Name: Sara BolandLab: Saturday, 8:00 a.m.

Lab Name: Acid Fast Staining

Purpose: Certain bacteria, specifically mycobacteria have a waxy outer layer which makes them resistant to staining. This is where the acid-fast staining procedure comes into play. One of the most well-known diseases caused by these acid-fast bacteria is Tuberculosis. Tuberculosis causes respiratory distress due to the infection of the alveoli with the M. tuberculosis bacteria. These bacteria are particularly virulent because their waxy exteriors make them resistant to phagocytosis and will instead replicate and kill the macrophages. Clusters of bacteria and macrophages both dead and alive cause the formation of tubercules. The tubercules along with the overwhelming inflammatory response to the M. tuberculosis bacteria can cause respiratory failure and lung damage. The Acid-Fast staining technique was developed to allow scientisst to study mycobacteria and their morphology. Mycobacteria have neither a Gram-positive nor a Gram-negative cell wall. Rather they have a third type which, while it does contain peptidoglycan, is surrounded by mycolic acid which makes it waxy and relatively impenetrable. The Acid-Fast staining technique consists of using Carbol Fuschin, a lipid-soluble dye which is them steamed to allow penetration of the mycobacterial cell wall. The slide is then washed with an acid-alcohol destain and stained again using Methylene Blue. Once the slide is rinsed in water, acid-fast bacteria should appear red under the microscope with all others appearing blue. The purpose of this lab is to introduce us to the technique of acid-fast staining as well as giving us an opportunity to view mycobacteria morphology under the microscope through the simulation of a TB scenario. I would expect one patient sample to be positive and one to be negative.

Materials:-microscope slides -innoculating loop-clothespins -flame source-water -wax pencil-microscope -appliocator swabs-broth culture of Patient A -broth culture of patient B-hot plate -water bath-Carbol Fuschin stain -3% acid-alcohol destain-Methylene Blue stain -rinse containerOrganisms:-Staphylococcus epidermidis -Mycobacterium nonchromogenicum

Procedure:

1. Sterilize work area with bleach solution2. Start water bath3. Label slides A and B4. Flame inoculating loop5. place water droplet on slide and add sample from test tube A6. Repeat steps 4 and 5 for B7. Air dry slides8. Heat fix slides9. Add Carbol Fuschin10. Allow to penetrate for 5 minutes11. Steam slides12. Cool and rinse13. Decolorize slides with acid-alcohol destain for 30 seconds14. Add Methylene blue and allow to penetrate for 60 seconds15. Rinse16. Sterilize work area17. Observe under microscope

Results:

Patient A Patient B