PHYSIOLOGY OF MICROORGANISMS

PHYSIOLOGY OF MICROORGANISMS
Methods of laboratory diagnosis
Methods of the bacterial cultivation
Identification of bacteria
Bacterial metabolism
Media for bacterial growth
Sterilization

Methods of laboratory diagnosis
Bacterioscopical (Microscopic examination)
Bacteriological (Culture method)
Detection sensitivity of bacteria to antibiotics
Serological
Biological
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.

Methods of the cultivation
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.

Methods of the cultivation
Stroke culture. It is made in tubes containing agar slopes. It is used for providing a pure growth of bacterium (for slide agglutination).

Methods of the cultivation
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

Methods of the cultivation
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

Identification of bacteria
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

Identification of bacteria
Culture character: Growth requirement, colonial characteristics in culture
Colony morphology descriptions

Colony morphology

Identification of bacteria
Metabolism: Capacity to form pigment and power of haemolysis is help for classification of bacteria
Staphylococcus
aureus
Micrococcus
roseus
Studying of
haemolysis

Colonies and pigments of bacteria

Identification of bacteria
Biochemical reactions: The more important and widely used tests are as under:
a) Sugar fermentation

Identification of bacteria
b) Indole production
c) Hydrogen sulfide production

Identification of bacteria
d) Other tests: Citrate utilization; Nitrate reduction; Methyl red test; Urease test; Catalase test; Oxidase reactions.
Positive Catalase Test
on Staphylococcus aureus
Negative Catalase Test
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

Identification of bacteria
Antigenic analysis: by using specific sera we can identify microorganism by agglutination reaction (Serologic Typing of Shigella).
No clumping of the bacteria
is seen in this circle
The clumping of the bacteria
is seen in this circle

Identification of bacteria
Bacteriophage typing: Phage brings about lysis of susceptible bacterial cells.

Identification of bacteria
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%) – bound water and free water
Dry matter (15-25%) – organic part and mineral substances (inorganic part)

Dry matter
Organic part
proteins – 50-80%
nucleic acid – 10-30%
carbohydrates – 12-18%
polysaccharides – 3-5%
lipids – 5-10%.
Inorganic part
nitrogen (N), carbon (C), oxygen (O), hydrogen (H), phosphorus (P), sulfur (S), sodium (Na), magnesium (Mg), potassium (K), calcium (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

Sterilization