Practical 3: Cell Biology

1. Mitosis in Onion Root Tip
2. Study of Meiosis (Permanent Slides)
3. Preparation of Barr Body Slide

1. Preparation of Temporary Stained Squash of Onion Root Tip to Study Mitosis

Principle: The meristematic cells at the tip of an onion root are actively dividing, making them an excellent material to observe the various stages of mitosis (somatic cell division).
Requirements: Onion bulb, jar/beaker, water, 1N Hydrochloric acid (HCl), Acetocarmine stain, glass slide, coverslip, spirit lamp, blotting paper, microscope.
Procedure:
1. Grow Roots: Place an onion bulb over the mouth of a jar filled with water. Roots will grow into the water in 3-5 days.
2. Fixation: Cut off 2-3 mm of the root tips (best done in the morning). Fix them in a fixative (like Carnoy's fluid) for 30 min, or use them directly.
3. Hydrolysis: Place a root tip in a drop of 1N HCl on a glass slide. Gently warm the slide over a spirit lamp for a few seconds (do not boil). This hydrolyzes the middle lamella, softening the tissue.
4. Staining: Blot away the excess HCl. Add 1-2 drops of Acetocarmine stain.
5. Squashing: Place a coverslip over the root tip. Place a piece of blotting paper over the coverslip and press firmly with your thumb to squash the cells into a thin layer. (Avoid side-to-side movement).
6. Observation: Observe under the microscope, first in low power to find a good region, then in high power to identify the stages.
Observation: Scan the slide for cells in different stages of division.

Identification of Mitotic Stages

Stage	Key Identification Points (Draw and Label)
Interphase	1. This is the resting (non-dividing) stage, but metabolically active. 2. A distinct, large nucleus is visible. 3. Chromatin appears as a tangled, thread-like network. 4. A dense nucleolus is clearly visible.
Prophase	1. Chromatin threads condense, shorten, and thicken to become visible as distinct chromosomes. 2. Each chromosome consists of two sister chromatids joined by a centromere. 3. The nuclear envelope and nucleolus begin to disappear.
Metaphase	1. The nuclear envelope is completely gone. 2. The fully condensed chromosomes align themselves at the center of the cell, forming the metaphase plate or equatorial plate. 3. Spindle fibers are attached to the centromeres.
Anaphase	1. The centromere of each chromosome splits. 2. The sister chromatids separate and are pulled towards opposite poles by the shortening spindle fibers. 3. The separated chromatids are now considered individual chromosomes.
Telophase	1. The chromosomes arrive at the opposite poles and begin to uncoil and decondense back into chromatin. 2. A new nuclear envelope forms around each set of chromosomes. 3. The nucleolus reappears. 4. A cell plate begins to form in the center of the cell, which will grow outwards to become the new cell wall, dividing the cell in two.

2. Study of Various Stages of Meiosis

Principle: To identify the stages of meiosis (reductional division) from prepared permanent slides. This is typically done using sections of Grasshopper testis (for spermatogenesis) or Lily anther.
Procedure: Observe the provided permanent slide under the microscope. Scan the slide to find and identify the different stages of Meiosis I and Meiosis II.
Observation: The key difference from mitosis is the pairing and separation of homologous chromosomes.

Identification of Meiotic Stages

Stage	Key Identification Points (Draw and Label)
Prophase I	This is the longest and most complex stage. Look for: Leptotene: Chromosomes appear as thin threads. Zygotene: Pairing of homologous chromosomes (synapsis) begins. Pachytene: Paired chromosomes (bivalents) are thick. Crossing over occurs (not visible, but is a key event). Diplotene: Homologous chromosomes start to separate, but remain attached at points called chiasmata (X-shaped structures).
Metaphase I	The homologous pairs (bivalents) align at the metaphase plate (equator). This is different from mitosis where single chromosomes align.
Anaphase I	Homologous chromosomes separate and move to opposite poles. Sister chromatids remain attached at their centromeres. This is the reductional step.
Metaphase II	(Looks like mitotic metaphase, but in a haploid cell). Single chromosomes (each with two chromatids) align at the metaphase plate.
Anaphase II	(Looks like mitotic anaphase). The centromeres split, and sister chromatids separate and move to opposite poles.
Telophase II	Four haploid nuclei are formed, resulting in four new haploid cells (gametes or spores).

3. Preparation of Permanent Slide to Show Barr Body

Principle: To identify the Barr body (sex chromatin), which is the inactive X chromosome found in the somatic cells of female mammals (XX). Male cells (XY) lack Barr bodies. This serves as a method for nuclear sexing.
Requirements: Sterile toothpick or spatula, clean glass slide, fixative (95% ethyl alcohol), stain (e.g., Papanicolaou stain, acetocarmine, or Feulgen stain), microscope.
Procedure:
1. Gently scrape the inside of the cheek to collect exfoliated epithelial cells.
2. Smear the cells in a small circle on a clean glass slide.
3. Immediately (before it dries) immerse the slide in the fixative (95% alcohol) for 15-30 minutes. This is a critical step.
4. Follow the standard procedure for the chosen stain (e.g., Papanicolaou staining, which is complex, or simpler staining with acetocarmine).
5. Mount with a coverslip.
6. Observe under the microscope, using the oil immersion lens (100x) for the best view.
Observation:
- Scan for large, healthy-looking epithelial cells with a clear, unfolded nucleus.
- Look for a small, dark-staining, distinct mass of chromatin, typically plano-convex (half-moon shaped).
- The Barr body will be located pressed against the inner surface of the nuclear envelope.
- It will only be visible in a certain percentage of cells (30-60%) from a female-sourced sample.
- A male-sourced sample will not show these structures.

Exam Tip: Do not confuse the Barr body with the nucleolus. The nucleolus is typically larger and located more centrally in the nucleus. The Barr body is small and always appressed to the nuclear membrane.