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381 PHT Lab #2

381 PHT Lab #2. STAINING. Staining of Bacteria. Bacteria cells are almost colorless and transparent A staining technique is often applied to the cells to color them → Their shape and size can be easily determined under the microscope. Principle of staining.

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381 PHT Lab #2

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  1. 381 PHTLab #2

  2. STAINING

  3. Staining of Bacteria • Bacteria cells are almost colorless and transparent • A staining technique is often applied to the cells to color them → Their shape and size can be easily determined under the microscope.

  4. Principle of staining • Stains → combine chemically with the bacterial protoplasm. • Commonly used stains are salts: • Basic dyes:colored cation + colorless anion e.g. methylene blue (methylene blue chloride) MB+ + Cl- • Acidic dyes:colored anion + colorless cation e.g. eosin ( Na+ + eosin-).

  5. Bacterial cells areslightly negatively charged( rich in nucleic acids bearing negative charges as phosphate groups) → combine withpositively charged basic dyes • Acidic dyesdo not stain the bacterial cell → can stain the background materialwith a contrasting color.

  6. Types of staining techniques Simple staining (use of a single stain) Differential staining (use of two contrasting stains separated by a decolorizing agent) For visualization of morphological shape & arrangement. Identification Visualization of structure Gram stain Acid fast stain Spore stain Capsule stain

  7. Smear Preparation preparation smear Smear Preparation

  8. Smear Preparation:Preparation and Fixation of Bacteria for Staining. Objective: To kill the microorganism & fix them to the slide to prevent them from being washed out during the process of staining.

  9. Smear preparation S Fixation

  10. Simple Staining

  11. Definition: It is the use of single basic dye to color the bacterial organism. e.g. methylene blue, crystal violet, safranin. All bacteria take the color of the dye. • Objective:- To show the morphological shapes and arrangement of bacterial cells.

  12. Simple Staining • Procedure:- MB 1-2 min

  13. Basic Shapes of Bacteria Bacilli Cocci

  14. Arrangements Cocci Irregular Clusters Tetrads Chains or Pairs Micrococci Streptococci Staphylococci

  15. Results • Type of staining: • Name of stain: • Shape of cells: • Arrangement of cells: • Color: • Name of m.o:

  16. Simple Staining • Type of staining:-Simple Stain • Name of dye:-Methylene blue • Shape of cells:-bacilli • Arrangement of cells:-strain • Color:-Blue • Name of m.o:-Bacillus

  17. Simple Staining • Type of staining:-Simple Stain • Name of dye:-Methylene blue • Shape of cells:-cocci • Arrangement of cells:clusters • Color:-Blue • Name of m.o:- Staphylococci

  18. Simple Staining • Type of staining:-Simple Stain • Name of dye:-Crystal violet. • Shape of cells:-cocci • Arrangement of cells:clusters • Color:-Purple • Name of m.o:- Staphylococci

  19. Gram Staining

  20. Gram Stain: • It is the most important differential stain used in bacteriology because • it classified bacteria into two major groups: b)Gram negative: Appears red after Gram’s stain • Gram positive: • Appears violet after Gram’s stain

  21. Crystal violet ↓ Iodine ↓ Alcohol ↓ Safranin

  22. Gram –ve E.coli Gram +ve S.aureus Step 1:Crystal Violet Step 2:Gram’s Iodine Step 3: Decolorization (Aceton-Alcohol) Step 4:Safranin Red

  23. Step 1:Crystal Violet Step 2:Gram’s Iodine Step 3: Decolorization (Aceton-Alcohol) Step 4:Safranin Red

  24. Gram’s -ve Bacteria Gram’s +ve Bacteria

  25. Gram’s -ve Bacteria Gram’s +ve Bacteria

  26. Gram-positive bacteria • Have a thick peptidoglycan layer surrounds the cell. • The stain gets trapped into this layer and the bacteria turned purple. • Retain the color of the primary stain (crystal violet) after decolorization with alcohol Gram-negative bacteria • have a thin peptidoglycan layer that does not retain crystal violet stain. • Instead, it has a thick lipid layer which dissolved easily upon decoulorization with Aceton-Alcohol. • Therefore, cells will be counterstained with safranin and turnedred.

  27. Gram Stain • Materials:- • Cultures of Staphylococcus aureus, Candida albican, Bacillus subtilis, E.coli • Gram stain: Crystal violet (primary stain) Gram’s iodine (mordant) Acetone-alcohol (decolorizing agent) Safranin (counter stain)

  28. Gram Stain Procedure: CV safranin iodine s 30 sec 30-60 sec 10 sec 2 min

  29. Results: Shape: Cocci Arrangment: irregularclusters Colour: Violet Gram’s reaction: Gram’s+ve Name of microorganism: Staphylococci

  30. Results: Shape: Oval Arrangment: Single Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Candida

  31. Results: Shape: Bacilli Arrangment: Chains Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Bacillus

  32. Results: Shape: Rods Arrangment: Single Colour: red Gram’s reaction: Gram’s –ve Name of microorganism: Gram negative bacilli

  33. Negative staining

  34. Negative staining (Indirect staining with acidic dye) • The negative staining technique does not stain the bacteria due ionic repulsion. but stain the background. • The bacteria will appear colorless against a dark background. • No heat fixation or strong chemicals are used→ the bacteria are less distorted than in other staining procedure. • Example: Nigrosine

  35. Negative staining Candida

  36. Negative staining Staphylococci

  37. Negative staining Bacillus

  38. Thank You

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