Acid fast stain and virus inoculation for foreign students

Acid-fast bacteria will appear red; non-acid-fast will appear blue.
Questions
• What’s the importance of waxy substance in the cell wall to TB besides acid fast stain?
host.
Principles
Differ from other infectious agents: • They are very small. • They need host cells for multiplication. • Contain a single type of nucleic acid, DNA or RNA. • Contain protein coat. Cultural materials of virus Embryonated eggs, living animals (or tissues from
Methods
• Mark air space of eggs under strong lighter. • Broken shell of eggs by grind stone. • Inoculate 0.5 ml degermed virus sample
suspension (through bacterial filter) into eggs at broken site. • Incubate eggs 72h under 37 degree. • Keep the eggs under 4 degree in freezer for identification. • Identify Virus titer by haemagglutination test (next week)
• Others: slides, alcohol lamp, inoculation loop, blotting paper
Methods
• 1. Heat-fix a smear of a bacterium as follows:
• 2. Flood the smear with carbol fuchsin (about 6 drops).
• According to structure of cell wall, does TB belong to G+ bacterium? Why?
cultivation & identification of virus: embryo-culture of virus
Objectives
• Learn methods of virus culture (using chicken embryo in this experiment).
• Carbol fuchsin is the primary stain in this procedure, and it contains phenol to help solubilize the cell wall. Heat is also applied during the primary stain to increase stain penetration. All cell types will take up the primary stain.
• Master technique of virus identification (next week).
Principles
The smallest infectious agents and the smallest form of life. Viruses can be viewed only by Electron microscope.
minute. • 8. Wash with water, blot dry, and observe
using oil immersion microscopy.
Overview of acid-fast staining process
Results
Acid-fast bacteria will appear red; non-acid-fast will appear blue.
Acid fast stain
Chen Qingxin Department of Immunology and
mircrobiology
objectives
• Master procedure and principles of acid fast stain:
– The acid-fast stain is a differential stain which distinguishes organisms with waxy cell walls that can resist decolorization with acid alcohol.
• The cells are then decolorized with acid-alcohol, which decolorizes all cells except the acid-fast ones. Methylene blue is then applied to counterstain any cells which have been decolorized. At the end of the staining process, acid-fast cells will be reddish-pink, and non-acid fast cells will be blue.
General characteristics of viruses: 1- The viral nucleic acid become active only
when it enters to the host cells. 2- Viruses are live when they multiply in the
Methods
• 5. Add the acid-alcohol (10 drops) decolorizing slowly dropwise until the dye no longer runs off from the smear.
• 6. Rinse with water. • 7. Counterstain with methylene blue for 1
Three parts are of use: 1. The amniotic cavity 2. The allantoic cavity 3. The chorio-allantoic membrane
The allantoic cavity is routinely used because it is technically much simpler to inoculate.
Materials
• Specimen (sputum from TB patients treated by 4%NaOH)
• Acid fast staining reagents:
– I. carbol fuchsin (primary stain) – II. acid-alcohol (decolorization) – III.methylene blu What’s the characters of virus? • How does virus infect host cell?
(Multiplication cycle of viruses)
• 3. Steam for 5 minutes by passing the slide through the flame of a alcohol lamp.
• 4. Allow the slide to cool and wash with water.
(Note: Acid-fast stains are performed on smears that have been heat-fixed.)
• These bacteria are termed "acid-fast" because they are able to resist decolorization with acid alcohol.
Structure of an Acid-Fast Cell Wall
Principles
• Know the importance of acid fast stain:
– The genus Mycobacterium and the genus Nocardia are acid-fast.
Principles
• Acid-fast bacteria have a waxy substance called mycolic acid in their cell walls which makes them impermeable to many staining procedures, including the Gram stain.
human and animals ) and cell culture.
Materials
• 9-10 day-old chicken embryos • Samples [new castle disease virus (NDV)]
The Fertile Egg(9-10 day-old)