Study of Various Stages of Meiosis in Testis of Cockroach (Squash Preparation)

 Study of Various Stages of Meiosis in Testis of Cockroach (Squash Preparation)

Aim

To prepare a squash of cockroach testis and identify the stages of meiosis (Prophase I: leptotene, zygotene, pachytene, diplotene, diakinesis; Metaphase I, Anaphase I, Telophase I; Interkinesis; Metaphase II, Anaphase II, Telophase II), and observe spermiogenesis.

Learning Outcomes

1. Dissect out cockroach testes safely and efficiently.
2. Prepare a clean meiotic squash using aceto-orcein/acetocarmine.
3. Recognize and sketch the cytological features of meiotic stages in spermatocytes.
4. Troubleshoot common issues (overstaining, thick squash, poor cell spread).

Principle

In adult male cockroach (Periplaneta americana), the testis is formed by multiple testicular follicles enclosed in a sac. Within each follicle, germ cells occur in “cysts” at synchronous stages: tip (spermatogonia), middle (primary/secondary spermatocytes—where meiosis occurs), basal/near vas deferens (spermatids → sperm). A squash prep in 45% acetic acid softens tissue and spreads cells; aceto-orcein or acetocarmine stains chromatin strongly so meiotic chromosomes are easily seen.

Materials and Reagents

Specimen & tools:
1. Adult male cockroach
2. Dissecting board, fine forceps, needles, scissors
3.  Petri dish, watch glasses, glass slides, coverslips
4. Filter paper/tissue, blotting paper

Solutions:
1. Insect Ringer/0.7–0.9% NaCl
2. 45% glacial acetic acid
3. 2% Aceto-orcein or Acetocarmine
4.  Fixative: Carnoy’s II (ethanol:acetic acid 3:1)
5. Distilled water
6. Nail varnish/DPX mountant

Safety:
1.  Lab coat, gloves, eye protection.
2. Work in ventilated area when using acetic acid/ethanol.
3. Dispose of biological waste and solvents as per biosafety rules.

Procedure

Dissection (10–15 min)

1. Anesthetize/euthanize the cockroach as per guidelines.
2. Position dorsal side up in a dissecting tray with a little saline. Pin the specimen.
3. Make a mid-dorsal incision from posterior towards the thorax; gently lift the tergites.
4. Locate testes: two yellowish, lobulated sacs in anterior abdomen, dorsal to gut and fat body.
5. Excise testes and transfer to saline.
6. Optional: Fix in Carnoy’s II for 10 min, then rinse in 45% acetic acid.

Slide Preparation (15–20 min)

1. Tease a small portion of a follicle in a drop of saline on a clean slide.
2. Add one drop of 45% acetic acid, wait 2–3 min.
3. Replace with 1–2 drops of aceto-orcein/acetocarmine.Heat gently until vapors appear (do not boil). Rest for 5 min.
4. Place coverslip, squash gently with pencil eraser.
5. Check under microscope (10×, then 40×).
6. Seal coverslip with DPX.

Microscopy and Identification

Use 10× to locate fields, then 40× and 100× oil for chromosomal detail. Prefer primary spermatocyte cysts (larger cells) for Prophase I and Metaphase I; secondary spermatocytes for Meiosis II; spermatids for spermiogenesis.

Diagnostic Features of Meiotic Stages

Prophase I:
1.  Leptotene: thin, thread-like chromosomes.
2. Zygotene: homologous pairing begins.
3. Pachytene: thick bivalents, nucleolus visible.
4.  Diplotene: homologues repel, chiasmata visible.
5. Diakinesis: condensed bivalents, nuclear envelope breaks.

Metaphase I: bivalents aligned at equator.
Anaphase I: homologous chromosomes separate.
Telophase I: haploid nuclei form.

Metaphase II: chromosomes align again.
Anaphase II: sister chromatids separate.
Telophase II: four haploid nuclei form.
Spermiogenesis: round spermatids elongate into sperm bundles.

Recording Results

Make labelled line diagrams of pachytene, diplotene, diakinesis, metaphase I, anaphase I, metaphase II, anaphase II, spermatids. Prepare a result table with field no., stage, features, and sketch no.

Viva Questions

1.Why use 45% acetic acid?

To soften tissue, lyse cytoplasm, and help spread cells while preserving chromatin for orcein/acetocarmine binding.

2. Why select the middle region of the follicle?

It is enriched in primary/secondary spermatocytes undergoing meiosis.

3. How do you distinguish Anaphase I from II?

I: homologues separate; II: sister chromatids separate.

4. What does chiasma represent?

Cytological manifestation of crossing over between non-sister chromatids.

5. Why prefer cockroach testis for meiosis study?

Large cell size, synchronous cysts, robust chromosomes that take stain well.

 

References

1. Gupta, P. K. (2017). Cytogenetics. Meerut: Rastogi Publications.

2. Sharma, A. K., & Sharma, A. (1980). Chromosome Techniques: Theory and Practice. London: Butterworths.

3. Chatterjee, C. C. (2018). Human Physiology, Volume 1. Kolkata: New Central Book Agency.

4. Wilson, E. B. (1925). The Cell in Development and Heredity. New York: Macmillan.

5. Verma, P. S., & Agarwal, V. K. (2019). Cell Biology, Genetics, Molecular Biology, Evolution and Ecology. New Delhi: S. Chand Publishing.

6. Plopper, G., Sharp, D., & Sikorski, E. (2013). Lewin’s Cells. Burlington, MA: Jones & Bartlett Learning.

7. Bhatia, K. N. (2000). Practical Zoology (Invertebrates and Chordates). New Delhi: Vishal Publications.

8. Wilson, J., & Hunt, T. (2019). Molecular Biology of the Cell: The Problems Book. New York: Garland Science.

9. Rieger, R., Michaelis, A., & Green, M. M. (2012). Glossary of Genetics: Classical and Molecular. Berlin: Springer.

10. Stern, H. (1936). Somatic Crossing-over and Segregation in Drosophila melanogaster. Genetics, 21(6), 625–730.