Lesson 10: CULTURE METHODS Flashcards
A population of bacteria grown in the laboratory is referred to as a
Culture
used to obtain a pure culture of an infectious agent, and also for studies leading to the identification of the pathogen.
Subculturing
Contains only single type of bacteria
Pure Culture
Contains two or more different bacteria
Mixed Culture
Periodic transfer of bacterial culture to new media to keep the bacterial population growing
Subculturing
using practices and procedures to prevent contamination from pathogens. It involves applying the strictest rules to minimize the risk of contamination
Aseptic technique
Indications for various culture methods:
- Isolate bacteria in pure culture and identify the same by performing various tests.
- Demonstrate biochemical, antigenic, and other phenotypic and genomic properties of the isolated colonies.
- Demonstrate susceptibility of the isolated bacteria to antibiotics, bacteriophages, bacteriocins, etc.
- Prepare antigens for various uses.
- Maintain stock culture.
- Estimate viable counts.
The piles of bacterial cells observed after an incubation period are called
Colonies
Complex media like EMB and Mc Conkey are use to detect what kind of bacteria
Gram Negative bacteria only
The most effective way to isolate a single type of bacteria from a source that contains many by diluting the individual cells by spreading them over the surface of an agar plate using a platinum or inoculating loop of 2–4 mm diameter. On the plate single cells reproduce and create millions of clones, which all pile up on top of the original cell.
Streak Plate Method
Procedure in Streak Plate Method
- a loopful of the inoculum is placed near the peripheral area of the plate.
- The inoculum is then spread with the loop to about one-fourth of the plate with close parallel strokes.
- From the primary inoculum, it is spread thinly over the plate by streaking with the loop in parallel lines.
- The inoculated culture plate is incubated at 37°C overnight
for demonstration of colonies.
Note: The loop is flamed and cooled in between the streaks to obtain isolated colonies.
Confluent growth occurs at the primary inoculum, but becomes progressively thinner, and well-separated colonies are demonstrated on the final streaks of the inoculum Single isolated colonies obtained by this method are very useful to study various properties of bacteria.
Streak Plate Method
Also called as carpet culture
Lawn Culture
Lawn or carpet culture method is used for
a. Antibiotic susceptibility testing by disk diffusion method
b. Bacteriophage typing
c. For preparation of bacterial antigens and vaccines
Liquid culture method is use for
a. blood culture and for sterility
b. dilution in the medium
c. large yields culture.
Note: liquid cultures does not provide a pure culture from mixed inocula—the major disadvantage, nor identify a bacteria.
After incubation, provides a uniform growth of the bacterium.
Lawn culture
Procedure of lawn culture method
- flood the surface of the plate with a liquid culture or suspension of the bacterium, pipetting off the excess inoculum and incubating the plate.
- Alternatively, the surface of the plate maybe inoculated by applying a swab soaked in the bacterial culture or suspension.
provides a pure growth of bacteria for carrying out slide agglutination and other diagnostic tests. It is carried out in tubes usually containing slanted nutrient agar slopes.
Stroke Culture
Antibiotic sensitivity testing
Lawn culture
The use of stab culture method
a. demonstration of gelatin liquefaction
b. demonstration of oxygen requirement of the bacterium under study
c. for the maintenance of stock cultures
d. to study motility of bacteria in semisolid agar
a suitable medium such as nutrient gelatin or glucose agar is punctured with a long, straight, charged wire into the center of the medium and withdrawing it in the same line to avoid splitting the medium. The medium is allowed to set, with the tube in the upright position, providing a flat surface at the top of the medium.
Stab culture method
used to determine approximate number of viable organisms in liquids, such as water or urine. It is used to quantitate bacteria in urine cultures and also to estimate the viable bacterial count in a suspension.
Pour plate culture methods
Pour plate culture method procedure
- Carried out in tubes, each containing 15 mL of molten agar.
- The molten agar in tubes is left to cool in a water bath at 45°C.
- The inoculum to be tested is diluted in serial dilution.
- Then 1 mL each of
diluted inoculum is added to each tube of molten agar and mixed well. - The contents of tubes are poured into sterile Petri dishes and allowed to set.
- After overnight incubation of these Petri dishes at 37°C, colonies are found to be distributed throughout the depth of the medium, which can be counted using a colony counter
In tubes, bottles or flasks may be inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes.
Liquid Culture