pureculturetechnic-160204060702.pdf

pureculturetechnic-160204060702.pdf

• 1.
  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.
• 2.
  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 low o Pure culture clone is 99.999% identical
• 3.
  HISTORY ROBERT KOCH (1843-1912) “Fatherof 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
• 4.
  Pure culture techniqueconsist 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 probability Something either sterile or it is not ; it is probability
• 5.
  The most commonmethod of sterilizing is autoclaving Heat-sensitive solutions are sterilized by filtration Glassware is sterilized by dry heat Bunsen 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……
• 6.
  STERLIZATION STERLIZATION www.waynemetalproductsinc.com
• 7.
  Biological safety cabineti.e. Laminar hood HEPA:(High– efficiency particulate air filters ) filters the exhaust air BIOLOGICAL SAFETY CABINET
• 8.
   Obtaining PureCultures •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
• 12.
  Isolation of pureculture 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 45 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
• 13.
  Different media arerequired for different microbes www.datuopinion.com  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
• 17.
  Proof of Purityof Cultures Assuming 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.
• 18.
  Maintenance and Preservation ofPure 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 Refrigeration Pure 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
• 19.
  Paraffin Method This isa 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. Cryopreservation Cryopreservation (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.