Gram Staining
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When was gram staining published?
1884
What is gram staining?
Gram staining is a technique which is based on a sequence of chemicals, starting with using crystal violet followed by an iodine treatment, washed out with alcohol, and finally counterstained. This procedure leads to gram positives appearing purple and gram negatives appearing red
Method of gram staining? [FULL]
• First, heat fixation is carried out, by passing the slide of bacteria cells over a heat source such as a Bunsen burner. This ensures that the bacteria and cells will stay on the slide during staining. • Second, crystal violet is used on the bacteria, followed by an iodine treatment. The crystal violet and iodine then form a complex (crystal violet/iodine complex). This is washed out with alcohol, and then a counterstain is used. • Gram-positive bacteria have cell walls with a thicker layer of peptidoglycan/murein (than Gram-negative bacteria), which retains the crystal violet/iodine complex within their cells, and when these gram positive bacteria are washed with alcohol, they stain purple. • On treatment with alcohol, the cell walls of Gram negative bacteria lose their outer lipopolysaccharide membrane, and the thin inner peptidoglycan layer is left exposed. This means that the crystal violet/iodine complexes are washed from the gram-negative cell along with the outer membrane. They then stain red with the counterstain safranin.
What are the main differences between gram positive and gram negative bacteria?
• Gram positive bacteria have cell walls with a thicker layer of peptidoglycan/murein than Gram-negative bacteria do. • Gram negative bacteria have an outer membrane (made up of lipopolysaccharide and protein), but gram positive bacteria don’t.
What causes a bacteria’s shape?
The shape of bacteria is due to their rigid cell wall which has a unique structure: it contains a 3D mesh of peptidoglycan (murein).
How can bacteria be distinguished from each other?
Bacteria can be distinguished from each other by their: -Size -Shape -staining characteristics, -metabolic, antigenic and genetic features.
The rigid cell wall contains a 3D mesh of…
peptidoglycan
How many cells are assumed to give rise to a colony?
1
What causes Gram-positive bacteria to retain the crystal violet/iodine complex (staining purple)?
Thicker peptidoglycan layer
What do bacteria rigid cell walls contain?
peptidoglycan/murein
What do Gram-negative bacteria lose on treatment with alcohol?
outer lipopolysaccharide membrane
What colour do Gram-negative bacteria stain?
Red
What colour do Gram-positive bacteria stain?
Purple
What counter stain makes Gram-negative bacteria red?
safranin
What is step 1 of gram staining?
First, heat fixation is carried out, by passing the slide of bacteria cells over a heat source such as a Bunsen burner flame. This ensures that the bacteria and cells stay on the slide during staining.
What is step 2 of gram staining?
Second, crystal violet is used on the bacteria, followed by an iodine treatment. The crystal violet and iodine then form a complex (crystal violet/iodine complex). This is washed out with alcohol, and then a counterstain is used.
What is step 3 of gram staining?
Gram-positive bacteria have cell walls with a thicker layer of peptidoglycan/murein (than Gram-negative bacteria), which retains the crystal violet/iodine complex within their cells, and when they are washed with alcohol – they stain purple.
What is step 4 of gram staining?
On treatment with alcohol, the Gram negative cell walls lose their outer lipopolysaccharide membrane, and the thin inner peptidoglycan layer is left exposed, this means that the crystal violet/iodine complexes are washed from the gram-negative cell along with the outer membrane – they then stain red with the counterstain safranin.
What causes the decolourisation process in Gram Staining?
Ethanol
Why cause the initial colour change in gram staining?
Crystal violet
Which type of bacteria retains the dyed colour? and why?
Gram-positive due to thicker peptidoglycan layer trapping crystal violet/iodine complex
What is removed from the gram-negative bacteria to facilitate gram staining?
Outer lipopolysaccharide membrane
What causes the final staining of the gram-negative bacteria in gram staining?
Safranin
What is the purple dye from gram staining formulated from?
Crystal violet/iodine complex