Microscope Lab Notes: Setup, Lenses, Diffusion Experiments, and Safety

Microscope Setup and Quick Start

Ensure the microscope is plugged in and turned on; confirm there is a light source radiating from the bottom. If there is no light, report it.
Clean the ocular lens (where your eyes go) with lens cleaning paper and lens cleaning solution before use; never use paper towels.
Understand the basic structure: head, arm, base. The base includes the coarse adjustment knob (the large, big round knob) and the fine adjustment (the smaller knob that sticks out).
The stage sits above the objective lenses and holds the slide with a metal clip that can be opened to place the slide and then released to hold it in place.
When sharing the microscope, clean the ocular area and ensure proper alignment before use.
Start with a good starting position to save time and to ensure you can return the microscope to the instructor after use.

Starting Position, Loading a Slide, and Light Alignment

Place the microscope on a stable surface and lower the stage to its lowest point using the coarse adjustment knob; stop once it auto-clicks and cannot go lower.
Use the fine adjustment knob to fine-tune focus once you have something in view. If your view is blurry, switch to the fine adjustment to sharpen the image.
Rotate the nosepiece to the scanning lens (shortest objective, 4x) to begin broad scanning. The objective lenses increase magnification the longer they are; the shorter lenses require more distance from the slide.
Load the slide with the label upright (top portion visible). Open the metal clip on the stage, place the slide on the stage, push it to the back of the clip, and release so the clip holds it in place.
Adjust the light so the slide is illuminated in the field of view:
- Use the condenser under the stage to regulate light entering the lens (fully open for more light or adjust as needed).
- Use the side dimmer to increase or decrease the overall brightness.
Ensure the slide is within the halo of light and that the light path is aligned with the object you want to observe (e.g., a flea or a letter “e”).

Lenses, Magnification, and Focusing Workflow

Ocular lenses: binocular setup; distance between the two lenses should match your eyes. Each eye views through its own lens. If you see a black line or a flicker, it’s just a visual cue (the pointer or eyelashes, not an issue).
Interpupillary adjustment and careful alignment are important for comfortable viewing and to avoid smashing glasses into the lenses.
Parfocal: once you focus with one objective, you should be able to switch to another objective with minimal adjustment; you should not have to move the stage when changing from one lens to another (to avoid crashing the slide into the lens).
The two side levers near the light adjust the light path to ensure the object is well illuminated in the field of view. The condenser controls the amount of light that goes through the slide; brightness can be adjusted with the side knob as needed.
Magnification progression: start with the scanning lens (shortest, 4x) to locate the specimen, then move to higher magnifications as needed. The general rule is to begin broad and zoom in progressively.
The order of objective lenses commonly used:
- Scanning lens: 4\times
- Low-power lens: 10\times
- High-dry lens: 40\times
- Oil immersion lens: 100\times
On the nosepiece, always start with the shortest objective first; higher magnifications require the slide to be closer to the lens and reduce the working distance.

Field of View, Working Distance, and Total Magnification

Field of view: the circular area visible through the oculars; when you center the feature (e.g., head or tail of the flea) it should be in the middle of this circle.
Working distance: the space between the slide and the objective lens; as magnification increases, this distance decreases. Do not move the stage when using higher magnifications unless you are on the 4x scanning lens.
Total magnification (TM) is the product of the ocular magnification and the objective magnification:
- Formula: TM = 10\times M_{\text{objective}}
- Example: with the 4x scanning lens, TM = 10\times 4 = 40, so the viewer sees the sample at 40x total magnification.
- In general, the ocular lens magnification is 10x, and you multiply by the objective lens magnification (4x, 10x, 40x, 100x).
Practical note: when moving from 4x to 40x, use the coarse adjustment to bring the stage up, then fine-tune with the fine adjustment to sharpen the image.
The gear is parfocal, so you should largely avoid moving the stage when changing lenses; if the image is lost, return to the 4x scanning lens to reacquire the image and then switch lenses again.

Practical Lab: Slide Observation and Focus (Flea/Letter “e” Demonstration)

You should be able to focus on a prepared slide and locate a specific feature (e.g., flea, head vs. tail, or letter “e”) using the following approach:
- Start with the 4x scanning lens and locate the specimen.
- Bring the feature roughly into the center of the field of view.
- Switch to the next higher lens (e.g., 10x) by using the nosepiece; avoid moving the stage too much; if the image goes out of focus, switch back to the 4x lens to reacquire, then try again.
- Use the coarse adjustment and then the fine adjustment to sharpen focus at each lens.
If the image becomes blurry or disappears, reassess by returning to the 4x scanning lens and re-focusing before attempting higher magnifications again.

Exercise One: Diffusion in Agar (Diffusion Plates)

Concept: Agar plate acts as a semi-permeable membrane with mostly water; solutes can move through depending on size and properties.
Dyes used: Permanganate (lighter, faster diffusion) and Methylene Blue (darker, slower diffusion).
Key factors affecting diffusion rate:
- Temperature: colder temperatures slow diffusion; warmer temperatures speed it up.
- Polarity, permeability, molecular weight – larger or less permeable molecules diffuse more slowly.
Observation: Over time, dyes diffuse from regions of higher concentration to lower concentration until equilibrium is reached.
Practical takeaway: This demonstrates diffusion as a model for how substances move through membranes.

Exercise Two: Dialysis Bag Diffusion Experiment

Setup: A dialysis bag (semi-permeable) contains small molecules: NaCl (sodium chloride), glucose, and starch (polymer). The bag is placed in a beaker with water and iodine outside.
Objective: Determine which solutes diffuse out of the bag and which outside solutes diffuse in.
Observations and indicators:
- Iodine outside the bag diffuses in and reacts with starch inside the bag to create a characteristic blue color; iodine outside and inside bag indicates diffusion is occurring. If the bag turns blue, iodine diffused into the bag; if the bag remains amber, starch did not diffuse (starch is too large).
- Glucose diffusion is tracked with Benedict's reagent in test tubes after diffusion and heating; a color change (toward green, yellow, orange, or red, depending on glucose amount) indicates glucose moved out of the bag into the outside solution.
- Sodium chloride diffusion is tracked with silver nitrate reagent (AgNO3) in another test tube; formation of a white precipitate (AgCl) indicates chloride ions diffused into the outside solution, i.e., NaCl diffused out of the bag.
Key reagents and indicators:
- Iodine: detects starch via a blue-black color when starch is present; outside iodine turns the solution amber if starch remains inside the bag.
- Benedict's solution: a blue reagent that, when heated with reducing sugars (like glucose), changes color indicating glucose presence; high amount leads to brick-red; color change indicates diffusion of glucose into the outside solution.
- Silver nitrate: a clear liquid that produces a white precipitate (AgCl) in the presence of chloride ions; used to detect diffusion of NaCl.
Procedure highlights:
- Pour approximately one inch of the outside beaker liquid into tall test tubes for testing.
- Add Benedict's solution to one test tube in a 1:1 ratio with the sample and place in a boiling water bath to observe color changes.
- In a separate test tube, add silver nitrate to detect chloride diffusion (white precipitate indicates diffusion).
- Observe the color changes over time to determine which solutes diffused out of the bag.
Notes on experimental issues:
- Some dialysis bags may leak; this does not invalidate the diffusion concept but indicates that diffusion seen may include leakage of starch, which can complicate interpretation.
- Positive results are defined by detectable color changes or precipitates indicating diffusion of the respective solutes.

Benedict’s Test Protocol and Interpretation (Glucose Diffusion)

Setup: For each sample, mix an equal volume of the outside liquid with Benedict’s reagent in a test tube (1:1 ratio).
Heat: Immerse the test tube in a boiling water bath to develop color changes; do not leave too long as Benedict’s reagent can darken with prolonged heating.
Interpretation: A color change (green to yellow to orange to red) indicates reducing sugars (like glucose) diffused into the outside solution.
Practical note: Different intensities of color change reflect the relative amount of glucose that diffused; any positive color change confirms diffusion occurred.

Silver Nitrate Test for Chloride Diffusion (NaCl)

Procedure: Add 2–3 drops of silver nitrate solution to the test tube containing the outside liquid that was in contact with the dialysis bag.
Observation: The appearance of a white precipitate (AgCl) indicates the presence of chloride ions that diffused from inside the bag to outside.
Conclusion: A positive AgNO3 test indicates diffusion of NaCl out of the dialysis bag.

Safety, Etiquette, and Cleanup in the Lab

No food or drinks in open containers in the lab; close water bottles; place them in drawers or cabinets when not in use.
If you need to leave the lab, you may step out and return, but ensure any liquids or containers are closed and stored.
Bathroom use: follow building directions; return to the lab and store materials properly.
After experiments: turn off hot plates and unplug them; return microscopes to their numbered cabinet spots; lower the stage to the 4x lens; remove slides; clean the workspace; sign out or sign back in as required.
Waste disposal: rinse and dispose of glassware and solutions in appropriate sinks; bag and dispose of dialysis bags as instructed; wipe down tables with Lysol and paper towels; wash hands after cleanup.
Sign-in and sign-out procedures: ensure you complete the appropriate paperwork or sign-in sheet before leaving.

Homework, Quiz Preparation, and Resources

Exercise two practical review list highlights topics that will appear on quizzes: parts of the microscope, and the parts to know; how to calculate total magnification; field of view; and working distance.
Quiz questions pull from a large pool; you may encounter questions where you identify the lens type from a diagram or image, or compute magnification and interpret field of view.
Use the resource list: PowerPoint slides and the lab manual for definitions and diagrams; use the Practice APR (assessment practice) before the quiz for thorough preparation.
Exercise four focuses on membrane transport concepts: diffusion, osmosis, and tonicity (hypertonic, hypotonic, isotonic).
Tonicity overview:
- Hypertonic: cell shrivels due to water loss.
- Hypotonic: cell may burst due to water gain.
- Isotonic: balanced water movement; cell maintains normal size.
Note: If tonicity is not covered yet in your course, read the manual’s introduction and work through the virtual tonicity/osmosis lab for additional context.

Upcoming Topics and Practice Opportunities

Next week: histology; about 15 slides will be used for practice; expect repeated use of the microscope and more slides to gain hands-on experience.
The practical exercises (1, 2, and 4) are designed to reinforce the connection between theory and hands-on technique.
While you won’t need to be an expert today, focus on turning on the microscope, loading a slide, focusing from 4x to higher magnifications, and understanding the basic terms and concepts (parfocality, field of view, working distance, total magnification).

Quick Reference: Common Terminology and Key Concepts

Ocular lens (eye piece): magnifies by M_{ ext{ocular}} = 10\times (pre-set to 10x on most models).
Objective lenses (on the nosepiece): commonly 4\times\ ( ext{scanning}), 10\times\ ( ext{low}), 40\times\ ( ext{high dry}), 100\times\ ( ext{oil immersion}).
Field of view: the circular area visible through the oculars.
Working distance: space between the slide and the objective lens; decreases with higher magnification.
Coarse adjustment knob: moves the stage up and down to bring the specimen into general focus.
Fine adjustment knob: finely tunes focus for a sharper image.
Parfocal: once focused on one objective, switching to another requires minimal adjustment.
Condenser: adjusts lighting to control light intensity through the slide.
Light dimmer: side control to adjust brightness.
Dry and immersion objectives: high power requires careful handling of the slide; oil immersion is used for very high magnification.
Important safety: avoid touching ocular lenses with dirty hands; clean lenses properly; keep workspace clean and organized.

Summary and Exam Readiness

You should be able to describe the major parts of the microscope, explain the purpose of coarse and fine adjustments, and outline the correct sequence for focusing from 4x to higher magnifications.
You should be able to calculate total magnification and explain how working distance changes with magnification.
You should be able to describe the diffusion experiments (agar, dialysis bag) and interpret results from Benedict’s test, iodine starch test, and silver nitrate test.
You should be familiar with lab safety, cleanup procedures, and the use of reagents and indicators used in diffusion experiments.