Practical Basic Microbiology and Immunology

1. Lab #2# results of sources of contamination and gram stain Practical Basic Microbiology and Immunology

2. Results of sources of contamination

3. Results • Description of colonies: 1- Shape: (Circular-irregular). 2-Size: -Small (pinpoint). -Medium (2-5 mm). -Large (>5 mm). 3-Color: -Colored (state the color you see). -Colorless. 4-Margin: (Entire- irregular). 5-Texture: (Soft- hard- mucoid). 6-Opticalcharacters: (Transparent- opaque- translucent). 7-Elevation: (Flat- convex- raised). 8-Surface: (Smooth, rough).

4. Gramstain

5. Gram stain Why do gram-positive cells keep their color while gram- negative cells lose it? Due to the difference in the structure of the cell wall. -ve: Loss of the crystal violet/ Iodine complex +ve: Retention of the crystal violet/ Iodine complex

6. Gram stain Principle of staining technique: • Primary stain:- Crystal Violet • Mordant(fixes the dye):- Iodine • Decolorizing agent:-Alcohol/Acetone • Counter stain;- Safranin

7. Gram stain Gram +ve Gram -ve

8. Gram stain • Procedure 1.Shake the suspension very well. 2.Transfere Aseptically3-4loopfulls to a clean glass slide marked from below. 3.Air or heat drying 4.Heat fixation 5.Cover film with CVleave for(30 sec)

9. Procedures {cont.} 6. Wash gently 7. Cover the film with Iodine leave for (2 min),discard 8.Add fresh Iodine leave for (1min)

10. Procedures {cont.} 9. Wash gently 10. Decolorize using : Alcohol /Acetone : for only 5 seconds

11. Procedures {cont.} 11. Wash gently 12. Cover film with Safranin &leave for (5 min)

12. Procedures {cont.} 13. Wash gently 14. Leave to dry 15. Add Oil 16. Use Oil Immersion lens

13. Gram stain {precautions} • Avoid excessivedecolorization • Avoid insufficientdecolorization • Stains are added on cool slides • Don’t dry by heat after staining

14. Staphylococcus aureus

15. Gram negative rods ,example: {Escherchia coli}

16. Bacillus subtilis