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Pure culture isolatin
- 2. Containing single species of microbe. Aseptic technique must be followed. No. of procedures to obtain pure culture.
- 3. To identify & study the characters of microbes. To detect the causative agent of diseases. Medical samples from clinics, samples associated with food products and the environmental samples contains mixed culture of microbes.
- 4. 1 gram of feces = Has over 1010 bacteria. That gram contains 20 different bacterial species. To identify the bacteria and antibiotic susceptibilities must be done. So pure culture is highly essential.
- 5. Technique was developed by German bacteriologist Robert Koch and his associates. Potato sliced with sterilized knife & boiled- Inoculated and streaked the bacteria over the potato surface-Incubated beneath the bell jars-Unfortunately bacteria not grown well.
- 6. Frederick Loeffler, an associate of Koch’s, developed meat extract peptone medium. He spread a mixture of Loeffler’s medium and gelatin over a glass plate-allowed it to harden-inoculated the surface in the same way as potato. Gelatin was melted & some were eaten by bacteria.
- 7. Minora Tarazaemon- 1882- discovered Agar as solidifying agent from extra seeweed soup. Used by the East Indies Dutch to make jellies and jams. Jersey–wife of Walther Hesse, Koch’s assistants-learned of agar from a Dutch acquaintance. Suggested to use agar as solidifying agent.
- 8. Use common sense to avoid contamination. Non-sterile objects must not touch the media and other equipments. Do not remove the lid of the Petri dish completely- lift it and hold it above the dish. Do not put test tube caps or Petri dish lids down. Sterilize your loop or inoculating needle by heating it until it glow.
- 9. 2 Methods Platting method Most portable number method
- 10. Most common method. Usage of petriplate. 3 types. Streak Plate Method, Spread Plate Method, Pour Plate Method.
- 11. Developed by Loeffler and Gaffkey in Robert Koch laboratory. Continuous dilution of the microbes by streaking, results in separation of individual cells. Mixed microbial culture is transferred to the edge of an agar plate and then a series of parallel non-overlapping streaks.
- 12. Different types of streak pattern was performed. Single streak Continuous streak T-Streak Radiant streak Quadrant streak
- 19. Suitable for obtaining pure colony isolation. Done for the microbial suspension containing 30-300 colonies over the agar plate. Done by sterile bent rod or spreader or L-rod.
- 20. Transfer 0.1ml of dilution of the serially diluted sample. On the centre of the pre-poured agar plate. Spread over by the L-Rod. After incubation calculate the no. of colonies. Suitable for heat sensitive microbes.
- 22. Original sample was diluted several times. The diluted sample of 0.1ml or 1 ml was mixed with liquid agar @ 45 degree celsius & poured on the petriplate. After incubation colonies appear on both within the agar & on the surface of the agar.
- 23. Suitable for aerobic, facultative aerobic & non-stringent microbes. More than 0.1ml can be used. Not suitable for heat sensitive microbes. Not suitable for diagnostic state.
- 26. One of the type of pure culture isolation. Used for the estimation of number of microorganisms in foods, waste water and other samples. Mainly done to determine the coliforms & E.coli of the sample. Also called as MPN method.
- 27. Coliform utilizes the lactose present in the medium & produces the acid & gas. Contains 3 phases Presumptive Confirmatory Completed
- 29. Presumtive Phase 15 test tubes-Each 5 are innoculated with 5ml,1ml & 0.1ml in a lactose broth incubated at 37 °C for 24 h. After incubation the test tubes are examined for gas production.
- 30. Confirmatory Phase All positive presumptive cultures used to inoculate tubes of 1ml in a brilliant green lactose bile broth incubation for 48 ± 3 hours at 35°C. Positive test tubes of gas production are further examined for completed phase.
- 31. Completed Phase 1 loopfull of suspension - streaked onto eosin methylene blue agar (EMB) - incubated at 37 °C for 24 h Positive – greenish metallic sheen Calculated per MPN table.
- 32. Single cell isolation method Isolation of 1 cell from mixed culture & then allowed to grow separately. 2 types Capillary pipette method Micromanipulator method
- 33. Sample containing microbe is placed on the cover slip. Each drop is searched under the microscope that containing single microbe. That drop is sucked using sterile capillary pipette. Transfer to fresh medium & starts multiplication.
- 35. INSTRUNMENT: Microscope + Micropipette Have adjustment that can move in all direction. Microscope search for single microbe. Sucked by the micropipette. Transferred to the culture medium Starts multiplication.
- 38. Micro-Inoculation culture method. Substitute for Pour plate & MPN method. Developed by Thailand Research team & succeeded in field works.
- 39. Micro inoculation culture (MIC) plates 96 well microtiter plate (5 mm width, 10 mm length and 1.5 mm thickness) 96 samples = 96 Petri dishes
- 40. 1 microwell = Chromocult® Coliform agar (CCA) + inoculation volume of 10 μl Incubated at 37 ºC for 12–15 hrs. Dark blue – E.coli Red – Coliform Finally re streaked by EMB agar medium for isolation.
- 41. DIMENSION MIC POUR PLATE MPN Detection time 12 - 18 h 2 – 3 days 3 – 4 days Medium usage 0.5 ml/well 25 ml/plate 9 ml/tube Inoculum size 0.01 ml 1ml 10ml/1 ml/0.1ml
- 42. Rapid & accurate reading Less costly Pour plate- $36 MIC- $6 (preparation, media, disposable material, utility, etc.) Mainly reduces the incubation time. High volume microbial enumeration samples.