"Microtomy and Microscopy in Plant Histology: Techniques and Applications" (Author: Prashant B. Kale (Ph.D.))

 "Microtomy and Microscopy in Plant Histology: Techniques and Applications"

Author: Prashant B. Kale (Ph.D.)

A microtome is a specialized precision cutting instrument, which accurately and repeatedly slices sections from a block of embedded tissue. Different kinds of microtomes are used to section paraffin and plastic-embedded tissues as well as the specialized microtomes used to section frozen tissues.

In the case of plants, microtomy is used to prepare thin sections of plant tissues for analysis, particularly for microscopic examination of cells and sub-cellular events. Basic steps for creating microtome sections for plant tissue analysis are explained as: (figure: microtome and cryotome instruments with important parts)

Materials and Equipment:

1. Plant specimens
2. Microtome (a specialized instrument for cutting thin sections)
3. Embedding medium (e.g., paraffin wax or resin)
4. Razor blades or microtome blades
5. Glass slides
6. Microscope
7. Staining solutions (optional)

Procedure:

1. Specimen Preparation:
• Collect the plant tissue you want to analyze.
• Fix the tissue using an appropriate fixative, such as formalin or ethanol, to preserve the cellular structures.
• Dehydrate the tissue by gradually transferring it through a series of alcohol solutions with increasing concentrations (e.g., 70%, 95%, and 100% ethanol). This process replaces water in the tissue with alcohol.
2. Embedding:
• Infiltrate the dehydrated tissue with an embedding medium. This is typically done with paraffin wax or resin.
• Place the tissue in a mold and fill it with molten embedding medium.
• Allow the medium to solidify; creating a block that contains the embedded tissue.
3. Sectioning:
• Mount the embedded tissue block onto the microtome.
• Adjust the microtome to the desired thickness for the sections. Typically, plant tissue sections are very thin, often in the range of 5-20 micrometers (µm).
• Use a razor blade or a microtome blade to cut thin sections from the embedded tissue block. The microtome allows precise control over section thickness.
4. Collection of Sections:
• Collect the thin sections onto glass slides.
• If the sections are not sticking to the slides, you can use a drop of water to help them adhere.
5. Staining (Optional):
• If necessary, stain the sections to enhance contrast and highlight specific structures. Common stains used in plant histology include hematoxylin and eosin (H&E) for general tissue structure and various special stains for specific cell components.
6. Mounting:
• Place a coverslip over the stained sections. You can use a mounting medium to secure the coverslip in place and prevent air bubbles.
7. Microscopic Examination:
• Examine the prepared sections under a microscope. This allows you to study the plant tissue's cellular structure, identify different cell types, and investigate any abnormalities or features of interest.
8. Documentation:
• Document your findings by taking photographs or making drawings of the microscopic images, noting any significant observations.

 

Applications for microtome and microscopy for plant studies

Microtomy and microscopy are essential techniques in plant studies, enabling scientists and researchers to investigate the internal structures of plant tissues and gain valuable insights into various aspects of plant biology. Here are some key applications of microtomy and microscopy in plant studies:

1. Plant Anatomy and Morphology:
• Microtomy and microscopy are used to study the anatomical features and morphology of plant tissues and organs. Researchers can examine the arrangement of cells, tissues, and specialized structures in roots, stems, leaves, flowers, and fruits.
2. Disease Diagnosis and Pathology:
• Microtomy allows the preparation of thin sections of plant tissues for microscopic examination. This is crucial for diagnosing diseases, infections, and pathogenic interactions within plant tissues, helping to identify pathogens and understand disease progression.
3. Root and Shoot Development:
• Researchers can study the development of roots and shoots, including the formation of root hairs, vascular tissues, and leaf structures. This is important for understanding plant growth and development.
4. Stomatal Studies:
• Microscopy is used to examine stomata, the tiny pores on plant leaves, and investigate their role in gas exchange and water regulation. Stomatal density, size, and distribution can provide insights into a plant's response to environmental conditions.
5. Xylem and Phloem Analysis:
• Microtomy and microscopy help in the analysis of xylem and phloem tissues, which are responsible for water and nutrient transport in plants. Researchers can study the structure of vessels, tracheids, and sieve elements in these tissues.
6. Cellular Ultrastructure:
• Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) allow researchers to explore the fine cellular ultrastructure of plant cells, including organelles like chloroplasts, mitochondria, and cell walls.
7. Plant Reproductive Biology:
• Microscopy is used to investigate various aspects of plant reproduction, such as pollen morphology, pollen tube growth, fertilization processes, and seed development.
8. Plant Stress Responses:
• Researchers can examine plant responses to abiotic and biotic stress factors, such as drought, salinity, and herbivory. Microscopy can reveal structural changes in stressed plant tissues.
9. Genetically Modified Organisms (GMOs):
• Microscopy is used to detect and confirm the presence of genetically modified traits in plant tissues, which is important for regulatory compliance and research purposes.
10. Plant Taxonomy and Identification:
• Microscopy aids in the identification and classification of plant species by examining unique cellular features, trichomes, and other characteristics.
11. Nutrient and Mineral Uptake:
• Microscopy is used to investigate the uptake and distribution of nutrients and minerals within plant tissues, helping to understand nutrient transport and deficiency symptoms.
12. Environmental and Ecological Studies:
• Researchers use microscopy to assess the impact of environmental factors, pollution, and climate change on plant tissues and ecosystems.