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)
The above medium is prepared and is autoclaved at 15 lbs. The solution
is poured into clean, sterilized petri dishes. After cooling, the petri dishes are
kept in the refrigerator for 10–20 hrs. Then, the petridish is kept for incubation
for 2–4 hrs for 37°C. The isolated E.coli
inoculum is treated with the nutrient
agar plate. The plates are inverted and incubated at 37°C for 24 hrs. After 24
hrs, whitish streaks indicate growth of E.coli
. A portion of the colony is stained
Staining of E.coli
To stain bacteria (E.coli
) using the Gram-staining method.
- E.coli culture
- Crystal violet
- Gram iodine solution
- Ethyl alcohol (98%)
- Staining tray
- Glass slide
- 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.