Gram stain procedure

Muhammad Abbas by Gram Stain Procedure

1. GRAM STAINING
Muhammad AbbasMuhammad Abbas
BS MLT
FSc MLT
Rehman Collage of Allied Health Sciences PeshawarRehman Collage of Allied Health Sciences Peshawar
Muhammad Abbas BS MLT " RMI Peshawar"

2. Objectives
Introduction to Gram stain
Reagents Used in Gram stain
Procedure of Gram stain
Result of Gram Stain
Muhammad Abbas BS MLT "
RMI Peshawar"

3. Introduction to Gram Stain
•The method is named after its inventor, the Danish
scientist Hans Christian Gram (1853–1938)
•a staining technique for the preliminary identification of
bacteria. i-e Gram positive bacteria or gram negative
Bacteria.
•Gram staining is a common technique used to
differentiate two large groups of bacteria based on their
different cell wall constituents. The Gram stain procedure
distinguishes between Gram positive and Gram negative
groups by coloring these cells red or violet
Muhammad Abbas BS MLT "
RMI Peshawar"

4. REAGENTS USED IN GRAM
STAIN
1.CRYSTAL VIOLET
2.GRAM IODINE (Iodine Acetone)
3.DECOLORIZER
4.GRAM SAFRANINE (Methylene Blue)
Muhammad Abbas BS MLT "
RMI Peshawar"

5. Muhammad Abbas BS MLT "
RMI Peshawar"

6. Procedure of the Gram stain
Make a smear on the slide from the bacteria colony.
Keep the slide in air to dry.
Then go to the staining area and apply the first step .
Muhammad Abbas BS MLT "
RMI Peshawar"

7. Procedure Cont….
Step 1: Crystal violet, let the dye over the smear up to 1
minute. After 1 minute wash the slide with tap water and
apply the second step.
Step 2: Put the Gram iodine dye in the second step for 1
minute and after 1 minute wash with tap water.
Step 3: the 3
rd
step is Decolorizer let the decolorizer dye
over the smear for 30 seconds only and then wash.
Step 4: The counter stain Gram Safranine put this dye for
1 minute and then wash with water .
Muhammad Abbas BS MLT "
RMI Peshawar"

8. Result of Gram Stain
After washing the slide clean the slid and wait to dry after dry
View under oil immersion lens of microscope (100x
magnification)
Those that retained the crystal violet dye, did so because of
their thick layer of peptidoglycan & were called Gram-positive
bacteria.
In contrast, Gram-negative bacteria do not retain the violet
dye due to thin layer and are colored red or pink.

Muhammad Abbas BS MLT "
RMI Peshawar"

9. Muhammad Abbas BS MLT "
RMI Peshawar"

10. Muhammad Abbas BS MLT "
RMI Peshawar"