MORPHOLOGICAL STUDY-PURE CULTURES
The identification of an organism is the process of determining its species. A routine systematic study of a pure culture includes morphology, cultural characteristics nutritional requirements, biochemical characteristics, physiological properties, serology, pathogenicity, sensitivity to bacteriophages and genetic characterization.
1. MORPHOLOGICAL CHARACTERIZATION
The size, shape and arrangement of cells is determined by microscopic examination of stained smears. Examination of living organisms in a hanging drop preparation reveals motility if present. Nonmotile organisms frequently display “Brownian movement”, a motion caused by the molecular activity of the fluid. The gram staining is of great value, because organisms are classified as gram positive and gram negative.
2. CULTURAL CHARACTERISTICS :-
One of the major features of bacteria is their appearence (growth characteristics following growth on various media. Under appropriate cultural conditions organisms show characteristic type of growth, and such observations are usefu in the identification of the organism. Growth characteristics are commonly observed on the following types of culture :-
- Colonies on solid media (plate cultures).
2 Growth in liquid media
- Growth on agar slants.
- Growth in agar slabs.
- Growth in gelatin stabs.
2.1. Colony characteristics on Solid media :- (Plate Culture)
(a) Size :-
Colonies range in size from small (Pinpoints) [a millimeter in diameter to medium to large [5 to 10 mm in diameter.] Individual species may have its own characteristic diameter.
(b) Form or Shape :-
The colony may develop various forms or shapes like circular (unbroken peripheral edge) or irregular (indented peripheral edges); filaments (mycelialtype) or rhizoid (root like spreading growth). It may be punctiform and spindle-shape also. Other forms may be amoeboid, toruloid and curled
The degree to which colony growth is raised on the agar surface is described as elevation. In criteria of elevation, the colony may be flat (elevation not discernible), raised (slightly elevated), convex (shril dom-shaped clevation), pulvinate (tall dom-shaped elevation) or umbosse (raised, with elevated convex central region).
(d) Margin or edge :- The outer periphery of bacterial colony may take one of several different patterns, depending on the species. Margin may be entire (sharply defined even). lobate (marked indentations), Undulate (wavy indentations), serrate (tooth like appearance) or filamentous (threadlike).
(e) Surface texture :: The colony surfag may be smooth (Shiny, glistening), rough (dull, granular) or mucoidlimy or gummy); and sometimes, some species may have highly wrinkled surface.
This characteristic can he judged by touching a transfer needle to the colony. Some bacterial species form colonics having a butyrous (butter-like) consistency or may have viscous (rubbery) consistency. Some organisms (e.g. Actionomycetes) may form dry, brittle or powdery colonies which break up when touched with the inoculating needle.
Figure: growth characteristics of microorganism.
(g) Opacity or optical features
The degree or amount of light transmitted through colony is described a opacity. Colonies may be opaque (no light transmission) or translucen (semi-transparent with partial transmission) or watery (full transmission) .
(h)Chromogenesis or Pigmentation:
Some bacteria produce and retain water-insoluble pigments intracellularly and thus causing the colonies to become coloured or pigmented.Some bacterial species produce pigments that are water-soluble; these diffuse into the surrounding agar and stain it. For example, Pseudomonas aeruginosa develops a blue water-soluble pigment known as pyocyanin. Some pigments are only sparingly water soluble and may precipitate in the medium.
For example, Pseudomonas chlororaphis forms a pigment called chlororaphin which accumulates in the form of green crystals around the colonies.
Certain water-soluble pigments are fluorescent; i.e. the agar medium around the colonies glows white or blue-green when exposed to ultra-violet light. For example, Pseudomonas aeruginosa produce not only the nonfluorescent pigment pyocyanin but also a fluorescent pigment know as pyoverdin.
For a bacterial strain to exhibit its characteristic pigmentation, special media, incubation temperatures, or some special conditions may required. For example, Mycobacterium kansasii develops its characteris yellow pigment (P-carotene) only when the colonies are exposed to light.
Figure 1. Growth Characterisitics of Microorganisms
2.2.Growth in liquid media (Broth culture)
The growth in liquid broth media may be scanty, moderate or abundant in quantitative sense. Qualitatively, the growth may be uniformly distributed throughout the medium(Uniform turbidity). Alternatively it may be flocculent (flaky aggregates dispersed throughout the medium) or pellicle (thick padlike growth on surface of liquid medium). Even sometimes, the growth may accumulate as a sediment which may be granular of viscous.
2.3.Growth on agar-slants and in agar-stabs :-
On agar slants, bacterial species may show characteristic forms and consistency after incubation. Following types of forms are observed commonly known as “Agar stroke forms of growth”.
(a) Filiform: continuous, thread like growth with smooth edges.
(b) Echinulate :- Continuous, thread like growth with irregular edges.
(c) Beaded Non- confluent to semi-confluent colonies.
(d) Effuse: Thin, spreading growth.
(e) Arborescent Tree- like growth.
(f) Rhizoid:- Roct like growth
Similar types of growth patterns are also observed more or less in agar stabs or in gelatin stabs.
These cultural (growth) characteristics are frequently typical of certain species, but are modified under different cultural conditions.
3. NUTRITIONAL REQUIREMENTS
The orgonisms are classified on the basis of their growth and nutritional requirements. Autotrophs are capable of synthesizing their cellular constituents form simple inorganic compounds in the medium; but heterotrophs fail to grow unless one or more essential metabolites, growth factors are provided in the medium. The growth of strict autotrophs is inhibited in the presence of organic compounds while facultative autotrophs can grow. Microorganisms can be divided into many nutritional groups on the basis of their nutritional requirements.
4. BIOCHEMICAL CHARACTERISTICS :-
Microorganisms are differentiated on the basis of various enzyme-catalysed metabolic reactions, presence or absence of certain enzymes, intermediary metabolites, or end products often give valuable information in identifying and classifying the organisms. A variety of biochemical tests, performed in appropriate media and under standardized conditions of growth are available.
5. PHYSIOLOGICAL PROPERTIES:-
Physiological tests which are commonly employed in the identification of an organism includes temperature range of growth, oxygen tolerance, pH range of growth, pigment production, tolerance and requirement of salts etc.
6. SEROLOGICAL PROPERTIES:-
Microorganisms contain numerous antigenic substances which stimulates animal to produce antibodies. These antibodies react specifically with antigens.Antibodies are found in the sera of inoculated animals. The sera are then used to delect corresponding antigens. For example, a known culture is inoculated into an animal to stimulle antibody production. The serum of the inoculated aninmal is mixed with a suspension of the unknown microorganism. If there is reaction, then the unknown culture is either identical with the known culture, or at least the two organisms are very closely related in antigenic properties
7. PATHOGENICITY –
The term pathogenicity refers to the ability of a microorganism to enter a host and produce disease. The value of pathogenicity in microbial identification is limited because suitable means for testing are not always available. Seenndly, some organisms loose their pathogenicity after prolonged laboratory cultivation. Pathogenicity can be tested in the laboratory by injecting a 24-hours culture into a susceptible animal and observing the symptoms of the disease.
8. GENETIC CHARACTERIZATION:-
In recent years microorganisms have been identified and classified on the basis of genetic make-up i.c. DNA base per cent composition or DNA hybridization and homology. Organisms with similar phenotypes usually posses similar DNA base compostion. Genetic similarity can also be estimated by measuring the extent of hybridization or recombination between DNA of different organisms. The degree of hybridization or genetic homology determines the similarity or dissimilarity among species.
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