Isolation & Preservation of Pure Culture.pptx

Isolation and preservation method for pure culture, cultivation of anaerobes, quantitative measurement of bacterial growth (total & viable count)

1. NAME-MUFTI MEHEBUB ROHAN. COURSE:~B.PHARM ROLL NO:~38405923028 REG NO:~233840210028. SUB:~PHARMACEUTICAL MICROBIOLOGY. SESSION:~2024-2025. TOPIC:~ Isolation and preservation method for pure culture, cultivation of anaerobes, quantitative measurement of bacterial growth (total & viable count) PANDAVESWAR SCHOOL OF PHARMACY

2. ISOLATION AND PRESERVATION OF PURE CULTURE:—- Pure culture techniques are used to isolate a single type of organism from a mixed culture, and are often used in research in molecular biology. Some techniques for obtaining pure cultures include The commonly used methods are: Streak plate method Pour plate method Spread plate method Roll tube method .

3. STREAK PLATE METHOD The method is based on the principle that by streaking, a dilution gradient gets established across the surface of the Petri plate as bacterial cells are deposited on the agar surface. Place a loopful of the inoculums near the periphery of the petri dish and cover with the close parallel streaks. Turn the plate at right angles and streak approx one half of the remaining portion Without overlapping the previous streaks Turn the plate to 180°c and streak the reminder of the plate, avoiding previous streaked areas.

4. POUR PLATE METHOD This technique is used to isolate microbial colonies by serial dilution and then counting the colony forming units (CFUs). Prepare a liquefied agar tube and allow to cool to 45°C. prepare a water blank. Transfer 1 ml of a suspension of a mixed culture of organism to water blank tube. Transfer 1 ml of the suspension from tube 1 to tube 2 and repeat the same process up to tube 5 to get the appropriate dilutions. Transfer 1 ml of the bacterial suspension each from tubes 1-5 sterile petri plates using sterile pipettes. Pour the liquefied agar medium in tubes into each petri plates using sterile pipettes. Pour the liquefied agar medium in tubes into each petri plate containing 1 ml of diluted suspension. Rotate the plates gently to ensure uniform distribution of bacterial cells in the medium and allow the medium to solidify. Incubate the plates for 24-48 hours at 37°C in an inverted position

5. SPREAD PLATE METHOD:— It is  a technique used to spread bacteria evenly over an agar plate so that the bacteria can be isolated and counted Take three nutrient agar plates and label them with name of the organism to be inoculated Aseptically inoculate the plates with a loopful of the given organism Place plate 1 on the turn table Sterilize the spreader by putting it first in ethanol in a beaker, then on the flame of Bunsen burner and cool the rod for 30 sec. Remove the lid of plate and spin the turn table. Touch the spreader gently on the surface of agar and move it forth and back to spread bacterial cells on the agar surface when the turn table is spinning When turn table stops spinning put the lid over the lower half of petri dish Sterilize the spreader again and repeat same process for the other two plates. Incubate all the plates at 37°C for 24 hrs.

6. ROLL TUBE METHOD:— This method is used for isolation of obligate anaerobes. A stoppered anaerobic culture tube coated with a pre-reduced agar medium containing oxygen free nitrogen is used for isolation. When the stopper is removed the tube is kept anaerobic continuously flusing it with oxygen free carbon di oxide from a gas cannula. Inoculation is done with transfer loop held against the agar surface as the tube is being rotated by a motor.

7. Presevation Methods:~~ Deep freezing method:—A long term preserve method , can be frozsen at (70-80)•c with glycerol. Refrigeration:— Short term method, can be kept in refrigerator or cold room or (0-4)•c. [3-4 Months for fungi & 2-3 weeks for bacteria]. Paraffin method:— Paraffin oil added in the culture media. Which inhibit the growth of bacteria. Coz its form a thin layer over the surface of culture media..

8. Anaerobic cultivation:— Anaerobic cultivation is  the process of growing organisms in an environment without oxygen ,. METHODS— Anaerobic chamber method:— Candle jar method:— Anaerobic jar method:—

9. Bacterial Growth:~~ Total bacterial count:- It determines the No. of both living and dead bacteria. The common methods Electronic cell counter . Counting chamber method Viable bacterial count:- It determines the No. of living bacteria only. The common methods: Pour plate method. Spread plate method.

10. Conclusion:— Isolation and preservation of pure cultures are essential for accurate microbial study and experimentation, ensuring the integrity of research and reliable results through effective techniques and proper storage conditions. Cultivating bacterial growth allows for detailed study of microbial behavior and interactions, essential for research, diagnostics, and biotechnology applications.

11. Reference:— PHARMACEUTICAL MICROBIOLOGY,   By Dr. N. K. JAIN,  Edition – 2022 (4th Edition) Manual of Systematic Bacteriology" (2nd Edition) edited by George M. Page 79-105