Physiology of Microorganisms-2012

PHYSIOLOGY OF MICROORGANISMS
1. Methods of
laboratory diagnosis
2. Methods of the
bacterial cultivation
3. Identification of
bacteria
4. Bacterial metabolism
5. Media for bacterial
growth
6. Sterilization
Methods of laboratory diagnosis
1. Bacterioscopical (Microscopic examination)
2. Bacteriological (Culture method)
3. Detection sensitivity of bacteria to antibiotics
4. Serological
5. Biological
6. DNA-technology test (PCR)
In the clinical laboratory it is necessary:
• isolate bacteria in pure culture;
• obtain sufficient growth of bacteria for
demonstration their properties such as study of
morphological, cultural, biochemical, antigenic
and pathogenic properties, bacteriophage and
bacteriocin susceptibility;
• determine a sensitivity
to antibiotics.
Methods of the cultivation
• Streak culture (surface plating). The inoculum is
spreaded thinly over the plate of a culture media in
series of parallel lines in different segment of the
plate. On inoculation well separated colonies are
obtained over the final series of streaks.
• Lawn or carpet culture. Lawn cultures are prepared
by flooding the surface or plate with suspension of
bacteria. It provides uniform surface growth of bacteria.
It is useful for bacteriophage typing and antibiotic
sensitivity test.
• Stroke culture. It is made in tubes containing
agar slopes. It is used for providing a pure
growth of bacterium (for slide agglutination).
• Stab culture. It is prepared by puncturing with
charged long straight wire (loop). Stab culture is
employed mainly for cultivation of anaerobes.
Pure plate culture
• Liquid culture in a tube, bottle or flask may be
inoculated by touching with a charged loop
Identification of bacteria
• Microscopic examination: It helps to detect a shape, a
size and an arrangement of microorganisms
• Staining reaction: On gram staining we can have two
groups of microorganisms: Gram positive and Gram
negative.
E. coli, Gram negative (A), Staphylococcus epidermidis,

Gram positive (B) and Bacillus cereus, Gram positive
• Motility: Some bacteria can move (Salmonella, E.
coli, Proteus, Pseudomonas, Vibrions, Clostridia).
Dark ground microscopy and Phase contrast
microscopy, special culture media use for
studying motility of bacteria
Special stain for flagella
• Culture character: Growth requirement, colonial
characteristics in culture
Colony morphology descriptions

Colony morphology
• Metabolism: Capacity to form pigment and power of
haemolysis is help for classification of bacteria
Staphylococcus
aureus
Micrococcus Studying of
roseus haemolysis
Colonies and pigments of bacteria
• Biochemical reactions: The more important
and widely used tests are as under:
• a) Sugar fermentation
• b) Indole production
• c) Hydrogen sulfide production
• d) Other tests: Citrate utilization; Nitrate reduction;
Methyl red test; Urease test; Catalase test; Oxidase
reactions.
Positive Catalase Test Negative Catalase Test

on Staphylococcus aureus on Streptococcus lactis
API-20 "Bio Merieux" (France) strip test
• Twenty tests are performed on this strip by a
simple procedure, saving time and money.
Escherichia
coli
Enterobacter
agglomerans
Edwardsiella
hoshinae
• Antigenic analysis: by using specific sera we
can identify microorganism by agglutination
reaction (Serologic Typing of Shigella).
The clumping of the bacteria No clumping of the bacteria

is seen in this circle is seen in this circle
• Bacteriophage typing: Phage brings about lysis of
susceptible bacterial cells.
• Pathogenicity: For pathogenicity test commonly
used laboratory animal models are guinea pig,
rabbit, rat and mouse.
• Resistance to antibiotics and other agents
• Metabolism is the
process of building up
chemical compounds in
the cell and their breaking
down during activity to
receive the required
energy and the building
elements.
• Metabolism comprises of
anabolism (assimilation)
and catabolism
(dissimilation)
Chemically, bacteria consist of:
• Water (75-85%) – • Dry matter (15-
bound water and 25%) – organic
free water part and mineral
substances
(inorganic part)
Dry matter
• Inorganic part
• Organic part
• nitrogen (N), carbon (C),
• proteins – 50-80%
oxygen (O), hydrogen
• nucleic acid – 10-30% (H), phosphorus (P),
• carbohydrates – 12-18% sulfur (S), sodium (Na),
magnesium (Mg),
• polysaccharides – 3-5%
potassium (K), calcium
• lipids – 5-10%. (Ca), iron (Fe) and other
Classification of bacteria based
on nutritional requirements
• Autotrophs are free-living, most of which can
use carbon dioxide as their carbon source. The
energy can be obtained from:
• sunlight – protoautotrophs (get energy from
photochemical reactions)
• inorganic compounds, by oxidation –
chemoautotrophs (get energy from chemical
reactions)
• Heterotrophs are generally parasitic bacteria,
requiring more complex organic compounds
than carbon dioxide, e.g. sugars, as their source
of carbon and energy.
The basic requirements of
culture media
• energy source;
• carbon source;
• nitrogen source;
• salts like sulphates,
phosphates, chlorides and
carbonates of sodium,
potassium, magnesium,
ferric, calcium and trace
elements, like copper, etc.;
• satisfactory pH 7.2-7.6;
• growth factor like vitamins.
Classification of Media
• A. On the basic of consistency:
• Solid media
• Liquid media
• Semisolid media
Classification of Media
• Nutrient media can be subdivided:
• 1. Simple media - meat-peptone broth (MPB),
meat-peptone agar (MPA)
• 2. Synthetic media
• 3. Complex media
• 4. Special media: a) Enriched media; b)
Enrichment media; c) Selective media; d)
Indicator and differential media; e) Sugar
media; f) Transport media.
• 5. Aerobic and anaerobic media -
according to type of respiration bacteria
subdivided into 4 groups:
• Obligate aerobes (Brucella)
• Microaerophils (H.pylori)
• Obligate Anaerobes (C.tetani)
• Facultative Anaerobes (E.coli)
Sterilization
TREAT- TEMPE- EFFECTIVENESS
MENT RATURE
Incineration >500 C Vaporizes organic material on
nonflammable surfaces but may destroy
many substances in the process.
Boiling 100 C Thirty minutes of boiling kills vegetative

forms of bacteria but may not kill
bacterial endospores. There are also
toxins that are not inactivated at 100C.
Intermittent 100 C Three 30-minute intervals of boiling,

boiling followed by periods of cooling kills
bacterial endospores.
Autoclave 121 C for 15 Kills all forms of life including bacterial
(steam minutes at endospores. The substance being
under 15 p.s.i. sterilized must be maintained at the
pressure) effective temperature for the entire time.
Sterilization
Dry heat 160 C for 2 Used for materials that must remain dry.
(hot air hours Good for glassware, metal, but not most
oven) plastic or rubber items.
Dry heat 170 C for 1 Same as above. Note that increasing

(hot air hour the temperature by 10 C shortens the
oven) sterilizing time by 50 %.
Pasteurizati 63-66 C for Kills most vegetative bacterial cells,

on (batch 30 minutes including pathogens such as
method) streptococci, staphylococci and
Mycobacterium tuberculosis.
Pasteurizati 72 C for 15 Effect on bacterial cells is similar to

on (flash seconds batch method. For milk, this method has
method) fewer undesirable effects on quality or
taste.
AUTOCLAVES (1) AND HOT AIR OVEN (2)

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PHYSIOLOGY OF MICROORGANISMS

E. coli, Gram negative (A), Staphylococcus epidermidis,

Colony morphology descriptions

Positive Catalase Test Negative Catalase Test

The clumping of the bacteria No clumping of the bacteria

Boiling 100 C Thirty minutes of boiling kills vegetative

Intermittent 100 C Three 30-minute intervals of boiling,

Dry heat 170 C for 1 Same as above. Note that increasing

Pasteurizati 63-66 C for Kills most vegetative bacterial cells,

Pasteurizati 72 C for 15 Effect on bacterial cells is similar to

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