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 • Simple staining

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 membranesof some species of red algaeor 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 procedure 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.

19. Microscopy

20. Staining • Even with the microscope, bacteria are difficult to see unless they are treated in a way that increases contrast between the organisms and their background. • The most common method to increase contrast is to stain part or all of the microbe.

21. Preparing heat fixed bacterial smear • A heat fixed smear is a thin layer of the bacterial specimen dried and fixed onto the slide. Procedure: 1- A circle should be marked on the under side of a slide with a permanent marker. Glass slide

22. 2- To prepare a smear from a suspended culture, by means of an inoculating loop, aseptically transfer 3- 4 loopfuls of the culture (after shaking), place directly on the slide and spread gently in 1 cm area. 3- Air dry or dry over the flame. Inoculation loop Dried bacterial smear

23. 4- Heat fixation : By passing the slide about five passes through the flame. 5 to 6 times • Heat fixation accomplishes three things: • (1) kills the organisms. • (2) Causes the organisms to adhere to the slide. • (3) Alters the organisms so that they more readily accept stains (dyes).

24. Heat fixation • Precautions during heat fixation of the film: • If the slide is not completely dry when you pass it through the flame, the organism will be boiled and destroyed. • If you heat-fix too much, the organisms may be incinerated, and you will see distorted cells and cellular remains. • If you heat-fix too little, the organism may not stick and will wash off the slide in subsequent steps. Fixation while Incomplete drying Or Too much fixation No Film Too little fixation

25. Types of dyes (stain) • Stains are chemicals containing chromophores, (groups that impart color). Their specificity is determined by their chemical structure and charge they carry. • Accordingly there are 3 types of dyes: • Basic dye(cationic dye):Is a stain that has positively charged chromophore. Examples of basic dyes are crystal violet,safranin, basic fuchsin and methylene blue. • Acid dye (anionic dye): Has a negatively charged chromophores.Examples of acid dyes are Nigrosine and sodium eosinate. • Neutral dye: Has both a negatively and positively charged chromophores (net charge is neutral) Example: Eosin methylene blue.

26. Mechanism of staining • The surface of bacteria is somewhat negatively charged. • When we use cationic dye (crystal violet) it is attracted by the negatively charged components of bacterial cells and stains the cells, while the background is unstained. • This is called Direct simple stain. • When we use an anionic dye (Nigrosine) it is repelled by the bacterial surface. It stains the background and leave the bacteria colorless. • This is called Negative stain

27. Staining • There are several staining methods that are used routinely with bacteria. These methods may be classified as: • Simple stain: use only one dye, it will react with different types of bacteria in an identical fashion.

28. Direct simple stain procedure • Prepare a heat fixed bacterial smear as previous. • Leave to cool • Using a dropper, cover the film with crystal violet (methylene blue or safranin). • Leave for 30 sec in case of using crystal violet, 3- 5 min for safranin or methylene blue.

29. Wash gently with water. • Dry between 2 filter papers. • Add oil and examine using oil immersion lens. NO COVER

30. NB: Oil must be cleaned from the oil immersion lens using organic solvent: xylene, after terminating sample examination

31. Microscopical examination Scheme for description of M.O