PHT 381PHT 381Lab# 5Lab# 5

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

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

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

Tissue culture :-Tissue culture :- is prepared from living tissues treated in a is prepared from living tissues treated in a

special way to get a sheet of cells on a flat special way to get a sheet of cells on a flat side of a container.side of a container.

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

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

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

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

For a culture medium to be successful For a culture medium to be successful

in growing the pathogen, it must:in growing the pathogen, it must:

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

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

Most common ingredients:-Most common ingredients:-

1- Essential elements and 1- Essential elements and nutrients.nutrients.

±±

2- Solidifying agents.2- Solidifying agents.

I- Essential Elements & nutrients:I- Essential Elements & nutrients: 1-A Source of Carbon:1-A Source of Carbon:

- - usually supplied in form of carbohydrates.usually supplied in form of carbohydrates.

2- A Source of Nitrogen:2- A Source of Nitrogen:

usually supplied in form ofusually supplied in form of peptone (partially digested protein)peptone (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 in normally present in peptone but added in traces [e.g; Na, K, Ca, Mn, Fe, Co, 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 or amino acids.e.g. glucose or 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:Buffers: added to resist any changed in PH of the added to resist any changed in PH of the

medium. medium.

(most bacteria grow best around pH 7)(most bacteria grow best around pH 7)

It also aids the growth of acid producer m.o It also aids the growth of acid producer m.o [because it may poison themselves with their [because it may poison themselves with their own acid].own acid].

Blood and milk are naturally occurring buffers.Blood and milk are naturally occurring buffers. Phosphates are used to buffer synthetic or Phosphates are used to buffer synthetic or

chemically defined media. chemically defined media.

A good solidifying agent:A good solidifying agent:

Not utilized by microorganismsNot utilized by microorganismsDoesn’t inhibit bacterial growth.Doesn’t inhibit bacterial growth.Doesn’t liquefy at room temprature.Doesn’t liquefy at room temprature.

1- Agar: 1- Agar: The most common solidifying agent, The most common solidifying agent,

used in conc of:used in conc of:

1.5 – 2% (to give solid medium) 1.5 – 2% (to give solid medium)

0.2 – 0.5% (semisolid). 0.2 – 0.5% (semisolid). Extracted from certain red marine Extracted from certain red marine

algae & is composed of complex algae & is composed of complex carbohydrates carbohydrates

Not easily broken down by most Not easily broken down by most common bacteria. common bacteria.

Doesn’t liquefy at room temprature.Doesn’t liquefy at room temprature.

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

broth in conc of 12 -15% as a test for broth in conc of 12 -15% as 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 II- According to the consistency of the medium:-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 III- According to the purpose of use:-of use:-

1.1. Simple [Basic] media;Simple [Basic] media; contain only the essential or basic contain only the essential or basic

nutrients which support the growth of nutrients which support the growth of most common bacteria. most common bacteria.

Support the growth of micro-Support the growth of micro-organisms which do not have special organisms which do not have special nutritional requirements. nutritional requirements.

✎✎ e.ge.g; ; Nutrient brothNutrient broth

(meat extract, peptone, NaCl)(meat extract, peptone, NaCl)Nutrient agarNutrient agar→→plate, slantplate, slant

( nutrient broth + agar)( 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 substances such as blood, enriched substances such as blood, serum, egg, …..serum, egg, …..

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

ExamplesExamples: : 1- blood agar (nutrient agar +10% 1- blood agar (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 agar (heated blood agar)2-Chocolate agar (heated blood agar)→ → for Neisseriafor Neisseria

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

3. Selective media;3. Selective media; Contains substances which inhibit the Contains substances which inhibit the

growth of undesired bacterial species.growth of undesired bacterial species.

These inhibitory substances include: dyes, These inhibitory substances include: dyes, heavy metals, chemicals, antimicrobial heavy metals, chemicals, antimicrobial agents, … agents, …

mostly used in case of mostly used in case of

mixture/ contaminated samples.mixture/ contaminated samples.

ExamplesExamples::

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

3- Thayer-Martin medium (chocolate 3- Thayer-Martin medium (chocolate agar+ antibiotics)→ Neisseria.agar+ antibiotics)→ Neisseria.

4- Cetrimide agar:4- Cetrimide agar: it is highly selective medium for it is highly selective medium for pseudomonas pseudomonas

speciesspecies. . It contains cetrimideIt contains cetrimide.. Cetrimide is a surfactant that inhibit the Cetrimide is a surfactant that inhibit the

growth of all organism except growth of all organism except pseudomonas.pseudomonas.

4. selective and differential media:4. selective and differential media: Contains substances which selectively inhibit Contains substances which selectively inhibit

the growth of undesired bacterial species.the growth of undesired bacterial species.

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

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

ExamplesExamples::

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

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

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

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

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

Fluid thioglycolate:Fluid thioglycolate:Anaerobic mediaAnaerobic media

Hematin & glutathione in meat particles Hematin & glutathione 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 yeasts are Culture of pathogenic yeasts are incubated at 37 ℃ incubated at 37 ℃

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