Culturing and Staining of E.coli (Gram’s Staining)
E.coli colonies were isolated from water and inoculated in nutrient agar.Composition of Nutrient Agar
- Peptones – 5 gm
- Beef extract – 3 gm
- NaCl – 5 gm
- Agar agar – 20 gm
- Distilled water – 300 mL
- pH (7.0 ± 0.2)
Staining of E.coli
Objective
To stain bacteria (E.coli) using the Gram-staining method.
Materials
- E.coli culture
- Crystal violet
- Gram iodine solution
- Ethyl alcohol (98%)
- Saffranine
- Staining tray
- Dropper
- Glass slide
- Microscope
- Prepare a smear of the culture on a glass slide and heat-fix it.
- Flood the smear with crystal violet for 1 minute.
- The rinses are washed under slow running water and flood the smear with iodine solution. Let it stand for 1 minute.
- Rinse the slide under slow running water and wash with 98% ethyl alcohol. Let it stand for 1 minute.
- Rinse the slide under slow running water and add a few drops of saffranine to the smear and keep it for 30 seconds.
- Wash the smear and dry the slide.
- Use a cover slip. Put a drop of cedar wood oil over the cover slip and observe under an oil immersion lens.
If the material is Gram-negative, it is decolorized by alcohol and stained by saffranine. If it is positive, it is stained by Gram iodine stain.