Preparation and Staining of Specimens

1. Preparation and Staining of Specimens

2. Often specimens must be fixed and stained to enhance their visibility and to distinguish morphological properties

3. Fixation • Preservation of internal and external features of cells • Cellular enzymes are inactivated • Cell structures are hardened • Organism dies AND adheres strongly to the glass slide

4. Two types of fixation: • Heat fixing: flame heating bacterial film, preservation of morphology but NOT internal structures • Chemical fixing: chemical fixatives penetrate cells and preserves intracellular components • Acetone • Ethanol • Acetic acid • Mercuric chloride • Formaldehyde • Glutaraldehyde

5. Dyes and Simple Staining • Dyes contain chromophore groups and bind to cells by ionic, covalent and hydrophobic forces • Basic dyes (positively charged) bind to negatively charged groups • Work best at high pH • Acidic dyes (negatively charged) bind to positively charged groups • Work best at low pH

6. Simple staining: • One reagent • Usually involves basic dyes • Crystal violet • Methylene blue • Carbol fuchsin • Differential staining: • Used to differentiate groups on bacteria • Examples: • Gram stain • Acid fast stain

7. Gram Stain • Gram-positive bacteria retain the crystal violet • Gram-negative bacteria become colorless • Developed by Christian Gram in 1884

8. Steps: • 1. Crystal violet, 30”, rinse 5” • 2. Gram’s Iodine (mordant that strengthens the association of crystal violet with the cell wall), 1’, rinse 5” • 3. Decolorization: 95% ethanol or isopropanol:acetone, 15-30”, rinse 5” • 4. Counterstain: Saffranin, 1’, rinse 5”, Gram-negative bacteria become pink/red, Gram-positive remain purple

9. Gram staining is the most widely used differential staining procedure because it divides bacterial species into two roughly equal groups - gram positive and gram negative

10. Acid-Fast Staining • Some organisms do not stain well with conventional dyes (e.g. Mycobacterium) • Harsher treatment required • Steps: • 1. Heating with basic fuchsin and phenol (Ziehl-Neelson technique) – 5’, cool, rinse 30” • 2. Decolorize: Acid/Alcohol 10-30”, rinse 30” • 3. Counterstain with methylene blue, 2 ‘, rinse 30” • Acid-fast cells remain red due to high mycolic acid content (lipid) • Non-acid-fast cells = blue (counterstain)

11. Acid-fast staining is a differential staining procedure that identifies two medically important species of bacteria - • Mycobacterium tuberculosis, the causative agent of tuberculosis, and • Mycobacterium leprae, the causative agent of leprosy (Hansen’s disease)

12. Staining Particular Structures

13. Negative staining • Widely used to visualize diffuse capsules surrounding the bacteria; those capsules are unstained by the procedure and appear colorless against a stained background • Morphology of cells determined • No heat treatment or harsh chemicals • Film spread, stained and dried • Heating would cause shrinkage – distortion of cellular morphology

14. Deposits generated around cell or dark background observed • Dyes (acidic stain – does not penetrate cells) • Nigrosin • India ink • Eosin blue

15. Spore staining • Detects endospores generated by Bacillus and Clostridium • Formed within the cell  dormant • Hardy in adverse conditions • Various sizes, shapes and locations • Steps (Schaffer-Fulton procedure): • Heat + malachite green • Rinse • Counterstain with saffranin

16. Capsule stain • Gently – no heat fixation or may get shrinkage • Steps: • Grow culture in skim milk broth • Crystal violet • Copper sulfate counterstain

17. Flagella stain • Flagella are very difficult to observe directly because of their size • Thickness is increased using mordants • Tannic acid • Potassium alum • Stained with Pararosaniline (Leifson method) or basic fuchsin (Gray method)