Acid fast staining

Acid fast staining technique is used to identify the acid fast bacteria and non - acid fast bacteria.

1. ACID FAST STAINING Presented By: A.R. Deborah (BP211501) I M. Sc. Applied Microbiology Sacred Heart College (Autonomous) Tirupattur Tamil Nadu, India. Presented To: Dr. P. Saranraj Head, Department of Microbiology Sacred Heart College (Autonomous) Tirupattur Tamil Nadu, India

2. PRINCIPLE Acid fast staining is the Differential staining technique which was first developed by Ziehl and modified by Neelsen . So this method is also called Ziehl - Neelsen staining techniques. Neelsen in 1883 used Ziehl’s Carbol - fuchsin and heat then decolorized with an acid alcohol, and counter stained with Methylene blue. A Ziehl-Neelsen staining technique was developed. Acid fast staining method is used for those microorganisms which are not staining by Simple or Gram staining method, particularly the member of genus Mycobacterium, Nocardia and Cryptosporidium. The presence of thick waxy wall (Mycolic acid) in the Mycobacterium sp . makes penetration by stains extremely difficult once the stains penetrates it cannot be readily removed even with Acid alcohol as a decolouring agent because of this properly this organisms are called Acid fast. Acid fast stain three different reagents, the primary stain is Carbol fuschin, the second stain is Acid alcohol (3 % hydrochloric acid and 95 % ethanol) and is used as decoloring agent. The final reagent is Methylene blue that act as Counter stain.

3. MATERIALS REQUIRED Bunsen burner Inoculating loop Glass slide Hot plate Microscope. Methylene blue Acid alcohol (3 % hydrochloric acid and 95 % ethanol) Carbol fuschin

4. PROCEDURE Clean and dry the microscopic slide. Prepare thin smear of given bacteria on a clean glass slide and Allow the smear to air dry and fixed with heat. Cover the smear with Carbon fuschin, heat fixed and allowed steam in a hot plate for 3 - 5 minutes and allow to Cool the slide and washed with tap water. Decolorized with Acid alcohol by adding reagent drop by drop for 10 – 15 minutes until Carbol fuschin filled to wash from the smear with tap water. Counter stain the smear with Methylene blue for 2 minutes and then washed the smear with tap water and dried properly. A ir dry the smear and observed under oil immersion objective. Observe the slide under Low power objective (10 x or 40 x) and High power objectives (100 x Oil immersion) of the microscope.

5. OBSERVATION AND RESULTS Acid fast: Bright red to intensive purple, Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded (Example - Mycobacterium, Nocardia and Cryptosporidium ). Non-acid fast: Blue color; In addition, background material should stain blue. Acid fast bacilli (Red) and Non – Acid fast bacilli (Blue)