Nucleus staining (Giemsa's method)

 Aim- To stain nuclear material by Giemsa's method. 

Theory- Bacteria do not contain true nucleus. They contain area near the center of the cell. i.e regarded as nuclear structure. These structure has been dedicated nuclear or nucleoid or chromosome. The bacterial nucleoid contain double stranded circular DNA and it can be visible under light microscope by special staining procedure such as Giemsa's method. There are some other method also used of staining of chromatin material which includes feulgens method, rainbow method, acridine orange method etc. 

Principle- The demonstration of nucleus require selective hydrolysis of RNA on the cytoplasm with affecting DNA. The acidic nature of bacteria and other organism has affinity towards the basic dyes the hydrolysis of RNA is achieved by acid treatment because of it is double stranded and contain triple bond between guanine and cytosine pair absent in RNA. The acid causes hydrolysis change in confirmation and the aldehyde group react with Giemsa's and give dark colour of DNA because of eiosin cytoplasm become faint pink or colourless. Bowines fixative is used because it doesn't allow change in colour. 

Bowines Fixative Composition- 

.Picric acid - 25ml

.Formaldehyde - 5ml

. Acitic acid- 5ml

.1N HCL- 9ml

Giemsa's stain-

.Geimsa powder- 0.3gm

.Methanal- 25ml

. Glycerol- 25ml

Diagram- 

Observation- As per diagram. 

Procedure- 

1. Take grease free slide. 

2. Smear the suspension with the help of sterile wire loop. Allow it to air dry (Do not heat fix). 

3. Apply Bowines fixitative for 30min. Slide kept in 1N HCL in water bath at 60c for min. Rinse the slide under water.

4. After washing slide apply the Giemsa's stain for 5 min rinse the stain with water. 

5.Allow it to air dry and observe under oil immersion lens. 

Result- 

In an microscopic field pink coloured cell wall and blue coloured nucleus is observed.