Cultivation of bacteria: culture media, pure culture.

1. The purposes of cultivation:

• Medical
- etiological diagnosis of bacterial infections
- antibiotic susceptibility testing
It is necessary to obtain "pure culture" (culture made up of identical bacteria) → indispensable for
identification and antibiotic sensitivity testing
• Industrial - obtaining antigens, vaccines, products of biosynthesis, bioconversion and fermentation

2. Definitions
Culture medium = aqueous solution of nutritive substances (nutrients, growth factors).
Nutrients = substances whose solutions can cross the cytosolic membrane.

3. Characteristics of a culture medium:

- it must be nutritious.
- it must ensure the osmotic pressure (usually isotonic medium) and pH (generally 7-7,5) (similar to the
internal environment).
- it must be sterile, in order to cultivate exclusively the microorganisms found in the sample
investigated.
- it must be transparent – in order to observe the culture.
- it must allow separation of microorganisms one from another, in a microbial product.

4. Optimum conditions of growth → Physico-chemical conditions necessary for cultivation:

a. Osmotic pressure
- exceptions: halophile bacteria (grow in a medium containing > 2% NaCl)

b. pH (Hydrogen concentration)
- most bacteria require an optimum pH around neutrality (7-7,5)
- exceptions:
 alkalinophiles = grow best in a strongly alkaline solution (8,5-9) Vibrio cholerae
 acidophiles = grow in a higly acid environment (5-5.5) Lactobacillus acidophilus

c. Temperature:
- most bateria, including all parasites of man are mesophiles, with optimal growth temperature of 37 ° C
(limits 25-45 ° C)
- psychrotrophic bacteria multiply rapidly at 20-30 ° C and grow slowly at refrigerator temperature
- psychrophilic bacteria grow best at low temperature (0-20 ° C), and are important in connection with
cold storage of food and blood
- thermophiles, found in hot springs, can multiply as high as 50 - 70 ° C; they are of no medical
importance.

d. Atmosphere of incubation/ Gaseous requirements:

- obligate aerobes = grow only in the presence of oxygen
- obligate anaerobes = grow only in the absence of oxygen → anaerobic jars are used = incubation
chambers that remove oxygen and replace it with carbon dioxide and hydrogene
- facultative anaerobes = able to grow in the presence or absence of oxygen
- microaerophiles = grown only in the presence of low concentrations of O2 (5%)
- CO2-dependent bacteria / carboxiphilic bacteria = have an absolute requirement for a higher
concentration of CO2 than in air (5-10%)

5. Classification of culture media

a. Source of materials → microbiological media currently are categorized as either chemically defined
or complex.
Chemically defined.
Complex media.

b. Consistency → Microbiological media may be prepared as either liquid (broth) or solid media.
When a solid medium is prepared, the corresponding broth is solidified by the addition of agar to the
broth. "
Semi - solid = used for mobility study and preservation of bacteria.

c. Nutritional value
Simple media - support the growth of non- fastidious bacteria.
Enriched media contain the nutrients (e.g. vitamin, amino acid, blood component, or carbon source)
necessary for the growth of a wide variety of organisms, including some of the more fastidious ones.
Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic
nutrients.
Chocolate agar is enriched with heat-treated blood which turns brown and gives the medium the color
for which it is named.

d. Purpose of use
- media used for identification of bacteria - study the biochemical activity of bacteria for classification
in a genus / species.
- media used for conservation of bacteria.
- media used for isolation of bacteria → may be categorized as enrichment, selective and differential.
• Differential media allow for the separation of organisms based on some observable change in
the appearance of the media.
• Selective media are solid media that allow for the selection of particular microorganisms that
may be present in a mixed culture. Selective media usually contain a component that inhibits the
growth of competing organisms.
• Enrichment media are liquid media that contain a component that enhances the growth of the
desired organism and inhibits the growth of competing organisms → designed to increase the
numbers of desired microorganisms to a detectable level.
•

6. Cultivation and isolation of bacteria in pure culture

Streak plate technique

To cultivate= to deposit material culture medium of bacterial inoculum.

Culture = all bacteria acquired through multiplication
Colony = culture mass, visible to the naked eye the surface of solid culture medium, obtained by
multiplying a single bacterial cell or group of cells called colony-forming units / CFU.
Colony = bacterial pure culture (clone)
Character description of culture:
1.Optimal cultivation conditions: bacteria that can be cultured vs. bacteria that cannot be cultured
2. Nutritional requirements: fastidious bacteria vs. non fastidious bacteria
3. Optimum temperature of cultivation: psychrotrophic / psychrophilic / mesophilic / thermophilic;
4.Atmosphere cultivation: strictly aerobic / strictly anaerobes / facultative anaerobes / microaerophiles /
carboxiphilic bacteria;
5.PH: neutral / acidophiles bacteria / alkalinophiles bacteria;
6.Osmotic pressure: bacteria growing on isotonic media/ halophile bacteria.

Description of culture aspect on solid and liquid media

- S (smooth) and R (rough) forms of culture

Form of Liquid media Solid media

culture

S homogeneous turbidity smooth, glossy, circular margins

R grown as a coarse, leaving clear supernatant the surface is rough

irregular shape margins

Appearance of colonies: diameter, circumference, relief, surface, color, transparency, consistency,

adherence to the environment;

Changes induced by the growing to culture media:

- Hemolysis on agar-blood: alpha / beta / gamma

- Differential media (lactose positive, lactose negative)
- Pigment production run in the medium
- Production of a particular odor.