1. 416PHT Lab#2 Gram’s stain Acid fast stain Spore stain

2. Identification of Bacteria
Microscopical Examination:
Examination of wet mount preparation.
Examination of stained preparation.
Macroscopical Examination:
Characters of colonies.
Hemolysis on blood agar.
Pigment production.

3. Identification of Bacteria
Biochemical Tests.
Additional Tests:
such as serological tests

4. Staining of Bacteria
Bacteria cells are almost colorless, and for this reason a staining technique is often applied to the cells to color them so that their shape and size can be easily determined under the microscope.

5. Staining of Bacteria Types of staining technique:- Simple staining
(use of a single stain)
Differential staining
(use of two contrasting stain)
For visualization of morphological
shape & arrangement.
Identification
Visualization
of structure
Gram
stain
Acid fast
stain
Spore
stain
Capsule
stain

6. Staining of Bacteria Principle of staining:-
Dye are generally salts in which one of the ions is colored.
Example: methylene blue (simple dye) is the salt of methylene blue chloride (MBC)
MBC MB + C
Dyes may be either:
Acidic dyes [ -ve]
Basic dyes [ +ve] + -

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

8. Preparing a smear for staining.
(The following procedure is used for all of our staining)
1. Flame (sterilize) your inoculating loop/needle before and after use. Heat from base to tip. Be sure to get the entire wire red hot.
Make sure that you are collecting your hair

9. 2. Prepare the smear .
a. With solid culture
(agar colony), place a small drop of distilled water on a clean slide. Drag the sterile inoculating needle tip through the edge of an isolated colony. Gently spread the mixture into a circle the size of a quarter.
b. With liquid culture
(A loop of liquid culture can be placed directly on the slide and spread out.)
3. Let the smear air dry completely. Do not apply heat while drying because this can lyse the cells

10. Smear preparation S
Fixation

11. Simple Staining Objective:-
To show the morphological shapes and arrangement of bacterial cells.
direct staining
Indirect staining with acidic dye (Negative staining)

12. Simple Staining Materials:- Cultures of S. aureus, B. subtilis Stain:
Methylene blue stain
Crystal violet

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

14. Basic Shapes of Bacteria
Cocci
Bacilli

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

16. Results Name of stain: Name of dye: Shape of cells:
Arrangement of cells:
Color:
Name of m.o:

17. Simple Staining Name of stain:- Simple Stain
Name of dye:- Methylene blue
Shape of cells:- bacilli
Arrangement of cells:- Chinese letter
Color:- Blue
Name of m.o:- Coryebacterium diphtheria

18. Simple Staining Name of stain:- simple stain
Name of stain:- Methylene blue
Shape of cells:- cocci
Arrangement of cells:- clusters
Color:- Blue
Name of m.o:- Staphylococcus aureus

19. Simple Staining Name of stain:- simple stain
Name of stain:- Crystal violet
Shape of cells:- cocci
Arrangement of cells:- clusters
Color:- purple
Name of m.o:- Staphylococcus aureus

20. Indirect staining with acidic dye (Negative staining)
The negative stain technique does not stain the bacteria but stain the background.
The bacteria will appear clear against a dark background.
No heat fixation or strong chemicals are used, so the bacteria less distorted than in other staining procedure.
Example: Nigrosine are acidic stain (negatively charged), so the –ve stain doesn’t stain the bacteria due ionic repulsion of bacterial cell wall

21. Negative staining
Candida albicans

22. Negative staining
S. aureus

23. Negative staining
B. subtilis

24. Shape Special arrangement Capsule formation Bacterial Morphology
Spore formation
Capsule formation
Motility
Staining affinity

25. Bacterial Shapes

26. Bacterial Arrangement Clusters (group). Chains. Pairs (diploids).
No special arrangement.

27. Clusters
Chains
Pairs
Gram-Stained Cocci

28. Gram-Stained Bacilli

29. Bacterial Spores Morphological characters of bacterial spores:
* Shape.
* Position.
* Staining.

30. Staining of Bacteria Types of staining technique:- Simple staining
(use of a single stain)
Differential staining
(use of two contrasting stain)
For visualization of morphological
shape & arrangement.
Identification
Visualization
of structure
Gram
stain
Acid fast
stain
Spore
stain
Capsule
stain

31. Smearing out of the sample

32. Smear Fixation

33. Principle of Differential Stains * Application of the primary stain.
* Decolourization.
*Application of the counter-stain.

34. “One of the most common mistakes is to decolorize a smear for too long a time period. Even Gram-positive cells can lose the crystal violet-iodine complex during prolonged decolorization.
Gram Staining

35. Gm+ve cocci
G-ve bacilli

36. 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

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

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

39. Gram Stain 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 Acetone-Alcohol.
Therefore, cells will be counterstained with safranin and turned red.

40. Gram Stain Materials:-
Cultures of S.aureus, C.albican, B.subtilis, E.coli
Crystal violet (primary stain)
Gram’s iodine (mordant)
Acetone-alcohol (decolorizing agent)
Safranin (counter stain)

41. Gram Staining Technique

42. Gram Stain [single] Procedure: CV s 30-60 sec 30 sec 10 sec 2 min
safranin
iodine s
30-60 sec
30 sec
10 sec
2 min

43. 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

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

45. Results: Shape: Cocci Arrangement: clusters Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism: Staphylococcus aureus (S. aureus)

46. Results: Shape: Oval Arrangement: Single Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism:
Candida albicans (C. albicans)

47. Results: Shape: Bacilli Arrangement: Chains Colour: Violet
Gram’s reaction: Gram +ve
Name of microorganism:
Bacillus subtilis (B. subtilis)

48. Results: Shape: Rods Arrangement: Single Colour: red
Gram’s reaction: Gram -ve
Name of microorganism:
Escherichia coli (E. coli)

49. Gram’s stain (mixture)

51. Staining of Bacteria Types of staining technique:-
Differential staining
(use of two contrasting stains
separated by a decolorizing agent)
Simple staining
(use of
a single basic stain)
Identification
Visualization
of structure
For visualization of morphological
shape & arrangement.
Gram
stain
Acid fast
stain
Spore
stain
Capsule
stain

52. Acid-Fast
Staining

53. Acid Fast Staining
Mycobacteria have a 3rd type of cell envelope (rather than the basic gram- related properties).
The cell wall of bacteria in this genus contain → considerable amounts of lipids → form an extremely hydrophobic external layer.

54. Acid Fast Staining
These organisms are not readily stainable with ordinary stains.
Staining of these bacteria needs exposure to a strong stain e.g., concentrated carbol fucsin
With application of heat.
Once they are properly stained, they resist decolorization by strong mineral acids or acid-alcohol→ so they are said to be
Acid-fast.

55. Acid Fast Staining
AFS is an important diagnostic value in identifying pathogenic members of genus Mycobacterium such as M. tuberculosis and M. leprae.

56. Materials:- Acid Fast Staining Culture of M. phelei
Acid-fast staining kit:
Conc. carbol fuchsin (primary dye)
Acid-alcohol (decolorizing agent)
Methylene blue (counter stain)

57. Acid Fast Stain \\\\ Procedure:- 5 min Carbol fuchsin MB 30-60 sec

58. Results Type of Staining: Acid fast stain Shape: beaded bacilli
Arrangement: Tree shaped
Colour: red
Name of microorganism: M.phelei

59. Spore Stain

60. The Spore Stain
Some bacteria (e.g., Bacillus and Clostridia) form resistant bodies in the cell known as endospors.
Bacterial spores are highly resistant to physical & chemical agents (primarily due to a thick tough spore coat).
They are not easily stained by routine staining.
Heat is required in spore staining to promote the penetration of the dye into the spore.
Once the spores stained they resist decolorization.

61. Materials :- The Spore Stain Culture of B. subtilis
Spore-staining kit:
Malachite green (primary stain)
Safranine (counter stain)

62. Spore Stain of Bacillus subtilis
Type of Staining: Spore stain
Shape: bacilli
Arrangement: Chains
Colour of spores: green
Colour of vegetative cells: red
Name of microorganism:
B. subtilis

66. Thank You