1. Microbial Biotechnology Reem Alsharief Lab 3

2. General Methods of Isolation and selection of Microorganism Microbial isolation: To separate (a pure strain) from a mixed bacterial or fungal culture.( to obtain a pure culture) A microbiological culture(or microbial culture) :is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions.

3. Purpose of Culturing To determine the type of organism and its abundance in the sample being tested. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined media.

4. To study the properties of bacteria To create antigens for laboratory use To test for Antibiotic sensitivity Typing with Bacteriophages and Bacteriocins susceptibility.

5. Bacterial Culture Streaking Bacterial culture streaking allows bacteria to reproduce on a culture medium in a controlled environment. The process involves spreading bacteria across an agar plate and allowing them to incubate at a certain temperature for a period of time. Bacterial streaking can be used to identify and isolate pure bacterial colonies from a mixed population.

6. A Streak Plate A single bacterial colony

7. Obtaining single colonies is important as it enables The size, The shape Colour of the individual colonies to be examined. It can also highlight the presence of contaminating micro-organisms

8. 1. Streak Plate Methods A.Crossing Shape ( The quadrant streak technique) Tools you need: Gloves Inoculating Loop (or sterile toothpick) Bunsen Flame Agar plates Culture media with microorganism

9. Technique: 1.Flame the inoculating loop until it is red hot and then allow it to cool 2.Remove a small amount of bacterial growth with a sterile inoculating loop (either from a broth, single colony from a plate or a slant) 3.Immediately streak the inoculating loop very gently over a quarter of the plate using back and forth motion

10. 4.Flame the loop again and allow it to cool. Going back to the edge of area 1 that you just streaked, extend the streaks into the second quarter of the plate (area 2). 5. flame the loop again and allow it to cool. Going back to the area that you just streaked (area 2), extends the streaks into the third quarter of the plate (area 3) 6.Flame the loop again and allow it to cool. Going back to the area that you just streaked (area 3), extends the streaks into the center fourth of the plate (area 4)

11. Quadrant streak

13. B. Zigzag or Snake shape technique Procedure: 1.While wearing gloves, sterilize an inoculating loop by placing it at an angle over a flame. The loop should turn orange before you remove it from the flame. A sterile toothpick may be substituted for the inoculating loop. Do not place toothpicks over a flame. 2.Remove the lid from a culture plate containing the desired microorganism. 3.Cool the inoculating loop by stabbing it into the agar in a spot that does not contain a bacterial colony.

14. 4. Pick a colony and scrape off a little of the bacteria using the loop. Be sure to close the lid. 5. Using a new agar plate, lift the lid just enough to insert the loop. 6. Streak the loop containing the bacteria at the top end of the agar plate moving in a zig-zag horizontal pattern until 1/3 of the plate is covered. 7. Sterilize the loop again in the flame and cool it at the edge of the agar away from the bacteria in the plate that you just streaked.

15. 8. Rotate the plate about 60 degrees and spread the bacteria from the end of the first streak into a second area using the same motion in step 6. 9. Sterilize the loop again using the procedure in step 7. 10. Rotate the plate about 60 degrees and spread the bacteria from the end of the second streak into a new area in the same pattern. 11. Sterilize the loop again. 12. Replace the lid and invert the plate. Incubate the plate overnight at 37 degrees Celsius (98.6 F̊). 13. You should see bacterial cells growing in streaks and in isolated areas

16. ZigZag Shape

18. 2. Serial dilution for bacterial enumeration An easier method is to spread bacteria over a wide area (i.e. nutrient agar plate) and count the number of colonies that grow. If the bacteria are spread out enough, each bacterial cell in the original sample should produce a single colony. Bacterial cell numbers need to be reduced, which is done by repeatedly diluting the amount of bacteria you have in your sample. A small amount of bacteria sample is mixed with a diluent solution (such sterile broth), and then successive dilutions are made. A small amount of each of the diluted bacteria samples is then spread onto an agar plate. The numbers of bacteria colonies that grow on each plate are counted.

19. Materials: Gloves. Mixed bacterial culture. 6 tubes of distilled water ( each tube contains 9ml of dis. Water) 12 Petri dishes ( 2 per 1 tube sample) Nutrient agar media ( cooled at 45 ̊C in a water bath) Pump Pipettes. Bunsen flame

20. Procedure: 1.One milliliter from the sample was transferred to 9 ml of distilled water in tube A and mixed. This was a 1/10 serial dilution. 2. One milliliter from tube A will be transferred to 9 ml of distilled water in tube B and mixed. This was another 1/10 serial dilution. The total dilution up to this point is 1/100. 3. Then 1 ml from tube B will be transferred to 9 ml of sterile media in tube C and mixed. This is a 1/10 serial dilution. The total dilution up to this point is 1/1000.

21. 4. Continue the serial of dilution until the total dilution reach up to 1/1000 000 (10 ̄⁶) Tip: change the Pipette each time you dilute the solution. 5. Pour about 15-20 ml of the nutrient agar into each dish and shake it slightly in a circular round. 6. Incubate the agar plates in a 37 degree incubator and you should see the bacterial colonies in 24-48 hours. 7. Count the number of bacteria colonies that appear on each of the plates that has between 30 and 200 colonies. Any plate which has more than 200 colonies is designated as "too many to count" (TMTC). Plates with fewer than 30 colonies do not have enough individuals to be statistically acceptable.

22. 8. To compute the estimated number of bacteria on the surface that you tested, use the following formula: B = N/D B = number of bacteria N = number of colonies counted on a plate D = dilution factor (either 1, 10 or 100)

23. Thank You