by the addition of iodine. According to this

822 Concise Book of Medical Laboratory Technology: Methods and Interpretations

school of thought it is the acidity of the cytoplasm

which helps the organism to retain the stain.

Method

1. Make a thin smear of the material or culture let dry at

room temperature. Heating, should be avoided as this

interferes with the staining reaction.

2. Pass the slide through a flame once or twice or until it

feels comfortably warm on the back of the hand.

3. Place the slide on the rack and flood with the crystal

violet or gentian violet stain—stain for 1 minute.

4. Wash off the stain with Gram’s or Lugol’s iodine and

leave the slide covered with iodine for 1 minute.

5. Rinse in water.

6. Pour on acetone or alcohol till no more blue color

comes from the slide (Acetone does this more quickly

than alcohol so care should be taken not to use acetone

for a longer period). (Serous and mucoid material are

more difficult to decolorize than saline suspensions

and require a longer exposure to the decolorizing

agent).

parasites and many are pathogenic to human beings. ¾ Pseudomonadaceae → Spirillaceae ¾ Enterobacteriaceae → Bacteroidaceae ¾ Corynebacteriaceae → Bacillaceae ¾ Lactobacillaceae → Neisseriaceae

 Results

Alkaline phosphatase activity : Reddish brown

Nuclei : Green.

α-Naphthyl Acetate Method for Nonspecific Esterase

This method employs α-naphthyl acetate as the substrate,

the enzyme releases α-naphthol during the hydrolysis

of the substrate. The α-naphthol is then coupled with a

suitable diazonium salt to produce an insoluble azo dye at

the site of enzyme activity.

Preparation of Solutions

a. Substrate solutions

α-naphthyl acetate 50 mg

Acetone 5 mL

b. Buffer solution

Sodium dihydrogen orthophosphate 2.75 g

Distilled water 100 mL

c. Sodium nitrite solution

Sodium nitrite 400 mg

Distilled water 10 mL

d. Pararosanilin HCl - Stock solution

Pararosanilin hydrochloride 2 g

2 N-hydrochloric acid 50 mL

e. Distilled water.

Preparation of Incubating Medium

Solution (A) 0.25 mL

Solution (B) 7.25 mL

Solution (C) 0.8 mL

Solution (D) 0.8 mL

A volume of 0.4 mL of solution C and D is mixed together

and allowed to stand before adding to incubating medium.

The pH should be 5.8 to 6.1.

Method

a. Incubate smears at 37°C for 20 minutes.

b. Wash in water.

c. Counterstain in 2% methyl green (chloroform extracted).

d. Wash well in water.

e. Dehydrate, clear and mount.

Results

Esterase : Reddish brown

Nuclei : Green.

Diaminobenzidine Method for Peroxidase

Preparation of Incubating Solution

3:3 - diaminobenzidine tetrahydrochloride 5 mg

Tris buffer (pH 7.6) 10 mL

1% hydrogen peroxide 0.1 mL

Cytology 817

Method

a. Rinse fixed smears in distilled water.

b. Transfer to incubating solution for 5 minutes at room

temperature.

c. Rinse in 3 changes of distilled water.

d. Dehydrate clear and mount in DPX.

Result

Peroxidase—fine brownish granules.

Peroxidase Stain

Fixative—95% ethanol : 90 mL

 40% Formaldehyde (HCHO): 10 mL

Preparation of Solutions

To 30 mL of 30% ethanol, add 0.9 g of Benzidine, mix well

and then add 3 mL of zinc sulfate (3.8%). A precipitate is

formed then add 3 mg of sodium acetate and 4.5 mL of INNaOH (4 g in 100 mL). The pH should be 6. Filter and store

at room temperature.

Method

a. Fix the smear with fixative for one minute.

b. Wash gently with tap water and soak out the excess

water.

c. Take 5 mL of staining solution and 3 drops of 3%

hydrogen peroxide, mix well and cover the slide for

30 sec at 20°C.

d. Wash the slide in running tap water for 10 seconds.

e. Counterstain with dituted Giemsa’s stain for 2 minutes.

f. Wash with tap water then dried and mounted in DPX.

Results

Peroxidase positive seen as green granules.

Oil Red O Method for Lipids

It is a useful preliminary method to indicate two major

lipid classes. For detailed morphology oil red O method is

used with Mayer’s hemalum.

Preparation of Solution

The working solution is prepared an hour in advance by

mixing three parts of a stock solution of oil red O (saturated

in 99% isopropanol) with two parts of distilled water and

filtering just before use.

Method

Fix the smears in formalin vapor for 5 minutes and wash in

running tap water for 10 minutes.

a. Rinse in 60% isopropanol.

b. Stain for 15 minutes in Oil Red O.

c. Differentiate in 60% isopropanol until a delipidized

control section appears colorless.

d. Wash in water and counterstain nuclei with Mayer’s

hemalum for 3 minutes.

e. Wash well in water.

f. Rinse in distilled water and mount in glycerin jelly.

Results

Unsaturated hydrophobic lipids and mineral

Oil stain : Red

Phospholipids stain : Pink

IMMUNOPEROXIDASE STAINING FOR

C YTO AND HISTOPATHOLOGY

Introduction

This is an immunohistochemical technique, aimed at

the specific histological localization of particular tissue

antigens by immunological method. These techniques

are being increasingly used for diagnostic histology. The

immunoperoxidase test which makes use of specific antibodies conjugated with horse radish peroxidase or alkaline

phosphatase enzymes are nowadays commonly used for

histochemical detection of various antigenic markers.

Direct method: The primary antibody conjugated with

enzyme, is used to react with the antigenic sites. The combined antibody-antigen complex with enzyme is developed

with specific substrate. Tissues/smears are examined under

light microscope to detect the substrate color at the antigenic

site.

Indirect method: It is more sensitive and commonly used.

The specific primary antibody is applied directly to the

tissues/smears. This is followed by the second antibody

(antispecies specific IgG) conjugated with enzyme. The

color of the reaction is developed by using specific substrate

and examined under light microscope.

Material

Peroxidase conjugated with antirabbit immunoglobulins

(Igs), PBS (pH 7.2/0.2M), DAB (3.3 Diamino benzidine-4

HCl), normal swine serum, hydrogen peroxidase, antigen

specific antibody raised in rabbit.

Indirect Method

1. Dewaxed paraffin sections are hydrated in usual

manner. Where prefixed smears are used, these are

washed with buffered distilled water.

818 Concise Book of Medical Laboratory Technology: Methods and Interpretations 2. Endogenous peroxidase activity is blocked with a fresh

3% solution of hydrogen peroxide in distilled water for

10–30 minutes or with acid alcohol for 15 minutes.

For Cryostat, use acid alcohol or phenylhydrazine

(5 × 103M) for 15–30 minutes.

3. Wash twice with phosphate buffer saline *(pH 7.2, 0.2M).

4. Expose sections/smears to normal swine serum diluted

1:5 with buffer at 22o

C, 5–10 minutes. Excess NSS/NGS

is removed, without washing prior to stage 4.

5. Sections/smears are treated with optimally diluted

primary rabbit antiserum at 22oC, 15–30 minutes or

24–48 hours at 4oC with highly diluted antiserum.

6. Treat sections/smears with horseradish peroxidase

labeled swine/antirabbit IgG 1:20-1:100 for 15–30

minutes at 22oC.

7. Wash twice with phosphate buffer saline* (pH 7.2). The

end product is revealed with a freshly made solution

of 0.05%, 3, 3-diaminobenzidine tetrahydrochloride

(DAB) in 0.01% H2O2 in wash buffer.

Sections/smears are counterstained with a weak hematoxylin, dehydrated, cleared in xylene and mounted in

DPX for the DAB or PDP reactions (brown to dark brown).

Aqueous mountants, e.g. glycerin gelatine, are used for the

carbazole reaction (red).

Solution A

(Sodium dihydrogen orthophosphate)

NaH2PO4. 2H2O—31.2 g for 1 liter.

Solution B

Disodium hydrogen phosphate

Na2HPO4. 2H2O—31.6 g for 1 liter.

Working solution:

Solution A 70 mL + Solution B 180 mL. Make the volume

up to 1 liter and dissolve 5.7 g NaCl in a liter and filter

before use.

Immunofluorescence

Principle

It is a histochemical or cytochemical technique for in

situ detection and localization of specific intracellular

antigens. Specific antibodies conjugated with fluorescent

dyes, such as fluorescein or rhodamine, are used to trace

the specific antigenic areas on the tissue smear or section.

This can be visualized under the fluorescent microscope,

as bright purple green/red color fluorescence.

Direct Immunofluorescence

In this method, conjugated antiserum is added directly to

the tissue sections or viable cell suspension.

Indirect Immunofluorescence

It is more sensitive and commonly used. The unlabeled,

specific antibody is applied directly to the tissue smears/

sections, followed by a second antibody treatment, i.e.

antispecies specific Ig conjugated with fluorescein or

rhodamine and examined under UV-microscope. Due to

the use of second antibody, the sensitivity and specificity

of the reaction is highly improved.

Material

FITC/antihuman Igs conjugate, phosphate buffer saline

(PBS), specific antibody, glycerol buffer, fluorescent

microscope. Smear or section of tissue.

Method

1. Reasonably diluted antibody put on the antigen slide

fixed in methanol for half an hour at room temperature

in moisture chamber.

2. Wash the slide twice in PBS pH 7.2. All washes are

carried out on a magnetic stirrer.

3. Incubate slides for 30 minutes with 1:20 diluted

FITC (Fluoroscein isothiocynate) conjugated with

Igs in PBS/pH 7.2* containing 0.01%. Evans blue

as counterstain at room temperature in a moisture

chamber.

4. Wash the slide twice in PBS pH 7.2.

5. Mount the slide with 90% glycerol buffer pH 8.6.

6. Examine the slide under UV-microscope. The antigen

positive areas of the cell will show purple green

fluorescence, whereas the negative area would appear

brick red.

AUTOMATION IN CYTOLOGY

A programable cytocentrifuge from WESCOR is available,

which can be used to prepare slide from any body fluid.

With the help of cytocentrifuge sample cells, one can

safely and quickly deposit a monolayer of cells on to a

microscope slide for staining or any other processing.

This can be used on any of the body fluids, such as CSF,

urine, synovial fluid, aspirates, washes, etc. and can be

programed as per the requirements.

* PBS (Phosphate buffer saline) pH 7.2/0.2 M

* PBS preparation described earlier.

27

Microbiology and Bacteriology

C H A P T E R

In the following pages maximum stress is laid on diagnostic

bacteriology.

CLASSIFICATION

Protophyta

Schizomycetes (Bacteria and related forms)

Actinomycetales

These members form elongated cells and have a tendency

to branch, produces spores, not all are pathogenic to man

¾ Actinomycetaceae

¾ Mycobacteriaceae

¾ Nitrobacteriaceae.

Eubacteriales

This represents the true bacteria forms, classifiable as

bacilli, cocci, or vibrios. Their staining reaction can either

be gram-positive or gram-negative. Some are motile and

possess peritrichous flagella. They multiply by binary

fission. Widely distributed they can be saprophytes,

parasites and many are pathogenic to human beings.

¾ Pseudomonadaceae → Spirillaceae

¾ Enterobacteriaceae → Bacteroidaceae

¾ Corynebacteriaceae → Bacillaceae

¾ Lactobacillaceae → Neisseriaceae

¾ Micrococcaceae → Brucellaceae.

Spirochetales

These are slender, spiral shaped cells, aflagellate but move

by flexing or whirling and spinning. Stainable by special

stains only, they are free-living and include saprophytic

and parasitic forms.

¾ Treponemataceae.

Sodium naphthyl phosphate 10 mg 0.2M Tris buffer (Stock solution A) pH 10 mL Diazonium salt (fast red TR) 10 mg The final pH of the incubating medium should be between 9.0 and 9.4. The sodium naphthyl phosphate is dissolved in the buffer, the diazonium salt is added and the

 

Preparation of Stains

i. Preparation of May-Grünwald stain: 0.3 g of powdered

dye is weighed out and transferred to a conical flast of

200–250 mL capacity. A volume of 100 mL of methanol

is added and the mixture is warmed to 50°C. The flask

is then allowed to cool to room temperature and is

shaken several times during the day. After standing

for 24 hours, the solution is filtered. It is then ready

for use, no ripening being required.

ii. Preparation of solution of Giemsa powder is dissolved

in 54 mL of glycerol and after cooling, mixed with

84 mL of methanol GR and filtered.

Staining Techniques

Air-dried smears are fixed in a jar of methanol for

5 minutes. Fixed smears are stained as follows:

1. With May-Grünwald stain, freshly diluted with an

equal part of phosphate buffer for 5 minutes.

2. With Giemsa’s stain, diluted with 9 parts of phosphate

buffer for 10 to 15 minutes.

3. Washed with phosphate buffer (pH 6.8).

4. Dried in air.

5. Mounted by a rectangular cover glass using DPX as

mountant.

Papanicolaou’s Stain with EA-36

Harris’s Hematoxylin

Prepared as follows:

Hematoxylin 1 g

Absolute alcohol 10 mL

Potassium alum 20 g

Distilled water 200 mL

Mercuric oxide 0.5 g

The hematoxylin is dissolved in absolute alcohol and

potassium alum in distilled water with the aid of heat. The

two solutions are mixed together. The mixture is boiled,

removed from the flame and mercuric oxide is added bit

by bit. The flask containing the solution is then immersed

into cold water bath. After cooling, it is filtered and stored

in a colored bottle.

Orange G-6

Orange G-6 solution is prepared as follows:

Orange G 0.5 g

95% ethyl alcohol 100 mL

Phosphotungstic acid 0.015 g

Eosin Azure-36 (EA-36)

The stock solutions of light green SF yellowish (A), eosin

yellow (B), are prepared as follows:

FIGS. 26.3A TO D: Two-step technique. Line drawing summary. (A) Finger

position. (B) Collection of aspirate, (C) Particle concentration smear,

and (D) Preparation of a monolayer smear are illustrated for a righthanded operator

A B

C D

814 Concise Book of Medical Laboratory Technology: Methods and Interpretations a. Light green—(SF)

Light green SF yellowish 0.5 g

95% ethyl alcohol 100 mL

b. Eosin yellow

Eosin yellow 0.5 g

95% ethyl alcohol 100 mL

From the stock solution, the working solution of EA-36

is prepared as follows:

Light green SF yellowish (A) 45 mL

Eosin yellow (B) 45 mL

Phosphotungstic acid 0.200 g

All these solutions are kept in the refrigerator when not

in use.

Automatic Staining

Automatic stainer can process a large number of smears

with excellent results.

Autostainer: Slides held in rack are automatically rotated

around baths containing stains and other reagents. Time

schedule for staining as mentioned previously in manual

process can be obtained by calibrating a timing dial.

The reagents are renewed weekly. The instrument has

the advantage that it can easily be adapted for staining

techniques. It uses laboratory prepared reagents and it

allows for complete adaptability in staining, times. After

staining, the slides are mounted with DPX.

Staining Method

Manual process

The fixed slides are transferred directly from the fixative

into the following solutions:

1. 80% ethyl alcohol 10 dips

2. 70% ethyl alcohol 10 dips

3. 50% ethyl alcohol 10 dips

4. Distilled water 3 minutes

5. Harris’s hematoxylin 1 minute

6. Running tap water 1 minute

7. Hydrochloric acid (0.5%) 5 dips

8. Running tap water 1 minute

9. Dilute solution of lithium carbonate 1 minute

10. Running tap water 1 minute

11. 50% ethyl alcohol 10 dips

12. 70% ethyl alcohol 10 dips

13. 80% ethyl alcohol 10 dips

14. 95% ethyl alcohol 10 dips

15. Orange G-6 1 minute

16. 95% ethyl alcohol 10 dips

17. 95% ethyl alcohol 10 dips

18. EA-36 4 minutes

19. 95% ethyl alcohol 10 dips

20. 95% ethyl alcohol 10 dips

21. Absolute alcohol 4 minutes

22. Xylene 5 minutes

Slides are then mounted with DPX.

Results

Nucleus—blue

Cytoplasm of superficial cell— pink

Cytoplasm of intermediate cell—bluish green

Red blood cells—orange.

Papanicolaou’s staining schedule for automated stainer

(total staining time about 30 minutes)

Step No. Time in minutes

1. H2O 1

2. Harris’s hematoxylin 3

3. H2O 1

4. 0.1% HCl in 70% Ethanol ¼

5. H2O ¼

6. 1% NH4OH in 70% Ethanol 1

7. 95% Ethanol 1

8. 95% Ethanol 1

9. 95% Ethanol 1

10. 95% Ethanol 1

11.  OG-Modified 2

12. 95% Ethanol 1

13. 95% Ethanol 1

15.  EA-modified 3

16. 100% Ethanol 1

17. 100% Ethanol 1

18. 100% Ethanol 1

19. 100% Ethanol 1

20. Xylol 1

21. Xylol 1

Papanicolaou’s staining procedure for manual set-up

(total staining time under 7 minutes)

Regressive

method

Progressive

method

1. H2O 30 dips 30 (seconds)

2. Harris’s Hematoxylin 60 dips 30

3. H2O 10 dips 10

Contd...

Cytology 815

4. H2O 10 dips 10

5. 0.1 % HCl in 70% Ethanol 5 dips Not Necessary

6. H2O 10 dips 10

7. 1% NH4OH in 70% alcohol 30 dips 30

8. 95 % Ethanol 10 dips 10

9. 95% Ethanol 10 dips 10

10. 95% Ethanol 10 dips 10

11. OG-Modified 30 dips 30

12. 95% Ethanol 10 dips 10

13. 95% Ethanol 10 dips 10

14. 95% Ethanol 10 dips 10

15. EA-Modified 60 dips 60

16. 100% Ethanol 10 dips 10

17. 100% Ethanol 10 dips 10

18. 100% Ethanol 10 dips 10

19. 100% Ethanol 10 dips 10

20. Xylol 10 dips 30

21. Xylol 10 dips 10

22. Xylol 10 dips 10

Combined Alcian Blue—PAS Technique

for Acid and Neutral Mucins

Acid mucin and neutral mucin are clearly separated by

this technique. It is also useful as a routine demonstration

technique for the presence of any mucin. The acid mucins

are first stained with alcian blue and are not available for

PAS reaction. Only the neutral mucin is stained by PAS

reaction which follows. In this way, a good color distinction

can be made between acid and neutral mucins.

Preparation of Stains

a. Alcian blue : 1 g

3% acetic acid : 100 mL

b. Schiff’s reagent.

Method

1. Wash the fixed smears in distilled water.

2. Stain smear with alcian blue solutions for 5 minutes.

3. Wash in water.

4. Treat with 1% periodic acid for 5 minutes.

5. Rinse in distilled water.

6. Place in Schiff’s reagent for 10 minutes.

7. Wash in running water for 10 minutes.

8. Stain nuclei with hematoxylin.

9. Wash in water.

10. Dehydrate, clear and mount.

Results

Acid mucin—blue

Neutral mucin—magenta

Nuclei—pale blue.

Naphthol ASBI Phosphate Method for

Acid Phosphatase

Acid phosphatase is demonstrated by an Azodye coupling

technique, which depends upon the hydrolysis of a

substrate containing Alpha-naphthoxl phosphate. As

hydrolysis occurs, the liberated naphthol couples with

a diazotized amine and forms an insoluble colored

precipitate.

Burstone (1958), recommended naphthol ASBI phosphate

as substrate—the primary reaction product, produced by the

enzyme hydrolyzing this substrate is extremely insoluble.

Preparation of Solutions

a. Substrate solutions

Naphthol ASBI phosphate 10 mg

Dimethyl formamide 1 mL

b. Buffer solution

Sodium acetate 1.17 g

Sodium barbitone 2.94 g

Distilled water 100 mL

c. Sodium nitrite solution

Sodium nitrite 400 mg

Distilled water 10 mL

d. Pararosanilin hydrochloride stock solution

Pararosanilin hydrochloride 1 g

Distilled water 20 mL

Concentrated HCl 5 mL

Heat gently, cool to room temperature and filter

e. Distilled water preparation of incubating solution

 Solution (A) 0.5 mL

 Solution (B) 2.5 mL

Preparation of incubating solution

 Solution (A) 0.5 mL

 Solution (B) 2.5 mL

 Solution (C) 0.8 mL

 Solution (D) 0.8 mL

 Solution (E) 6 mL

Contd...

816 Concise Book of Medical Laboratory Technology: Methods and Interpretations A volume of 0.4 mL of solution C and solution D mixed

together and allowed to stand for 2 minutes before adding

to incubating solution.

The pH should be between 4.7 and 5.0 it is adjusted with

0.1 N NaOH.

Method

1. Incubate smears at 37°C for 60 minutes.

2. Wash in water.

3. Counterstain in 2% methyl green (chloroform

extracted).

4. Wash in running water.

5. Dehydrate clear and mount.

Results

Acid phosphatase activity : Red

Nuclei : Green

Alkaline Phosphatase: Azo Dye Coupling Method

Using Alpha Naphthyl Phosphate

Fixation

Formol calcium at 4oC.

Formol vapor.

Preparation of Incubating Medium

Sodium naphthyl phosphate 10 mg

0.2M Tris buffer (Stock solution A) pH 10 mL

Diazonium salt (fast red TR) 10 mg

The final pH of the incubating medium should be

between 9.0 and 9.4. The sodium naphthyl phosphate is

dissolved in the buffer, the diazonium salt is added and the

solution well mixed. The solution is then filtered and used

immediately.

Method

a. After fixation, incubate the smears at room temperature

for 10–60 minutes.

b. Wash in distilled water.

c. Counterstain in 2% methyl green (chloroform extracted).

d. Wash in running tap water.

e. Mount in glycerin jelly.