1. Lab 2

2. STAINING

3. Types of staining techniques
Special stain
Simple staining
(use of a single stain)
Differential staining
Spore
stain
Capsule
stain
Identification
For demonstrate morphological
shape & arrangement.
Gram
stain
Acid fast
stain

4. Gram Staining

5. GRAM STAINING
Gram's Stain is a widely used method of staining bacteria as an aid to their identification. It was originally devised by Hans Christian Gram, a Danish doctor.

6. 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

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

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

9. 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.

10. Crystal violet ↓
Iodine
Alcohol
Safranin

11. Solutions used
Gram positive
Gram negative
1-Crystal violet
Cells violet
2-Iodine
Increase the affinity of attraction between the cell and stain
C.V.I complex ,cells remain violet
3-Alcohol- decolorizing agent to remove the stain from the cells
Cell wall dehydrate-pores shrinks- permeability of cell wall and membrane decreases –C.V.I. complex cannot pas out of cell wall, cells remain violet
Lipid extracred from the cell wall – pores enlarged -C.V.I. complex removed from cells and cells become colorless
4-Safranin
To give different color of the intial stain
Cells remain violet
Cells red

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

13. Gram Stain procedure Cultures of bacteria Gram stain solutions:
Crystal violet (primary stain)
Gram’s iodine (mordant)
Alcohol (decolorizing agent)
Safranin (Secondary stain)or
(counter stain)

14. The Gram staining method
1. A small sample of a bacterial culture is removed from a culture. In this example it is being taken from a broth culture of the pure microbe but it could be removed from a culture on solid medium .

15. The Gram staining method
2. The bacterial suspension is smeared onto a clean glass slide. If the bacteria have been removed from a culture on solid media it will have to be mixed with a drop of distilled water.

16. The Gram staining method
3. The bacterial dried slowly at first and then, when dry, heated for a few seconds for fixation

17. The Gram staining method
4. Cover the surface of the slide with Crystal Violet stain and let sit for ( one minute ).

18. The Gram staining method
5. Washing the slide with water.

19. The Gram staining method
6..Cover the slide with Gram’s Iodine and time for ( one minute).
Then Washing with water!

20. The Gram staining method
7. Cover the slide with Gram’s decolorizer and time for seconds.
And wash with water

21. The Gram staining method
8. Cover the slide with the counterstain, Safranin, and let sit for seconds

22. The Gram staining method
9. washing with water

23. The Gram staining method
10.drying the slide and read with the oil immersion lens of the microscope. Look for Gram-negative and Gram-positive bacteria.

24. The Gram staining method
Typical Gram-positive bacteria

25. The Gram staining method
Typical Gram-negative bacteria

26. Gram Positive may be appear as
Gram Negative:
Using old culture.
2.Wrong in Technique Steps.
a. If not treated with iodine.
b. If alcohol stay on slide more time.
c. Acidic PH of medium.

27. Thank You