Aim: To study nuclear material Staining.
Bacteria are prokaryotic which possess nuclear material and circular DNA. In the case of Eukaryotic, genetic material is membrane-bound. The nucleus present in prokaryotes that is not well defined is called the nucleoid. There is a lack of nuclear membrane. In bacteria extrachromosomal DNA plasmid is present. The plasmid is small circular extrachromosomal DNA that shows self-replication properties. Plasmid DNA may give a bacterium the power to synthesize new products and also antibiotic resistance properties. Cytoplasm present in cells shows a high affinity for stains and also interferes with the observation of nuclear material. So the hydrolysis is carried out with HCl and is then stained with Giemsa stain.
Giemsa stain is used for nuclear material visualization. The stain is used to differentiate nuclear material and cytoplasmic material. The cytoplasm of bacteria possessing a strong affinity for nuclear stain gets hydrolyzed with hydrochloric acid. Finally, it is stained with Giemsa stain which differentiates nuclear material and cytoplasmic material.
- Bacterial culture: 24 hours fresh bacterial culture
- Chemicals: 1N HCl, Giemsa stain
- Apparatus: Glass Slide, inoculating loop, water bath, microscope
- Take a clean glass slide and prepare the smear on the slide.
- Heat fixed smear on the flame of the burner.
- Place the smear in the beaker containing HCl solution.
- Keep the beaker in a water bath at 50°C for 10 minutes.
- Wash the slide with gentle tap water.
- Flood the smear with Giemsa stain for 2-3 minutes and wash with gentle tap water.
- Blot dries the smear.
- Add oil on the smear and observe under oil immersion lens.
Examine the nuclear material on the smear under an oil immersion lens.
The nuclear bodies may appear purple and surrounded by colourless zone of cytoplasm, while the cell membrane appears faint purple colour.
Nuclear staining is used to differentiate cytoplasmic material from nuclear material.
- Giemsa stain should be freshly prepared.
- Do not over-dry the smear.
- Cultures used should be 24 hours old.