Magnification calculations

Microscope lenses and a drawing have related but distinct magnifications. Magnification is a ratio, so it has no length unit.

Total microscope magnification

The objective forms a magnified image, which the eyepiece magnifies again.

A $\times10$ eyepiece and $\times40$ objective give $10\times40=400$, written $\times400$. Do not add the lens powers.

Image or drawing magnification

Use the same length unit before dividing. A $48\,\mathrm{mm}$ drawing of an $80\,\mathrm{\mu m}$ cell has image size $48\,000\,\mathrm{\mu m}$. Therefore $M=48\,000/80=600$, or $\times600$. Check: $80\,\mathrm{\mu m}\times600=48\,000\,\mathrm{\mu m}=48\,\mathrm{mm}$.

To find actual size, divide image size by magnification. A cell measuring $30\,\mathrm{mm}$ on a $\times250$ micrograph is actually $30/250=0.12\,\mathrm{mm}=120\,\mathrm{\mu m}$ long. The actual object is smaller than its magnified image.

To find image size, multiply. A $25\,\mathrm{\mu m}$ structure at $\times800$ has image length $800\times25=20\,000\,\mathrm{\mu m}=20\,\mathrm{mm}$. Checking $20/0.025=800$ recovers the magnification.

An image's printed magnification only applies at its stated reproduction size. Resizing it changes image magnification. A scale bar resized with the image is therefore a more dependable size reference.

Core practical: preparing cell slides

The purpose is to prepare and observe a thin plant-cell specimen and a thin animal-cell
specimen, identify only the structures that are visible, estimate cell size, and make
labelled scientific drawings.

Useful apparatus includes a compound light microscope and lamp, clean slides and coverslips,
forceps, pipettes, a mounted needle or clean toothpick, paper towel, a transparent ruler,
onion bulb epidermis, iodine solution, a sterile cheek swab or spatula, methylene blue and
disinfectant.

This is primarily an observational comparison, not an investigation of how one variable
causes another to change. There is no manipulated independent variable when each slide is
simply observed. If the observations are compared, specimen type is the comparison variable;
the recorded outcomes are visible structures, cell arrangement and estimated size. For a
fair comparison of apparent detail, keep the microscope, total magnification, illumination,
field-selection rule and approximate specimen thickness as consistent as possible. The two
stains are deliberately different because they suit different specimens; stain identity is
not a controlled variable.

[DIAGRAM: asset_name: Biology 1.6 - Light microscope; asset_slug: biology_1_3_1_6_microscopes_scale_and_cell_observation__diagram_01; description: Labelled compound light microscope: eyepiece, rotating nosepiece, objectives, stage and clips, glass slide with coverslip, light source, coarse and fine focus controls.]

Preparing onion epidermis

1. Put on eye protection. Add one small drop of dilute iodine solution to the centre of a
   clean slide.
2. Use forceps to peel a very small piece of the transparent epidermis from the inside of an
   onion-bulb layer. A thin, single layer lets light pass through and prevents cells hiding
   one another.
3. Lay the epidermis flat in the iodine drop. The stain increases contrast, making some
   structures easier to distinguish.
4. Touch one edge of a coverslip to the drop. Support it at about 45 degrees with a mounted
   needle, then lower it slowly. Air is pushed ahead of the descending edge instead of being
   trapped as bubbles.
5. Touch paper towel to liquid outside the coverslip to remove excess without pressing on the
   glass.

Preparing cheek cells

1. Place a small drop of water on a second clean slide.
2. Gently stroke the inside of your own cheek with one sterile swab or spatula to collect
   loose cells. Do not scratch the tissue and never share the sampler.
3. Stir the sampled end in the water drop, then place it immediately in disinfectant or the
   teacher's designated biological-waste container.
4. Wearing eye protection and gloves, add one small drop of methylene blue. This increases
   contrast; cheek-cell nuclei usually stain dark blue.
5. Lower a coverslip from about 45 degrees and blot only excess liquid, as for the onion
   slide.

Hazards, routes and controls

Focusing, observing and recording

Using the microscope

1. Rotate the lowest-power objective into place and switch on or position the lamp. Low
   power gives the widest field, so the specimen is easier to locate.
2. Secure the slide on the stage and centre the stained specimen over the light opening.
3. Looking from the side, use coarse focus to bring the objective close to the coverslip
   without touching it. Then look through the eyepiece and move the lens and slide slowly
   apart until the image appears. Sharpen it with fine focus.
4. Adjust illumination so boundaries are distinct rather than washed out. Centre a useful
   region before changing objective.
5. Record the eyepiece power, objective power and calculated total magnification. Measure the
   low-power field diameter with a transparent ruler, or use a field diameter supplied for
   that microscope.
6. Move to the next objective for more detail. At high power use fine focus only, because the
   working distance between objective and coverslip is small.
7. Observe at least three non-overlapping fields and several cells in each. Record folded
   tissue, bubbles or damaged cells rather than silently treating them as typical.

Increasing objective power narrows the field of view and usually reduces brightness. It may
make already resolved detail easier to inspect, but a larger image is not evidence of higher
resolution by itself.

What may be observed

An observation is what the image directly shows, such as a dark oval region. Naming that
region as a nucleus is an interpretation, supported by its staining and position. If a
structure is not visible, it may be too small, have too little contrast, lie outside the focal
plane or be absent from that specimen. "Not seen" does not automatically mean "not present."

Scientific drawings from observations

A scientific drawing records evidence rather than decorating it.

• Draw two or three representative adjacent cells large enough to show their proportions.
• Use a sharp pencil and single, clear, continuous lines. Do not sketch repeatedly, colour or
  shade.
• Draw only structures actually observed; do not turn the view into a memorised ideal cell.
• Give the specimen a title. Use ruled, uncrossed label lines without arrowheads; each line
  must touch the feature it identifies.
• State the total microscope magnification and, where available, an estimated size or scale.
  Do not claim the drawing's page size is the microscope's total magnification.

A useful results table is:

Make estimates in several fields because cells vary and edge cells may be only partly
visible. Report the spread of estimates or a representative mean, and keep the raw field
counts so another person can judge the result.

Putting a size on the observation

Suppose the field diameter is $600\,\mathrm{\mu m}$ and about ten cells span it. Estimate cell width as $600/10=60\,\mathrm{\mu m}$. Repeating this in different fields reveals natural variation and reduces dependence on one unusual field. Average the separate width estimates; keep the raw counts and field diameter.

Evaluating microscope evidence

Evaluation should connect a specific limitation to its effect and then to a change that
addresses that effect.

Repeating fields improves repeatability by showing whether similar observations can be
made again. Keeping magnification and illumination consistent improves the validity of a
comparison. A known field diameter and clearly located boundaries improve the likely
accuracy of a size estimate. These improvements solve different problems, so "repeat it"
is not a complete response to every limitation.