PHT 381 Lab# 5 Culture Media

A microbiological culture medium is the food we use for culturing bacteria and fungi. The culture medium is an artificial preparation that contains the essential elements and nutrients in a proper concentration needed by the microorganism (most bacteria &fungi) to grow.

Strict intracellular organisms (e. g Strict intracellular organisms (e.g., some bacteria & all viruses)→ only cultures of living eukaryotic cells. Tissue culture :- is prepared from living tissues treated in a special way to get a sheet of cells on a flat side of a container.

Composition of culture media

Requirements for growth of extracellular micro-organisms : 1-All the elements present in their organic matter ( carbon, nitrogen, hydrogen, oxygen, phosphorus, sulpher). 2- Ions required for energetics & catalysis (K, Na, Mg, ……) 3- A source of energy (fermentation /respiration).

For a culture medium to be successful in growing the pathogen, it must: 1- provide all essential nutrients, ions, and moisture, 2- maintain the correct pH and osmotic pressure.

1- Essential elements and nutrients. ± 2- Solidifying agents. Most common ingredients:- 1- Essential elements and nutrients. ± 2- Solidifying agents.

Essential Elements and Nutrients

I- Essential Elements & nutrients: 1-A Source of Carbon: - usually supplied in form of carbohydrates. 2- A Source of Nitrogen: usually supplied in form of peptone (partially digested protein) meat extract, yeast extract, amino acids & inorganic compound.

e.g. glucose or amino acids. 3-Inorganic salts: normally present in peptone but added in traces [e.g; Na, K, Ca, Mn, Fe, Co, Zn…….etc] II- A source of energy: e.g. glucose or amino acids.

IV- Maintainance of pH& osmotic pressure: III- moisture: Distilled water should be used in preparing media protoplasm consists of 70-85% water all the enzymatically controlled chemical reactions that occur within the cell occur only in the presence of an adequate amount of water) IV- Maintainance of pH& osmotic pressure: ***NaCl : is added to render the medium isotonic especially in medium containing blood to prevent lysis of the RBCs

*** Buffers: added to resist any changed in PH of the medium. (most bacteria grow best around pH 7) It also aids the growth of acid producer m.o [because it may poison themselves with their own acid]. Blood and milk are naturally occurring buffers. Phosphates are used to buffer synthetic or chemically defined media.

Solidifying Agents

A good solidifying agent: Not utilized by microorganisms Doesn’t inhibit bacterial growth. Doesn’t liquefy at room temprature.

1- Agar: The most common solidifying agent, used in conc of: 1.5 – 2% (to give solid medium) 0.2 – 0.5% (semisolid). Extracted from certain red marine algae & is composed of complex carbohydrates Not easily broken down by most common bacteria. Doesn’t liquefy at room temprature.

2. Gelatin: It is a protein derivative added to nutrient broth in conc of 12 -15% as a test for proteolytic activity of bacteria. hydrolyzed by few organisms, liquefy at room temperature.

Classification of Media

I- According to its content:- 1- Nonsynthetic Complex medium; contains extracts or digests of plant or animal tissues such as beef, peptone & soyabean digest. These components are inexact in composition.

2- Synthetic Chemically defined medium; contains known chemical compounds that are precisely defined, such as: amino acids, sugar, Vitamins & salts.

II- According to the consistency of the medium:- 1- Liquid (broth)→ **used for propagation of large numbers of organisms fermentation studies, other tests **disadvantages: No special characteristic for the growth, No isolation of m.o. in mixture

2-Solid (containing agar)→ used for developing surface colony growth of bacteria and molds. 3- Semisolid (containing low conc of agar)→ for determining the motility of bacteria.

III- According to the purpose of use:- Simple [Basic] media; contain only the essential or basic nutrients which support the growth of most common bacteria. Support the growth of micro-organisms which do not have special nutritional requirements.

✎ e.g; Nutrient broth (meat extract, peptone, NaCl) Nutrient agar→plate, slant ( nutrient broth + agar)

Nutrient agar plate Nutrient agar slant Nutrient broth

2- Enriched media; In addition to basic nutrients, they contain enriched substances such as blood, serum, egg, ….. They support the growth of fastidious organisms (their growth require highly nutritive substances)→ can’t grow on ordinary simple media.

Examples: 1- blood agar (nutrient agar +10% blood): It is also a differential medium (complete”β”, partial, or non hemolytic growth) 2-Chocolate agar (heated blood agar)→ for Neisseria 3-Serum agar (serum + glucose broth)→ for Corynaebacterium diphtheriae.

3. Selective media; Contains substances which inhibit the growth of undesired bacterial species. These inhibitory substances include: dyes, heavy metals, chemicals, antimicrobial agents, … mostly used in case of mixture/ contaminated samples.

3- Thayer-Martin medium (chocolate agar+ antibiotics)→ Neisseria. Examples: 1- L-J medium: (beaten eggs, mineral salts, malachite green)→ for Mycobacterium tuberculosis. 2- Blood tellurite medium (blood agar + K tellurite)→ for Corynaebacterium diphtheriae. 3- Thayer-Martin medium (chocolate agar+ antibiotics)→ Neisseria. 4- Cetrimide agar: it is highly selective medium for pseudomonas species. It contains cetrimide. Cetrimide is a surfactant that inhibit the growth of all organism except pseudomonas.

4. selective and differential media: Contains substances which selectively inhibit the growth of undesired bacterial species. Contain also substances which are affected by the growth of the organismsuch as pH indicators which indicates acid production during sugar fermentation. → this enable us to differentiate between the species which are able to grow on these media.

1- Mannitol salt agar; Examples: selective for Staphylococcus species (contains high conc. of salt (about 7%). Test sugar: mannitol pH indicator: phenol red (red in alkaline, yellow in acidic pH). **Staph.aureus is the only species of staphylococci that can ferment mannitol→ acid production → yellow color around the growth (resulting from changing the color of the indicator).

Growth with yellow colour around the colonies S.aureus Growth without change in the colour of the medium S.epidermidis

2. MacConkey agar; selective medium for gram –ve bacteria ( bile salt & crystal violet inhibit the growth of gram +ve bacteria). Test sugar: lactose. pH indicator: neutral red ( yellow in alkaline, pink in acidic pH). Gram –ve bacteria classified to: Lactose fermenter (Pink colonies) Lactose non-fermenter (pale colonies)

Lactose fermenter Pink colonies Lactose non-fermenter Pale colonies

Anaerobic culture media

Anaerobic cultivation is essential for obligate anaerobes (will not grow unless O2 is absent). This can be achieved through: 1- Removal of O2& replacing it with an inert gas→ Blood agar plates in Anaerobic Jar. 2- Special anaerobic media containing a reducing agent.

Anaerobic media Cooked meat medium: Fluid thioglycolate: Sodium thioglycolate act as a reducing agent. Cooked meat medium: Hematin & glutathione in meat particles act as reducing agents.

Media for Fungi

** Sabouraud dextrose agar. similar in composition as those of bacteria EXCEPT: 1-They are acidic in nature (pH 5.5-6.5). 2-contain relatively high concentrations of sugars e.g; dextrose and maltose.

Culture of pathogenic yeasts are incubated at 37 ℃ While cultures of superficial mycoses are usually incubated at lower temperature than bacteria (about 25℃).