pure culture isolation 1 Pure Culture Isolation Subject

pure culture isolation 1. Pure. Culture. Isolation Subject : Fundamentals of Bacteriology. 2. Introduction • Bacteria exist is nature in mixed forms • To study every species independently, it is necessary to obtain their pure culture • The two major steps of obtaining a pure culture as follows : �Firstly, the culture has to be diluted until the various individual microorganisms are separated far apart on agar surface that after incubation they form visible colonies isolated from the colonies of other microorganisms. This plate is called an isolation plate �Secondly, an isolated colony has to be aseptically picked off the isolation plate

3. Points to be taken into consideration during the inoculation are : �The inoculation loop has to be sterilized before every inoculation of colonies from the agar plate �Every time the loop is sterilized by heat it must be cooled before inoculating the next colony • There are several methods of isolating the pure culture of bacteria, like, streak plate method, pour plate method, spread plate method and serial dilution 4. Streak. Plate. Method • The most common technique of isolation of bacterial cells on the surface of agar plate is the streaking method • It is a simple and a rapid way of isolating cells of bacteria on the agar plate surface through mechanical means • As the loop is streaked on the agar surface the bacterial cells are rubbed off until individual separate organisms are deposited on the agar 5. • After incubation the area at the beginning of the streaking will show confluent growth of bacterial cells, whereas, the area at the end of the streak will show discrete colonies of bacterial cells

6. 1. Flame the loop. 2. Without setting the loop down, open the first culture tube and flame the mouth. Do not set the cap on the bench. The cap should be held in the same hand as the loop. 3. Insert the loop into the culture medium, then withdraw it. 4. Flame the mouth of the first culture tube again, and replace the cap. 7. �Attempts to identify bacteria in a clinical sample cannot be done unless isolated colonies are used. �To obtain well-isolated colonies, it is essential to disperse the inoculum (sample) on the surface of an enriched agar plate so that individual bacteria are well separated from each other. �Contaminants = other microorganisms present in the sample �Isolated colois= a population of millions of cells that are identical and are descendent from a single founder cell �Stock Culture = a culture that already contains cells. � It is used a source of cells from which to inoculate new cultures. �culture medium: rich/selective �growth inhibitors �liquid/solid � temperature �source of energy �sources of carbon, nitrogen, . . . �Aseptic technique: � sterilization of medium and equipment � proper handling

8. Indications for culture - �Isolate bacteria in pure cultures. �Demonstrate their properties. �Obtain sufficient growth for preparation of antigens & for other tests. �Typing bacterial isolates. �Antibiotic sensitivity. �Estimate viable counts. �Maintain stock cultures. 9. �STREAK PLATE TECHNIQUE �POUR PLATE TECHNIQUE �SPREAD PLATE TECHNIQUE 10. 1. With the loop, spread the inoculum back and forth across the upper 1/4 of the plate, keeping the lines of inoculation very close together (area 1 in figure). 2. Isolated colonies are not expected in this area. Do not use strong pressure, which will break the surface of the agar. Use the end of the loop, not its side when streaking. Dispose of the loop in the biohazard bucket on the bench. 3. Turn plate approximately 900. Streak the plate as indicated in the figure (area 2) across about 1/4 of the plate. Dispose of the loop. 4. Repeat step 2 one or two times more. 5. In area 3 and/or 4 single colonies should appear. 6. Label plates on the bottom and incubate inverted at 370 C

11. 1 ml of appropriately diluted inoculum is added to 15 ml of molten agar and poured on petridish. • Colonies appear through out the depth of medium. • Used to estimate viable count, recommended method for quantitative urine cultures. 12. • Prepare LAF bench with required material. • Dilute the sample and shake gently to suspend organisms. • Insert the loop into the test tube and remove loop full of broth with bacterial culture using aseptic techniques. • Put the loop full of culture on the sterile agar plate. • Spread the culture with L- shaped glass on the surface of agar by rotating the Petri. Plate and L-shaped glass both. • Keep the lid over the plate as much as possible. • Incubate to develop the culture.