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).