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PURE CULTUREPURE CULTURE TECHNIQUE TECHNIQUE
Ritesh Ranjan www.sunpmi.com
PURE CULTURE TECHNIQUES OF MICROBES
Presented by:Ritesh RanjanRitesh Ranjan
Email : [email protected] : [email protected]
Pure Culture Technique
Culture : Act of cultivating microorganisms or the microorganisms that are cultivated
Mixed culture : more than one microorganism Pure culture : containing a single species of organism. A pure culture is usually derived from a mixed culture (one
containing many species) by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample many times before inoculating the new medium.
Why important ? Pure cultures are important in microbiology for the following reasons Once purified, the isolated species can then be cultivated with the knowledge that
only the desired microorganism is being grown. A pure culture can be correctly identified for accurate studying and testing, and
diagnosis in a clinical environment. Testing/experimenting with a pure culture ensures that the same results can be
achieved regardless of how many time the test is repeated.
o Pure culture spontaneous mutation rate is lowo Pure culture clone is 99.999% identical
HISTORY
ROBERT KOCH (1843-1912) “Father of Practical Bacteriology”.
LOUIS PASTEUR (1822-1895) “Father of Microbiology”
Agar was discovered around 1658 by Minoya Tarozaemon in Japan
Agar was first used in microbiology in 1882 by the German microbiologist Walther Hesse ,an assistant working in Robert Koch”s laboratory
Pure culture technique consist of three interrelated techniques
Sterilization of growth media and glassware Introducing desired cells into sterile growth media or removing
samples from pure cultures without accidentally introducing contaminating microbes ,and
Isolating singles cells or, their progeny , to obtain pure culture
Something either sterile or it is not ; it is probabilitySomething either sterile or it is not ; it is probability
The most common method of sterilizing is autoclaving
Heat-sensitive solutions are sterilized by filtration
Glassware is sterilized by dry heatBunsen burner flames help to prevent
contamination during transfer into or out of containers
Some pathogenic microbes require special containment facilities .
BSC ( biological safety cabinet) is important in various aspect .
Sterilization continue……
STERLIZATIONSTERLIZATION
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Biological safety cabinet i.e. Laminar hood HEPA:(High– efficiency particulate air filters ) filters the exhaust air
BIOLOGICAL SAFETY CABINET
Obtaining Pure Cultures
•Cultures composed of cells arising from a single progenitor•Progenitor is termed a CFU•Aseptic technique prevents contamination of sterile substances or objects•Common isolation techniques
Streak plates method Spread plate method Serial dilution method Pour plates method
Isolation of pure culture continue….
Solid agar is prepared by adding agar, a complex polysaccharide derived from marine algae , to liquid media
The agar is dissolve at high temperature of the autoclaved and remains liquid as it cool down to a temperature about 45̊ C and below to this temperature gets solidify.
Solid media pour in petri plates To obtain pure cultures microbes are normally
streak onto solid media Inoculating loop are use for streaking Then the plate is incubate at desired
temperature . Then after some times ( generally 24 hours) colonies are visible wherever a microbial cell capable of growth on particular medium was deposited on the agar surface.
Then colony observation and selection of pure colony are follow
Different media are required for different microbes
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Different cell have widely varying requirement for their growth.
Media also called rich media or complex media
Media is mixture of many different organic compounds , including all of the amino acids, purine , pyrimidine , vitamins (enzyme cofactors) , etc.
Rich media generally contain growth factors , nutrition , and other supporting compound for growth of microorganism
Minimal media ( sometimes mineral media) is another media contains mineral salts , such as sulfur , nitrogen , and phosphorus
Proof of Purity of CulturesAssuming that one has isolated a pure culture, how does one establish that it is pure?
A pure culture is one in which the cells are all of one kind, i.e., demonstrate "likeness". Hence, the proof of purity of cultures consists of demonstrating the "likeness" of microorganisms in the culture. It is based on certain criteria as follows:
1.The microorganisms look alike microscopically and stain in the same fashion.2. When plated, all the colonies formed look alike.3. Streaks, stabs, etc. are uniform.4. Several isolated colonies perform identically, i.e., ferment the same sugars, and so on.
Maintenance and Preservation of Pure Cultures
Once a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure cultures free from contamination.
Since repeated sub culturing is time consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time.
Methods include preservation of pure cultures are following
RefrigerationPure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped
Paraffin MethodThis is a simple and most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin in poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for several years.CryopreservationCryopreservation (i.e., freezing in liquid nitrogen at -196°C) helps survival of pure cultures for long storage times. In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol that prevent the formation of ice crystals and promote cell survival.Lyophilization (Freeze-Drying)In this method, the culture is rapidly frozen at a very low temperature (-70°C) and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the microbes go into dormant state and retain viability for years. Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in refrigerators.
