Different Types Of Culture Methods

What Is Culture Method

Culture methods are used to determine the type of organisms and culture methods also used to determine antibiotic susceptibility culture method to help in the preparation of antigens for serodiagnosis.

Methods Of Culture

  1. Streak Culture
  2. Lawn Culture
  3. Stroke Culture
  4. Stab Culture
  5. Pour Plate Culture
  6. Liquid Culture

1. Streak Culture Method

It is the routiné method employed for bacterial isolation in pure culture. A platinum or nichrome wire loop of 2-4 mm internal diameter is used. Due to the high cost of platinum, loops for routine laboratory work are made of nichrome wire.

This loop is first sterilized in the Bunsen flame by making it red hot and cooled by touching an uninoculated part of the medium. Then a loopful of the specimen is smeared onto the surface of a dried plate near the peripheral area.
This is named as primary inoculum. From the primary inoculum, it is spread thinly over the plate by streaking with the loop in parallel lines. It is done to obtain isolated colonies over the final series of streaks. The culture plate is incubated at 37°C overnight.

2. Lawn Culture Method

This type of culture method is employed in antibiotic sensitivity testing and in bacteriophage typing. Lawn cultures are obtained by flooding the surface of the plate with a liquid culture or suspension of the bacterium. The culture plate is kept for a minute and then excess material is poured off. Alternatively, the culture plate may be inoculated by a sterile swab soaked in liquid bacterial culture or suspension. The plate is then incubated at 37°C overnight to obtain bacterial colonies.

3. Stroke Culture Method

Stroke culture is done in tubes containing agar slope. It is employed for providing a pure growth of the bacterium for slide agglutination and other diagnostic tests. A commonly used agar slope is the nutrient agar slope.

4. Stab Culture Method

Stab culture is performed by a straight wire, charged with culture material, by puncturing deep inside the agar. The technique is employed to demonstrate gelatin liquefaction and to maintain stock cultures for the preservation of bacteria.

5. Pour Plate Culture Method

Tubes containing 15 ml of agar medium in each are melted and kept to cool in a water bath at 45-50°C. The inoculum to be tested is diluted in serial dilutions. One ml of each diluted inoculum is added to each tube of molten agar, mixed well and the contents of the tube poured into a sterile Petri dish and allowed to solidify.

These plates are incubated at 37-degree celsius overnight Colonies will be seen throughout the depth of the medium and can be counted. This culture method is used to estimate viable bacterial count in a suspension or to quantitate bacteria in urine culture.

6. Liquid Culture Method

Liquid cultures in test tubes, screw-capped bottles, or flasks may be inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes. This type of culture method is adopted for blood culture and for sterility tests, where the number of bacteria in the inocula are expected to be small.

Incubation of Culture Media

Most of the pathogenic organisms grow best at 37°C i.e. body temperature of human beings. The inoculated culture media are incubated at 37°C in an incubator.

Anaerobic Culture  Methods

Anaerobic bacteria grow only in the absence of oxygen (anaerobic conditions). These anaerobic conditions or anaerobiosis can be established by various methods.

Methods of Anaerobiosis

  • Displacement of oxygen by inert gases
  • By displacement and combustion of Oxygen
  • Absorption of oxygen by biological methods
  • By Reducing Agents
  • Anaerobic Chamber

Displacement of Oxygen

Displacement of oxygen by inert gases like hydrogen, nitrogen, carbon dioxide, or helium is sometimes employed. A popular, but ineffective, the method is the use of the candle. It is expected that burning candles will use up all the oxygen inside before it is extinguished but some amount of oxygen is always left behind.

By Displacement and Combustion of Oxygen

Anaerobiosis obtained by Mclntosh and Fildes anaerobic jar is the most reliable and widely used method.

Procedure

Mclntosh and Filde’s anaerobic jar consists of a stout glass or metal jar with a metal lid that can be clamped air-tight with a screw. The lid is fitted with two tubes with taps, one acting as an inlet for the introduction of gas and the other as the outlet. The lid also contains two terminals which can be connected to an electrical supply.

A catalyst is suspended under the lid by stout wires which are connected with the terminals to heat the catalyst for its activity. Nowadays catalyst at room temperature is used. Culture plates inoculated with specimens are placed inside the anaerobic jar with an indicator. The lid is clamped tight.
The air inside is evacuated. The outlet tube is then closed and hydrogen gas is passed through the inlet tube till reduced atmospheric pressure is brought to normal the atmospheric pressure which is monitored on the vacuum gauze as zero.

Reduced methylene blue is generally used as an indicator of anaerobiosis in the jar. It remains colorless in anaerobic conditions but turns blue on exposure to oxygen.

Absorption Of Oxygen by Biological Methods

Biological Methods

This has been attempted by incubating aerobic organisms along with anaerobic bacteria. Two blood agar plates are taken-one is inoculated with Pseudomonas aeruginosa (aerobic bacteria) and the other with a specimen of anaerobic bacteria. Two plates are placed one over the other and sealed along the rims and are incubated.

Based on the above principle, the two most widely employed anaerobic liquid culture media are:

1. Thioglycollate Broth

It contains nutrient broth and 1% thioglycollate.

2. Cooked Meat Broth (CMB)

It is also known as Robertson’s cooked meat (RCM) medium. It contains nutrient broth and pieces of fat-free minced cooked meat of ox heart.

Principle

  • Unsaturated fatty acids present in meat utilize oxygen for autooxidation, this reaction is catalyzed by haematin in the meat.
  • Glutathione and cysteine both are present in meat also utilize oxygen. 
  • Sulphydryl compounds also contribute to a reduced oxidation-reduction (OR) potential.

Procedure

  • Before inoculation, the medium is boiled in a water bath at 80°C for 30 minutes to make it oxygen-free.
  • For strict anaerobiosis, the surface of the CMB medium may be covered with a layer of sterile liquid paraffin.

Anaerobic Chamber

It is an anaerobic inoculation system. It provides an oxygen-free environment for inoculating culture media and for their incubation. It is fitted with airtight rubber gloves to insert hands for working with specimens. These anaerobic chambers contain a catalyst, desiccant, hydrogen gas, chambers contain carbon dioxide gas, nitrogen gas, and an indicator.
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