Study Notes for General Botany Laboratory (BIOL 101)

GENERAL BOTANY LABORATORY BACKGROUND

  • Course: BIOL 101 General Botany

  • Adapted from the 39th New Phytologist Symposium

  • Location: Polytechnic University of the Philippines, College of Science, Department of Biology

  • Prepared by: Danielito L. Dollete, MSc. and Armin S. Coronado, PhD

LAB ACTIVITY 5: PHYSIOLOGY OF PLANT CELLS

Overview

  • Focus Area: Plant Cell Cycle

METHODOLOGY: PROCEDURE

Introduction to Cell Cycle

  • Key Quote: "Omnis cellula e cellula" (Every cell from a cell) by Rudolf Virchow

    • This principle emphasizes that all cells arise from pre-existing cells.

  • The Cell Cycle includes key phases:

    • Interphase: The cell prepares for division and replicates its genetic material.

    • Mitotic Phase (M phase): Involves the actual division of the cell.

Purposes of the Cell Cycle

  1. Reproduction:

    • In prokaryotes and single-celled eukaryotes, division is a form of reproduction.

  2. Growth and Development:

    • For multicellular organisms, cell division is crucial for growth and specialization.

  3. Tissue Renewal:

    • Regularly replacing old cells is essential for maintaining healthy tissues.

Key Text Reference

  • Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Reece, J. B. Campbell Biology (10th ed., p. 233). Pearson Education, Inc.

PRELAB: CELL CYCLE

Interphase Stages

  • G1 Phase: Cell growth occurs as the cell prepares for DNA replication.

  • S Phase: The cell replicates its genetic material (DNA).

  • G2 Phase: Further cell growth and preparation for mitosis.

Mitosis (M Phase)

Description by Walther Flemming

  • Mitosis is the process by which somatic cells divide, resulting in two identical daughter cells.

  • This phase is the shortest in the cell cycle and includes:

    • Mitosis + Cytokinesis: Signals the completion of interphase.

    • Visible Changes: Reorganization of the nucleus.

    • Suspension of Cellular Activities: Most cellular processes, including protein synthesis, pause.

  • Mitosis is divided into four primary stages:

    1. Prophase

    2. Metaphase

    3. Anaphase

    4. Telophase

Methodology: Procedure for Mitosis Observation

Preparing Onion Root Tips

  1. Selection: Choose healthy onion bulbs.

  2. Water Setup: only basal plate should touch water to prevent rotting.

  3. Light Placement: Position in light but out of direct sunlight.

  4. Water Maintenance: Regularly check and replace water.

  5. Root Growth Observation: Roots should reach 1-2 cm in length for harvesting.

  6. Harvesting: Cut tips of roots for fixation and avoid excessively long roots.

  7. Fixation: Immerse root tips in Farmer’s fixative for 12-24 hours.

Dehydration and Paraffin Infiltration

  1. Dehydration Steps: Use a series of ethanol solutions (70%, 95%, absolute) to dehydrate tissues.

  2. Clearing Tissues: Clear with xylene and then infiltrate with molten paraffin wax.

Embedding Procedure

  1. Mold Preparation: Create a paper mold for paraffin block.

  2. Inserting Sample: Place the root tip in the mold and cover it with more molten paraffin.

  3. Solidification: Cool down the mold for removing it gently afterward.

  4. Trimming: Shape paraffin block for even sectioning.

Sectioning with Microtome

  1. Preparation: Ensure the microtome and blade are clean and sharp.

  2. Setting Up Microtome: Align tissue properly with knife edge and adjust settings for section thickness.

  3. Maintaining Conditions: Ensure appropriate temperature to avoid shattering of wax during sectioning.

Mounting Paraffin Ribbons onto Slides

  1. Clean Slides: Thorough cleaning is necessary for adherence.

  2. Albumin Coating: Enhance adhesion with albuminized slides.

  3. Labeling: Keep a proper record of slides indicating sample IDs and dates.

  4. Floating Sections: Place tissue ribbons on distilled water for floating.

  5. Drying and Fixation: Place in warmer for proper adhesion before deparaffinization.

Deparaffinization and Rehydration

  1. Xylene Immersion: Remove paraffin wax completely through xylene immersions.

  2. Ethanol Series: Gradually rehydrate through hydration series (100%, 95%, 70%).

  3. Final Rinse: Clean thoroughly with distilled water.

Staining and Microscopy

  1. Staining: Immerse in 2% aceto-orcein or acetocarmine.

  2. Rinsing and Observation: Rinse and observe staining depth.

  3. Preparation for Mounting: Dehydrate and clear sections before applying mounting media.

Squashing Technique

  1. Slide Preparation: Use solutions to facilitate services on slide.

  2. Squashing: Obtain thin layers by carefully separating cells from root tips.

Calculating Mitotic Index

  1. Sampling: Focus on mitotic zones, recording stages across multiple fields.

  2. Calculations: Develop ratios and indices for stage duration and standard deviation.

Lab Report Preparation

  1. Photomicrograph Objectives: Analyze various stages of mitosis.

  2. Documentation: Investigate and discuss findings based on visual data.

  3. Submission Deadline: January 18, 2026, through online platforms.