Observing Cells: Using Stains - Antibiotics in Action

Biology Activity
Observing Cells
Using Stains

Introduction

To understand an infectious disease, one has to study microbes intensely. This is essential to identifying which specific microbe causes a disease. Among the many microbes one might find living in a sick patient's body, only one is usually causing the disease, and the rest are usually harmless. The work of Louis Pasteur (1822-96), Robert Koch (1843-1910), and Paul Ehrlich (1854-1915), among others in the 1800s, showed how important it is to be able to distinguish between the many different bacteria and fungi that one might find in a patient.
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Procedure

Bacteria made visible by staining.

Sometimes distinguishing one critter from another requires the use of dyes or stains that highlight various structural features of the organism. Many stains color some kinds of microbe, while leaving others alone. The Gram stain (actually a series of staining and washing techniques) is often used to help classify bacteria. Also used to classify bacteria are biochemical behaviors such as the production of a gas, a change in pH, and the production of distinctive excretion products such as ammonia (NH₃), nitrate (NO₃^-), nitrite (NO₂^-) among others.

methylene blue chloride salt

In time, the use of these stains led to the eventual development of some early drugs for destroying suspected disease-producing bacteria. Paul Ehrlich had a particular interest in dyes that were targeting specific cells and structures within cells. His cousin Karl Weigert introduced him to aniline dyes that Weigert was using to stain blood cells and tissues so they could be distinguished from each other under the microscope. Ehrlich perfected these techniques and used them to stain living tissue. You will use one of those stains, methylene blue, in the lab that follows. Ehrlich injected methylene blue into living rats. Later they were dissected to reveal nerve endings clearly stained blue. Since the dye targeted only one kind of cell, perhaps other compounds would target only bacteria cells, without harming the patient's tissues in the vicinity. He began the pursuit for compounds that would act as “magic bullets,” substances that would seek out specific disease-causing organisms much the same way biological stains would seek out specific cellular structures.

In his scientific lifetime, Ehrlich was responsible for developing a number of compounds to treat specific disease conditions. For example, he developed trypan-red for killing the trypanosome, a protozoan responsible for producing sleeping sickness. Another magic bullet Ehrlich developed was Salvarsan (1909) used to treat syphillis. Salvarsan was the commercial name of a compound with a very long name, dihydroxydiaminoaresenobenzenedihydrochloride! Salvarsan remained the drug of choice against syphilis until the advent of antibiotics in the 1940s.

salvarsan

Ehrlich showed that the molecular structures of drugs determine how they behave in the body. Therefore, chemical compounds can be constructed to attack specific targets in particular living cells, be they bacteria or tissue cells in the human body. That certain dyes and other chemical compounds could help to eliminate disease-causing bacteria became a modus operandi in the early part of the 1900s as other chemists began looking for additional dyes that had curative properties.
Procedure

Cells from plants, animals, and bacteria will be used in this exercise to show how each responds differently to different dyes. Several stains including methylene blue (see Introduction above), iodine, and a mix of stains called Gram stain will be used. The Gram stain is named after Danish physician Hans Christian Gram (1853-1938), who first noticed the different effects on bacteria using a combination of several stains and a clearing agent, ethyl alcohol. As always, remember that success in this lab depends upon a certain degree of patience as well as careful reading and following procedural instructions!

Prepare a wet mount of onion cells on a clean glass slide. To do this, peel thin, transparent single layers of inner epidermis of an onion using a forceps. Some practice is needed to obtain very thin samples of tissue. One trick is to “dig into” the onion a bit with the forceps, then pull sideways and upward until you find a cellophane-like piece of tissue beginning to come off with the small “hunk” of onion.

Transfer only the transparent epidermis sample to a glass slide. Add a drop or two of water and a coverslip. The coverslip should lay tightly on the glass slide. If it appears to be floating, place a small piece of paper towel on one edge of the coverslip and draw off some of the water until the coverslip adheres tightly to the slide. If there is not enough fluid, large air bubbles will be noticeable. You can add small amounts of water without removing the coverslip. To do this, place a single drop of water on one end of the coverslip and draw the water under the coverslip by placing a piece of paper towel on the opposite end of the coverslip.

Examine the tissue with the low power of your microscope. Note the shape of the cells as well as any structures within the cell (in the cytoplasm). You may even see motion within the cytoplasm.

While you continue to view the field, have your partner apply a drop of iodine stain (potassium iodide + iodine) to one edge of the coverslip and draw in the stain using a piece of paper towel on the opposite edge (see Step #2). Note any changes that occur. Are specific parts of the cell stained? Which ones?

Repeat the preparation of a new slide with onion epidermis and a coverslip. This time add a drop of methylene blue stain to one edge of the coverslip and draw under with a piece of paper towel on the opposite end. Observe what structures are stained in the onion epidermal cells. How do the staining effects compare with those of iodine that you used previously?

Prepare a new wet mount of a different type of cell, the cells from the inside of your mouth. To do this, first obtain 10 cm³ (ml) of sterile 0.9% saline solution (NaCl) from your teacher. This should be in a new, clean paper medicine cup. Rinse your mouth with the salt solution for 60 seconds.

Expel the salt solution that now contains your cheek cells into a clean test tube 20 mm x 125–250 mm in size.
Using a clean Berel pipette, stir the saline solution, then remove a pipette full of solution. Over a clean glass slide, add a drop or two of the salt solution to the slide and add a coverslip.
Using low power, examine the prepared slide for cheek cells. If no cells appear, switch to high power and continue to look for individual cells. Controlling the light is crucial (too bright may make the cells hard to find). If no cells are found, prepare a new slide with another several drops of salt/cheek cell solution.
When you find cheek cells, examine them on high power, noting (and drawing) their shape and any visible cell structures.
Using the same slide from Step #10, place a drop of methylene blue stain on one edge of the coverslip and draw the stain under the coverslip by again placing a piece of paper towel on the opposite end of the coverslip. Examine the cells under high power, noting any differences in what structures are visible compared with the nonstained slide preparation in Step #10. Are there new structures visible that were not visible before staining?
Repeat the preparation of a new slide of cheek cells with coverslip. Check for the presence of cheek cells, first under low power, then switch to high power for additional scanning. If cells are found, add a drop of iodine stain on one edge of the coverslip, drawing the stain into the cheek cells by placing a piece of paper towel on the opposite end of the coverslip.
Observe the iodine-stained cells. How does their appearance compare with the cheek cells stained with methylene blue? With onion epidermal cells stained with iodine? With methylene blue? Could iodine and methylene blue be used to distinguish cheek cells from onion cells (animal from plant)? What are the differences?

For more information, at other Web sites...

Paul Ehrlich: Pharmaceutical Achiever — part of the Pharmaceutical Achievers module “Magic Bullets: Chemistry vs. Cancer.”

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References

Bowden, Mary Ellen. Pharmaceutical Achievers. Philadelphia: Chemical Heritage Foundation, 2002.
Podolsky, M. Lawrence. Cures out of Chaos. Amsterdam: Harwood Academic Publishers, 1997.

Image Credits

Bacteria made visible by staining: Courtesty Brookhaven National Laboratory.

Copyright ©2002 The Chemical Heritage Foundation