Culture Methods
Question 1: Write a short note on Anaerobiosis.
Answer:
Anaerobiosis Anaerobiosis means anaerobic conditions.
Anaerobic bacteria grow in anaerobic conditions which are achieved by the following methods:
- Production of vacuum
- Displacement of oxygen
- Displacement and combustion of oxygen
- Absorption of oxygen by chemical or biological methods
- By reducing agents
- Anaerobic chamber.
- Anaerobiosis Production Of Vacuum: Cultivation of bacteria in a vacuum is done, i.e. in a vacuum desiccator, this method is unsatisfactory and these days is not in use.
- Anaerobiosis Of Displacement Of Oxygen In this inert gases are used such as hydrogen, nitrogen, and carbon dioxide for the displacement of oxygen. The most popular method for the displacement of oxygen is the use of a candle.
- The candle is kept in air-tight containers loaded with inoculated plates and it is expected that the burning candle uses all oxygen inside before it gets extinguished but some amount of oxygen is left behind.
- Anaerobiosis Of Displacement And Combustion Of Oxygen:
- Anaerobiosis obtained by McIntosh and Filde’s anaerobic jar is the most reliable and widely used method. Culture plates with specimens are inoculated with the specimen and placed in an anaerobic jar with an indicator.
- The lid is clamped tight. The outlet tube is connected to a vacuum pump and the inlet tube is closed. The air inside is evacuated and outlet tube is closed and hydrogen gas is passed through the inlet tube till atmospheric pressure is brought back to normal.
- Terminals are switched to heat catalysts. Catalysts help to combine hydrogen and residual oxygen to form water. The indicator used is methylene blue which remains colorless in anaerobic conditions and colorful in aerobic conditions.’
- Anaerobiosis Absorption Of Oxygen By Chemical Or Biological Methods: The following are absorption of oxygen by chemical methods:
- Pyrogallol: The large tube containing a solution of sodium hydroxide and pyrogallic acid is placed inside the airtight jar and produces anaerobic conditions.
- Chromium and sulphuric acid: A mixture of two chemicals in the presence of oxygen produces chrome sulfate which produces anaerobiosis.
- Gas-pak: As inoculated plates are placed in an airtight jar packet of gas-pak with water is added inside and the lid is tightly closed which produces anaerobiosis.
- Anaerobiosis Of Biological Methods: These are used while incubating anaerobic organisms with aerobic organisms.
- Anaerobiosis Of Reducing Agents: Oxygen in culture media is reduced by various methods such as glucose, thioglycolate, cooked meat pieces, cysteine, and ascorbic acid.
- Anaerobiosis Of Anaerobic Chamber: They provide an excellent anaerobic environment. These contain a catalyst, desiccant, hydrogen gas, carbon dioxide, nitrogen gas, and an indicator.
Question 2. Describe culture techniques, various culture media, and their uses in the isolation of organisms.
Answer:
Cultural Techniques The following are the cultural techniques:
- Streak culture
- Lawn culture
- Stroke culture
- Stab culture
- Pour plate culture
Liquid culture.
- Streak Culture
- It is also known as surface plating.
- Pure culture.
- In this, a nichrome wire loop of 2 to 4 mm diameter is used.
- The loop should be first sterilized in the flame by making it red hot and it is cooled by touching the inoculated part of the medium.
- Now a loopful of specimens is smeared on the surface of the dried plate near the peripheral area. It is known as primary inoculum.
- From the primary inoculum, the specimen is spread thinly over the plate by streaking with loops in parallel lines.
- The loop is flamed and cooled in between the different sets of streaks. This is done to obtain isolated colonies over a series of streaks.
- The culture plate should be incubated at 37°C for overnight.
- Well-separated colonies are obtained at the final streak.
- Lawn Culture
- It is carried out in antibiotic sensitivity testing and in bacteriophage typing.
- These cultures are obtained by flooding the surface of the plate with liquid culture or suspension of the bacterium.
- The culture plate should be kept for 60 seconds and then excess material is poured off
- Now, the culture plate is inoculated by a sterile swab soaked in liquid bacterial culture.
- The plate is now incubated at 37°C overnight to obtain bacterial colonies.
- Stroke Culture
- It is carried out in tubes containing agar slope.
- This technique is employed for giving a pure growth of bacterium for slide agglutination and other diagnostic tests.
- Stab Culture
- It is done by the straight wire which is charged with culture material by puncturing in the agar.
- This technique is done to demonstrate gelatin liquefaction, the oxygen requirement of bacterium, and maintaining stock culture for preservation of bacteria.
- Pour Plate Culture
- Tubes containing 15 mL of agar medium are melted and cooled in a water bath for 45 to 50°C.
- The inoculum to be tested is diluted in serial dilution.
- 1 mL of each diluted inoculum is added to each tube of molten agar which is mixed well and the contents of the tube are poured into a sterile petri dish and allowed to solidify
- Now, all the plates should be incubated at 37°C overnight.
- Colonies are seen all through the depth of the medium and are counted by using a colony counter.
- Liquid Culture
- These cultures in test tubes, screw-capped bottles, or flasks are inoculated by touching with the charged loop or by adding inoculum with syringes.
- They are preferred for specimens containing antibiotics and other antibacterial substances as they are ineffective by dilution in the medium.
Anaerobiosis means anaerobic conditions.
Anaerobic bacteria grow in anaerobic conditions which are achieved by the following methods:
- Production of vacuum
- Displacement of oxygen
- Displacement and combustion of oxygen
- Absorption of oxygen by chemical or biological methods
- By reducing agents
- Anaerobic chamber.
- Anaerobiosis Production Of Vacuum: Cultivation of bacteria in a vacuum is done, i.e. in a vacuum desiccator, this method is unsatisfactory and these days is not in use.
- Anaerobiosis Of Displacement Of Oxygen In this inert gases, such as hydrogen, nitrogen, and carbon dioxide for the displacement of oxygen. The most popular method for the displacement of oxygen is the use of a candle.
- The candle is kept in an air-tight container loaded with inoculated plates and it is expected that the burning candle uses all oxygen inside before it gets extinguished but some amount of oxygen is left behind.
- Anaerobiosis Of Displacement And Combustion Of Oxygen: Anaerobiosis obtained by McIntosh and Filde’s anaerobic jar is the most reliable and widely used method. Culture plates with specimens are inoculated with the specimen and placed in an anaerobic jar with an indicator.
- The lid is clamped tight. The outlet tube is connected to a vacuum pump and the inlet tube is closed. The air inside is evacuated and outlet tube is closed and hydrogen gas is passed through the inlet tube till atmospheric pressure is brought back to normal.
- Terminals are switched to heat catalysts. Catalysts help to combine hydrogen and residual oxygen to form water. The indicator used is methylene blue which remains colorless in anaerobic conditions and colorful in aerobic conditions.’
- Anaerobiosis Absorption Of Oxygen By Chemical Or Biological Methods: The following are absorption of oxygen by chemical methods:
- Pyrogallol: The large tube containing a solution of sodium hydroxide and pyrogallic acid is placed inside the airtight jar and produces anaerobic conditions.
- Chromium and sulphuric acid: A mixture of two chemicals in the presence of oxygen produces chrome sulfate which produces anaerobiosis.
- Gas-pak: As inoculated plates are placed in an airtight jar packet of gas-pak with water is added inside and the lid is tightly closed which produces anaerobiosis.
- Anaerobiosis Of Biological Methods: These are used while incubating anaerobic organisms with aerobic organisms.
- Anaerobiosis Of Reducing Agents: Oxygen in culture media is reduced by various methods such as glucose, thioglycolate, cooked meat pieces, cysteine, and ascorbic acid.
- Anaerobiosis Of Anaerobic Chamber: They provide an excellent anaerobic environment. These contain a catalyst, desiccant, hydrogen gas, carbon dioxide, nitrogen gas, and an indicator.
Cultural Media: The following is the classification of cultural media:
- Based on the physical state:
- Liquid media
- Semisolid media
- Solid media.
- Based on the presence of molecular oxygen and reducing substances in media:
- Aerobic media
- Anaerobic media.
- Based on nutritional factors:
- Simple media
- Complex media
- Synthetic media
- Special media:
- Enriched media
- Enrichment media
- Selective media
- Differential media
- Indicator media
- Transport media
- Sugar media.
Based on Physical State:
- 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.
- 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.
- 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:
- Simple Media:
- It is also known as basal media.
- The nutrient broth is an excellent example.
- Nutrient broth consists of peptone, meat extract, sodium chloride, and water.
- 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.
- 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.
- 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.
- Enriched Media:
- Aerobic Media: Aerobic media were used to grow aerobic organisms.
- Nutritional Factors Anaerobic Media:
- These media are used to grow anaerobic organisms,
- Example: Robertson’s cooked meat medium.
Use of Culture Media in Isolation of Organisms:
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