2. Cultivation:
the process of growing microorganisms in culture by
taking bacteria from the infection site in (vivo) and
grow them in artificial enveromintal in the laboratory
(in vitro). Isolates of new bacteria from the
environment must experiment with many nutrients and
growth conditions .
3. To culture the newly isolated bacteria in the lab. this need to:
1-Use of Media: (Solid , liquid and semi solid media )
A- solid media: (2% agar) includes:
1- General media :support the growth of a broad range of organisms.
General growth media usually have complex constituents. Examples of General
Growth media ( Trypticase-soy and Nutrient media).
2-Enriched media :is designed to support the growth of organisms with unusual
growth requirements. Such organisms are referred to as fastidious. Special
supplements added will vary dependent upon the particular requirements of the fastidious
organism .An example of an enriched medium is T-soy medium supplemented with sheep
blood (when agar is added to this medium it is commonly called “blood agar”). Most
commonly used medium. 5-10% defibrinated sheep or horse blood is added to melted
agar at 45-50°C. Blood acts as an enrichment material and also as an indicator .
Chocolate agar is enriched with heat-treated blood (40–45 °C), which turns brown and gives
the medium the color for which it is named. It is used for culture of pneumococcus,
gonococcus, meningococcus and Haemophilus. Heating the blood inactivates inhibitor of
growths.
4. 3-Selective media: will permit the growth of one type of bacteria while preventing
the growth of other types. This will facilitate the isolation of a desired species.
-MacConkey’s medium is selective medium for Gram-negative. It contains bile salts
and crystal violet, these will inhibit the growth of Gram-positive organisms.
- mannitol salt agar which inhibits the growth of salt intolerant organisms. It is
consider selective medium for Staphylococcus Spp.
-Eosin methylene blue (EMB) contains dyes that are toxic for Gram positive bacteria
and bile salt which is toxic for Gram negative bacteria other than coliforms. EMB is
the selective and differential medium for coliforms.
4-Differential media: Differential media or (indicator media) distinguish one
microorganism type from another growing on the same media. This type of media
uses the biochemical characteristics of a microorganism growing in the presence of
specific nutrients or indicators (such as neutral red, phenol red, eosin , or methylene
blue) added to the medium to visibly indicate the defining characteristics of a
microorganism.
5.
6.
7. Advantage of Differential media
1-Some bacterial colonies growing on blood agar are differentiated by hemolysis
patterns
1-Greening color - alpha hemolysis ex (S. pneumoniae)
2- Clearing - beta hemolysis ex(Streptococcus pyogenes , S. agalactiae )
3-no hemolysis -gamma
2- Using MacConkey’s medium, lactose fermenting and lactose non-fermenting
bacteria can be distinguished. Organisms that are able to ferment lactose will
produce an acid end-product that causes a change in the pH of the surrounding
media. A pH indicator (neutral red) , is present in the media and changes from
yellow to red in an acidic environment. Lactose fermenting bacteria growing on
MacConkey’s media will appear pink whereas non-lactose fermenters will be
yellow.
3- Mannitol salt agar allows discrimination among salt tolerant organisms. Those
that ferment mannitol will produce acid turning the pH indicator (phenol red) to a
yellow color. Those that cannot ferment mannitol will leave the media as red color
of phenol red at nuetral pH. Example Staph . aureus (yellow),while Staph.
epidermides (red).
10. B- liquid media (no agar)
Organisms can be grown in liquid media (broth). For example,Nutrient media is referred to as Nutrient
Broth when in the liquid form, and Nutrient Agar when in the solid form. Example (Nutrient Broth ,
Brain heart infusion).
3-Semi solid agar (0.5% agar) : media used for bacterial motility test.
2-Growth environmental Conditions
To grow bacteria in the lab, environmental conditions , as well as nutrients, must be
Considered . Bacteria may be isolated from a variety of environments. For cultivation of bacteria in the
lab, the conditions of the environments must be mimicked.
A-Temperature:some microorganisms that grow optimally between 20oC and 45 oC, suitable temp to
grow in human body is 37 oC .
B-Salt tolerance:Some bacteria require relatively high concentrations of salt for growth (10-20%); these
organisms are called halophiles. An example is Staphylococcus aureus. S.aureus is found on skin, which
often has a high salt concentration (10% NaCl).
C-Oxygen An organism that requires oxygen for growth or growth cannot occur in the presence of
oxygen or may be the growing under either aerobic or anaerobic conditions .The requirement for
oxygen relates to the energy metabolism of an organism and the presence of enzymes that destroy toxic
products of oxygen.
11. D-PH:
Most organisms have a fairly narrow optimal pH range. The optimal pH must be
empirically determined for each species. Most organisms (neutralophiles) grow
best at a pH of 6.0–8.0, although some forms (acidophiles) have optima as low
as pH 3.0 and others (alkaliphiles) have optima as high as pH 10.5.
E- carbon , nitrogen and water
3-Growth nutirant
Generally , culture media and the components can be divided into different roles
or functions:
1- Nutrients: proteins (peptone /peptides/amino-acids).
2- Energy: carbohydrates (lactose).
3 -Essential metals and minerals: calcium, magnesium, iron, trace metals:
phosphates, sulphates etc.
4- Buffering agents: phosphates, acetates etc.
5 -Indicators for pH change: phenol red
6- Selective agents: antimicrobial agents ,chemicals (Crystal Violet, bile salts).
7 Gelling agent: usually agar.
12. Agar
a galactan obtained from marine algae is used as the
hardening agent in solid media. Broth, with added agar
powder, is heated to 121oC in an autoclave, dissolving the
agar and sterilizing the medium. The molten medium may be
poured into plates or tubes. The agar-media will remain liquid
at temperatures above 45oC. Below 45oC the agar will
harden, and supply a solid surface for the growth of bacteria.
Agar plates and slants may be inoculated after they have
solidified.
