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  Section: Biotechnology Methods » Cell Biology and Genetics
 
 
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Culturing and Staining of E.coli (Gram’s Staining)

 
     
 
Content
Cell Biology and Genetics
  Cell Cycles
  Meiosis in Flower Buds of Allium Cepa-Acetocarmine Stain
  Meiosis in Grasshopper Testis (Poecilocerus Pictus)
  Mitosis in Onion Root Tip (Allium Cepa)
  Differential Staining of Blood
  Buccal Epithelial Smear and Barr Body
  Vital Staining of DNA and RNA in Paramecium
  Induction of Polyploidy
  Mounting of Genitalia in Drosophila Melanogaster
  Mounting of Genitalia in the Silk Moth Bombyx Mori
  Mounting of the Sex Comb in Drosophila Melanogaster
  Mounting of the Mouth Parts of the Mosquito
  Normal Human Karyotyping
  Karyotyping
  Black and White Film Development and Printing for Karyotype Analysis
  Study of Drumsticks in the Neutrophils of Females
  Study of the Malaria Parasite
  Vital Staining of DNA and RNA in Paramecium
  Sex-Linked Inheritance in Drosophila Melanogaster
  Preparation of Somatic Chromosomes from Rat Bone Marrow
  Chromosomal Aberrations
  Study of Phenocopy
  Study of Mendelian Traits
  Estimation of Number of Erythrocytes [RBC] in Human Blood
  Estimation of Number of Leucocytes (WBC) in Human Blood
  Culturing Techniques and Handling of Flies
  Life Cycle of the Mosquito (Culex Pipiens)
  Life Cycle of the Silkworm (Bombyx Mori)
  Vital Staining of Earthworm Ovary
  Culturing and Observation of Paramecium
  Culturing and Staining of E.coli (Gram’s Staining)
  Breeding Experiments in Drosophila Melanogaster
  Preparation of Salivary Gland Chromosomes
  Observation of Mutants in Drosophila Melanogaster
  ABO Blood Grouping and Rh Factor in Humans
  Determination of Blood Group and Rh Factor
  Demonstration of the Law of Independent Assortment
  Demonstration of Law of Segregation

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)
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 and mounted.

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
Procedure
  1. Prepare a smear of the culture on a glass slide and heat-fix it.
  2. Flood the smear with crystal violet for 1 minute.
  3. The rinses are washed under slow running water and flood the smear with iodine solution. Let it stand for 1 minute.
  4. Rinse the slide under slow running water and wash with 98% ethyl alcohol. Let it stand for 1 minute.
  5. Rinse the slide under slow running water and add a few drops of saffranine to the smear and keep it for 30 seconds.
  6. Wash the smear and dry the slide.
  7. Use a cover slip. Put a drop of cedar wood oil over the cover slip and observe under an oil immersion lens.
Result
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.

 
     
 
 
     




     
 
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