Link to the article: Microb hunting with your Microscope (Popular Science, Sept 1934)

• Observing dust samples: Students should collect house-dust and bring it to class to be observed under the stereo or compound microscope. Careful, some people may be allergic to dust!
• Observing sand and soil samples: Students should collect sand and soil samples to be observed under the stereo microscope.
• Observing textile fibers: Observing various fibers obtained from clothing (cotton, polyester, nylon etc.). Different colors and textures become visible under the microscope.
• Which printer is the best? Students bring in print-outs of different pictures on different types of paper. The printing resolution can be observed under the stereo microscope.
• Observing water life: A large jar is filled with pond water and a little soil. Algae and other organisms will (hopefully) develop over the course of a few weeks. Do not let the water rot!
• Fungi from cheese: Camembert, Brie, etc. contain edible molds (not hazardous) and can be used. Much safer than rotting food and observing the molds.
• Vegetables and fruits: The teacher cuts the tomatoes and mushrooms in various ways, they can be observed under the stereo microscope. Do not eat the food afterward, you never know what chemicals were left behind on the microscope by previous classes…..
• Hair samples: Each student donates one hair and then they have to match them with the hair left behind on the “crime site”. This is a playful approach into forensics and gives the observation some purpose. Maybe a competition between different groups is also a nice idea. The teacher may have to prepare a set of permanent slides with some hair samples.
• Coins: Coins collect many scratches (and dirt) over the years. How can the scratches be quantified? Is it possible to predict the age of a coin by looking at the number of scratches? The year is imprinted in the coin.
• Observing human cheek cells: This is a classic, really. Using a cotton swab, some epithelium cells from the inside of the mouth are collected and transferred to a microscopic slide.

Things NOT to observe – Some specimens or samples should not be observed in a classroom setting:

• Spoiled food material: they contain hazardous bacteria and fungi. Spores are unhealthy to breath in.
• Body parts: Samples taken from wounds (pus etc).
• Blood samples or other body fluids.
• Urine: Some students (often boys…) may be interested in observing their own urine. Fresh urine should be free of microorganisms (unless there is an infection) and it is not an interesting sample to be observed.
• Animal wastes: Excrements of animals are prone to contain parasites and are a clear health hazard.
• Polluted water Water from polluted rivers, lakes may contain toxic substances and harmful microorganisms. Leave stuff like this to university-level students, who (should) know appropriate safety procedures.

Materials: A large glass jar, fresh and unfertilized garden soil, water, hot plate, celophane foil

Method:

• Fill the glass jar with a few centimeters of the garden soil.
• Add non-chlorinated tap water to the soil and fill the jar with the water (3/4 full).
• Boil the soil-water mixture for about 30 min. This will kill off bacteria in the soil and will extract nutrients from the soil. Bacterial spores may survive the boiling, as they are heat-resistant. This is not a problem, though. These bacteria will serve as a food for other microorganisms later on.
• Cool the water to room temperature and let the soil settle to the bottom of the glass jar. Do not filter the soil away. The soil will continue to supply nutrients and will act as a buffer.
• Add a small amount of pond water which contains algae. Do not add too many algae. You may want to scrape off some algae from rocks or take a few algal filaments floating in a pond.
• Cover the jar with celophane foil. This will allow for gas exchange and prevent dirt and dust falling into the water. It also reduces evaporation.
• Wait a few weeks for the algae and ciliates to develop. With a bit of luck, paramecia will grow and form white clouds in the water. The color of the water may also change, an indicator for algal growth.
• Store the jar in a bright place but not in direct sunlight.
• Using a pipette, extract some of the microorganisms to be observed under the microscope.

Troubleshooting:

• Microorganisms do not form: This is probably due to the fact that there were none or not enough in the pond water which was added.
• The water starts to smell bad: This may be due to the system becoming anaerobic. Make sure that enough oxygen is able to enter the water. Paramecia and other ciliates are probably dead by now…..

The heat-fixed specimen can be stained using regular (non-toxic) ink. The ink is then carefully rinsed off with water.

Materials: a small amount of yogurt, water, hot plate, ink from a fountain pen or a marker, alcohol

Method 1:

1. Suspend a small amount of yogurt (tip of a knife) in a few ml of water.
2. Spread a drop of this suspension on a slide and let it dry completely at room temperature. Be patent here, do not accelerate the drying process by heating the slide.
3. Briefly place the dry (!) slide on the hot plate with the bacteria facing the top. If you “boil” the bacteria, then they may pop open and lose their shape, and will not accept the stain. So make sure that all the water is gone before heat-fixing.
4. Remove the slide. You should be just able to place the slide on your palm without burning yourself. If it hurts (or if you burn yourself) then the slide was heated too much and you have to retry and place a new suspension on the slide to dry. In this case the bacteria were burned and may have lost their shape. The heat treatment fixes the bacteria to the slide, so that they will not be washed off. In microbiological labs, the heat fixing process is usually conducted with a gas burner, but this may be too dangerous for schools.
5. Place a drop of ink on the specimen and wait for about 10 minutes. Carefully rinse the ink off by slowly pouring water or alcohol (depending on ink) over the slide. Continue this washing step until no more ink is given off, but do not over-wash. Also do not pour the washing liquid directly over the bacteria, but rather let it flow over it.
6. Let the slide dry.
7. If the bacteria are observed with oil immersion, then it is not necessary to place a cover glass on top of the sample. Instead place a drop of oil directly on the stained bacteria. This is only for experienced students, there is the danger that the wrong objectives are rotated into the oil….
8. The safest method would be to use water and a cover glass and to start observation with the low magnification objectives (In this case, of course, it is not necessary to let the slide dry after the washing).

Method 2: this method is easier and does not need a heat-fixing step.

1. Take a knife tip of yogurt and directly add 1-2 drops of water-based blue fountain pen ink. Do not use calligraphy ink. This type of ink is composed of suspended ink particles which can not be taken up by the bacteria.
2. There is no need for a washing step. The bacteria will accumulate the ink and will become darker than the surrounding medium.
3. Take a small drop and place on the slide for microscopic investigation. The drop has to be sufficiently small to form a very thin film between the slide and cover glass.
4. You should be able to see blue clusters of bacteria. Individual bacteria are probably too small to show a blue stain, but the diffraction pattern should make them visible. Some clusters may not have taken up the ink. The ability to take up the ink may be an indicator if the bacteria are still alive.

About the ink:

• Different types of inks contain different substances that may be more or less suitable for staining. I recommend you to experiment. The teacher could also try to dissolve some black or blue marker ink in some alcohol and then use this solution for staining. Inks used for calligraphy will most certainly not work. They contain suspended particles (carbon?) which are not able to enter the cells. Also be careful when using commercial stains. Some of them are designed to stain DNA and this is then not suitable for the use in schools (carcinogenic) – read the instructions that accompany the stain.
• If you use ink which is soluble in alcohol, then you may need to include a (brief) washing step with alcohol to remove excess ink. Experiment first.

Troubleshooting:

Problem: The unstained bacteria are not visible.
Solution: They are transparent, close the condenser aperture diaphragm all the way. You will then see diffraction patterns around the bacteria.

Problem: You see a blue mass but not individual cells.
Solution 1: The suspension was to dense. Dilute the suspension with more water, or if you directly observe the yogurt, make the drop smaller.
Solution 2: Remove more ink by rinsing longer.

Make a cut beneath the red layer and firmly press the red part of the onion against the edge of the knife, without cutting yourself...

Carefully tear off the layer of red cells. Remove the thick part of the onion (where the cut was made) and only observe the thin layer. Many cells will probably break open during this process and be useless, we only need a few intact cells.

The top image shows the cells before plasmolysis. The cells are filled with a red pigment and appear pink. The bottom image shows the same cells after the addition of saturated salt water. Intact cells will lose much of the water due to osmosis. The concentration of the pigment rises resulting in a darker color. The shape of the cell wall remains unaffected.

Materials: kitchen knife, red onions, salt, tap water, microscopic slides, cover slips

Method – Obtaining a single layer of red onion cells.
For this experiment, we can not use the onion skin which is found between the layers of the onion. We need a single layer of pigmented cells. These cells, however, do not separate easily.

1. We need a thin layer of cells of the red part of the onion. It is not possible to directly cut a single cell layer, so we need to use the “peeling method” to obtain a single layer of cells. Obtain a small piece of onion about (1cm x 1cm). The onion layer is about 2mm thick.
2. With the red side of the onion facing you, cut beneath the red layer, about half way into the onion. This cut does not have to be very thin. There will be about 1mm of onion between the knife and the red pigmented layer.
3. Press the onion firmly against the knife with your thumb.
4. Now tear off or peel away the red part of the onion. The red layer will become thin. Some red pigment may be released from broken cells.
5. Cut away and discard the thick part of the onion (the place where the initial cut was placed).
6. Observe the remaining cells (the thin, peeled part) under the microscope (using a glass slide, water and cover slip, of course.
7. Only consider those cells that are filled with the red pigment. White cells are broken and have lost the red pigment.

Method – Plasmolysis.

1. Make a saturated solution of salt-water
2. Using a pipette, add one drop of this solution to the specimen. The salt water should flow beneath the cover slip. There should be no need to remove the cover slip to add the salt water
3. If there is too much water beneath the cover slip, then the salt water will not flow between the cover slip and the slide. In this case use tissue paper to withdraw water from one side of the cover slip while adding the salt solution at the other side.
4. Observe what happens to the red pigment inside the cells.

Explanation: Water from the cells moves to the surrounding salt water. The shape of the cells does not change, the cell wall maintains the cell shape. The cell content (the red part of the cell) starts to shrivel up. At the same time it is possible to see that the intensity of the red pigment increases because it becomes more concentrated as water is removed (the red pigment is not able to move out of the cell). The process can be reversed when the salt water is removed and when distilled water is added.