Culture Media Question And Answers

Robertson’s cooked meat medium is an anaerobic media.

• Type: Enriched (Due to cooked media, Indicator (Blackening of meat by clostridia)
• Composition: Cooked meat from fresh OX heart, nutrient broth, or peptone infusion broth.
• Meat particles: Consists of unsaturated fatty acids, these unsaturated fatty acids utilize oxygen for their oxidation. It also consists of reducing substances such as glutathione and cysteine which utilize oxygen and help in the growth of anaerobes.
• Sterilization: Autoclave: 121°C, 15 lb pressure/sq inch for 20 minutes.
•  pH:  Adjusted at 7.8
• Water is made alkaline to neutralize the lactic acid of meat and to desaturate fatty acids in meat.

Read And Learn More: Microbiology Question And Answers

Robertson’s Cooked Of  Uses:

•  It is used for anaerobic culture
• For the preservation of the stock culture of aerobic organisms.
• Valuable medium for preserving cultures of delicate organisms.
• May be used as a recovery medium for spores.

Robertson’s Cooked Of Cautions:

• Use a tall column of cooked meat.
• Nutrient broth’s upper level should be at least half an inch above the meat’s level. Meat is covered with 10 mL nutrient broth.
• Put a half-inch thick layer of sterile liquid paraffin on the surface of the broth to prevent contact with air.
• Before inoculation, boil the tube at 80°C for 20 minutes to drive off oxygen as well as to kill other organisms.
• Air bubbles should be avoided.

Question 2. Write a note on artificial culture media.
Answer:

Artificial Culture Media: Artificial culture media is an artificial environment with essential nutrients at optimum quantity and optimum pH which is suitable for bacterial growth in vitro.

The artificial media are as follows:

• Basic/Basal media: Natural agar is used. It consists of essential amino acids, meat/yeast extracts, and NaCl.
• Synthetic/Defied media: They are added to produce definite chemical products, for example, simple peptone water, casein hydrolysate, Tween 80, etc.
• Selective media: It is a solid media that suppresses or inhibits the growth of all except particular species or strains of bacteria, for example. Lowenstein-Jensen medium for M. tuberculosis.
• Enriched Media: The media is prepared by the addition of special nutrients like blood, serum, and egg to a basal medium.
  • It is used to grow bacteria that are more exacting in their nutritional demands.
  •  Examples: Blood agar, chocolate agar, serum agar, and egg media.
• Enrichment Media:
  • Substances that have a stimulating effect on bacteria to be grown or an inhibitory effect on those who have to be suppressed should be incorporated into the medium.
  • If such substances are added to a liquid medium this leads to an absolute increase in the quantity of wanted bacteria relative to other bacteria.
  • These media are known as enrichment media, an example of Tetrathionate broth allows S. typhi to grow freely, and selenite-F broth allows dysentery bacilli to grow freely.
  • In materials consisting of more than one bacterium, a bacterium of importance is to be isolated and is often overgrown by unwanted bacteria
  • Usually, non-pathogenic or commensal bacteria overgrow pathogenic ones.
•  Indicator media: This media is prepared by incorporation of some indicator substance that is changed visibly, indicated by some visible changes in the media, as a result of some particular and distinctive metabolic activities of a particular organism.
  • It is used to detect colonies of particular bacterial species, for example.
  • Wilson and Blair medium containing sulfite which is reduced to sulfide by Salmonella typhi-producing black colonies.
• Differential media: It is a medium that has substances incorporated in it enabling it to bring out different characteristics of bacteria and helping to distinguish them. Example:  MacConkey medium consists of peptone, lactose, sodium taurocholate, natural red, and agar. It diffrentiate lactose fermenters from non-lactose fermenters.
• Transport media: It is a media to protect and preserve pathogens during delays in transport as delicate organisms may not survive the time taken for transporting specimens to the laboratory,
  • Example: Stuart’s transport medium: It is a non-nutrient soft agar gel that consists of a reducing agent for preventing oxidation and charcoal to neutralize bacterial inhibitors. It is used to transport gonococci and other bacteria.
•  Sugar media: Sugar denotes any fermentable substance. Sugar media consist of 1% sugar in peptone water and appropriate indicator. Example: Hiss serum sugar used for pneumococci.
•  Anaerobic media: This is used to grow anaerobic organisms  Example of Robertson’s cooked meat medium.

Question 3. Name the anaerobic culture and anaerobic organism.
Answer:

Anaerobic cultures are as follows:

• Production of vacuum
• Displacement of oxygen
• Displacement and combustion of oxygen
• Absorption of oxygen by chemical or biological methods
• By reducing agents
• Anaerobic chamber.

Anaerobic Organisms: These are those organisms that do not require oxygen for growth. They are classified as:

• Cocci:
  • Gram-positive: Peptostreptococci, anaerobic streptococci.
  • Gram-negative: Vellinella.
• Bacilli:
  • Sporulating: Clostridia.
  • Non-sporulating
    • Gram-positive: Actinomyces, Lactobacillus, Eubacterium, and Propionibacterium.
    • Gram-negative: Bacteroides, fusobacteria and leptotrichia.
• Spirochetes: Treponema, Borrelia.

Question 4. Write a short note on blood agar.

Answer:

Blood agar is an enriched medium in which blood is an additive nutrient to the basal medium.

• It is composed of nutrient agar, sheep blood (5-10%)
• It is used as a routine culture.
• As blood agar acts as an enriched medium it is helpful for the growth of bacteria which have important nutritional value.
• A blood agar also serves as a differential media.
• It differentiates between hemolytic and nonhemolytic organisms.
• Blood agar also acts as indicator media. It shows hemolysis and indicates the presence of hemolytic.
• Organisms, so it acts as an indicator medium.

Question 5.  Write a short note on selective media.
Answer:

Selective media contain substances that inhibit all but a few types of bacteria and facilitate the isolation of a particular species.

• These media are used to isolate a particular bacteria from specimens where mixed bacterial flora is expected. Selective media are solid in contrast to enrichment media which are liquid.
• Examples of selective media are: 
  • Bile salt agar for Vibrio cholera
  • deoxychocolate citrate agar for Shigella and
  • SalmonellaThiosulfate – citrate – bile salts – sucrose (TCBS) agarWilson–Blair medium for Salmonella.

Question 6. Differentiate between enriched media vs. enrichment media.
Answer:

Differentiate between enriched media vs. enrichment media:

Question – 7: Write notes on the classification of culture media.
Answer:

Following is the classification of cultural media:

1. Based on the physical state:
   • Liquid media
   • Semisolid media
   • Solid media.
2. Based on the presence of molecular oxygen and reducing substances in media:
   • Aerobic media
   • Anaerobic media.
3. Based on nutritional factors:
   • Simple media
   • Complex media
   • Synthetic media
4. Special media:
   • Enriched media
   • Enrichment media
   • Selective media
   • Differential media
   • Indicator media
   • Transport media
   • Sugar media.

Based on Physical State:

1. Liquid Media:
   • It is liquid in nature.
   • Liquid media does not exhibit specific characteristics on the basis of which identification is done.
   • It does not permit the isolation of different types of bacteria from mixed populations.
   • Liquid Media Uses:
     • For further testing of pure culture of bacteria.
     • For making bulk cultures to prepare antigens or vaccines.
     • For obtaining the growth of organisms from blood or water.
     • When large volumes are to be tested.
2. Semisolid Media:
   • This is neither liquid nor solid.
   • It exists between liquid and solid phases.
   • Semisolid Media Uses:
     • To study bacterial motility
     • For cultivating anaerobic and microaerophilic organisms.
3. Solid Media: Its nature is solid and produces discrete visible growth.
   • Solid Media Uses:
     • To gain pure growth
     • For studying distinctive colony morphology and other characteristic features.

Culture Media Based On Nutritional Factors:

1. Simple Media:
   • It is also known as basal media.
   • Nutrient broth is an excellent example.
   • Nutrient broth consists of peptone, meat extract, sodium chloride, and water.
2. Complex Media:
   • Media other than simple media are complex media.
   • They have added ingredients for bringing certain.
   • Characteristics or for providing special nutrients required for the growth of bacteria.
3. Synthetic Media or Defied Media:
   • Prepared from pure chemical substances and exact composition of media is known.
   • Used for special studies such as metabolic requirements.
   • The best example is Dubo’s media with Tween 80.
4. Special Media:
   • Enriched Media:
     • This media is prepared by the addition of special nutrients like blood, serum, and egg to a basal medium.
     •  It is used to grow bacteria that are more exacting in their nutritional demands. Examples are: 
     • Blood agar enriched with blood
     • Chocolate agar enriched with heated blood
     • Loeffl’s serum slope enriched with serum and
     • Lowenstein-Jensen medium is enriched with egg.
   • Enrichment Media:
     • Substances that have a stimulating effect on bacteria to be grown or an inhibitory effect on those that have to be suppressed should be incorporated into the medium.
     • If such substances are added to liquid medium this leads to an absolute increase in the quantity of wanted bacteria relative to other bacteria.
     • These media are known as enrichment media, an example of  Tetrathionate broth allows S. typhi to grow freely, and selenite F broth allows dysentery bacilli to grow freely.
     • In materials consisting of more than one bacterium, a bacterium of importance is to be isolated and is often overgrown by unwanted bacteria.
     • It is useful in situations where non-pathogenic or commensal bacteria tend to overgrow pathogenic ones.
   • Selective Media:
     • Selective media contain substances that inhibit all but a few types of bacteria and facilitate the isolation of a particular species.
     • These media are used to isolate a particular bacteria from specimens where mixed bacterial flora is expected.
     • Selective media are solid in contrast to enrichment media which are liquid. Examples of selective media are:
     •  Deoxycholate citrate agar (DCA)—the addition of deoxycholate acts as a selective agent for enteric bacilli (Salmonella, Shigella).
     • Bile salt agar (BSA) for Vibrio cholerae, Wilson-Blair medium for Salmonella.
   • Differential Media:
     • A medium that has substances incorporated in it enables it to bring out different characteristics of bacteria.
     • And helping to distinguish between them is known as differential media.
     • MacConkeys medium which has peptone, lactose, agar, sodium taurocholate, and natural red.
     • The lactose fermenters form pink-colored colonies, whereas nonlactose fermenters produce colorless or pale colonies.
   • Indicator Media:
     • This media contains an indicator that changes color when a particular bacterium is grown in that.
     • Wison and Blair medium containing sulphite which is reduced to sulfide by Salmonella typhi-producing black colonies.
     • Blood agar showing hemolysis indicates the presence of hemolytic organisms, so it is also an indicator medium.
   • Transport Media:
     • In the case of delicate organisms which may not survive the time taken for transporting the specimen to the laboratory or may be overgrown by non-pathogens.
     • Special media are developed for transporting the specimens they are known as transport media.
     • An example is Stuart’s medium which is a non-nutrient soft agar gel containing a reducing agent to prevent oxidation and charcoal to neutralize certain bacterial inhibitors used for gonococci.
   • Sugar Media:
     • Sugar is a fermentable substance.
     • Sugar media contain 1% sugar in peptone water along with an indicator.
     • A small tube is kept inverted in the larger sugar tube to detect gas production.
     • Colorless medium turns pink with the production of acid by bacteria and gas production is indicated by gas bubbles accumulated in Durham’s tube.
     • Certain bacteria are exacting in their growth requirements and need serum for their growth
     • Example: Hiss serum sugars for Pneumococcus.
5. Aerobic Media: Aerobic media were used to grow aerobic organisms.
6. Nutritional Factors  Anaerobic Media:
   • These media are used to grow anaerobic organisms,
   • Example:  Robertson’s cooked meat medium.

Question 8.  Write in brief on culture media.
Or
Write a short note on cultural media.
Answer: The food material on which an organism is grown is known as culture medium and the growth of an organism is known as culture.

Ideal Characteristics of an Ideal Culture Medium: Characteristics of an ideal culture medium are as follows:

• It should provide satisfactory growth from small inoculums
• It should show rapid growth.
• It should be cheap and easy to prepare
• It should be easily reproducible
• It should demonstrate all the characteristics in which the microbiologist is interested.

Requirements of Cultural Media: The following are the basic requirements of cultural media:

• Energy source
• Carbon source
• Nitrogen source
• Salts, i.e. sulfates, phosphates, chlorides, and carbonates of potassium, sodium, magnesium, calcium, iron, and trace elements such as manganese and copper
• pH should be satisfactory
• It should have adequate oxidation-reduction potential
• It consists of growth factors such as tryptophan for Salmonella typhi and glutathione for gonococci.

Ingredients of Culture Media: The following are the ingredients of culture media:

1. Water:
   • Water is very essential for the existence of living cells.
   • Demineralized distilled water is used in the culture medium.
2. Media Peptones:
   • It is a golden granular hygroscopic powder that is obtained from meat, casein firing, or soya flour.
   • It is also a complex mixture of partially digested proteins which consists of proteoses, amino acids, polypeptides, phosphates, minerals (K, Mg, Ca, Fe, Zn), and accessory growth factors (riboflavin, nicotinic acid)
   •  The functions of peptones are they act as nitrogen sources, carbon sources, and buffers.
3. Meat Extracts:
   • Meat extracts are gained by a special procedure which is commercially available as ‘Lab lemco’.
   • Lab lemco consists of protein degradation products, carbohydrates, inorganic salts, enzyme exciters, and growth factors that are rich in vitamin B complex.
   • The functions of meat extract are they act as a source of growth factors, inorganic salts, etc.
4. Yeast Extract:
   • Yeast extract is the extract of yeast cells.
   • Yeast extract consists of proteins, amino acids, growth factors (vitamin B), carbohydrates, and inorganic salts like potassium and phosphates.
   • The function of yeast extract is that it acts as a source of growth factors and hence excellent stimulator of growth.
5. Electrolyte:
   • Mainly the electrolyte is sodium chloride or other electrolytes.
   • The function of electrolytes is to maintain the osmotic pressure.
6. Media  Agar:
   1. Agar is the dried mucilaginous substance which is obtained from seaweeds.
   2. It is available in long shreds or in powder form.
   3. It consists mainly of long-chain polysaccharides, protein-material, and inorganic salts
   4. It does not provide any of the nutrition to the growing organism
   5. The function of agar is that it melts at 98°C and generally solidifies at 42°C, so it is used as a solidifying agent.
   6. It shows the difference in properties which varies from manufacturer to manufacturer, batch-to-batch variations are also present, i.e.
   7. Japanese agar: It yields gel at a concentration of 2%
   8. New Zealand agar: It yields gel at a concentration of 1%

Types Of Culture Media:

1. Based on the physical state:
   • Liquid media
   • Semisolid media
   • Solid media.
2. Based on the presence of molecular oxygen and reducing substances in media:
   1. Aerobic media
   2. Anaerobic media.
3. Based on nutritional factors:
   • Simple media
   • Complex media
   • Synthetic media
4. Special media:
   • Enriched media
   • Enrichment media
   • Selective media
   • Differential media
   • Indicator media
   • Transport media

Based on Physical State:

1. Liquid Media:
   • It is liquid in nature.
   • Liquid media does not exhibit specific characteristics on the basis of which identification is done.
   • It does not permit the isolation of different types of bacteria from mixed populations.
   • Liquid Media Uses:
     • For further testing of pure culture of bacteria.
     • For making bulk cultures to prepare antigens or vaccines.
     • For obtaining the growth of organisms from blood or water.
     • When large volumes are to be tested.
2. Semisolid Media:
   • This is neither liquid nor solid.
   • It exists between liquid and solid phases.
   • Semisolid Media Uses:
     • To study bacterial motility
     • For cultivating anaerobic and microaerophilic organisms.
3. Solid Media: Its nature is solid and produces discrete visible growth.
   • Solid Media Uses:
     • To gain pure growth
     • For studying distinctive colony morphology and other characteristic features.

Culture Media Based On Nutritional Factors:

1. Simple Media:
   • It is also known as basal media.
   • Nutrient broth is an excellent example.
   • Nutrient broth consists of peptone, meat extract, sodium chloride, and water.
2. Complex Media:
   • Media other than simple media are complex media.
   • They have added ingredients for bringing certain.
   • Characteristics or for providing special nutrients required for the growth of bacteria.
3. Synthetic Media or Defied Media:
   • Prepared from pure chemical substances and exact composition of media is known.
   • Used for special studies such as metabolic requirements.
   • The best example is Dubo’s media with Tween 80.
4. Special Media:
   • Enriched Media:
     • This media is prepared by the addition of special nutrients like blood, serum, and egg to a basal medium.
     •  It is used to grow bacteria that are more exacting in their nutritional demands. Examples are: 
     • Blood agar enriched with blood
     • Chocolate agar enriched with heated blood
     • Loeffl’s serum slope enriched with serum and
     • Lowenstein-Jensen medium is enriched with egg.
   • Enrichment Media:
     • Substances that have a stimulating effect on bacteria to be grown or an inhibitory effect on those that have to be suppressed should be incorporated into the medium.
     • If such substances are added to liquid medium this leads to an absolute increase in the quantity of wanted bacteria relative to other bacteria.
     • These media are known as enrichment media, an example of  Tetrathionate broth allow S. typhi to grow freely, and selenite F broth allows dysentery bacilli to grow freely.
     • In materials consisting of more than one bacterium, a bacterium of importance is to be isolated and is often overgrown by unwanted bacteria.
     • It is useful in situations where non-pathogenic or commensal bacteria tend to overgrow pathogenic ones.
   • Selective Media:
     • Selective media contain substances that inhibit all but a few types of bacteria and facilitate the isolation of a particular species.
     • These media are used to isolate a particular bacteria from specimens where mixed bacterial flora is expected.
     • Selective media are solid in contrast to enrichment media which are liquid. Examples of selective media are:
     •  Deoxycholate citrate agar (DCA)—the addition of deoxycholate acts as a selective agent for enteric bacilli (Salmonella, Shigella).
     • Bile salt agar (BSA) for Vibrio cholerae, Wilson-Blair medium for Salmonella.
   • Differential Media:
     • A medium that has substances incorporated in it enables it to bring out different characteristics of bacteria.
     • And helping to distinguish between them is known as differential media.
     • MacConkeys medium which has peptone, lactose, agar, sodium taurocholate, and natural red.
     • The lactose fermenters form pink-colored colonies, whereas nonlactose fermenters produce colorless or pale colonies.
   • Indicator Media:
     • This media contains an indicator that changes color when a particular bacterium is grown in that.
     • Wison and Blair medium containing sulphite which is reduced to sulfide by Salmonella typhi-producing black colonies.
     • Blood agar showing hemolysis indicates the presence of hemolytic organisms, so it is also an indicator medium.
   • Transport Media:
     • In the case of delicate organisms which may not survive the time taken for transporting the specimen to the laboratory or may be overgrown by non-pathogens.
     • Special media are developed for transporting the specimens they are known as transport media.
     • An example is Stuart’s medium which is a non-nutrient soft agar gel containing a reducing agent to prevent oxidation and charcoal to neutralize certain bacterial inhibitors used for gonococci.
   • Sugar Media:
     • Sugar is a fermentable substance.
     • Sugar media contain 1% sugar in peptone water along with an indicator.
     • A small tube is kept inverted in the larger sugar tube to detect gas production.
     • Colorless medium turns pink with the production of acid by bacteria and gas production is indicated by gas bubbles accumulated in Durham’s tube.
     • Certain bacteria are exacting in their growth requirements and need serum for their growth
     • Example: Hiss serum sugars for Pneumococcus.
5. Aerobic Media: Aerobic media were used to grow aerobic organisms.
6.  Nutritional Factors  Anaerobic Media: These media are used to grow anaerobic organisms,
   • Example:  Robertson’s cooked meat medium.

Question 9. Mention four selective media.
Answer: Selective media are the solid media that are incorporated with inhibitory substances that inhibit the growth of a large number of organisms and selectively permit the growth of required organisms in a large number.

Following are the selective media along with their composition and isolation of bacteria: