Introduction to Microscopy, Squash Preparations, and Dissection
Welcome to one of the most hands-on parts of your Biology B course! In this chapter, we step away from the textbooks and look directly at life itself. Whether you are measuring the tiny structures inside a cell or exploring the breathing system of an insect, these "Core Practicals" are the foundation of biological observation. These skills are not just for the lab; they are frequently tested in your exams, especially in Paper 3, where you'll need to explain how and why we use these techniques.
Don't worry if the math or the delicate nature of the work feels intimidating at first. We will break every process down into simple, logical steps.
Core Practical 2: Use of the Light Microscope
The light microscope is your window into the microscopic world. To use it effectively for your Edexcel A Level, you need to master two things: calibration (measuring things) and scientific drawing.
Magnification vs. Resolution
It is important to know the difference between these two terms:
1. Magnification: How many times larger the image is compared to the real object.
2. Resolution: The ability to distinguish between two separate points. Higher resolution means more detail and a clearer image.
Calculating Magnification
You can calculate magnification using this simple formula triangle:
\( \text{Magnification} = \frac{\text{Image size}}{\text{Actual size}} \)
Top Tip: Always make sure the units for "Image size" and "Actual size" are the same before you divide! Usually, we convert everything to micrometres \( (\mu m) \).
\( 1\ mm = 1000\ \mu m \)
Calibration: Using Micrometers
A microscope doesn't have a built-in ruler, so we have to "calibrate" it using two tools:
1. Eyepiece Graticule: A glass disc with a scale (0–100) fitted into the eyepiece. It has no units because its "size" changes depending on the magnification.
2. Stage Micrometer: A slide with a very accurate scale engraved on it (usually \( 1\ mm \) long with divisions of \( 0.01\ mm \)).
How to calibrate:
1. Line up the eyepiece graticule scale with the stage micrometer scale.
2. Count how many graticule divisions fit into a known distance on the stage micrometer.
3. Calculate the value of one graticule unit. For example:
If \( 10 \) divisions on the stage micrometer (\( 0.1\ mm \)) equal \( 40 \) units on the graticule:
\( 1\ \text{graticule unit} = \frac{0.1\ mm}{40} = 0.0025\ mm\ \text{(or } 2.5\ \mu m) \).
Biological Drawings
When drawing cells from a specialised tissue, examiners look for specific standards:
• Use a sharp HB pencil.
• Use clear, continuous lines (no "sketching" or "shaggy" lines).
• No shading or coloring.
• Label lines must be straight, drawn with a ruler, and must not have arrowheads.
• Include a scale bar and a title.
Key Takeaway: Calibration is like setting a scale on a map. Once you know what one "graticule unit" is worth at a specific magnification, you can measure any cell on your slide.
Core Practical 3: Root Tip Squash Preparation
This practical allows you to see mitosis in action. We use the tips of plant roots (like onions or garlic) because this is where the meristem is located—the area of active cell division.
The Procedure
1. Cut: Take a small sample (\( 5\ mm \)) of the root tip.
2. Fix and Soften: Place the tip in hydrochloric acid (HCl). This breaks down the calcium pectate in the middle lamellae (the "glue" holding plant cells together), allowing the cells to be separated.
3. Stain: Add a stain like acetic orcein or toluidine blue. This binds to the DNA, making the chromosomes visible under the microscope.
4. Squash: Place a coverslip over the sample and press down firmly with your thumb.
Warning: Do not smear the coverslip sideways; press straight down. You want a layer of tissue that is only one cell thick so light can pass through.
The Mitotic Index
You may be asked to calculate how quickly the tissue is growing by finding the Mitotic Index:
\( \text{Mitotic Index} = \frac{\text{Number of cells containing visible chromosomes}}{\text{Total number of cells in the field of view}} \times 100 \)
Did you know? We use the very tip of the root because, further up the plant, cells are just getting longer (elongating) rather than dividing.
Core Practical 7: Dissection of an Insect
To understand gas exchange, we dissect an insect (like a locust) to view its tracheal system. This requires careful work and respect for the organism.
Ethics and Safety
• Ethical use: Ensure the organism is handled humanely according to school and exam board guidelines.
• Safety: Use sharp tools (scalpels/scissors) with care. Always cut away from your body.
The Dissection Process
1. Fixing: Fix the insect to a dissection plate using pins through the legs/thorax.
2. Opening the exoskeleton: Carefully cut along the side of the abdomen and remove a strip of the exoskeleton.
3. Observation: Look for the tracheae. These appear as tiny, silvery-white tubes. They look silvery because they are filled with air.
4. Microscopic view: You can remove a small piece of tracheal tissue and view it under a microscope to see the chitin rings (spirals) that keep the tubes open.
Key Structures to Identify
• Spiracles: Small holes on the outside of the body where air enters.
• Tracheae: The main large tubes.
• Tracheoles: The tiny, branched tubes that deliver oxygen directly to the cells.
Key Takeaway: The "silvery" appearance of the tracheae is the best way to distinguish them from other internal tissues like muscle or nerves.
Common Mistakes to Avoid
• Units: Forgetting to convert \( mm \) to \( \mu m \) in magnification calculations. (Always check this first!)
• The Squash: Pushing the coverslip sideways in the root tip practical, which breaks the chromosomes and makes them impossible to see.
• Microscopy: Starting with the high-power objective lens. (Always start with the lowest power to find your sample.)
• Dissection: Cutting too deeply and damaging the internal organs you are trying to see.
Quick Review
• Core Practical 2: Focuses on using the stage micrometer to calibrate the eyepiece graticule and making accurate biological drawings.
• Core Practical 3: Uses acid to soften tissue and stain to see chromosomes during mitosis. We calculate a mitotic index.
• Core Practical 7: Involves identifying the tracheal system of an insect, looking for spiracles and silvery tracheae.
Note: For more on the theory of cell structures or the details of mitosis, see the "Cells, Viruses and Reproduction of Living Things" chapter. For details on gas exchange theory, see "Exchange and Transport".