1. Practical No. 5 Staining Department of Microbiology
College of Medicine

2. Natural stain : too expensive. Synthetic stain :
Staining: Bacteria are too small, colorless(Transperant).Therefore they must be stained to study :morphology, size, shape, arrangment of microorganisms.
Types of bacterial stain ( origin) :
Natural stain : too expensive.
Synthetic stain :
a- Basic stains: The stain molecule in such type of stains posses (+ve) charge after ionization, such as; crystal violet, methyle violet, methylene blue,safranin……etc .
The basic stains will be attracted to the bacterial cell and +ve stained and take the color of the stain used.
b- Acidic stains: In such stains,the stain molecule will be(–ve) charge after ionization, such as;india ink and nigrosin.
The stain molecule of the acidic stains will be repelled from the bacterial cell and a clear zone around the cell will be seen.
There are Three staining procedures applied depending on the purpose of use :

3. A. Simple staining: This procedure is simple,easy,time saving and economic. It is applied by using only a single stain and all bacteria in the specimen will be stained by one color.
1-use a clean slide & mark underneath side of slid.
2-place a loopful of broth culture in the center,spread and flame loop.
3-Air dry the slid &heat fix by passing a slid rapidly throughout flame.
4-Flood smear with simple stain,basic stains,such as;crystal violet,methyl violet,leave3 m
5-Wash with tap water, drain excess water & dry in air.
6-Examine under microscope using the low , high and oil immersion lens.

4. GRAM POSITIVE appear VIOLET
G+ve
G-ve
GRAM NEGATIVE
are RED in color.

5. B.Differential staining:This was first applied by the Danish scientist Christian Gram1884.
Where by this procedure the bacteria are classified into two main groups, which are called Gram +ve and Gram – ve;each group has its specific biochemical and physiological characters.
Gram positive(+ ve):retain the color of primary stain after using alcohol(dark blue to violet
Gram negative(-ve):those that can’t retain 1st stain when washed by alcohol(decolorizing agent)but then take the color for 2nd stains or counter stain(safranine or carbol fuchsin)red
Procedure for Gram staining :
Prepare a fixed smear after marking our slide.
Flood the smear with crystal violet stain for 1 minute.
Wash with tap water .

6. Cover the smear with Gram iodin (KL+I) ; (mordant) for 1 minute: The stain now is combined with iodin forming what is called Crystal Violet Iodin complex (CVI complex).This complex will be fixed in bacteria in its cell wall more than the stain alone.At this step all types of bacteria present in the sample smear are colored by one color which is the dark violet color.
Wash with tap water and get rid of the excess water on the slide.
Decolorize with (which is absolute alcohol 95% or absolute alcohol + acetone) for 5 sec.
Wash with running water immediately. Cover the smear with the counter stain which is safranine in order to color the bacteria that have lost the stain during the use of alcohol. Leave for 1 minute and wash with tap water.
Leave to dry at room temperature & Examine by oile immersion lens.

7. Gram
Staining
Technique

9. Cell wall
In addition to conferring rigidity upon bacteria, the cell wall protects against osmotic damage
Chemically, the rigid part of the cell wall is peptidoglycan
Cell wall first described by Gram in It is used to study morphologic appearance of bacteria. Gram's stain differentiates all bacteria into two distinct groups:
a. Gram-positive organisms
b. Gram-negative organisms

11. Thank You