School Science/Staining onion cells without methylene blue

Methylene Blue may not be suited for storing in classrooms (especially for younger students) due to health hazards if swallowed. The following method allows for a nice staining of onion cell nuclei using normal fountain pen ink. I have used this staining method with students, and after a short demonstration the students were able to conduct the experiment themselves. Only very little alcohol and ink is needed for each student (max 5 ml alcohol and 5 drops of ink per student or group). Success, of course, also depends on microscopy experience, maturity and class size.

Materials

 * 1) alcohol
 * 2) water based ink, not the one used for calligraphy, as this one contains particles of carbon that can not enter the cells. Blue fountain pen ink worked fine for me
 * 3) water
 * 4) slides and cover slip
 * 5) microscope
 * 6) onion
 * 7) small beakers for alcohol and water
 * 8) tweezers

Method

 * 1) Take a piece of onion and peel off the membrane using tweezers or your fingernails.
 * 2) Put the membrane into alcohol for about 30 s. This will pull out the water of the cells. The membrane will become stiff a bit.
 * 3) Put the membrane into beaker with ink for about 30 s. The ink is waterbased and will therefore enter the dehydrated cells. The membrane will stain dark blue.
 * 4) Wash the membrane in water until no more ink comes out (about 1 min). Both cytoplasm and the nuclei are still stained blue. We need to remove the excess ink from the cytoplasm.
 * 5) Put the membrane into a small beaker with alcohol for 30 s. Excessive fluid (and ink) is removed from the cytoplasm. The nucei are retaining their color.
 * 6) Put the membrane into clear water for 30 s. The cytoplasm swells up again.
 * 7) Repeat the washing steps 5 and 6 once more. After each washing the cells become lighter as they lose more color.
 * 8) Put the membrane on a slide, add a drop of water and put on the cover slip.

The nuclei of the cells should be clearly visible. If the cytoplasm is still to dark (the whole cell is blue), repeat the washing steps 5 and 6 again. If the nuclei are not visible, then the cells were probably washed too many times and the color was removed from the nuclei as well. In this case shorten the washing times in step 5 and 6 or do not repeat these steps too often. If done well, it is even possible to see two darker dots in each nucleus (the nucleoli?).