The organism ferments sugar and produces acid and, in certain groups, gas. Acid production is indicated by a color change of the medium, due to inclusion of a pH indicator. Gas production is shown by placing a small Durham’s tube upside down in the medium during its production. Before

 

Negative Staining

Negative Staining is a technique by which organisms

remain unstained against a dark background.

India Ink Method

A small quantity of India ink 10% nigrosin is mixed with

the material on a slide. A smear is made by means of

another slide and the preparation is allowed to dry. The

smear is examined and the spirochetes are seen as clear

transparent objects against a dark brown background.

Capsules may also be demonstrated by this method.

Motility of Bacteria

Hanging Drop Method

This method is used to observe the morphology but also

demonstrates the motility of organisms. A special slide

with a concave center is used or else a ring of plasticine

can be placed on the slide. A drop of the culture of

bacterial suspension is placed on a coverslip. Vaseline is

placed near the concave area of the slide approximately

the corners of the coverslip. The slide is placed over the

coverslip so that the drop of culture is directly under the

concave area and the Vaseline adheres to the coverslip.

The slide is then quickly inverted and placed under

the microscope. Motile organisms will be seen darting

through the medium in which they are suspended. Motility

should be differentiated from Brownian movement which

is caused by bombardment of the molecules of the fluid.

In motility, the organisms move in a definite direction,

whereas in Brownian movement they show no direction.

CULTURE

Four factors are to be taken into account

1. Media providing optimum growth

2. Temperature

3. Atmosphere

4. Cultural characteristics, e.g. size, shape and pigmentation of colonies.

Media

Media can be (a) basic (b) enrichment (c) selective, and

(d) indicator media.

1. Basic Media

These contain the necessary constituents for growth—

meat extract, peptone and salt, and these are nutrient

broth (liquid) or nutrient agar (solid). Many organisms

would grow on these types of media and need no other

factors.

2. Enrichment Media

These are used for organisms, which need an additional

source of nutrition. This can be done by adding blood

or serum to the nutrient agar or broth. An enrichment

medium used for growth of the Mycobacterium tuberculosis

contains eggs.

3. Differential and Selective Media

These media by virtue of their chemical composition

inhibit the growth of some organisms while at the same

time support the growth of others. Examples: eosin methylene blue agar and MacConkey agar contain lactose and

dye or an indicator in the decolorized state. Bacteria,

which ferment lactose with the production of acid will

produce red color or colonies with metallic sheen differentiates the lactose fermenting coliform bacilli from colonies

of lactose non-fermenting organisms. Some media, which

are used are also highly selective in their action on other

organisms. Such media as SS agar, deoxycholate citrate

agar and bismuth sulfite agar will inhibit the growth of

the majority of coliform bacilli along with many strains of

proteus and will permit the successful isolation of enteric

pathogens. Tellurite glycerin agar and mannitol salt agar

are selective media for the isolation of coagulase positive

Staphylococcus from material containing other organisms.

Phenyl-ethyl-alcohol agar is a selective medium for the

isolation of gram-positive cocci in specimens or cultures

contaminated with gram-negative organisms particularly

proteus. Infusion agar containing potassium tellurite

and blood/serum inhibits the growth of normal throat

commensals and encourages the growth of C. diphtheriae.

Some medias make use of the selective antimicrobial

activity of some antibiotics and are useful for isolating

certain pathogenic organisms from material containing mixed flora. Sabouraud dextrose agar containing

cycloheximide and chloramphenicol will support the

growth of dermatophytes and most fungi, while markedly

inhibiting the growth of many saprophytic fungi and

bacteria.

4. Indicator Media

These are largely used for biochemical reactions. The

most common example is sugar media containing various

carbohydrates such as glucose, lactose, maltose, etc.

Christensen’s urea medium is used mainly in the iden-

826 Concise Book of Medical Laboratory Technology: Methods and Interpretations

tification of Proteus, which has the ability to hydrolyze

the urea, and consequently because of the presence of

phenolphthalein in the medium, a change of color is

produced.

Temperature

Most bacteria, pathogenic in humans, give optimum

growth when incubated at body temperature, i.e.

37°C. Some saprophytes, however, grow best at lower

temperatures, even as low as 4oC (cryophilic) and others at

high temperatures. The latter are known as thermophilic

bacteria and are used in testing effectiveness of sterilization

techniques.

Atmosphere

Most organisms need oxygen for growth and are incubated

in normal atmospheric conditions. Some pathogens, e.g.

tetanus bacilli, will grow only in the absence of oxygen. This

is achieved by using McIntosh and Fildes’ jar, a thick metal or

glass jar with a metal lid which can be clamped down tightly

by bolts. On this lid are 2 holes-one an air inlet and the other

an outlet. There are also 2 electric terminals. On the underside

of the lid is a piece of asbestos saturated with palladium and

covered by wire gauze. This is connected to the terminals,

and acts as a catalyst in combining any oxygen still present

after evacuation of the jar with the hydrogen, which is passed

into the jar.

The method is given below.

1. Keep the plates upside down in the jar.

2. Place in the jar an indicator—equal parts of 10% NaOH,

6% glucose and 0.5% methylene blue, boiled until the

solution becomes colorless. It should remain colorless

throughout incubation. If it turns to its original blue

color during incubation, complete anaerobiosis

(oxygenless state) has not been achieved.

3. Tightly clamp down the lid.

4. Open the air outlet valve and close the air inlet valve.

5. Attach the apparatus to an exhaust pump, and slowly

evacuate the jar (If a glass jar is used, it should be

evacuated while enclosed in a padded box to avoid

danger of explosion).

6. Allow hydrogen obtained from hydrogen cylinders or

Kipp’s apparatus in through the inlet valve after closing

the outlet valve.

7. Attach the terminals to the main current and leave for

20 minutes. This heats the palladiumized asbestos to

assist the combination of hydrogen with any remaining

oxygen.

8. Allow a little more hydrogen in via the inlet valve.

9. Put the jar in the incubator overnight. The present day

McIntosh-Filde’s jars have room temperature catalysts

and need no electrical charge. They are left at room

temperature for 15–30 minutes before allowing more

hydrogen into the jar. There are other, less complicated

methods of achieving anaerobiosis (i.e. an oxygenless

state), e.g.

a. Boil a tube of nutrient broth and layer over it

sterile Vaseline. The boiling removes the oxygen

and the Vaseline prevents more entering as the

broth cools. The tube is inoculated using a sterile

Pasteur pipette.

b. A sterile iron nail placed in glucose broth which

has been treated as in method (1), will maintain

anaerobic conditions for some time.

c. Robertson’s cooked meat medium and Brewer’s

thioglycollate broth are frequently used in the

culture of anaerobic organisms.

Some organisms are not anaerobic, but do grow better

when the amount of oxygen has been reduced. One simple

technique is to place the plates in a tin or wide mouthed

bottle with a tight fitting lid. A candle is lit inside the

container and the lid replaced firmly. The candle flame

will use off the oxygen and give an atmosphere of 5–10%

CO2. The container is placed in the incubator.

Cultural Characteristics

Bacteria grown artificially (in vitro) on agar plates are

described as colonies. These colonies vary in size, shape,

pigment production, and hemolysis on blood agar

depending on the type of media.

Colonies are described as:

1. Shape

Circular, regular, radiating or rhizard.

2. Surface

Smooth, rough, fine, granular shiny, dull, etc.

3. Size

Usually colonies are 2–3 mm in diameter, smaller ones

may be less than 1 mm.

4. Contiguity

Colonies may be discrete or swarming.

5. Consistency

May be mucoid, tenacious dry or adherent to the medium.

6. Pigmentation

Some organisms produce pigmented colonies (Staphylococci, Pseudomonas).

Microbiology and Bacteriology 827

7. Opacity

On nutrient agar they may be transparent, translucent or

opaque.

8. Elevation

Colonies may be raised, low convex, umbilicated or dome

shaped.

9. Media Changes

Colonial growth may bring about color changes in the

media themselves, e.g. hemolysis on blood agar by

hemolytic streptococci. With Pseudomonas, the green

pigment produced may diffuse into the medium.

Biochemical Reactions

Organisms that are alike in microscopic and cultural

characteristic are often differentiated by their reactions in

various biochemical tests.

1. Sugar Fermentation

Specific carbohydrate fermentation is a property of some

organisms when grown in sugar media. Sugars most

frequently employed are glucose, sucrose, lactose, mannite,

maltose and dulcite. Usually, these are incorporated into

peptone water, but for the more delicate organisms, Hiss’s

serum water must be used. Meningococci and gonococci

will only react in solid serum-sugar media. Each sugar

medium has a colored stopper and a set ‘color scheme’

may be established for the following sugars.

Glucose (green), Lactose (red), Sucrose (blue), Mannite

(mauve), Maltose (blue and white), Dulcite (pink).

The organism ferments sugar and produces acid and, in

certain groups, gas. Acid production is indicated by a color

change of the medium, due to inclusion of a pH indicator.

Gas production is shown by placing a small Durham’s tube

upside down in the medium during its production. Before

inoculating the medium the tube should be completely

filled with the medium. If gas is produced, small bubbles

of gas will be seen in the inverted tube.

2. Other Biochemical Tests

Organisms may further be identified biochemically by

their production of indole, change in pH (as shown by

the methyl red test), by their utilization of citrate and

by another test called the Voges-Proskauer reaction.

These 4 tests are especially useful in the differentiation of

intestinal pathogens.

Loeffler’s Flagella Mordant Tannic acid 20% aqueous 100 mL Ferrous sulfate crystals 20 g Loeffler’s Flagella Stain 10% alcoholic solution of Basic fuchsin 10 mL Distilled water 40 mL Microbiology and Bacteriology 825

 

7. Rinse in water again.

8. Stain with one of the following counterstains: Safranin,

Neutral red, or 1:10 Carbol fuchsin.

9. Rinse in water and allow it to dry by standing it

vertically, or by blotting it with filter paper.

Results

Because the gram-positive organisms retain the crystal

violet after decolorization, they appear dark blue in color.

The gram-negative organisms are decolorized and take up

the counterstain and therefore, appear pink in color.

Reagents

1. Crystal violet—0.5% solution in distilled water.

2. Iodine-(Lugol’s)—10 g iodine, 20 g potassium iodide

in 1000 mL of distilled water. Dissolve the potassium

iodide in 250 mL water and then add 10 g of iodine.

When dissolved make up to 1000 mL with distilled

water (This solution is three times stronger than Gram’s

iodine and is preferable).

3. Acetone.

4. Counterstain.

a. 1 g Neutral red

2 mL 1% Acetic acid

Distilled water to make 1000 mL

b. Safranin

1.7 g safranin

50 mL alcohol

Distilled water to make 500 mL

c. Dilute carbolfuchsin

1:10 dilution of strong carbol fuchsin.

Ziehl-Neelsen Stain

This stain is another method of categorizing certain

bacteria, depending on their ability to resist decolorization

by acid and alcohol. A very strong stain is used, basic

fuchsin in a phenol solution and heat is applied in order

that the stain can penetrate the waxy covering certain

bacteria.

Method

a. Make a smear of the material and allow to dry at room

temperature.

b. Flood the whole slide with strong carbol fuchsin and

heat gently underneath the slide until steam is seen

rising from the slide (Do not overheat, avoid boiling

of the stain).

c. Rinse in water and flood the slide with 25% sulfuric

acid. Leave this until the smear is pale pink in color.

d. Rinse in water and pour on alcohol for a few minutes.

e. Counterstain with malachite green, methylene blue or

picric acid.

f. Dry by standing the slide vertically—do not blot dry as

the tubercle organisms may get attached to the paper

and later may get transferred to another slide.

Results

The tubercle bacillus resists decolorizing by acid and

alcohol (i.e. it is both acid and alcohol fast) it will remain

bright red while all other organisms and material will take

on the color of the counterstain.

Troubleshooting (AFB-Staining)

Problem: False positive results

Possible causes Solutions

1. Sputum collected without washing the Patient should wash their mouth thoroughly while procuring sputum to minimize

mouth or in an unclean container specimen contamination with food particles, mouthwash or oral drugs.

 Patient should be asked to collect sputum in a clean container free from waxes,

inorganic materials and artefacts

 Artefacts may be mistaken for acid-fast bacilli

2. Oil immersion lens is not cleaned during Oil immersion lens should be cleaned after every observation to avoid contaminaobservation of slides ting other slides

Microbiology and Bacteriology 823

3. Contaminated water with acid-fast bacteria used Use clean, non-contaminated water for washing of slides during staining

for washing of slides during staining procedure

4. Carbol fuchsin held on the slide for long Allow the stain to stand for exactly 5 minutes with the application of heat. While

time with improper heating heating, ensure that the stain is not boiled. Heat only till steam starts rising

 from the slide. Leave the slide to cool for 2 minutes before decolorizing

5. Less decolorization done for thick smear The number of times for decolorization is to be increased for thick smears.

preparation Decolorization is to be carried out till the pink color disappears and the smear

appears colorless

Problem: False negative results

Possible causes Solutions

1. Sputum collected inadequately, i.e. only Thick yellowish green mucoid sputum collected from an early morning deep

the saliva productive cough should be used as a specimen

2. Failure to select suitable sputum portion Select a suitable portion, i.e. thick yellowish green mucoid portion of the sputum

for smear preparation preparation

3. Longer time duration given for counter- Allow the counterstain B to stain for 15–20 seconds before washing.

stain, i.e. more than 30 seconds

4. Inadequate examination of the smear Smear should be examined thoroughly from one edge to the other covering

100 fields or more

Modified Ziehl-Neelsen’s Stain

Used for leprosy where the bacteria are less acid fast. The

method is as mentioned above except that 5% Sulfuric acid

is used instead of 25%.

Reagents

Carbol fuchsin:

Basic fuchsin 10 g

Alcohol—100 mL

5% aqueous phenol—1000 mL.

Decolorizing agents:

25% sulfuric acid, or

5% sulfuric acid (for M. leprae) or

Acid-alcohol 3% HCI in alcohol.

Counterstains:

Loeffler’s methylene blue or

Malachite green—0.05% aqueous solution or

Methylene blue—0.1% aqueous solution or

Picric acid-saturated aqueous solution.

Special Stains

Used to stain flagella, capsules, spores and granules.

Stains for Diphtheria Bacillus

Ponder’s Stain

Toluidine blue 0.02 g

Glacial acetic acid 1 mL

Absolute alcohol 2 mL

Distilled water to make 100 mL.

Method

Spread the stain on the film for 1 minute and wash in tap water.

Result

Dark blue granules in pale blue bacillus.

Albert’s Stain

Solution I Toluidine blue 0.15 g

Malachite green 0.2 g

Glacial acetic acid 1 mL

95% alcohol 2 mL

Distilled water 100 mL

Dissolve the dyes in alcohol and add to the water and

acetic acid. Let stand for one day and filter.

Solution II Iodine 2 g

Potassium iodide 3 g

Distilled water 300 mL

Method

Apply solution I for 3 to 5 minutes, wash in tap water, blot

and dry. Apply solution II for one minute, wash, blot and

dry.

Result

The granules stain bluish black, the cytoplasm green and

other organism light green.

Modified Neisser’s Method

Neisser’s methylene blue

Methylene blue 1 g

Ethyl alcohol (95%) 50 mL

Glacial acetic acid 50 mL

Distilled water 1000 mL.

Method

a. Stain with Neisser’s methylene blue for 3 minutes.

b. Wash off with iodine solution used in Gram’s method

and leave some solution on the slide for 1 minute.

824 Concise Book of Medical Laboratory Technology: Methods and Interpretations

c. Wash in water and counterstain with neutral red

solution used in Gram’s method for 3 minutes.

d. Wash in water and dry.

Result

The bacilli show deep blue granules, the remainder of the

organism assumes a pink color.

Staining of Capsules

Hiss’s Method

a. Saturated alcoholic solution of basic fuchsin or gentian

violet 1 part to distilled water 19 parts

b. 20% aqueous copper sulfate solution.

Method

Place a few drops of solution (a) on slide. Heat to steaming

and leave on slide 30 seconds.

Wash off with solution (b).

Result

Capsule appears as faint blue halo around dark purple cell.

India Ink Method

The capsule is seen as a clear halo around the microorganism against the black background. This method may

be used for demonstrating cryptococci.

Staining of Spores

Modified Ziehl-Neelsen Method

1. Ziehl-Neelsen carbol fuchsin

2. Sulfuric acid 0.5% or methylated spirit

3. Loeffler’s methylene blue.

Method

1. Stain with carbol fuchsin for 5–10 minutes, heating

until steam rises.

2. Wash in tap water.

3. Decolorize with 0.5% sulfuric acid or methylated spirit.

If the acid is stronger than 1%, spores of many bacilli

are decolorized.

4. Wash in tap water. Now the smear is examined and if

both bacilli and spores are red, it is decolorized again.

If the spores alone are stained, it is counterstained. Let

the counterstain to act for 2 minutes. Wash in water,

blot and dry.

Result

The spores are stained bright red and the bacilli blue.

Staining of Spirochetes

Fontana’s Method

a. Fixative Acetic acid 1 mL

 Formalin 2 mL

 Distilled water 100 mL

b. Mordant Phenol 1 g

 Tannic acid 5 g

 Distilled water 100 g

c. Ammoniated silver nitrate

 Add 10% ammonia to 0.5% solution of silver nitrate in

distilled water until the precipitate formed just dissolves.

Now add more silver nitrate solution drop by drop until

the precipitate returns and does not redissolve.

Method

1. Treat the film 3 times, 30 seconds each time, with the

fixative.

2. Wash off the fixative with absolute alcohol to act for

3 minutes.

3. Drain off the excess of alcohol and carefully burn off

the remainder until the film is dry.

4. Pour on the mordant, heating till steam rises and allow

to act for 30 seconds.

5. Wash well in distilled water and again dry the slide.

6. Treat with ammoniated silver nitrate, heating till steam

rises, for half minute, when the film becomes brown

in color.

7. Wash well in distilled water and dry.

Result

The spirochetes are stained brownish black on a brownish

yellow background.

Staining of Fungi

Lactophenol Cotton Blue

Phenol crystals 20 g

Lactic acid 20 mL

Glycerol 40 mL

Cotton blue/methylene blue 0.05 g

Distilled water 20 mL

Dissolve the phenol crystals in the liquids by gently

heating and then add the dye.

Take a portion from the fungal growth and place it on

a drop of lactophenol cotton blue on a slide. Then place a

cover slip over the drop and press gently. Blot to remove

excess stain. Seal with varnish or nail polish.

Staining of Flagella

Loeffler’s Method

Loeffler’s Flagella Mordant

Tannic acid 20% aqueous 100 mL

Ferrous sulfate crystals 20 g

Loeffler’s Flagella Stain

10% alcoholic solution of

Basic fuchsin 10 mL

Distilled water 40 mL

Microbiology and Bacteriology 825

Method

Flood the smear with the mordant for 5 minutes. Wash

with distilled water. Add heated Loeffler’s flagella stain

and allow to act for 3 minutes. Wash with distilled water

and dry (The slides should be very clean).

Result

Organisms stain red and flagella pink.

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).