Preparation of Permanent Slide to Show the Presence of Barr Body
Aim
To prepare a permanent slide of human female buccal epithelial cells or blood smear to demonstrate the presence of the Barr body (sex chromatin).
Learning Outcomes
Ø Understand the concept of sex chromatin (Barr body) and its genetic basis.
ØLearn the collection and staining technique of buccal smear / blood smear.
ØIdentify Barr body under microscope and differentiate it from other nuclear structures.
ØAcquire skills in preparing a permanent slide using cytological stains.
Principle
In females, one of the two X chromosomes in somatic cells is inactivated during early embryogenesis (Lyon’s hypothesis). The inactivated chromosome forms a dense heterochromatic body attached to the nuclear membrane, visible in interphase nuclei. This structure is called the Barr body. Females (XX) show one Barr body in each somatic cell nucleus, while males (XY) lack it. Thus, Barr body demonstration is a classical method of sex determination and cytogenetic diagnosis.
Materials and Reagents
Specimen & Tools:
1. Female volunteer (cheek cell smear or blood smear)
2. Sterile toothpick / wooden spatula / cotton swab
3. Microscope slides and coverslips
4. Fine forceps
Reagents:
1. Fixative: Carnoy’s II (ethanol:acetic acid 3:1) OR methanol:acetic acid (3:1)
2. Stains: Acetocarmine / Aceto-orcein / Feulgen reaction / Giemsa stain
3. Mountant: DPX or Canada balsam
Safety:
1. Obtain informed consent for sample collection.
2. Wear gloves, lab coat.
3. Dispose of biological material properly.
Procedure
A. Buccal Smear Method
1. Rinse mouth with water.
2. Gently scrape the inside of cheek with spatula/cotton swab.
3. Transfer material onto slide.
4. Fix with Carnoy’s II fixative (5–10 min).
5. Rinse and apply stain (acetocarmine/Feulgen, 10–15 min).
6. Wash, add DPX, place coverslip carefully.
7. Dry for permanent mount.
B. Blood Smear Method (Alternative)
1. Place small drop of blood on slide and spread into thin smear.
2. Fix in methanol for 10 min.
3. Stain with Giemsa for 20–30 min.
4. Wash, dry, and mount with DPX.
Observation
Under microscope, the nucleus of female cells shows a small dense dark-stained mass of chromatin at the nuclear periphery (Barr body). Males lack Barr bodies. In neutrophils of blood smear, the Barr body appears as a 'drumstick appendage'.
Result
A permanent stained slide of female buccal epithelial cells/blood cells was prepared. The Barr body (sex chromatin) was observed as a small dark-stained mass at the nuclear periphery.
Precautions
1. Obtain samples only from healthy volunteers with consent.
2. Ensure smear is thin and even.
3. Avoid over-staining and wash gently.
4. Handle human samples with biosafety care.
Viva Questions
1. What is a Barr body?
A condensed, inactivated X chromosome seen in female somatic cell nuclei.
2. Who discovered the Barr body?
Murray Barr and E. Bertram (1949).
3. How many Barr bodies are seen in: XX female? XXX female? XY male?
XX: 1, XXX: 2, XY: 0.
4. What is the genetic basis of Barr body formation?
X-inactivation (Lyon’s hypothesis).
5. Where else can Barr body be demonstrated?
Buccal smear, blood smear (neutrophil drumstick), hair follicle cells.
References
1. Verma, P. S., & Agarwal, V. K. (2019). Cell Biology, Genetics, Molecular Biology, Evolution and Ecology. S. Chand Publishing.
2. Gupta, P. K. (2017). Cytogenetics. Rastogi Publications.
3. Gardner, E. J., Simmons, M. J., & Snustad, D. P. (2012). Principles of Genetics. Wiley.
4. Barr, M. L., & Bertram, E. G. (1949). A morphological distinction between neurons of the male and female... Nature, 163, 676.
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