Microtatobiotes (The smallest living things)
Rickettsiales: Most of these are intracellular pathogens,
and filtrable forms and need special methods of culture.
These are the Molds and Yeasts.
Like other living cells, all bacteria possess the cell membrane,
cytoplasm and a nucleus. Special characteristics are seen
This is a protective outer covering layer possessed by some
These assist in locomotion, their arrangement may vary.
Under unfavorable conditions for growth sporing occurs.
Spores are non-reproductive. Upon return of favorable
distinctive placement within the cell. They may be central
subterminal or terminal. Knowing their location assists in
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
with Sudan black. Polysaccharide granules stainable by
iodine (like glycogen or starch) can be seen in cytoplasm
a. Cocci in cluster—Staphylococci
b. Cocci in chain—Streptococci
d. Cocci in groups of four—Tetrad
e. Cocci in groups of eight—Sarcino.
These are cylindrical or rod-shaped organisms. They can
a. Length of the cell equalling its breadth, called
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
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
Bacterial reproduction occurs by a simple process of
Bacterial physiology and biochemistry are studied by
observing cultures grown in the laboratory on artificially
concentration, oxygen, carbon dioxide, temperature and
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.
For bacterial growth moisture is essential. Drying in the air
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
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.
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%).
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
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.
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).
These injurious products of bacteria are of two types:
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).
a. Proteolytic enzymes: An enzyme responsible for
decomposition of dead animal and vegetable matter
b. Coagulase: This is often demonstrated during the
study of biochemical properties of some pathogenic
c. Amylase: This enzyme is capable of splitting starch and
is not much used in the study of bacteria.
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.
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
2. It must regularly be isolated and it must be grown in
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.
Saturated solution of methylene blue in alcohol 30 mL.
Potassium hydroxide 0.01% in distilled water—100 mL.
Stain for 3 minutes after making and fixing the smear. This
stain does not readily overstain.
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.
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.
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.
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
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