1. PHT 381 Lab# 4

3. A Culture medium:- ❊ An artificial preparation which contains the essential elements and nutrients needed by the m.o to grow. (most bacteria &fungi) ❊ Strict intracellular organisms (e.g., some bacteria & all viruses) → only cultures of living eukaryotic cells.

4. ❊ It may be: Liquid (broth) Solid (containing agar) Semisolid (containing low conc of agar) Most common ingredients:- 1.Essential elements and nutrients. 2.Solidifying agents.

5. Inoculation: Culturing of sterile media with m.o [Inoculation loop]. Culturing of sterile media with m.o [Inoculation loop]. Incubation: Incubation: Placing the culture into the incubator at optimum temperature for growth. Placing the culture into the incubator at optimum temperature for growth.

6. Growth: Multiplication ( ↑ number) to quantities sufficient to be seen by naked eye.. Multiplication ( ↑ number) to quantities sufficient to be seen by naked eye.. Bacterial growth in the lab has 2 main forms: 1- Development of Colonies ( the macroscopic products of 20-30 cell divisions of a single bacterium on solid media) 1- Development of Colonies ( the macroscopic products of 20-30 cell divisions of a single bacterium on solid media) 2- Turbidity (macroscopic clumps) of a clear fluid medium. 2- Turbidity (macroscopic clumps) of a clear fluid medium.

7. Bacterial Growth

8. 1- Macroscopical Examination (colony morphology): Characters of colonies. Hemolysis on blood agar. Pigment production. 2- Microscopical Examination: Examination of wet mount preparation. Examination of stained preparation.

9. 3-Biochemical Tests: (The ability to attack various substances e.g., carbohydrate breakdown; or to produce particular metabolic products e.g., enzymes. 4-Additional Tests: such as seriological tests

10. Colony vs. Cell Colonies morphology (Macroscopical examination) Cells (Microcopical examination)

11. Colony vs. Cell Colonies morphology (Macroscopical examination) Cells (Microcopical examination

14. Contamination: Introduction of undesirable m.o. Asepsis: Processes designed to prevent m.o. from reaching a protected environment. Aseptic technique: Practices used by microbiologists to exclude all organisms from contaminating media or contacting living tissues.

15. An aseptic technique must be used when inoculating culture media to: 1- prevent contamination of cultures 2- prevent infection of laboratory workers and enviroment.

16. Isolation of Pure Colonies of Microorganism “Streak Plate Method” In natural environments, bacteria & other m.o exist in mixed populations. To study the cultural, morphological, and physiological characteristics of an individual organism, it is essential, first of all, that the organisms are separated from other species i.e. we must have pure culture of the micro- organism.

17. “Streak Plate Technique” Streak plate method is one of the most frequently used methods of getting a pure culture from a mixed culture. The individual cells are separated from each other by certain distance on the surface of the agar. After incubation, each single deposited cell divide many times and finally form visible mass of growth “COLONY”.

18. The streak Plate Method The culture prepared from a single type of colony is regarded as a pure culture. The streak Plate Method is used for:  Checking the purity of a bacterial culture.  Isolating individual species from a mixture culture.

20. The streak Plate Method Objective:- for isolation of individual species of a mixed broth culture. Materials:-  Nutrient agar plate.  Mixed broth culture of Serratia marcescens and Staph. aureus.

21. The streak Plate Method Procedure: S & S Aseptic technique Invert the plate and Incubate for 24h at 37 ℃ Drop of the culture Flam & Cool

22. The streak Plate Method

24. Description of Colonies

27. Sources of Contamination Objective: To identify some of the sources of contamination present in the lab. ✔ in order to avoid them Contamination from hands. Contamination from breath. Contamination from air. Contamination from bench.

28. Sources of Contamination  Sterile nutrient agar plate  a- unwashed b- washed with water c- disinfected with alcohol d- control  incubate the plate Inverted at 37°c for 24 hr.  Record the appearance of the plate a b c d 1.Contamination from hands:

29. Sources of Contamination 2. Contamination from breath:  Take a sterile nutrient agar plate  Hold it in front of your mouth  Cough and breath vigorously  Invert the plate and incubate for 1 day at 37 °c  Record the appearance of the plate.

30. Sources of Contamination 3.Contamination from air:  Expose one sterile nutrient agar plate on the bench for 30 min  Invert the plate and incubate for 1 day at 37 °c  Record the colonial appearance 30 min

31. Sources of Contamination 4.Contamination from bench:  Take a sterile nutrient agar plate and mark out 2 sections on its base  Take a swab from unclean part of the bench and press it over one section  Take another swab from cleaned part with disinfectant and press it over the second section  Invert the plate and incubate for 1 day at 37 °c  Record the result. uc c /////////