Cultivation of Bacteria Dr. Suhad Faisal Hatem

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 .

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. 3

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.

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).

MacConkey agar(lactose ferment is pink and non lactose ferment is yellow) 8

Streptococcus hemolysis types on blood agar

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.

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.

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.

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