1. Cultivation of baceriaBy
Dr. Emad AbdElhameed Morad
Lecturer of Medical Microbiology and Immunology

2. INOCULATION PROCEDURE Dr. Emad AbdElhameed Morad
Plating out technique:
Aims at spreading out the bacteria on the surface of the medium.
Each bacterium will divide to give a separate colony.
The loop is sterilized and loaded with the bacterial mixture present in the culture or the specimen. Then, it is smeared over area A to give the main inoculum.
The loop is then sterilized and after cooling, drawn into three parallel lines B across a fresh part of the medium.
The procedure is repeated as shown in the figure.
The plate is incubated at 37 degree for 24 hours.
INOCULATION PROCEDURE
Dr. Emad AbdElhameed Morad
12/1/2018

3. Dr. Emad AbdElhameed Morad
Plating out technique
Dr. Emad AbdElhameed Morad
12/1/2018

4. Dr. Emad AbdElhameed Morad
Liquid media.
Solid media.
Simple media
Enriched media
Selective media
Differential (indicator media)
Sugar media.
Enrichment media.
Cultivation of anaerobic bacteria (culture media & anaerobic jar)
CULTURE MEDIA
Dr. Emad AbdElhameed Morad
12/1/2018

5. Dr. Emad AbdElhameed Morad
Peptone water:
1% peptone + 0.5%NaCl.
Base for indole test.
Base for sugar media.
Liquid media
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

6. Dr. Emad AbdElhameed Morad
Nutrient broth (meat infusion broth):
Meat extract + 1% peptone + 0.5% NaCl.
pH is adjusted to 7.2 and sterilized in the autoclave.
Base for nutrient agar medium.
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

7. Nutrient broth with bacterial growth Dr. Emad AbdElhameed Morad
12/1/2018

8. Dr. Emad AbdElhameed Morad
Nutrient agar:
Nutrient broth + 2 – 3% agar.
Agar is sea weeds that is dissolved by heating at 100 degree and become s solidified at 45 degree.
Base for blood agar.
Detect exopigment production by pseudomonas.
Simple
Solid media
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

9. Dr. Emad AbdElhameed Morad
Nutrient agar
Dr. Emad AbdElhameed Morad
12/1/2018

10. Nutrient agar showing pseudomonas Dr. Emad AbdElhameed Morad
12/1/2018

11. Dr. Emad AbdElhameed Morad
Nutrient slope agar:
Nutrient broth + 2– 3% agar.
Poured in tubes. It has a sloping surface.
Preservation of bacteria.
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

12. Dr. Emad AbdElhameed Morad
Deep soft agar:
Nutrient broth – 1% agar.
Poured in tubes. It has horizontal upper surface.
Detection of bacterial motility.
Cultivation of anaerobes.
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

13. Dr. Emad AbdElhameed Morad
Bacterial motility
Dr. Emad AbdElhameed Morad
12/1/2018

14. Dr. Emad AbdElhameed Morad
Blood agar:
Nutrient agar + 10% sheep or horse blood.
The sterile blood is added to the nutrient agar at 55 degree to avoid hemolysis of red blood cells.
Base for chocolate agar.
Differentiates between bacteria according to the type of hemolysis produced: beta, alpha and gamma hemolysis.
Enriched
Solid media
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

15. Dr. Emad AbdElhameed Morad
Blood agar
Dr. Emad AbdElhameed Morad
12/1/2018

16. Blood agar showing beta hemolysis Dr. Emad AbdElhameed Morad
12/1/2018

17. Blood agar showing alpha hemolysis Dr. Emad AbdElhameed Morad
12/1/2018

18. Dr. Emad AbdElhameed Morad
Chocolate agar:
The same as blood agar.
But, the temperature of the medium is raised to 100 degree. During heating, red blood cells become ruptured with release of hematin.
Cultivation of highly fastidious bacteria as neisseria and hemophilus.
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

19. Dr. Emad AbdElhameed Morad
Chocolate agar
Dr. Emad AbdElhameed Morad
12/1/2018

20. Dr. Emad AbdElhameed Morad
Loffler’ serum:
3 parts horse serum + 1 part glucose broth.
Serum is the solidifying agent.
Cultivation of diphtheria bacilli.
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

21. Dr. Emad AbdElhameed Morad
Loffler’s serum
Dr. Emad AbdElhameed Morad
12/1/2018

22. Dr. Emad AbdElhameed Morad
Lowenstein-Jensen medium:
Beaten eggs + mineral salts + malachite green.
Malachite green inhibits bacteria other than tubercle bacilli.
Cultivation of tubercle bacilli.
Selective
Solid media
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

23. Lownstein Jensen medium Dr. Emad AbdElhameed Morad
12/1/2018

24. Dr. Emad AbdElhameed Morad
TCBS medium:
Thiosulphate, citrate, bile as selective agents.
Sucrose as test sugar.
Thymol blue and bromothymol blue as pH indicators.
Cultivation of Vibrio cholerae.
Vibrio cholerae grow s as yellow colonies on TCBS.
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

25. TCBS with Vibrio cholerae Dr. Emad AbdElhameed Morad
TCBS medium
TCBS with Vibrio cholerae
Dr. Emad AbdElhameed Morad
12/1/2018

26. Dr. Emad AbdElhameed Morad
MacConkey’s medium:
Peptone : nutrient.
Sodium taurocholate: inhibits non intestinal bacteria.
Lactose + neutral red indicator.
Differentiates enterobacteriacae into:
lactose fermenters which give rose pink colonies.
Non lactose fermenters which give pale colonies.
Differential
Solid media
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

27. Dr. Emad AbdElhameed Morad
MacConkey’s agar
Dr. Emad AbdElhameed Morad
12/1/2018

28. MacConkey’s agar with both lactose and non lactose fermenters
Dr. Emad AbdElhameed Morad
12/1/2018

29. Dr. Emad AbdElhameed Morad
Triple sugar iron (TSI) agar:
Glucose 0.1%, Lactose 1%, sucrose 1% are the test sugars.
Phenol red is the pH indicator.
Ferrous sulphate to detect H2 S production.
0.5% agar (soft agar cracks on gas production).
Differentiates between:
Bacteria according to their effect on sugars and production of H2S.
Composition
Uses
Dr. Emad AbdElhameed Morad
12/1/2018

30. Dr. Emad AbdElhameed Morad
Triple sugar iron
A B C D E F G H
Dr. Emad AbdElhameed Morad
12/1/2018

31. Dr. Emad AbdElhameed Morad
(From left to right)
Uninoculated control.
Red slant and red butt, no black color= no fermentation of glucose, sucrose or lactose. No Hydrogen sulfide produced.
Red slant and black butt = no lactose or sucrose fermentation, H2S has been produced.
Red slant with yellow butt = no lactose or sucrose fermentation, glucose is fermented, no H2S has been produced.
Yellow slant, yellow butt and black coloration = Lactose, sucrose and glucose fermented, and H2S has been produced.
F, G. Yellow slant, yellow butt and lifting and/or cracking of media, no black coloration = Lactose, sucrose and glucose fermented, H2S has not been produced but gas has been produced.
H. Yellow slant, yellow butt and no lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented, H2S has not been produced nor has gas been.
Dr. Emad AbdElhameed Morad
12/1/2018

32. Dr. Emad AbdElhameed Morad
Quiz A: B: C: D:
Dr. Emad AbdElhameed Morad
12/1/2018

33. Dr. Emad AbdElhameed Morad
Six tubes.
Each tube contains peptone water + specific sugar in a concentration 1%.
The sugars are glucose, lactose, maltose, mannite, sucrose, salicin.
Each tube contains also Andrade’s indicator which changes to pink if acid is produced.
An inverted small tube (Durham’s tube) is immersed in the medium and gas production is revealed by collection of bubbles at its apex.
Sugar media
Composition
Dr. Emad AbdElhameed Morad
12/1/2018

34. Uses
Differentiate between bacteria according to their fermentative effect on different sugars and whether the fermentation is accompanied by production of gas or not.
Fermentation of glucose, maltose, mannite with acid only
Fermentation of all sugars with acid + gas
Dr. Emad AbdElhameed Morad
12/1/2018

35. Dr. Emad AbdElhameed Morad
These are fluid media that contain substances that enhance the growth of some bacteria and not others.
Examples:
Selenite broth: an enrichment medium used for isolation of salmonella and shigella.
Tetrathionate broth: an enrichment medium used for isoaltion of salmonella.
Enrichment media
Dr. Emad AbdElhameed Morad
12/1/2018

36. Cultivation of anaerobes
Culture media for anaerobes
Anaerobic jar
Dr. Emad AbdElhameed Morad
12/1/2018

37. Culture media for anaerobes
Examples of anaerobic bacteria: clostridia, bacteriodes, lactobacilli.
Meat contains hematin, glutathione and lipids which reduce the oxygen in the medium.
Culture media for anaerobes
Robertson cooked meat medium
Thioglycolate broth
Deep soft agar
Dr. Emad AbdElhameed Morad
12/1/2018

38. Robertson cooked meat medium Dr. Emad AbdElhameed Morad
12/1/2018

39. Dr. Emad AbdElhameed Morad
It is a plastic jar with tightly fitted lid.
Hydrogen is introduced from hydrogen generator envelope.
Hydrogen reacts with the O2 in the jar in the presence of a catalyst suspended from the lid forming water.
Anaerobic indicator such as methylene blue is placed in the jar. It is colorless under anaerobic conditions and changes into blue in the presence of O2.
Anaerobic jar
Dr. Emad AbdElhameed Morad
12/1/2018

40. Dr. Emad AbdElhameed Morad
Anaerobic jar
Dr. Emad AbdElhameed Morad
12/1/2018

41. Dr. Emad AbdElhameed Morad
Thank You
Dr. Emad AbdElhameed Morad
12/1/2018