Experiment 02Meristematic Cytology • B.Sc. / M.Sc.
Mitosis in Onion Root Tip (Allium cepa) and Mitotic Index
🎯 Aim: To prepare a temporary acetocarmine squash mount of the apical meristem of Allium cepa root tips, identify all four mitotic phases (Prophase, Metaphase, Anaphase, Telophase), and quantitatively calculate the Mitotic Index (MI) and phase frequency distribution.
🔬 Theoretical Principle & Cytological Mechanism
The root apical meristem of onion (Allium cepa, $2n = 16$) provides an ideal model for studying eukaryotic somatic cell division due to rapid cell proliferation and large, easily stained chromosomes. Root tip tissues are harvested during peak mitotic diurnal cycles (typically between 9:00 AM and 10:30 AM). Fixation in Carnoy’s fluid (absolute ethanol : glacial acetic acid in a 3:1 v/v ratio) precipitates nucleoproteins and preserves native chromosome architecture while inhibiting endogenous nucleases.
Because plant cells are cemented together by a calcium- and magnesium-pectate middle lamella, physical squashing into a single monolayer requires mild acid hydrolysis (1N HCl at 60°C for 6–8 minutes). Acid hydrolysis selectively dissolves the pectin matrix and hydrolyzes purine-deoxyribose glycosidic bonds, exposing reactive aldehyde groups for basic dye condensation.
Acetocarmine stain (carmine dissolved in 45% boiling acetic acid) acts as a basic nuclear dye. The carminic acid chromophore complexes with ferric ions (often added as mordant) and binds electrostatically to the negatively charged phosphodiester backbone of chromatin, rendering chromosomes deep magenta-crimson while cytoplasm remains virtually colorless.
text{Mitotic Index (MI, %)} = frac{text{Total Number of Dividing Cells } (P + M + A + T)}{text{Total Number of Counted Cells } (N)} times 100Where $P = text{Prophase}$, $M = text{Metaphase}$, $A = text{Anaphase}$, $T = text{Telophase}$, and $N ge 500text{ cells}$.
Figure 2.1: Allium Cepa Root Tip Zonation & Cytological Mitotic Phases
🧪 Reagents, Materials & Solution Recipes
Reagents & Fixatives
- Carnoy’s Fixative: 3 parts absolute ethanol + 1 part glacial acetic acid (freshly prepared).
- 1% Acetocarmine: Dissolve 1.0 g certified carmine powder in 100 mL boiling 45% glacial acetic acid; reflux 15 min, cool, add trace ferric acetate ($1text{ drop } 1% text{FeCl}_3$), filter.
- Hydrochloric Acid (1N HCl): 82.5 mL conc. HCl ($37%$) in $917.5,text{mL} text{dH}_2text{O}$.
- 45% Glacial acetic acid & 70% ethanol storage fluid.
Equipment & Glassware
- Water bath maintained at precisely $60^circtext{C} pm 1^circtext{C}$
- Rooted onion bulbs (hydroponic culture over water for 48–72 hr)
- Spirit lamp, fine dissecting needles, watch glasses, scalpel
- Whatman No. 1 filter paper strips, glass slides & No. 1 coverslips
📋 Step-by-Step Experimental Protocol
- Root Harvesting & Fixation: Excise 2–3 mm apical root tips using a fresh razor blade between 9:00 AM and 10:30 AM. Immediately submerge in freshly prepared Carnoy’s fixative for 2–12 hours at room temperature. Transfer to 70% ethanol at 4°C for long-term storage.
- Acid Hydrolysis: Transfer fixed root tips into a watch glass containing 1N HCl. Place in a pre-warmed water bath at exactly 60°C for 6–8 minutes. (Crucial: Over-hydrolysis disintegrates chromatin; under-hydrolysis prevents single-layer squashing).
- Staining: Remove root tips from acid, rinse briefly in distilled water, and transfer to a watch glass containing 1% acetocarmine. Gently warm the watch glass over a spirit lamp flame for 5–10 seconds until vapor rises (do NOT allow the liquid to boil). Let stain for 10–15 minutes.
- Micro-Dissection of Meristem: Transfer one stained root tip onto a clean glass slide. Using a scalpel under a magnifying lens, cut off and discard all tissue above the terminal 1.0 mm whitish-pink meristematic cap. Add a fresh microdrop of 45% acetic acid.
- Maceration & Squashing: Tease the meristematic cap with two dissecting needles into fine pulp. Apply a clean coverslip, taking care to avoid air bubbles. Fold a piece of filter paper over the slide and press vertically downwards with the ball of the thumb. Apply firm, steady vertical force without any lateral sliding.
- Microscopic Screening & Counting: Scan under 10× to locate evenly spread areas. Switch to 40× objective. Count all cells systematically field by field across multiple non-overlapping optical fields until a minimum of 500 cells are scored. Classify every cell as Interphase, Prophase, Metaphase, Anaphase, or Telophase.
📊 Morphological Stage Criteria & Quantitative Data Table
| Mitotic Stage | Cytological Diagnostic Markers | Observed Count | Phase Frequency (%) |
|---|---|---|---|
| Interphase | Intact spherical nuclear envelope; granular chromatin; prominent nucleoli; no visible distinct chromosomes. | 435 | 87.0% |
| Prophase | Condensing chromatin into distinct thread-like chromosomes; nucleolus gradually disappears; nuclear envelope breaks down. | 28 | 5.6% |
| Metaphase | Thick, contracted chromosomes aligned precisely along the equatorial metaphase plate; kinetochores attached to spindle microtubules. | 18 | 3.6% |
| Anaphase | Centromeres divide; sister chromatids separate and migrate poleward as V-, L-, or J-shaped daughter chromosomes. | 11 | 2.2% |
| Telophase | Daughter chromosome sets reach opposite poles, decondense; nuclear envelope reassembles; phragmoplast/cell plate forms centrally. | 8 | 1.6% |
| Total Cells Scored | Dividing Cells = 28 + 18 + 11 + 8 = 65 | 500 | Mitotic Index = 13.0% |
text{Calculated Mitotic Index (MI)} = frac{65}{500} times 100 = 13.0%Phase Durations (assuming $T_c = 20,text{hr}$): Prophase $= 5.6% times 20 = 1.12,text{hr}$; Metaphase $= 3.6% times 20 = 0.72,text{hr}$; Anaphase $= 2.2% times 20 = 0.44,text{hr}$; Telophase $= 1.6% times 20 = 0.32,text{hr}$.
⚠️ Key Precautions & Troubleshooting
- Lateral Movement: Any sideways sliding of the coverslip during thumb pressure folds the cell monolayer into overlapping rolls, ruining mitotic stage identification.
- Water Bath Temperature: Strict adherence to $60^circtext{C}$ is vital; temperatures $> 62^circtext{C}$ degrade chromatin morphology, while $< 58^circtext{C}$ will fail to soften cell walls.
- Mordanting: Traces of iron ions catalyze intense dye binding; if chromosomes appear pale pink, dip a clean dissecting needle into ferric chloride and touch the acetocarmine drop before warming.
💬 Viva-Voce Questions & Model Answers
Q1: Why are root tips harvested specifically in the morning (9:00 AM – 10:30 AM)?
Plant meristematic cell division follows a diurnal circadian rhythm. The maximum frequency of mitotic spindle assembly and chromosome division peaks during early daylight hours due to accumulated photosynthetic energy metabolites and auxin signaling.
Q2: What is the specific chemical function of glacial acetic acid in Carnoy’s fixative?
Absolute ethanol causes marked cell and protein shrinkage. Glacial acetic acid rapidly penetrates tissues and causes cellular swelling, precisely counteracting ethanol shrinkage. It also precipitates nucleic acids and nucleoproteins while maintaining chromosome coil plasticity.
Q3: How does colchicine affect the Mitotic Index?
Colchicine binds to β-tubulin dimers and prevents microtubule polymerization, blocking spindle apparatus formation. Cells enter mitosis normally but cannot proceed past metaphase (mitotic spindle checkpoint arrest), causing an artificial accumulation of cells in metaphase (C-metaphase) and an elevated Mitotic Index.
