PHT 381PHT 381Lab# 5Lab# 5

A microbiological culture mediumA microbiological culture medium is is the the foodfood we use for culturing bacteria and fungi. we use for culturing bacteria and fungi.

The culture mediumThe culture medium is an is an artificial artificial preparationpreparation that contains the that contains the essential essential elements and nutrientselements and nutrients in in a proper a proper concentrationconcentration needed by the microorganism needed by the microorganism (most bacteria &fungi) to grow.(most bacteria &fungi) to grow.

Strict intracellular organismsStrict intracellular organisms (e.g., some (e.g., some bacteria & all viruses)→ only bacteria & all viruses)→ only cultures of cultures of living eukaryotic cellsliving eukaryotic cells..

Tissue cultureTissue culture

is prepared from living tissues treated is prepared from living tissues treated in a special way to get a sheet of cells in a special way to get a sheet of cells on a flat side of a container.on a flat side of a container.

Requirements for growth of extracellular Requirements for growth of extracellular micro-organisms :micro-organisms :

1-All the elements present in their organic 1-All the elements present in their organic mattermatter ( carbon, nitrogen, hydrogen, ( carbon, nitrogen, hydrogen, oxygen, phosphorus, sulpher).oxygen, phosphorus, sulpher).

2- Ions2- Ions required for energetics & catalysis required for energetics & catalysis (K, Na, Mg, ……)(K, Na, Mg, ……)

3- A source of energy3- A source of energy (fermentation (fermentation /respiration)./respiration).

For a culture medium to be successful in For a culture medium to be successful in growing the pathogen,growing the pathogen, it must: it must:

1- provide 1- provide all essential nutrients, ions, and all essential nutrients, ions, and moisturemoisture,,

2- maintain the correct 2- maintain the correct pH and osmotic pH and osmotic pressure.pressure.

Most common ingredients:-

1- Essential elements and nutrients.

±2- Solidifying agents.

I- Essential Elements & I- Essential Elements & nutrients:nutrients:

1-A Source of Carbon: 1-A Source of Carbon: usually supplied in form of usually supplied in form of

carbohydrates.carbohydrates. 2- A Source of Nitrogen:2- A Source of Nitrogen: usually supplied in form ofusually supplied in form of• peptonepeptone (partially digested protein) (partially digested protein)• meat extract,meat extract,• yeast extract, yeast extract, • amino acids & inorganic compound.amino acids & inorganic compound.

3-Inorganic salts: 3-Inorganic salts:

normally present in peptone but added normally present in peptone but added in traces [e.g; Na, K, Ca, Mn, Fe, Co, in traces [e.g; Na, K, Ca, Mn, Fe, Co, Zn…….etc]Zn…….etc]

II- A source of energy:II- A source of energy:

e.g. glucose e.g. glucose or or amino acids.amino acids.

III- moisture:III- moisture: Distilled waterDistilled water should be used in preparing should be used in preparing

mediamedia• protoplasm consists of 70-85% waterprotoplasm consists of 70-85% water• all the enzymatically controlled chemical reactions that all the enzymatically controlled chemical reactions that

occur within the cell occur only in the presence of an occur within the cell occur only in the presence of an adequate amount of water)adequate amount of water)

IV- Maintainance of pH& osmotic IV- Maintainance of pH& osmotic pressure: pressure:

***NaCl :***NaCl :is added to render the medium isotonic is added to render the medium isotonic

especially in medium containing blood to especially in medium containing blood to prevent lysis of the RBCs 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.

A good solidifying agent:A good solidifying agent:

Not utilizedNot utilized by microorganisms by microorganisms Doesn’t inhibit bacterial growth.Doesn’t inhibit bacterial growth. Doesn’t liquefyDoesn’t liquefy at room temprature. 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 liquefyDoesn’t liquefy at room temprature. at room temprature.

2. Gelatin: 2. Gelatin: It is It is a protein derivativea protein derivative added to nutrient added to nutrient

broth in conc of 12 -15% as broth in conc of 12 -15% as a test for a test for proteolytic activity of bacteria. proteolytic activity of bacteria.

hydrolyzed by few organisms,hydrolyzed by few organisms, liquefy at room temperature.liquefy at room temperature.

I- According to its content:-I- According to its content:-

1- Nonsynthetic Complex medium; 1- Nonsynthetic Complex medium;

• contains extracts or digests of plant contains extracts or digests of plant or animal tissues such as beef, or animal tissues such as beef, peptone & soyabean digest.peptone & soyabean digest.

• These components are inexact in These components are inexact in composition.composition.

2- Synthetic Chemically defined medium;2- Synthetic Chemically defined medium;

• contains known chemical contains known chemical compounds that are precisely defined, compounds that are precisely defined, such assuch as::

amino acids, amino acids, sugarsugar,, Vitamins & salts.Vitamins & salts.

II- According to the consistency of the medium:-

1- Liquid (broth)1- Liquid (broth)→→ **used for**used for propagation of large numbers propagation of large numbers

of organismsof organisms fermentation studies, fermentation studies, other testsother tests**disadvantages: **disadvantages:

No special characteristic for the No special characteristic for the growth,growth,

No isolation of m.o. in mixtureNo isolation of m.o. in mixture

2-Solid 2-Solid (containing agar)(containing agar)→ →

used for developing surface used for developing surface colony growth of bacteria and colony growth of bacteria and molds.molds.

3- Semisolid3- Semisolid (containing low conc of (containing low conc of agar)agar)→ for determining the → for determining the motility of bacteria.motility of bacteria.

III- According to the purpose of use:-

1. 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 plateNutrient agar plate Nutrient agar slantNutrient agar slant

Nutrient brothNutrient broth

2- Enriched media;2- Enriched media;

In addition to basic nutrients, they contain In addition to basic nutrients, they contain enriched substancesenriched substances such as blood, such as blood, serum, egg, …..serum, egg, …..

They support the growth of They support the growth of fastidious fastidious organismsorganisms (their growth require highly (their growth require highly nutritive substances)nutritive substances)→→ can’t grow on can’t grow on ordinary simple media.ordinary simple media.

Examples: Examples: 1- blood agar1- blood agar (nutrient agar +10% (nutrient agar +10%

blood):blood):It is also a differential medium It is also a differential medium

(complete”(complete”ββ”, partial, or non ”, partial, or non hemolytic growth)hemolytic growth)

2-Chocolate agar2-Chocolate agar (heated blood agar)(heated blood agar)→ → for for NeisseriaNeisseria

3-Serum agar3-Serum agar (serum + glucose (serum + glucose broth)broth)→ for → for Corynaebacterium Corynaebacterium diphtheriae.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.

Examples:Examples:

1- L-J medium:1- L-J medium: (beaten eggs, mineral salts, malachite green)(beaten eggs, mineral salts, malachite green)→ →

for for Mycobacterium tuberculosis.Mycobacterium tuberculosis.

2- Blood tellurite medium2- Blood tellurite medium (blood agar + (blood agar + K tellurite)→ for K tellurite)→ for Corynaebacterium Corynaebacterium diphtheriae.diphtheriae.

3- Thayer-Martin medium3- Thayer-Martin medium (chocolate (chocolate agar+ antibiotics)→ agar+ antibiotics)→ Neisseria.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:4. selective and differential media: Contains substances which selectively

inhibit the growth of undesired bacterial species.

Contain also substances which are affected Contain also substances which are affected by the growth of the organismsuch as by the growth of the organismsuch as pH pH indicatorsindicators which indicates acid production which indicates acid production during sugar fermentationduring sugar fermentation..

→ → this enable us to differentiate between the this enable us to differentiate between the species which are able to grow on these species which are able to grow on these media.media.

Examples:Examples:

1- Mannitol salt agar; 1- Mannitol salt agar; • selective for selective for Staphylococcus speciesStaphylococcus species (contains (contains

high conc. of salt (about 7%).high conc. of salt (about 7%).

• Test sugar: mannitolTest sugar: mannitol• pH indicator: phenol red (red in alkaline, yellow in pH indicator: phenol red (red in alkaline, yellow in

acidic pH).acidic pH). **Staph.aureus**Staph.aureus is the only species of staphylococci that can is the only species of staphylococci that can

ferment mannitolferment mannitol→→ acid production acid production →→ yellow yellow color around the growthcolor around the growth

(resulting from changing the color of the indicator). (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; 2. MacConkey agar; • selective medium for selective medium for gram –vegram –ve bacteriabacteria ( bile ( bile

salt & crystal violet inhibit the growth of salt & crystal violet inhibit the growth of gram +ve bacteria).gram +ve bacteria).

• Test sugar: lactose.Test sugar: lactose.• pH indicator: neutral red ( yellow in alkaline, pH indicator: neutral red ( yellow in alkaline,

pink in acidic pH).pink in acidic pH).

Gram –ve bacteria classified to:Gram –ve bacteria classified to:

Lactose fermenter (Pink colonies)

Lactose non-fermenter (pale colonies)

Lactose fermenter Pink colonies

Lactose non-fermenter Pale colonies

Anaerobic cultivationAnaerobic cultivation is essential for is essential for obligate anaerobesobligate anaerobes (will not grow (will not grow unless O2 is absent).unless O2 is absent).

This can be achieved through:This can be achieved through:

1- Removal of O21- Removal of O2& replacing it with an & replacing it with an inert gas→ Blood agar plates in inert gas→ Blood agar plates in Anaerobic Jar.Anaerobic Jar.

2- Special anaerobic media2- Special anaerobic media containing containing a a reducing agent.reducing agent.

Sodium thioglycolateSodium thioglycolate act as a reducing agent. act as a reducing agent.

Fluid thioglycolate:Fluid thioglycolate:Anaerobic mediaAnaerobic media

Hematin & glutathioneHematin & glutathione in meat particles in meat particles act as reducing agents.act as reducing agents.

Cooked meat medium:Cooked meat medium:

** Sabouraud dextrose ** Sabouraud dextrose agar. agar.

similar in composition as those similar in composition as those of bacteria of bacteria EXCEPT:EXCEPT:

1-They are 1-They are acidicacidic in nature (pH in nature (pH 5.5-6.5).5.5-6.5).

2-contain 2-contain relatively high relatively high concentrations of sugarsconcentrations of sugars e.g; e.g; dextrose and maltose.dextrose and maltose.

• Culture of pathogenic yeastsCulture of pathogenic yeasts are are incubated atincubated at 37 ℃ 37 ℃

• While While cultures of superficial cultures of superficial mycosesmycoses are usually incubated at are usually incubated at lower temperature than bacteria lower temperature than bacteria (about(about 25℃). 25℃).