This laboratory session introduces participants to microscopy through the observation of human saliva samples. Students will prepare and examine two slides: one with unstained saliva and another treated with Lugol’s iodine solution. This comparison allows for the exploration of cell morphology, the composition of saliva, and the effects of staining on microscopic visualization.
The main objective is to identify different components present in saliva—such as epithelial cells, mucus, and microorganisms—and to understand how Lugol’s iodine enhances visibility of certain cellular features, particularly the nucleus.
Educational Goals
- Microscopy skills: Develop proper microscope operation skills, including focusing, adjusting magnification, and slide handling.
- Biological observation: Identify and differentiate structures found in saliva, such as epithelial cells, mucus, and bacterial colonies.
- Application of Lugol staining: Understand how Lugol’s iodine binds to certain biological molecules to improve contrast and visibility.
- Observation and documentation: Record and interpret visual differences between unstained and stained samples accurately.
- Analytical reasoning: Relate observed cellular structures to their biological functions and composition.
Significance and Lessons Learned
- Biological insights: Observing saliva under the microscope provides an understanding of cell diversity and the coexistence of human and microbial structures.
- Technical skills: Reinforces safe handling of slides, microscope use, and correct application of stains.
- Analytical development: Encourages detailed observation and critical thinking to connect visual information with biological meaning.
- Laboratory discipline: Stresses the importance of precision, cleanliness, and adherence to safety protocols.
Protocol
In front of you is an optical microscope with 4 objectives. In order to make use easier, certain features are disabled and are used in more advanced laboratories.
Please note that total magnification is calculated by multiplying the magnification of the objective (for example, 4x) with that of the eyepiece. The microscope used in this laboratory has a 10x magnification eyepiece. For example, an objective with a 4x magnification will result in a total magnification of 40x.
Preparation
1. Turn on the microscope by pressing the switch on the front of the device. You will turn on the illuminator, so that the transmitted light passes through the sample from below, which is ideal for transparent specimens.
2. Place two clean slides on your work area.
Preparation of the first slide
1. Place a drop of saliva on the first slide.
2. Cover the slide with a coverslip.
3. Carefully blot the excess saliva with absorbent paper.
Preparation of the second slide
1. Place a drop of saliva on the second slide.
2. Place a drop of Lugol on that same slide.
3. Cover the slide with a coverslip.
4. Carefully blot the excess Lugol with absorbent paper.
Observations
Observation of the first slide (saliva only)
1. Place the first slide on the microscope stage.
2. Click the « Microscope « button on the tablet to view the microscope image.
You can save an image of the observed view by clicking the « Save image « button, located in the lower left area of the « Microscope « section.
3. Adjust the magnification by touching the microscope objectives. Begin observation at 40x magnification (red objective – labeled Plan 4/0.10).
4. Refine the focus with the coarse adjustment knobs located on the left and right of the microscope.
5. Gradually increase the magnification from 40x to 100x (yellow objective – labeled Plan 10/0.25), then to 400x (blue objective – labeled Plan 40/0.65), adjusting the focus as required.
Note: A magnification of 1000x (white objective – labeled Plan 100/1.25) requires the use of oil between the objective and the coverslip. We therefore do not use this objective in this laboratory.
Observation of the second slide (saliva and Lugol)
1. Replace the first slide with the second slide containing the saliva and the Lugol.
2. Starting at 100X magnification (yellow objective – labeled Plan 10/0.25), note the presence of cell nuclei, which should appear colored brown by the Lugol.
* Do not forget to record the important observations !
Turn off the microscope
1. Turn off the microscope by pressing the switch on the front of the device.
Observation questions
1. What do you notice in the unstained saliva slide (without Lugol) ?
2. What is the purpose of Lugol in the experiment ?
3. Which part of the cell becomes more visible after adding Lugol, and why ?
4. Why must you always begin observation at low magnification (40x) before moving to 100x or 400x ?
5. Based on your observations, which structures in saliva belong to a living being (human cells) and which are non living elements (mucus, debris, etc.) ?
6. For each magnification, describe the differences in observation between a saliva sample and a human epithelium sample taken from the cheek.
Anticipated Outcomes
Results are found in this document
Participants will observe that saliva contains a mixture of epithelial cells, mucus, bacteria, and debris. In the unstained slide, the cells will appear transparent with faint outlines, while the Lugol-stained sample will exhibit enhanced contrast, revealing cell nuclei and granular structures more clearly. This experiment emphasizes the role of staining in microscopy and the complexity of biological fluids.
By conducting this experiment, students will strengthen their laboratory skills, understand the purpose of staining, and improve their ability to analyze and describe biological samples.
Observation questions
What do you notice in the non-stained saliva blade (without Lugol)?
In the unstained saliva slide, I can see transparent or barely visible cells. The epithelial cells are flat and irregular in shape, with faint outlines and hardly visible nuclei. There may also be mucus strands, small debris, or tiny dots that could be bacteria. The overall view looks clear and colorless.
What is the usefulness of Lugol in the experiment?
The Lugol-stained slide shows the cells more clearly. The cytoplasm and nuclei appear yellow or brown, making them easier to distinguish. The contrast is much stronger, so the cell boundaries and internal structures are more visible. The unstained slide looks much paler and less detailed.
Which part of the cell becomes more visible after the addition of Lugol, and why?
The nucleus becomes more visible because Lugol’s iodine reacts with certain molecules like glycogen and stains them yellow brown. This helps to highlight the nucleus and cytoplasmic details that were hard to see before. Lugol increases contrast so we can see the internal organization of the cells.
Why should we always start the observation at low magnification (40X) before moving to 100X or 400X?
Starting with low magnification helps to find the area of interest easily and prevents damage to the slide or coverslip. At 40X, the field of view is larger, so it’s easier to locate the sample. Once the image is centered and focused, it’s safe to move to higher magnifications for more detail.
Based on your observations, which structures of saliva belong to a living being (human cells) and which are non-living elements (mucus, debris, etc.)?
The large, flat epithelial cells with visible nuclei are from living human tissue. The mucus threads, food particles, and small debris are non-living components. Bacteria may also be present — they are living but not human. So, the sample contains both living (cells, bacteria) and non-living (mucus, debris) material.
For each magnification, describe the observational differences between a saliva sample and a sample of human epithelium taken from the cheek.
Saliva sample at 40x
- At this low magnification, the field appears uneven, with several irregular patches and filamentous or amorphous material scattered across the slide.
- This is mucus, composed of glycoproteins secreted by salivary glands. It’s slightly stained by Lugol, giving it a pale yellow-brown hue.
Differences from cheek epithelium
- The cheek smear showed large, flat, continuous sheets of polygonal cells.
- The saliva sample looks more disorganized — no uniform cellular layer, and more gel-like streaks.
- You may see a few epithelial cells floating among mucus, but they’re isolated, not in clusters.
Saliva sample at 100x
- At 100x, several small, faintly stained cells become visible among mucus strands.
- Some are flattened epithelial cells similar to the cheek cells, but others are small round cells — likely leukocytes (white blood cells) or bacteria clumps.
What stands out
- The Lugol stain gives the mucus a yellow-brown tint, but smaller cells appear paler.
- The cheek cells at this magnification were large and polygonal; in contrast, the saliva field is heterogeneous, with particles of different sizes and densities.
Interpretation
- This shows that saliva is not purely epithelial — it’s a complex suspension containing mucus, immune cells, and microorganisms.
Saliva sample at 400x
At 400x, details become clearer
- You can likely see clusters of bacteria as tiny dots or short rods, often adhering to mucus filaments or cell debris.
- The epithelial cells present are often damaged or folded, not as intact and transparent as in your cheek sample.
- Some granular inclusions may appear within mucus — possibly food debris or denatured proteins reacting with iodine.
Comparison to cheek 400x
- Cheek: clean background, large defined cells with visible nucleus.
- Saliva: crowded background, smaller irregular structures, microorganisms present.
If you observe movement in unstained saliva at this magnification, you might even see motile bacteria or protozoans (though Lugol fixation usually halts motion).
Summary of Assignment by Grade Range
Grades 3–5 (Ages 8–10)
- At this introductory level, students are introduced to the basic concept of microscopy and the idea that living organisms are made up of very small units called cells. They will explore saliva as a sample to understand that even simple body fluids contain living elements.
- Students will practice how to handle a microscope safely, observe at low magnification, and describe what they see in simple terms. The emphasis is on curiosity, observation, and the difference between what is visible with and without staining.
- Teachers will guide them in identifying large particles, bubbles, or simple shapes, encouraging careful observation and description.
Grades 6–8 (Ages 11–13)
- Students at this level will refine their ability to prepare slides independently and safely apply Lugol’s iodine. They will begin recognizing distinct cellular structures, such as the cell membrane, nucleus, and cytoplasm, while also noting non-cellular elements like mucus strands or bacteria.
- The goal is to deepen understanding of the relationship between cell structure and function while strengthening microscopy technique. Learners will be expected to create labeled sketches, compare the clarity of stained versus unstained samples, and record observations systematically.
- Discussions may include why iodine binds to certain cellular substances and how staining enhances biological study.
Grades 9–12 (Ages 14–18)
- At this advanced stage, students perform a detailed comparative analysis of stained and unstained saliva samples, and optionally compare them with cheek epithelial cells. They will describe differences in cell morphology, cell wall visibility, and the appearance of nuclei.
- Students will explore how Lugol’s iodine interacts chemically with glycogen or other cytoplasmic materials to reveal cell structures more clearly. This stage emphasizes critical analysis, accurate measurement of cell size under various magnifications, and the production of full scientific reports with observations, drawings, and conclusions.
- The experiment also provides an opportunity to discuss how microscopy contributes to medical and biological sciences by revealing the microscopic organization of human tissues.
Laboratory essentials
Instruments
- Beaker (50 ml)
- Droppers
- Microscope
- Microscope blades
- Microscope sliders
- Paper towel
- Tweezers
Products
- Human saliva in suspension
- Lugol 2% solution