Sputum staining

1. Sputum Staining Guidelines
• A Guide for Medical Students
• Presented by: [Your Name/Institution]
• Date

2. Objectives
• - Understand the purpose of sputum staining
• - Learn types of sputum stains
• - Know proper collection and preparation
techniques
• - Identify common findings in stained samples

3. Indications for Sputum Staining
• - Suspected pulmonary infection (e.g., TB,
pneumonia)
• - Monitoring known infections
• - Identifying causative organisms
• - Diagnostic workup of chronic cough

4. Types of Sputum Stains
• - Gram stain
• - Ziehl-Neelsen stain (Acid-Fast Bacilli)
• - Auramine-rhodamine stain (fluorescent)
• - Giemsa stain (for parasites, Pneumocystis)

5. Sputum Sample Collection
• - Collected early morning before eating or
drinking
• - Rinse mouth with water beforehand
• - Instruct patient to take deep cough from
lungs
• - Use sterile, leak-proof container

6. Sputum Sample Criteria
• - Should be mucoid or purulent, not saliva
• - Bartlett's grading system can assess quality
• - Inadequate sample: high epithelial cell count

7. Gram Staining Technique
• - Fix smear by heat
• - Apply crystal violet (1 min), rinse
• - Apply iodine (1 min), rinse
• - Decolorize with alcohol (10–30 sec), rinse
• - Counterstain with safranin (1 min), rinse
• - Examine under microscope

8. Ziehl-Neelsen (ZN) Stain
• - Used for Mycobacterium tuberculosis
• - Carbol fuchsin (heat to steam), rinse
• - Decolorize with acid-alcohol, rinse
• - Counterstain with methylene blue, rinse
• - Acid-fast bacilli appear red on blue
background

9. Auramine-Rhodamine Stain
• - Fluorescent stain for AFB
• - Smear is stained with auramine-rhodamine
dye
• - Viewed under fluorescent microscope
• - More sensitive than ZN stain

10. Interpretation of Results
• - Gram-positive cocci: e.g., Streptococcus
pneumoniae
• - Gram-negative rods: e.g., Klebsiella,
Pseudomonas
• - Acid-fast bacilli: suspect TB
• - Mixed flora: possible contamination

11. Common Pitfalls
• - Salivary contamination
• - Inadequate staining technique
• - Delayed processing
• - Over-decolorization in AFB staining

12. Summary
• - Proper sample collection is key
• - Choose appropriate stain based on clinical
suspicion
• - Interpret with clinical correlation
• - Staining is essential for diagnosis and
treatment

13. References
• - WHO TB Laboratory Manual
• - Bailey & Scott's Diagnostic Microbiology
• - CDC Laboratory Methods
• - Medical Microbiology textbooks