Copy of Acid fast staining and results - M smegmatis and Staphylococcus epi PDF

Preview

Summary

Download Copy of Acid fast staining and results - M smegmatis and Staphylococcus epi PDF

Description

Heat fixing: Add 2 drops of water on a slide. Put a small amount of Staphylococcus epidermidis in one and Mycobacterium smegmatis in the other Heat fix the slide until the smears are dry Acid fast Staining: 1. Put the kinyoun carbolfuschin over the heat fixed bacteria and let it stand for 5 min 2. Wash with distilled water, do not blot dry 3. Wash with acid-alcohol (let it trickle down the slide) for 20 seconds* 4. Wash with distilled water, do not blot dry 5. stain with brilliant green or methylene blue (they are both blue in color) for 30 seconds 6. wash with distilled water and blot dry with paper towels Observe under 100x objective with oil immersion Explanations: Mycobacteria have very thick and waxy cell wall. It is resistant to being washed by acid-alcohol (hence the name “acid-fast”. The mycobacteria should retain the red dye (carbolfuschin) after being washed briefly with acid-alcohol. For all other bacteria that do not have thick waxy cell wall, acid-alcohol will wash away the carbolfuschin very quickly and the bacteria will only show the color of the second stain (Brilliant Green- which is blue in color) *Standard clinical procedure for performing acid fast for diagnosing Mycobacterium tuberculosis or M. leprae infections calls for 1 min of destaining with alcohol. However, the M. smegmatis we grow in the lab does not have as thick/waxy a cell wall and most likely will not retain the carbofuschin after 1 minute of washing with acid alcohol. Therefore, I opt to reduce the washing time to demonstrate a better result for this lab. Caution: acid alcohol is 95% alcohol supplemented with 3% hydrochloric acid (HCl), it is very caustic and will burn your skin. You must wear a lab coat and gloves when handling this reagent. If you spill any on your skin, wash immediately with plenty of water. Results: Morphology: Color: Acid Fast Status:

Staphylococcus epidermidis round shape (cocci) blue negative

Mycobacterium smegmatis rod shape (bacilli) mostly red positive

Clincal application: Acid fast test is used to diagnose Mycobacterial diseases. The most common disease this test can diagnose is Mycobacterium tuberculosis. Other diseases that can be diagnosed with this tests include Mycobacterium leprae, Mycobacterium avium, and Cryotosporidium parvum

Mycobacterium smegatis

Mycobacterium smegatis

Staphylococcus epidermidis...