ZOO: DSM-351 Practical
Unit 1: Cell Biology
1. Preparation of Temporary Stained Squash for Mitosis
Mitosis is the process of somatic cell division resulting in two genetically identical daughter cells. Onion root tips are ideal for this study as they contain actively dividing meristematic cells.
Materials Required
- Onion root tips (fixed in Carnoy's solution).
- 1N HCl (for hydrolysis).
- Acetocarmine or Aceto-orcein stain.
- Slides, coverslips, and spirit lamp.
Step-by-Step Procedure
- Hydrolysis: Place the fixed root tips in 1N HCl at 60 degrees Celsius for 5-10 minutes to soften the cell walls.
- Staining: Transfer the hydrolyzed tips to a slide and add a drop of Acetocarmine. Warm gently over a flame (do not boil).
- Squashing: Place a coverslip over the tip. Tap gently with a needle or use thumb pressure to spread the cells into a single layer.
- Observation: Examine under a microscope to identify Prophase, Metaphase, Anaphase, and Telophase.
2. Study of Various Stages of Meiosis
Meiosis is the reductional division occurring in germ cells to produce haploid gametes. It involves two successive divisions: Meiosis I and Meiosis II.
Key Stages for Identification
- Prophase I: Includes specialized sub-stages: Leptotene, Zygotene, Pachytene, Diplotene, and Diakinesis.
- Metaphase I: Homologous pairs (bivalents) align at the equatorial plate.
- Anaphase I: Homologous chromosomes move to opposite poles (Reductional step).
- Meiosis II: Similar to mitosis, resulting in the separation of sister chromatids.
3. Preparation of Permanent Slide for Barr Body
A Barr body is an inactivated X chromosome found in the somatic cells of female mammals. It appears as a small, densely stained structure near the nuclear envelope.
Objective
To demonstrate the presence of a Barr body in human female cells, specifically from hair follicles.
Procedure
- Pluck a fresh hair from a human female, ensuring the hair follicle (bulb) is intact.
- Fix the follicle in a suitable fixative (e.g., Ethanol-Acetic acid).
- Stain with a DNA-specific stain such as Acetocarmine or Feulgen stain.
- Prepare a permanent mount using DPX or a similar mounting medium.
- Under high power, look for a dark, condensed mass attached to the inner surface of the nuclear membrane.
Lyon's Hypothesis: Only one X chromosome is active in the somatic cells of females; the other is heterochromatinized and becomes the Barr Body.
Exam Focus: Tips and Common Practical Pitfalls
Exam Tip: When drawing Mitosis stages, pay close attention to the position of centromeres during Anaphase. They should be leading toward the poles while the arms of the chromosomes trail behind.
Common Mistake: Over-staining or under-hydrolyzing the root tip often leads to a dark mass where individual chromosomes cannot be seen. Always ensure the root tip is soft enough to "spread" easily.
Frequently Asked Questions (FAQs)
- Q: Why is 1N HCl used in the mitosis squash?
A: To break down the middle lamella (pectin) between cells, allowing them to separate easily during the squash.
- Q: Can Barr bodies be found in males?
A: Generally no, unless the male has a chromosomal abnormality like Klinefelter Syndrome (XXY).
- Q: What is the best tissue to study meiosis?
A: In animals, the testis is commonly used; in plants, young flower buds (anthers) are used.