STAINING TECHNIQUE
STAINING :-
staining techniques used in microscope technique the microscopic image. Staining or dyes are widely used in the scientific filed to highlight the structure of the biological specimen such as cell tissue,etc. The most widely used staining procedure in microbiology in the gram stain techniques discovered by the denis scientist and physician han's Christian gram in 1884
WHAT IS STAIN:- it is a dye used in coloring to microscope object and tissue or applying pigment to a tissue or to microscope object to see their components part.
TYPES OF STAINING TECHNIQUE
GRAM STAINING -
Gram staining is developed by of Danish scientist & physician HANS
CHRISTIAN & GRAM in (1884). This method is divided bacteria into 2 groups; Gram positive & Gram negative, based on the nature of cell.
THIS METHOD CONSIST OF FOUR STEPS-
1ST STEP-
PRIMARY STAIN- The air dryad and heat fixed smear is stained with crystal violet for 60 seconds.
2nd STEP-
DE-COLORIZED- Wash the slide with water and de-colorized with 70% ethyl alcohol for 15 seconds.
3RD STEP-
MODERN - Wash the slide with water , add Gram iodine 10% and keep for 60 seconds.
4TH STEP-
COUNTER STAIN- Wash the slide with water and counter stain with SAFRANIN.
METHODS OF GRAM STAINING INVOLVE SIX BASIC STEPS-
(1). Preparation of smear.
(2). Fixing of the smear by heating.
(3). Staining with crystal violet.
(4). Use of iodine.
(5). Treatment with alcohol acetone.
(6). Use of the counter stain (SAFRANIN).
RESULT-
Gram positive cell appear PURPLE in color & gram negative cell appear PINK & RED in color.
ACID FAST STAINING-
Acid fast staining develop by PAUL EHRLICH in (1882) and was modified later ZIELH NELSON and their also known as ZIELH NELSON STAINING.
' The main aim of this technique is to different between acid fast and non- acid fast bacterial growth '.
(1). ACID FAST BACTERIA- If they retain the stain after the application of strong acid and appear red color.
NON-ACID FAST BACTERIA - If they do not retain the stain are counter stain by methyl blue.
PROCEDURE -
1ST STEP - Heat fixed smear is covered with carbo.
2ND STEP - Wait for 12-15 minutes without heating.
3RD STEP - Wash with water.
4TH STEP - De- colorized with 5% sulphuric acid.
5TH STEP - Counter stain with methylene blue.
6TH STEP- Wash with water.
7TH STEP- Heating of the slide.
8TH STEP- Examine under microscope or oil- emulsion lens.
RESULT -
The organisms appear bright red in blue background.
HANGING DROP PREPARATION
HANGING drop preparation method is used to examine the motility rate of bacteria in culture medium.hanging drop method is a confirmatory method to examine and identify in an organism in motile or non motile. it can also be used for the basis of its characteristic motility
procedure
1 clean the cavity slide under the water
2 cavity of slide
3 dry with bibulous paper
4 dry completely slide by showing over flame
5 apply petroleum jelly around the cavity
6 fresh broth culture bacteria less than 24 hr
7 transfer loop of bacteria to the center of the cover slip as a small drop
8 invert cavity slide and place on the cover slip in such the cavity cover the drop
9 press the slide and cover gently so that the cavity is sealed
10 quickly invert the slide such that the drop hangs into the cavity without touching
11 observe under the using microscope
important of hanging drop method
STAINING :-
staining techniques used in microscope technique the microscopic image. Staining or dyes are widely used in the scientific filed to highlight the structure of the biological specimen such as cell tissue,etc. The most widely used staining procedure in microbiology in the gram stain techniques discovered by the denis scientist and physician han's Christian gram in 1884
WHAT IS STAIN:- it is a dye used in coloring to microscope object and tissue or applying pigment to a tissue or to microscope object to see their components part.
TYPES OF STAINING TECHNIQUE
- GRAM STAINING
- ACID FAST STAINING
- HANGING DROP PREPARATION
GRAM STAINING -
Gram staining is developed by of Danish scientist & physician HANS
CHRISTIAN & GRAM in (1884). This method is divided bacteria into 2 groups; Gram positive & Gram negative, based on the nature of cell.
THIS METHOD CONSIST OF FOUR STEPS-
1ST STEP-
PRIMARY STAIN- The air dryad and heat fixed smear is stained with crystal violet for 60 seconds.
2nd STEP-
DE-COLORIZED- Wash the slide with water and de-colorized with 70% ethyl alcohol for 15 seconds.
3RD STEP-
MODERN - Wash the slide with water , add Gram iodine 10% and keep for 60 seconds.
4TH STEP-
COUNTER STAIN- Wash the slide with water and counter stain with SAFRANIN.
METHODS OF GRAM STAINING INVOLVE SIX BASIC STEPS-
(1). Preparation of smear.
(2). Fixing of the smear by heating.
(3). Staining with crystal violet.
(4). Use of iodine.
(5). Treatment with alcohol acetone.
(6). Use of the counter stain (SAFRANIN).
RESULT-
Gram positive cell appear PURPLE in color & gram negative cell appear PINK & RED in color.
ACID FAST STAINING-
Acid fast staining develop by PAUL EHRLICH in (1882) and was modified later ZIELH NELSON and their also known as ZIELH NELSON STAINING.
' The main aim of this technique is to different between acid fast and non- acid fast bacterial growth '.
(1). ACID FAST BACTERIA- If they retain the stain after the application of strong acid and appear red color.
NON-ACID FAST BACTERIA - If they do not retain the stain are counter stain by methyl blue.
PROCEDURE -
1ST STEP - Heat fixed smear is covered with carbo.
2ND STEP - Wait for 12-15 minutes without heating.
3RD STEP - Wash with water.
4TH STEP - De- colorized with 5% sulphuric acid.
5TH STEP - Counter stain with methylene blue.
6TH STEP- Wash with water.
7TH STEP- Heating of the slide.
8TH STEP- Examine under microscope or oil- emulsion lens.
RESULT -
The organisms appear bright red in blue background.
HANGING DROP PREPARATION
HANGING drop preparation method is used to examine the motility rate of bacteria in culture medium.hanging drop method is a confirmatory method to examine and identify in an organism in motile or non motile. it can also be used for the basis of its characteristic motility
procedure
1 clean the cavity slide under the water
2 cavity of slide
3 dry with bibulous paper
4 dry completely slide by showing over flame
5 apply petroleum jelly around the cavity
6 fresh broth culture bacteria less than 24 hr
7 transfer loop of bacteria to the center of the cover slip as a small drop
8 invert cavity slide and place on the cover slip in such the cavity cover the drop
9 press the slide and cover gently so that the cavity is sealed
10 quickly invert the slide such that the drop hangs into the cavity without touching
11 observe under the using microscope
important of hanging drop method
- its help to identify external character of bacteria
- it help to observe motility of bacteria
- diagnosis of disease
- treatment and control of disease
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