What is gram staining?
- invented by Hans Christian gram in 1884
- it allows identification of a bacterial cell from gram positive and gram negative
- gram positive bacteria have a thicker peptidoglycan cell wall and trap more of a crystal video stain and will appear purple under the microscope
- gram negative bacteria have thinner peptidoglycan cell walls and cannot trap crystal violet stain, a counter stain called safranin can confirm their identity and show as pink/red
Why is gram staining important in medicine?
- Penicillin is more effective at stopping the growth of the thick cell wall of gram positive bacteria
- penicillin is effective in killing gram positive bacteria but penicillin is less effective at killing gram negative bacteria as they have a thinner cell wall
- makes it possible to determine whether to treat a bacterial infection with penicillin or a different antibiotic
What is the procedure of gram staining?
- Heat a sample of bacteria on a slide with a Bunsen burner so the bacteria cannot be washed away
- apply Crystal Violet stain (floods slide) to make them appear purple
- apply iodine to the sample, iodine binds to the crystal video stain
- apply ethanol to wash away excess crystal violet stain and decolourise the rest of the slide
- apply safranin to the sample, this is taken up by the bacteria cells, the gram negative bacteria show up pink/red, in gram positive this is hidden by the Crystal violet
Prepare slides - dry mount
-specimens cut into thin slices, they are either whole or cut (sectioned)
- specimen placed in centre of slide and coverslip
Prepare slides - wet mount
-Specimens suspended in a liquid (water or oil)
- coverslip placed at an angle
Prepare slides - squash slides
-Wet mount is first prepared, lens tissue is used to press down cover slip
- squash the sample between two slides to ensure there is no damage to the cover slip
- good for soft samples
Prepare slide - smear slides
- Edge of slide used to smear sample
- thin even coating
- cover slip placed over sample
Staining - crystal violet/ methylene blue
-Positive charged dyes attracted to negative charged materials in cytoplasm
-stains cell components
Staining - Congo red/ nigrosin
-Negative charged dyes, repelled by negative charged cytosol
-cells left unstained stand out against stained background
Staining - gram stain technique
- Separates bacteria into gram positive and negative, crystal violet and iodine put on slides, wash with ethanol
-Gram positive will appear purpose and gram negative will lose stain due to thinner cell wall and will be stained with safranin (counterstain)
Staining - acid fast technique
-Differentiates species of mycobacterium from other bacteria
-Lipid solvent carries carbolfuchsin dye into cells
-Cells washed with acid solution, Mycobacterium is not affected and retain carbolfuchsin stain which is red, other bacteria lose the stain and are exposed to methylene blue
Stages involved in making pre prepared slides
FIXING - chemicals preserve specimens in as near natural state as possible
SECTIONING- specimens dehydrated with alcohol, placed in mould with wax to form hard block
STAINING - Specimens treated with multiple stains to show structues
MOUNTING - specimens are secured to a slide and cover slips placed on top
Risk management for slides
-Stains could be toxic
-CLEAPSS support for practical work in schools, provide student safety sheets that identify risks in schools, more slides are pre prepared in schools due to the danger of stains
Why is staining used?
All stains increase the contrast between cell structures and back-ground
