11.Media preparation (1).pptx

media preperation

1. Preparation of Media for Bacterial Growth Presented by Abdul Waheed

2. Introduction Culture media are mediums that provide essential nutrients and minerals to support the growth of microorganisms in the laboratory In the process of culturing bacteria, there are three steps The preparation of a suitable culture medium The initial removal of other organisms from the medium and its containers by sterilization 3. Adjustment of pH of the medium

3. PREPARATION OF MEDIA The basis for almost all of the bacteriological media is some kind of extract of meat (broth), which provides most of the substances required for bacterial growth The media may be solid or in liquid form Gelatin is an albumin-like material derived from tendons and cartilage Agar is prepared from dried seaweed

4. NUTRIENT AGAR Ingredients: Lab- lemco powder 1.0g Yeast extract 2.0g Peptone 5.0g Sodium Chloride 5.0g Agar 15.0g Distilled water 1L

5. Preparation Suspend 28g of nutrient agar powder (CM0003B) in 1L of distilled water. 2. Mix and dissolve them completely. 3.Sterilize by autoclaving at 121°C for 15 minutes. 4.Pour the liquid into the petri dish and wait for the medium to solidify 5.When these ingredients are dissolved in a steamer 6.The pH is adjusted to between 7.2-7.6

6. Nutrient Broth The formula for the nutrient broth is the same, except that agar is not added in it Therefore, the medium remains in liquid form It is dispensed in sterile, screw-capped tubes

7. Adjustment of pH The pH of a medium is a matter of great importance if good growth of the organisms is to be obtained and it must be adjusted before the medium is used The amount of N/10 HCl or N/10 NaOH that is to correct the pH of the 5 ml sample is then determined by titration

8. The inoculation of culture media Sterilize the wire loops and other instruments in a flame, before and after use. Protect yourself from the dangers of aerosols. Masks should be used. 2. Flame the mouth of the culture bottles and tubes after removing and before replacing their covers. 3. De-contaminate the table before you start working and after you have finished the day‘s work.

9. Continue… 4. Air currents should be reduced to a minimum by closing windows and doors and restricting the movement of people in the room. 5. During the inoculation, a culture medium should be uncovered for only a few seconds. 6. Place the lighted Bunsen burner and inoculating instruments to the right of the bench, and cultures and media to the back and the left. (If the operator is right-handed).

10. CONTINUE… 7. Media for seeding should be labelled, indicating the inoculum and the date with glass-marking pen, before seeding the plate. 8. Labelling should be on the bottom of the petri dishes, on tubes and on bottles rather than on lids or caps. 9. During inoculation, the right hand holding the inoculating instrument charged with the culture material from the specimen should be moved as little as possible and the left hand should bring the media to it.

11. Steaking method

12. Incubation Turn the plates upside down and put them in a warm place. The ideal temperature for incubation is 32° C or 90° F. Bacterial growth should start to become visible in about 2 -3 days.

13. Preparation of slide .  Place one needle of solid bacterial growth or two loops of liquid bacterial growth in the center of a clean slide. If working from a solid medium, add one drop (and only one drop)of water to your specimen with a water bottle. If using a broth medium, do not add the water.

14. Continue… Now, with your inoculating loop, mix the specimen with the watercompletely and spread the mixture out to cover about half of the total slide area. Place the slide on a slide warmer and wait for it to dry. The smear is now ready for the staining procedure

15. Staining The Gram staining process includes four basic steps, including: Applying a primary stain (crystal violet). Adding a mordant (Gram’s iodine). Rapid decolorization with ethanol, acetone or a mixture of both. Counterstaining with safranin.

16. Procedure

17. Microscopic Examination If the bacteria was colored purple, it means you likely have a Gram-positive infection. If the bacteria was colored pink or red, it means you likely have a Gram-negative infection

18. Results

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