Practical Basic Microbiology and Immunology

1. Practical Basic Microbiology and Immunology

2. Microbiology • Microbiology is the science dealing with microorganisms. • We are going to study microorganisms: Microscopically Macroscopically

3. Our session today • Culture media. • Aseptic technique. • Sources of contamination.

4. Culture Medium • Definition: It is a medium used for growing microorganisms. • Forms: liquid, solid, semi solid.

5. Essential requirements in culture media • Any culture medium must contains: -A source of energy. -Sources of carbon, nitrogen, sulfur, phosphorus. -Minerals, e.g., Ca2+ ,Mg2+,Na+. -Vitamins and growth factors. - Water.

6. Basic media • Culture media may be in liquid form ex: Nutrient broth : - Composed of nutrients + water. • Or may be in a solid form ex: Nutrient agar: -Similar to nutrient broth but supplemented with solidifying agent (1-2% agar).

7. For solid medium, A solidifying agent is incorporated in the media, Agar (1-2%). • Agar agar: It is an un-branched polysaccharide obtained from the cell membranes of some species of red algae or seaweed. • Agar is not digested by M.O. • Agar melts in a water bath at 98˚C, and solidifies at 42˚C.

8. Solid culture media Plate (Petri dish) deep Slant

9. M.O are widely distributed around us, How to avoid contamination of our work with pure culture?

10. Aseptic technique • Is the procedures used by microbiologists to prevent: • Microbial contamination of themselves, which may result in infection. • Contamination of the environment they are working in. • Contamination of the specimen they are working on, which is especially important when a pure culture is desired.

11. Aseptic technique involves: 1- Working in 20 cm diameter around a blue flame (sterile zone). 2- Never leave a culture dish open, even for a short time ,when it is necessary to open a dish, keep the lid close to the dish, and keep the lid between your face and the agar surface.

12. Aseptic technique involves (cont.): 3- For most bacterial cultures you will use a sterile loop or needle to inoculate or to obtain an inoculum. 4- Flame a loop or needle to red-hot just prior to use, burning off any organic material ,Cool the loop prior to touching a culture. Inoculation loop

13. Aseptic technique • Pass the neck of a culture tube or any container with a culture or sterile contents through a flame before taking culture from it • Use sterile glass wares • Use sterile culture media

14. Practical work Sources of contamination • Materials one sterile Petri-dish one molten nutrient agar tube • Procedure 1- Pour the nutrient agar tube at the suitable temperature aseptically into the provided plate. 2- Leave the nutrient agar plate to solidify.

15. Pouring the nutrient agar

16. Practical work Sources of contamination(cont.) 3- Expose the agar plate to one of the following source of contamination: - Air (outside the aseptic zone) - Forced air - Skin touch - Cough (Inside the aseptic zone) - A piece of hair or cloth - Drop of water

17. Practical work Sources of contamination(cont.) 4- A student in each bench will perform a control plate. 5- Label the cover of the plate with your name, no., source of contamination. 6- Incubate the plate inverted , except for water containing plates.

18. Incubation • The plate is incubated, usually for 24 to 36 hours, to allow the microorganism to reproduce. • At the end of incubation there should be enough microorganism to form visible colonies.