1. 381 PHT Lab #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 Basic dyes: colored cation + colorless anion
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 are slightly negatively charged ( rich in nucleic acids bearing negative charges as phosphate groups)
→ combine with positively charged basic dyes
Acidic dyes do not stain the bacterial cell → can stain the background material with 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

8. Smear
Preparation
preparation smear
Smear Preparation

9. 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.

10. Smear preparation S
Fixation

12. Simple Staining

13. Definition: Objective:-
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.

14. Simple Staining
Procedure:- MB
1-2 min

15. Basic Shapes of Bacteria
Cocci
Bacilli

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

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

18. 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

19. 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

20. 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

22. Gram Staining

23. Appears violet after Gram’s stain
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

24. Crystal violet ↓
Iodine
Alcohol
Safranin

25. 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

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

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

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

29. 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 turned red.

30. 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)

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

32. Results: Shape: Cocci Arrangment: irregular clusters Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism: Staphylococci

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

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

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

37. Negative staining

38. 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

39. Negative staining
Candida

40. Negative staining
Staphylococci

41. Negative staining
Bacillus

47. Thank You