Basic Microbiology, Protocols

Culture medium Definition, Preparation, Classification

Content hide 1 Culture Medium Definition 2 Classification of culture media/Types of Culture Medium 3 Preparation of Culture Media/Medium 4 Composition/Preparation of...

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Culture medium Definition, Preparation, Classification

The food supply upon which microorganisms thrive in laboratories is termed as a culture medium or culture media.

Culture Medium Definition

The nutrient preparation on or in which a culture (microorganism) is grown in the laboratory is known as culture medium or culture media.

  • A culture medium is a solid or liquid preparation utilized to grow, transport, and store microorganisms. To be useful, the medium must hold all the nutrients the microorganism needs for germination.
  • All the microorganisms demand basic requirements, although there is a variation as to the application of organic and inorganic compounds.
  • Depending upon the species to be grown, culture media vary in form and composition.
  • Some media carry a solution of inorganic salts and may be supplied with one or more organic compounds while other media contain miscellaneous components such as extracts or digests of plant and animal tissues.
  • Many of the studies of microbiology are based on the capacity to grow and maintain microorganisms in the laboratory, and this is achievable only if proper culture media are convenient. 
  • The selection of a suitable culture medium further depends on the Information about the microorganism’s natural territory because its nutritional demands reveal its natural surroundings.
  • A satisfactory medium contains energy sources, carbon, nitrogen, phosphorus, sulfur and various minerals, etc which is required for a microorganism to be grown.
  • During the preparation of a culture medium for any microorganism, the main intention is to implement a stable mixture of the expected nutrients at concentrations that will allow immeasurable growth.
  • Additionally, the culturing of microorganisms needs the precise control of different environmental agents which usually are kept within narrow boundaries.

Classification of culture media/Types of Culture Medium

Many special-purpose media are required to promote the identification, enumeration, and isolation of specific types of bacteria. To satisfy these requirements, the microbiologists have prepared numerous media, on the basis of their purpose or function, composition may be classified as follows;

Classification of culture medium based on physical state

Based on physical state the cultural medium is divide into three classes such as;

  1. Liquid Medium/Broth
  • When only nutrients are dissolved in a solvent without any solidifying agent, then these types of media are known as Liquid Media or Broth.
  • They are also called broth, used for the growth and production of microbes.
  • The germination of cells becomes apparent in the form of a minute size on the tip of the broth.
  • Mostly bacteria and fungi are cultured in liquid broth. 
  • Some examples of liquid broth are Nutrient broth, glucose broth, beef extract, skimmed milk, peptone solution.
  • Beef extract (a beef derivative which is a reservoir of organic carbon, nitrogen, vitamins, and inorganic salts) and peptone solution (a semi-digested protein) are considered as the most widely used liquid medium in bacteriological laboratory.
  1. Solid Medium
  • The Solid media may either constantly remain solid such as potato slices, coagulated blood serum, coagulated egg or can be melted for example nutrient-agar medium, nutrient-gelatin medium, potato dextrose agar medium.
  • They are also known as agar medium.
  • Solid Medium contains 1.5%-2% solidifying agents such as agar with other nutrients.
  • Other solidifying agents are also used in a solid medium such as silica gel, gelatin (10-20%), etc.
  • These types of media are accepted for composing agar slants or slopes and agar stab.
  • They are used for colony identification, colony characterization, isolation and enumeration of microbial cells, and antibiotics sensitivity tests.
  • Agar is a heterogeneous polysaccharide (carbohydrate) composed of 3, 6-anhydro-L-galactose, and D-galactopyranose, free of nitrogen, produced from various red algae belonging to genera Gelidium, Gracilaria, Gigartina and Pterocladia.
  • Agar melts on warming to 96°C and solidifies within a jelly on reducing to 40-45°C.
  • The hardened medium is deposited in a Petri dish which produces an artificial habitat proper for a speedy growth of fungi.
  • While in the liquefied phase, solid media can be exerted in test tubes, which are either left to cool and solidify in a slanted state creating agar slants, or left to freeze in an upward manner creating agar deep tubes.
  1. Semi-solid Medium
  • These types of medium remain in semi-solid condition.
  • It is prepared by adding half quantity of agar (1/2 than needed for solid medium) i.e. approximately 0.5% in the medium.
  • Semi-solid Medium is mainly used for the cultivation of microaerophilic bacteria and the study of bacterial motility.
  • This type of medium can be selective or differential media.
  • The selective media encourage the growth of one organism and hinders the growth of the other organism while differential media help to distinguish organisms growing collectively.
  • An example of Semi-solid Medium is Cystine trypticase-agar medium.

Classification of culture media based on Chemical Nature

Based on the chemical nature the culture media is divided into four classes such as;

  1. Non-Synthetic Media or Complex Medium
  • The medium that contains ingredients of unknown composition is known as non-synthetic medium, undefined medium, or complex medium.
  • It is composed of sugar, yeast, beef extract, vitamins, peptones, salts, etc.
  • Example: Some examples of non-synthetic media are Potato-Dextrose-Agar (GM-25), Soil-Extract-Agar (SM-1), Oatmeal-Agar (GM-24), Malt-Extract-Agar (GM-19b), Waksman’s medium (GM- 40).
  1. Synthetic Media or Defined Media
  • The medium which contains some ingredients of known chemical composition is known as synthetic medium.
  • Synthetic Media are valuable for nutritional and metabolic experiments.
  • Example: An example of a synthetic medium is E. Coli broth, Czakek’s Dox medium (GM-9), and Richard’s solution (GM-27).
  1. Semi-Synthetic Medium
  • The medium that contains some known and some unknown chemical ingredients is known as Semi-Synthetic Medium.
  • Example: An example of Semi-Synthetic Medium is Tryotic soy sugar [Trypticase (15g), soya peptone (5g), NaCl (5g), Distilled water (1lite), pH 7.1]
  1. Natural Medium/Empirical
  • When a natural product is used for growing bacteria, this type of natural product is known as a natural medium.
  • Some examples of natural products are milk, wine, blood, vegetable juices, yeast extract, egg, etc.
  • Some natural media may be made of a synthetic medium expanded by tomato juice, carrot strips, or plant stems.
  • Natural media are usually very useful and support sporulation in fungi that may contrarily remain sterile.
  • Example: An example of Natural Medium/Empirical is Blood Agar [Infusion from beef heart (500g), Tryptone (10g), NaCl (5g), Agar (15g), Distilled water (1000ml), pH 7.3]

Classification of Medium Based on purpose or use

Based on the purpose or uses the medium is classified into the following groups;

  1. Selective Medium
  • The selective medium inhibits the growth of undesired microorganisms while grants and encourages the growth of desired microorganisms to develop characteristic colonies.
  • The medium shows selective action due to the presence of certain chemicals to it. For example, the addition of crystal violet stain in the culture medium selectively hinders the germination of gram-positive bacteria and grants and encourages the germination of gram-negative bacteria.
  • Example: Some examples of selective Medium are MacConkey-Agar for E. coli, Deoxycholate-Citrate-Agar (DCA) for Salmonella and Shigella, Wilson and Blair’s medium for Salmonella, and Mannitol-Salt-Agar medium for pathogenic staphylococci.
  • The high concentration of sodium chloride is present within the Mannitol-Salt-Agar medium which prevents the growth of bacteria other than pathogenic staphylococci.

The Selective Medium is prepared by incorporation of –

  1. A specific Nutrient: Cellulose as carbon source.
  2. Antibiotic: antibacterial and antifungal. antibiotics inhibit the respective groups.
  3. Dye: Rose-bengal is a bacteriostatic chemical that is added to prevent the growth of bacteria.
  4. Adjusting the pH value: The growth of specific microorganisms is supported and the growth of others is hindered by adjusting the pH.
  1. Differential Medium or Indicator Media
  • A differential (indicator) medium is one that makes a noticeable difference between various groups of bacteria breeding in the medium and also allow experimental classification of microorganisms based on their biological properties. 
  • In one word, a medium which contains certain reagents or supplements which allows differentiation of various kinds of microorganisms on the basis of visual differences in their growth pattern.
  • Example: Some examples of differential medium are Blood-Agar medium, MacConkey-Agar medium, and Christensen’s medium.
  • Hemolytic and non-hemolytic bacteria are distinguished by using a Blood-Agar medium. Hemolytic bacteria such as many streptococci and staphylococci isolated from throats created clear zones throughout their colonies because of red blood cell disruption. Non-hemolytic bacteria do not form this type of clear zone around their colonies.
  • Lactose fermenting bacteria can be distinguished by using MacConkey-Agar medium. This medium contains lactose and neutral red, hence when the Lactose fermenting bacteria after germination on this medium creates acid and in acidic pH the neutral red converts into red color.  E. coli produces red or pink colonies on MacConkey-Agar medium.
  • Urease producing bacteria (e.g., Proteus, Klebsiella) can be identified by using Christensen’s medium. This medium contains urea and phenol red, hence urease-producing bacteria break down the urea into ammonia and CO2. Ammonia converts the medium to alkaline and in alkaline pH the medium produces pink color because phenol redshifts in red color in alkaline medium.
  1. Enriched Medium
  • Enriched media contain specific growth substances such as blood, serum, and egg with the nutrient agar to satisfy the nutritional stresses of metabolically fastidious microorganisms 
  • Example: Some examples of Enriched Medium are blood agar for isolation of Streptococcus, chocolate agar for isolation of Neisseria and Haemophilus, Bordet-Gengou for isolation of Bordetella, and Loeffer’s serum slope for the isolation of Corynebacterium diphtheriae.
Culture Medium - MacConkey-Agar and Blood Agar
Culture Medium – MacConkey-Agar and Blood Agar
  1. Assay Medium
  • Media of designated composition are utilized for the assay of vitamins, amino acids, and antibiotics.
  • In this medium, the rate of growth is directly proportional to the nutrient concentration.
  • Example: Biotin assay medium, used for vitamin assay. Seed agar medium used for antibiotics assay.
  1. Enumeration Medium
  • Specific types of medium are used to determine the microbial content of such materials as milk (lactose agar), soil extract agar, and sewage water (lactose broth) to favor the type of organism found in this material.
  1. Biochemical Medium
  • This medium is utilized to distinguish between two microorganisms on the basis of their biochemical activities.
  • This type of medium is effective in the study of metabolic processes and the classification of microorganisms.
  • Example: Some examples of Biochemical Medium are Peptone broth used for testing for the production of hydrogen sulfide (H2S) by lead acetate, that terms black.
  1. Maintenance Medium
  • Maintenance Medium is used for maintenance of viability and physiological characteristics of a culture requiring a medium in which a large number of microbes can be grown and used for routine cultivation and maintenance of a wide variety of organisms.
  • Example: Fogg’s Medium use for algae, Potatoes dextrose agar used for fungi.
  1. Transport Medium
  • In the medical field, the transport medium is used for different purposes.
  • During the transport of pathogenic microorganisms from the hospital to the laboratory transport mediums are used to maintain them.
  • This medium ideally maintains the viability of all organisms in the specimen without altering their concentration.
  • These types of medium only contain buffers and salts.
  • When the pathological laboratory is far away from the patient, the delicate pathogenic microorganism (e.g., Neisseria gonorrhoeae that causes gonorrhoea) may not last, or the normal microorganisms (e.g., Escherichia coli) may overgrow pathogenic microorganisms (e.g., Salmonella, Shigella, Vibrio cholerae) even ere the transportation of clinical sample to the testing laboratory. To evade this, culture media have been devised to sustain the viability of the pathogen.
  • Example: 
    • Stuart’s medium: Stuart’s medium utilized to keep the viability of gonococci bacteria.
    • Pike’s medium: Pike’s medium is utilized to store Streptococcus pyogenes.
    • Glycerol-saline medium: Glycerol-saline medium is utilized to inhibit normal intestinal microflora from overgrowing the enteric fever bacilli.
    • Bile-Peptone medium: Bile-Peptone medium is utilized to sustain the viability of cholera causing bacteria.
    • Venkatraman Ramakrishnan Medium: V.R medium is used for transport of stools of cholera patients.
  1. Characterization Medium
  • This medium is utilized to determine or characterize the type of growth produced by bacteria as well as to determine their ability to produce certain chemical changes.
  • Example: Gelatin stab is used for gelatin liquefaction test.
  1. Minimal Medium
  • The medium which lacks certain growth factors is known as Minimal Medium.
  • Minimal Medium is mainly used for genetic experiments.
  1. Ashby Medium
  • The medium which lacks any nitrogenous compound is known as the Ashby medium.
  • Nitrogen-fixing bacteria are cultivated by using this type of medium.

Anaerobic media:

  • Anaerobic bacteria require specialized media for extension because they demand low oxygen content, diminished oxidation-reduction potential, and more nutrients.
  • This type of Media ought to be enriched by nutrients such as hemin and vitamin K.
  • Such media may also have to be overcome by physical or chemical means.
  • Boiling the medium helps to discharge any suspended oxygen.
  • The addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine, or red hot iron filings can provide a medium reduction. Before applying the medium needs to be sterilized in a water bath to dismiss any suspended oxygen and then sealed using sterilized liquid paraffin.
  • Robertson Cooked Meat (RCM) medium that is generally applied to develop Clostridium spps carries a 2.5 cm column of bullock heart content and 15 ml of nutrient broth.
  • Thioglycollate broth includes sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate.
  • An oxidation-reduction potential indicator (Methylene blue or resazurin ) is added to the medium. Following disgraced conditions, methylene blue is transparent.

Preparation of Culture Media/Medium

Preparation of Culture medium
Preparation of Culture medium | Image Author: SouravBio (microbiologynote.com)

Commercially all mediums are available in powdered form. The preparation of culture medium or bacteriological media can be done by these following steps;

  1. Dissolved the desired ingredients or the complete dehydrated medium into an appropriate volume of distilled water.
  2. Then determine the pH of the medium by using a pH meter and adjust it if necessary.
  3. Add agar to this medium and boil it to dissolve the agar in case a solid medium is required.
  4. Then dispensed the medium into tubes or flasks.
  5. After that sterilized them by using an autoclave. Some heat-labile media or specific ingredients are sterilized by using filtration.

Composition/Preparation of Certain Culture Medium

Name of Culture Media Preparation of Culture Medium
Tributyrin agarProvided available for use. Give Heat to melt and dispense aseptically. May be made by adding 1% tributyrin to nutrient agar.
Sugar peptone waterMix 10 g of peptone, 5 g of NaCl, 5 g of sugar and 20 cm³ of Universal indicator with 1 liter of distilled water; Maintain the pH 7.4. Manage as needed and sterilize.
Nutrient agarDissolve 28 g of nutrient agar powder in 1 liter of distilled water. Then boil the medium to dissolve the agar completely. Dispense as required and sterilize.
Nutrient brothMix 13 g of nutrient broth powder to 1 liter of distilled water. Then stir it well. Dispense as required and sterilize.
Malt extract agarDissolve 18g agar powder in 1 liter of distilled water. hen boil the medium to dissolve the agar completely. Mix 15g malt extract per liter. stir it well. Dispense as required and sterilize.
Mannitol yeast extract agarDissolve 10 g agar in 1 liter of distilled water. Boil the media to dissolve the agar. Mix 0.5 g K2HPO4 , 0.2g MgSO4.7H2O, 0.2 g NaCl, 0.2 g CaCl2.6H2O, 10 g mannitol and 0.4 g yeast extract. Dispense as required and sterilize.
Mannitol yeast extract brothFollow the above step, without agar.
Glucose nutrient brothPrepare nutrient broth as previously guided and add 10 g per liter of glucose.
Glucose yeast extract brothMix 10 g of peptone, 5 g of NaCl, 3 g of yeast extract to 1 liter of distilled water. Dispense as required and sterilize.
Glucose yeast extract lemco brothMix10 g of Lemco (meat extract) to glucose yeast extract broth.
Milk agarPrepare nutrient agar as previously guided but using only 900 cm³ of distilled water. Dissolve 20 g of condensed skimmed milk in 100 cm³ of distilled water. Sterilize individually. Shift the milk to the agar aseptically after cooling to 45-50 °C. Dispense aseptically.
Starch agarDissolve 15 g of nutrient agar in 100 cm³ distilled water. Boil the media to dissolve the agar. Heat 40 g of soluble starch in 100 cm³ of distilled water to develop a suspension. Permit to cool and then mix with the nutrient agar solution. Dispense and sterilize.
Iodine solutionSuspend 1 g of iodine crystals and 2 g of potassium iodine in 300 cm³ of distilled water.
Cellulose broth (for Trichoderma reesei)Add 0.1 g CaCl2, 0.5 g (NH4)2SO4, 0.5 g yeast extract powder, 0.5 g asparagine, 10 g carboxymethylcellulose, 1.0 g KH2PO4 into 800 cm3 distilled water. Then maintain the pH6.2. Mix the ingredients properly, heat gently, and stir until dissolved.

Propertie of some Culture Medium with Application

Ashby’s Mannitol Agar

  • Type: chemically defined.
  • Special Ingredients: N-free, containing glucose.
  • Use for Organisms: heterotrophic, free living, N-fixing bacteria Azotobacter.

Brain Heart infusion agar

  • Type: non-chemically defined.
  • Special Ingredients: Glucose.
  • Use for Organisms: streptococci and pneumococci.

Czapek-Dox agar

  • Type: non-chemically defined.
  • Special Ingredients: Sucrose.
  • Use for Organisms: Moulds specially aspergillus and penicillium.

Malt extract Medium defined

  • Type: non-chemically defined.
  • Special Ingredients: Vitamins.
  • Use for Organisms: yeast and acetobacter.

Nutrient Agar

  • Type: non-chemically defined.
  • Special Ingredients: Peptone.
  • Use for Organisms: Many non-fastidious bacteria, including E. coli, B. cereus, S. aureus and P. aeruginosa Algae.

George’s Medium

  • Type: mineral salt medium.
  • Special Ingredients: Peptone.
  • Use for Organisms: Algae.

Waksman’s Sulphur medium

  • Type: chemically defined.
  • Special Ingredients: Sulphur energy source bacteria.
  • Use for Organisms: Autotrophic, sulphur-oxidizing bacteria, Thiobacillus.

Winogradsky’s Medium

  • Type: chemically defined, inorganic medium.
  • Special Ingredients: NH4+ as energy source.
  • Use for Organisms: Autotrophic, nitrifying bacteria, nitrosomonas and nitrobacter.

Blood Agar

  • Type: Enriched and differential medium.
  • Special Ingredients: Blood.
  • Use for Organisms: Many fast growing bacteria including streptococcus and Neisseria gonorrhoeae gives evidence of hemolysis.

Chocolate Agar

  • Type: Enriched medium.
  • Special Ingredients: Blood agar is heated to disrupt the blood cells.
  • Use for Organisms: Neisseria.

Filde’s medium

  • Type: Nutrient medium.
  • Special Ingredients: Contains enzymatic digest of blood.
  • Use for Organisms: Haemophilus and clostridium tetani.

Eosin Methylene blue (EMB) agar

  • Type: Selective and differential medium.
  • Special Ingredients: Eosin and methylene blue.
  • Use for Organisms: For differentiating E. coli and Aerobacter aerogenes.

MacConkey’s Agar Medium

  • Type: Selective and differential medium.
  • Special Ingredients: Bile salts crystal violet and neutral red sodium chloride, phenol red.
  • Use for Organisms: for determining the number of coliforms in water and faeces.

Mannitol salt agar medium

  • Type: Selective and differential medium.
  • Special Ingredients: Sodium chloride phenol red.
  • Use for Organisms: For the isolation of Salmonella and shigella, staphylococci.

Selenite broth

  • Type: Enrichment medium.
  • Special Ingredients: Sodium selenite.
  • Use for Organisms: Salmonella.

Thayer-martin agar

  • Type: Selective medium.
  • Special Ingredients: Antibiotics.
  • Use for Organisms: Neisseria.

Chopped meat glucose broth

  • Type: Selective medium.
  • Special Ingredients: Cysteine.
  • Use for Organisms: Anaerobes.

Sabouraud’s agar

  • Type: Selective medium.
  • Special Ingredients: Glucose.
  • Use for Organisms: Pathogenic fungi.

How to make tissue culture medium?

The plant tissue culture medium is composed of two distinct parameters such as the plant species and the part or type of plant material you will use (tissues, protoplasts, cells, etc.). To prepare a culture medium follow these steps;

  1. Dissolve a powdered medium within an appropriate amount of water.
  2. If you are preparing for a 1-liter medium, then load a beaker by 800ml distilled water. Gradually add the powered medium into the beaker.
  3. Then add 30g sucrose.
  4. Fix the PH at 5.8.
  5. Then add agar to the beaker (8g).
  6. Add hormone (if using).
  7. Add 200 mL distilled water.
  8. Autoclave media.
  9. Distribute the liquefying media within sterile tubes and make confirm that each tube is marked.
  10. The media should load up about a third of the tubes.
  11. The media should be left in a sterile atmosphere, where it is observed till it can be used to culture cells seven days later.

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