Culture Methods

CULTURE TECHNIQUES:

           

In clinical laboratory indications for culture are:

• Isolation of bacteria in pure culture.
• To demonstrate their properties.
• To obtain sufficient pure growth for preparation of antigen and for other tests.
• For typing bacterial strain isolate by method like bacteriophage bacteriocin susceptibility.
• To determine sensitivity to antibiotics.
• To estimate viable counts.
• To maintain stock culture.

 

Culture Methods for bacteria in Bacteriology

CULTURE METHODS:

 

• Streak culture
  • It is the method routinely employed for the isolation of bacteria in pure culture. A platinum loop is charged with specimen to be cultured and is placed on the surface of dried plate of solid media towards peripheral area. The inoculum is spreaded thinly over the plate in series of parallel lines in different segment of plate. On incubation confluent growth at the site of primary inoculum appears, while well-separated colonies appear over the final series of streaks.

 

• Lawn culture
  • It is prepared by flooding the surface or plate with suspension of bacteria. It provides uniform surface growth of bacteria. It is useful for bacteriophage typing and antibiotic sensitivity test.

 

• Stroke culture

  

  • It is made in tubes containing agar. It is used for providing a pure growth of bacterium for slide agglutination.

 

• Stab culture
  • It is prepared by puncturing with charged Nichrome straight wire. Stab culture is employed for demonstration of gelatin liquefaction and for maintaining stock culture.

 

• Pour Plate culture
  • Agar medium is melted and appropriate dilutions of inoculum is added to molten agar and mixed well. Then it is poured in petri dish and allowed to set and after incubation bacterial colonies will appear throughout the depth of the medium. This method gives viable count in a suspension. It is the recommended method for quantitative urine culture.

 

• Liquid culture
  • liquid culture in a tube, bottle or flask may be inoculated by touching with a charged loop. Liquid culture is preferred when large and quick yield is required.

 

 

Selections of temperature and environmental conditions:

Once medium is selected, temperature and environmental conditions must also be considered.

• Environmental conditions are chosen according to the growth requirements of the indigenous flora or pathogens suspected for the body site from which the specimen is taken.
• Fastidious organisms may require increased CO₂ or an anaerobic environment for growth.
• Most routine bacterial culture plates are incubated at 35⁰ C to 37⁰ C for 48 hours.
• Broth cultures when included are routinely held 5 to 7 days.

 

Primary isolation media for unusual and fastidious bacteria:

Temperature requirements and length of incubation vary for individual organisms. Unusual organisms may require special processing and selections of medium beyond the routine.

 

METHODS OF ANAEROBIC CULTURE:      

Obligate anaerobes grow only in absence of free oxygen. These bacteria lack mechanism of oxidation through respiratory enzymes like cytochrome oxidase, catalase and peroxidase resulting in H₂O₂ accumulation, which is toxic for the growth of anaerobic bacteria.

 

Techniques:

Various methods are available for achieving Anaerobiosis on the basis of following principles.

• Displacement of oxygen.
• Absorption of oxygen by chemical or biological means.
• Reduction of oxygen.

 

1. Gaspak system:
   • It is the method of choice for preparing anaerobic jars. It is available as a disposable envelope, containing chemicals, which generate hydrogen and carbon dioxide on the addition of water. After the inoculated plates are kept in the jar, the Gaspak envelope, with water added, is placed inside and the lid screwed tight. Hydrogen and carbon dioxide are liberated and the presence of a cold catalyst in the envelope permits the combination of hydrogen and oxygen to produce an anaerobic environment. The Gaspak is simple and effective.

 

2. McIntosh and Fildes anaerobic jar:
   • It is the most reliable and widely used anaerobic method. It consists of glass or metal jar with metal lid, which can be clamped airtight with screw. The lid has two tubes, one act as a gas inlet and the other one as outlet. Additionally lid has two terminals, which can be connected to electrical supply. Inoculated culture plates are inside jar Outlet tube is connected to vacuum pump and air inside is evacuated. The outlet tap is closed and inlet tube is connected with hydrogen, electric terminals are connected so that palladinised asbestos is heated. This act as catalyst for combination of hydrogen and residual oxygen. It ensures complete anaerobiosis. It carries the risk of explosion.

 

3. Robertson’s cooked meat medium:
   • It is probably most widely used fluid medium for culture of anaerobes. It consists of fat-free minced cooked meat in broth, with a layer of sterile vaseline over it. It permits the growth of even strict anaerobes and indicates their saccharolytic or proteolytic activities, by the meat being turned red or black, respectively.

 

4. Anaerobic chamber:
   • It is used for fastidious anaerobes, particularly for quantitative cultures. The anaerobic chamber is an airtight, glass-fronted cabinet filled with inert gas, with an entry lock for the introduction and removal of materials, and gloves for the hands. Pre-reduced media are required for isolation of anaerobes

An indicator should be employed for verifying anaerobic conditions in various anaerobic techniques. Reduced methylene blue is generally used. It remains colorless anaerobically but turns blue on exposure to oxygen.

 

CAPNOPHILIC ORGANISMS:

 

Candle jar

Here inoculated plates are placed inside airtight container and lighted candle is kept before lid is sealed. A burning candle use up all the oxygen before it gets extinguished, some oxygen is left behind. It provides concentration of carbon dioxide, which stimulates growth of capnophilic organisms like S. pyogenes, N. meningitides, H. influenza etc.