pureculturetechnic.pptx Automated Microbial identification

Automated Microbial identification
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1. PURE CULTURE TECHNIQUES OF MICROBES

2. 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.

3. 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. Pure culture spontaneous mutation rate is low Pure culture clone is 99.999% identical

4. 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

5. 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 probability

6. The most common method 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……

7. STERLIZATION www.waynemetalproductsinc.com

8. Biological safety cabinet i.e. Laminar hood HEPA:(High– efficiency particulate air filters ) filters the exhaust air BIOLOGICAL SAFETY CABINET

9. 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

10. Single cell isolation methods Capillary pipette method Special methods of isolation on of pure culture Micromanipulator method Enrichment culture method The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation.

11. Techniques to isolate microorganisms in pure cultures or axenic cultures Streak- plate technique of isolation The method of serial dilutions broth brotb Pour-plate/spread- plate techniques of isolation 9 m 1.0 ml to each Patrl Fewer cotonlea

12. O lnoculate empty plate. O Add nutrient O Swirl IO MÏX. Colonies grow on and in medium. 1.0 or 0.1 ml figure_0ö_17 labeled

13. Isolation of pure culture continue…. Solid aga r 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

14. Different media are required 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

15. In the clinical (and laboratory) seQing there are functiona1 types of growth Enriched media - contain complex organic substanæs that ceMain species MUST have to grow - these organisms are oten termed Selective media - contain agents that inhibit growth of certain microbes Di¥erential media - contain growth agents that promote di&rent phenotype of di&rent organisms on same media

16. Amino acids Cholesterol Hems NADH Niacin (nicotinic acid, vitamin Bg Pantothenic acid (vitamin B/ Para- aminobenzoic acid (PABA) Purines, pyrimidines Pyridoxine (vitamin Bt Riboflavin (vitamin By) Thiamine (vitamin B Components of proteins Used Dy mycoplasmas (bacteria) for cell membranes Functional portion of cytochromes in electron transj>ort system Electron carrier Precursor of NAD“ and Component of coenzyme A Precursor of folic acid, which is involvecJ in metabo ism of one- carbon compounds and nucleic acid synthesis Components of nucleic acids Utilized in transamination syntheses of amino acicfs Precursor of FAD Utilized in some decarboxylation reactions

17. Proof of Purity of 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: The microorganisms look alike microscopically and stain in the same fashion. When plated, all the colonies formed look alike. Streaks, stabs, etc. are uniform. Several isolated colonies perform identically, i.e., ferment the same sugars, and so on.

18. 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 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 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. 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.