Test Catageory
فئة التحليل
Microbiology
Refrence Books
كتب مراجع
1. Bailey & Scott’s Diagnostic Microbiology
2. Laboratory Manual and Workbook in Microbiology
Skills Required
المهارات المطلوبة
1. Smear preparation
2. Staining technique
3. Decolorization control
4. Microscope operation
5. Organism identification
Video Resources
مصادر فيديو
Principle
The primary stain crystal violet penetrates all bacterial cell walls. The addition of a mordant reinforces the crystal violet stain. The decolorizing agent removes the dye from Gram negative bacteria but not from Gram positive bacteria. A secondary counterstain is applied that will stain Gram negative bacteria but not those retaining crystal violet.
Solutions & Equipments
1. New, clean slides
2. Sterile inoculation loops
3. Crystal violet stain
4. Gram’s iodine
5. Decolorizer
6. Safranin counterstain
7. Light microscope
Sample Type
Pus
Sputum
Cerebrospinal fluid
Tissue biopsies
Body fluids
Procedure
1. Prepare a thin smear on a new, clean slide and allow to air dry completely
2. Heat fix smear
3. Flood slide with crystal violet for 1 minute
4. Gently rinse with water
5. Flood slide with Gram’s iodine for 1 minute
6. Decolorize with alcohol-acetone solution for 10-20 seconds
7. Gently rinse with water
8. Counterstain with safranin for 45-60 seconds
9. Gently rinse with water and allow to air dry
10. Examine slide under microscope using oil immersion lens
– Gram positive organisms will appear purple
– Gram negative organisms will appear pink or red
Results
Identification of Gram positive bacteria (purple) or Gram negative bacteria (red/pink) under the microscope.
Interpretation
Positive: Presence of Gram positive or Gram negative bacteria aids in diagnosis and identifies appropriate antimicrobial therapy based on organism type.
Negative: A negative stain does not definitively rule out presence of organisms. Repeat testing or culture may be indicated.
Notes:
– Use known positive and negative control organisms to ensure proper decolorization and staining.
– Do not over-decolorize, as Gram positive organisms may appear Gram negative.
– Check microscope and immersion oil quality.
– Take precautions when handling potential pathogenic cultures.
M. Sulieman
mohammad@mlsgaate.com
The primary stain crystal violet penetrates all bacterial cell walls. The addition of a mordant reinforces the crystal violet stain.
