1. Faculty of Medicine and Health Sciences Microbiology Lab second semester 2013 prepared by: Mohammad Al-Qadi E-mail: m.qadi@najah.edu

2. Spore staining and Acid fast staining Lab # 6

3. Acid Fast stain Differential staining technique that classifies Bacteria to Acid fast +ve or Acid fast –ve (non Acid fast) Mycobacterium & Nocardia contain large amounts of lipid substances within their cell walls called mycolic acids. The method used are Ziehl–Neelsen method called Hot stain. (carbol fuchsin, acid alcohol, and methylene blue). Kinyoun modification (cold stain) Acid-fast bacilli will be bright red after staining.

4. Mechanism explanation Initially, Carbol Fuchsin stains every cell. When they are treated with acid-alcohol, only non-acid-fast bacteria get destained since they don't have a thick, waxy lipid layer like acid-fast bacteria. This waxy lipid layer resists decolorization. When counter stain is applied, non-acid-fast bacteria pick it up and appear blue when viewed under the microscope. Acid-fast bacteria retains Carbol Fuchsin so they appear red.

5. Ziehl–Neelsen acid fast staining procedure heat-fixed bacterial smear. Cover the smear with carbolfuchsin in 5% Phenol solution). Heat the slide for 5-10 minutes. (Do not allow the smear to dry out during the heating process. Keep the smear covered with carbolfuchsin by adding more stain) Allow the slide to cool down Rinse (wash) the slide gently with water. Decolorize the slide with acid-alcohol for 30 second. Rinse the slide gently with water. Counterstain with methylene blue for 2 minutes. Rinse the slide gently with water. Blot-dry the slide carefully with tissue paper. Observe the slide under the microscope using oil immersion lens Acid – fast cells – red color Non - acid - fast cell – blue color

6. Ziehl–Neelsen acid fast staining procedure

7. Acid Fast stain principle

8. Acid-Fast Staining

9. Endospores stain Endospores are formed by two genera of medically important bacteria: Bacillus & Clostridia. ( Endospores are also formed by a number of nonpathogenic genera ) By forming endospores, bacteria can survive in hostile conditions in which the bacteria can not find all the necessary chemical and physical requirements needed for survival such as water, Oxygen, nutrients, appropriate temperature and pH. Bacterial endospores are resistant to: Desiccation, Radiation, Chemicals and extreme environmental temperatures. (Endospores are killed by autocalving) Bacteria can form endospores in approximately 6 to 8 hours after being exposed to adverse conditions. Vegetative cell: normally-growing bacterial cell that replicates and is metabolically active. Endospores: metabolically inactive and dehydrated. Bacterial endospores are more or less “dormant cells”. They can remain viable for thousands of years. When endospores are exposed to favorable conditions, they germinate into vegetative (metabolically active) cells.

10. Endospore (Spore) Staining ( Schaeffer – Fulton) Endospore – special resistant structure that protects bacteria Cannot be stained by ordinary methods – stain cannot penetrate the wall of endospore Heat fixed smear Malachite green –(primary stain) heat by steaming –5 min (stains the vegetative cells and the endospores) Wash with water – 30 seconds (The water acts as a decolorizer for the vegetative cells, but the stain is not released by the endospores and free spores.) Safranin – counterstain- 2 min (to give color to the vegetative cells ) Wash with water Endospore – green color / Cells – red color e.g. Bacillus, Clostridium

11. Notes Young cultures of spore-forming microbes may not demonstrate any endospores because the vegetative cells may not have been subjected to sufficient stress to stimulate sporulation. To improve the odds that spore formers can be detected, most methods suggest using cultures that are 18 to 36 hours old.

12. Important link Endospore Stain Protocol http://www.microbelibrary.org/component/re source/laboratory-test/3112-endospore-stain- protocol http://www.microbelibrary.org/component/re source/laboratory-test/3112-endospore-stain- protocol