This presentation summarizes the technique of Gram staining for bacteria. Gram staining distinguishes between Gram-positive and Gram-negative bacteria based on differences in their cell wall structures. The procedure involves staining a bacterial smear with crystal violet dye, adding iodine as a mordant, decolorizing with alcohol, and counterstaining with safranin. Gram-positive bacteria retain the crystal violet dye and appear purple or blue, while Gram-negative bacteria take up the counterstain and appear red or pink. The presentation outlines the history, principles, staining procedure and reagents, and provides examples of Gram-positive and Gram-negative bacteria.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level.Staining and fluorescent tagging can serve similar purposes. Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level.Staining and fluorescent tagging can serve similar purposes. Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis.
Capsule is an layer around the bacteria cell which gives bacteria the protection and pathogenicity. Staining such an layer is difficult with the normal stains so it is necessary to stain the background and the cell itself which makes the capsule appear colourless.
Acid fast staining is differential staining technique which differentiate bacteria into two group- acid fast bacteria and non acid bacteria. It used to identify acid-fast organisms such as members of the genus Mycobacterium .
Gram staining Principle, Procedure, Reagents required for Gram Staining and t...Zunaira Gillani
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this ppt well describes the principle, procedure, modification, usage and limitations of gram's staining.
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Capsule is an layer around the bacteria cell which gives bacteria the protection and pathogenicity. Staining such an layer is difficult with the normal stains so it is necessary to stain the background and the cell itself which makes the capsule appear colourless.
Acid fast staining is differential staining technique which differentiate bacteria into two group- acid fast bacteria and non acid bacteria. It used to identify acid-fast organisms such as members of the genus Mycobacterium .
Gram staining Principle, Procedure, Reagents required for Gram Staining and t...Zunaira Gillani
Gram staining Principle, Procedure, Reagents required for Gram Staining and their Functions, Peptidoglycan Structural difference in Gram positive and Gram Negative.
this ppt well describes the principle, procedure, modification, usage and limitations of gram's staining.
it is a differential staining method used in bacteriology laboratory
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Gram stain is technique used to differntiate gram positive and gram negative bacteria.
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1. H
Presentation
On
TECHNIQUE OF GRAM STAINING FOR BACTERIA
Master of science in Agriculture
Submitted By
Dayashankar Baghel
M.Sc (previous)
Dept. of Agricultural
Microbiology
Submitted To
Dr. Anurag Tomar
(Scientist)
Dept. of Soil Science and
Agricultural Chemistry
IGKV Raipur
2. Outline of presentation
• History
• Introduction
• Objectives
• Principles
• Gram positive vs Gram negative
• How to perform gram staining
• Procedure
• Interpretation
• Examples
• References
3. History
The Gram stain was devised by
the Danish physician, Hans Christian Gram,
While working in Berlin in 1883.
He later published this procedure in 1884.
At the time, Dr. Gram was studying lung tissue
sections from pateints who had died of pneumonia.
4. Introduction
What is Gram Staining?
• The Gram staining is almost always the first step in the identification of 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.
• Gram positive bacteria stain violet and Gram negative bacteria stain red or pink
because the presence of different layer of peptidoglycan in their cell walls.
5. Objectives
• To differentiate between the two major categories of bacteria: Gram positive and
Gram negative.
• To understand how the Gram stain reaction affects Gram positive and Gram
negative bacteria based on the biochemical and structural differences of their cell
walls.
Gram-positive Gram-negative
6. Principles
• The structure of the organism ‘s cell wall determines whether the organism is
gram positive or negative.
• When stained with a primary stain and fixed by a mordant, some bacteria are
able to retain the primary stain by resisting declorization while other get
decolorized by decolorizer.
• Those bacteria which retain the primary stain are called Gram positive.
• Those bacteria which get decolorized and then get counterstained are called Gram
negative.
7. Principles continue
1. Crystal violet – all bacteria take crystal violet- so all appears violet.
2. Iodine – Crystal Violet-iodine(CV-I) complex is formed.
3. Ethyl alcohol- bacteria with high lipid content loose CV-I complex
(appear colourless) but bacteria with less lipid content retains CV-I complex ( appear
violet/blue).
4. Safraninee- only colourless bacteria takes – appear pink/red
11. How to perform Gram stain
Material required
• Clean glass slides
• Inoculating loop
• Bunsen burner
• Microscope
• Lens paper and lens cleaner
• Immersion oil
• Distilled water
• 18 to 24 hour cultures of organisms
13. Procedure – Preparation of smear
Wash the slides with water and wipe the slides with spirit or alcohol. After
cleaning, dry the slides.
Drawing a circle on the underside of the slide using a glassware-marking pen
may be helpful to clearly designate the area in which you will prepare the smear.
: With a sterile cooled loop, place a drop of sterile water on the slide.
Sterilize and cool the loop again and pick up a very small sample of a bacterial
colony and gently stir into the drop of water on the slide to create smear.
Dry the smear thoroughly in cool air and pass the entire slide through the flame
of a Bunsen burner two to three times.
Now the smear is ready to be stained.
15. Final staining procedure
After making a smear smear flood the slide with crystal violate sol. for upto 1 min.
Wash off briefly with tap water & drain.
Flood the slide with gram’s iodine sol. & allow to act as a mordant for about 1 min.
Wash off with tap water & drain.
Decolourise the smear with acetone for 10-30 sec. taking care not to
overdecolourise & immediately wash off with water
Flood the slide with safranin sol. & counterstain for about 30 sec, wash off with tap
water, drain & blot dry with filter paper & examine under oil immersion objective.
18. Interpretation
• Gram positive bacteria: Stain dark purple due to retaining the primary dye called
Crystall Violet in the cell wall.
Example- Staphylococcusus
• Gram negative bacteria: Stain red or pink due to retaining the counter staining
dye called safranin.
Example- Eschiaerichia coli
21. Safety precautions during Gram staining
• Use young, vigorous cultures rather than older cultures for your experiment.
• Properly adjust the flame of the Bunsen burner. The proper flame is a small blue
cone
• Allow your loop to cool before you try to pick up your organism. .
• Try to prepare a single cell layer of organism (a thin smear). Otherwise the all
cells will appear as gram positive in thick area.
• Decolorisation step should not exceed the time limit.
• Always prefer to observe under 10X first. This will give you an idea of the
location of a good area for observation. After this you may prefer to switch over to
40X.
• Do not ever observe at a specimen at 100X without oil.
