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Staining techniques
Staining is a technique used to improve contrast and study samples at the microscopic level. There are several types of stains including acidic, basic, and neutral stains. Basic staining techniques involve preparing a smear, applying a dye, rinsing, and examining under a microscope. Specific techniques are used for simple/positive staining, negative staining, Gram staining, capsule staining, acid-fast staining, and endospore staining to identify cell morphology and structure. Proper staining allows visualization of cellular and structural features that aid in identification and disease diagnosis.
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Staining techniques
- 1. Basic Staining Techniquesin Microbiology Mr. Vikas Lalji Gupta Asst. Professor, VIVA College,MH-India
- 2. Staining • Staining isa technique used to improve the contrast of samples at microscopic level. • Stains and dyes are commonly used to study the tissue or cells under the microscope. • In the medical fields of histopathology, hematology, and cytopathology which will be focus on the study and diagnose disease at a microscopic level. • Biological staining is also used to mark cells in flow cytometry. • Staining is not restricted to only biological materials, it can also be used to study the structure of other materials like lamellar structures of semi-crystalline polymers.
- 3. Types of stain/dye •Acidic – Eosin, Nigrosin, India ink. • Basic – Malachite green, crystal violet, basic fuschin, safranin, methylene blue. • Neutral – Giemsa stain.
- 4. Prepare a bacterialsmear and heat fix it Add basic dye on the smear for approximately 1 minute Rinse the slide gently under running tap water Carefully blot dry with Filter paper. Observe the slide under the microscope, using proper microscope technique. Simple/Positive Staining Simple staining provide a quick and easy way to determine cell shape, size, and arrangement.
- 5. Negative Staining Negative stainingis an excellent way to determine an organism’s cellular morphology. The nigrosine provides a dark background against which the shapes of the unstained cells are clearly visible. Place a single drop of Nigrosin on a clean glass slide Take a loopful of culture suspension and mix it into the drop of Nigrosin Place the end of another clean slide at an angle to the end of the slide containing the organism and spread the drop out into a film. Allow the film to air dry. Observe the slide under the microscope
- 6. Gram Staining Prepare a bacterial smearand heat fix it Flood the smear with crystal violet for 1 minute. Rinse the slide gently under running tap water Flood the smear with iodine for 1 minute. Rinse the slide gently under running tap water Decolorize with decolorizer (acetone/alcohol) for 30-45 seconds Rinse the slide gently under running tap water Counterstain with safranin for 1 minute Rinse the slide gently with water Carefully blot the slide dry with filter paper Observe the slide under the microscope With proper staining technique… Gram positive bacteria will stain purple. Gram negative bacteria will stain red/pink.
- 7. Capsule Staining Place asingle drop of India ink on a clean microscope slide Take a loopful of K. pneumoniae culture suspension and mix it into the drop. Place the end of another clean slide at an angle to the end of the slide containing the organism and spread the drop out into a film Allow the film to air dry Saturate the slide with crystal violet for 1 minute Rinse the slide gently under running tap water Allow the slide to air dry Observe the slide under the microscope The background will be dark. The bacterial cells will be stained purple. The capsule will appear clear against the dark background.
- 8. Acid-Fast Staining Prepare a bacterial smear Placea small piece of filter paper over the smear Add carbolfuchsin on the filter paper Heat the slide gently over the burner for 5 minutes If carbolfuchsin dry then add more carbolfuchsin Remove the filter paper from the slide Rinse the slide gently under running tap water Decolorize the slide with acid-alcohol Rinse the slide gently under running tap water Counterstain with methylene blue for 1 minute Rinse the slide gently under running tap water Observe the slide under the microscope Acid-fast cells will stain fuchsia. Non-acid-fast cells will stain blue.
- 9. Endospore Staining Prepare a bacterial smearof Bacillus Place a small piece of Filter paper over the smear Saturate the paper with malachite green Heat the slide gently over the burner for 10 minutes If malachite greendry then add more malachite green Remove the filter paper from the slide Rinse the slide gently under running tap water Counterstain with safranin for 1 minute Rinse the slide gently under running tap water Carefully blot the slide dry with filter paper Observe the slide under the microscope Endospores will stain green. Parent cells will stain red.
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