Nuclear Staining by Giemsa’s Method and its Details

Nuclear Staining by Giemsa’s Method and its Details

Aim

To stain bacteria nuclear material by Giemsa’s staining technique.

Approach

DNA is a principle genetic material of all micro-organisms except some viruses.Bacterial cell lacks well organised nucleus.It is very essential structure of a living cell.Nucleus performs important functions like growth,metabolic activities,multiplication and transfer of heridatory characters.Nuclear material of bacteria is also called as nucleochromatin body.Bacteria cell have haploid nuclear content and they posses single copy of double stranded DNA material.Besides DNA some bacteria may posses extra chromosomal material called as plasmid. The nuclear material of bacteria is stained by using Giemsa’s method where as other method’s that can be used are Feulgen’s method and Robinow’s method.

Requirements

  1. Bouin’s fixative
  2. 1 N HCL
  3. Water bath
  4. Giemsa’s stain
  5. Cell suspension

Procedure

  1. A clean grease free slide it taken and a thick smear is prepared by using a sterile wireloop.
  2. The slide is allowed to air dry and then the smear is fixed by using Bouin’s fixative for about 15 minutes here we avoid heat fixation step.
  3. Further after chemical fixation the slide is treated with 1 N HCL solution in water bath at 60°C for about 5 minutes.
  4. After 5 mins the slide is rinse with water and flooded with Giemsa’s stain for about 3 to 5 minutes after that slide is observe under oil immersion lens.

Flow chart- Procedure of Nuclear Staining technique

Nuclear Staining by Geimsa's method
Nuclear Staining by Giemsa’s method

Mechanism

Giemsa’s stain is prepared by mixture of two stains that are methylene blue ( basic dye) and eosin( acidic dye) so the resulting stain has properties of both dye.The chromatide of a cell is highly acidic in nature so when it reacts with Giemsa’s stain it gives a reddish purple color to DNA.The RNA of a cell is removed by acid hydrolysis step in this step the smear is treated with 1 N HCL solution in a water bath of about 60°C temperature for 5 minutes during this step due to heat and acid the double bonds between the base pairs of RNA molecule get week and break.Where as DNA material has triple bonds in between some base pairs so DNA molecule  doesn’t get hydrolyzed and hence only DNA molecule get stain and cytoplasm remains colourless.

Observation

The cytoplasm appears colourless where as nuclear material appears purple in colour.

 

 

3 Comments

  1. k. choudhury October 29, 2015 Reply
  2. k. choudhury October 29, 2015 Reply
  3. noreen October 5, 2015 Reply

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