MicrobeHunter.com » staining

Heat-fixing and staining human cheek cells

Oliver — Wed, 05 Jan 2011 12:50:07 +0000

Observing human cells is a good introductory activity to learn heat-fixing and staining. I will not waste many introductory words here. Here is the method:

Heat fixing

Heat fixing essentially “bakes” the cells to the glass slide much like a fried egg sticking to a frying pan. Heat fixing is absolutely essential before staining. Otherwise the staining procedure will wash away the cells.

Take a cotton swab and rub the inside of your cheeks to collect epithelium cells.
Smear these cells on a microscopy slide
Completely air-dry the slide, without applying heat. This should not take long because there is not much liquid on the slide anyway. If you heat the slide before it is completely dry, then you end up “boiling apart” the cells. The vapor pressure inside the cells will burst them…
Heat fix the dried slide by quickly pulling it through a Bunsen burner (2x), but in a way that the cells do not touch the flame. Pull it through the flame with the cells on top and the flame below. The slide should be pretty hot but you should still be capable of holding it in the palm of your hand without burning yourself. You should just be capable of holding the slide. Too high a temperature and you destroy the cells on the slide (and on your skin!). Too low a temperature and the cells will not stick to the glass slide.

Staining

Apply a drop of the stain (eg. methylene blue) to the heat fixed but cold specimen slide. Allow the stain to work for a few minutes and then carefully rinse away the stain with water. Do not apply the water stream to the cells directly. Allow the water to run over the cells from the top of the slide. Air dry the slide and observe in the microscope.

Staining bacteria

Oliver — Fri, 15 Jan 2010 11:00:31 +0000

Here is yet another link to an article from Popular Science magazine. It deals with the isolation, fixing and staining of bacteria. I would not recommend the use of some of the solvents that they use (such as xylol) with children, however. They also describe a blood smear preparation, what I do not recommend for schools (it may not even be allowed in some countries). Still, the article gives a very nice introduction into several preparatory techniques. The article stretches over several pages, click the link at the end of the pages to continue reading. The fact that the article was published 75 years ago, in 1934, does not matter. The preparatory method stayed the same.

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

Stains and reagents for microscopy

Oliver — Tue, 12 Jan 2010 13:30:32 +0000

I found an article in Popular Science Magazine (see link below) which gives a general overview of different stains that can be used in microscopy. The article divides the stains into three categories:

Common household chemicals: this includes Iodine, for example. They are very readily available.
Substances used mostly for microscopy: Methylene blue, Hematoxyline, and Eosine belong to this group.
Commercial substances: they are sold by companies specializing in microscopic chemicals.

The article also provides a step-by-step guide on how to stain a blood sample (don’t do this in schools due to danger of infection).

Link to the article: Help Your Microscope with Stains and Reagents (Popular Science, March 1937)

Staining of Onion Cell Nuclei

Oliver — Fri, 12 Dec 2008 22:13:57 +0000

The nuclei of onion cells stain blue.

This is a simple preparatory technique that allows students to observe the otherwise difficult to see nucleus of onion cells. There is no need to employ, possibly harmful, DNA staining chemicals.

Materials: Onion, tap water, alcohol, fountain pen ink, several small beakers or film containers.

Method:

Using a sharp knife, cut out about 1 cm² of onion material. This may be done by the teacher to reduce the risk of injury.
The individual layers of the onion are separated by a thin skin. Peel this skin off using your fingernails or tweezers. The skin can be found on the inside part of each layer.
Place the skin into pure alcohol for about 30 seconds. This procedure removes water from the cells.
Place the skin into the ink. The ink, which is water-based, will enter the cells and strain the onion skin deep blue.
Using tweezers, transfer the skin into pure water and rinse it for about 30 seconds or until no more ink is given off. The skin will still have retained its blue color.
We now start the washing steps. We have to remove excess ink from the cells. Transfer the skin into pure alcohol for about 30 seconds. Some of the ink will be removed staining the alcohol slightly blue.
The skin is transferred into pure water for about 30 seconds.
The skin is carefully spread on a microscope slide and a cover slip is placed on top. Excess water is removed with filter paper
If the cytoplasm of the cells is still too dark, then it is necessary to repeat the washing steps 6 and 7 for a second time.

Troubleshooting:

Problem: The cells are too blue and too dark.
Solution: The cells were insufficiently washed. Prolong the washing times or introduce another washing cycle.

Problem: The nuclei are not stained, or not stained enough.
Solution: There could be several reasons for this.

The onion was not placed into the alcohol. Therefore the water inside the cell was not removed and the ink could not enter the cell.
The onion was not submerged long enough in the ink or the onion was not fully covered by ink on all sides
The onion was washed too intensively. This is the most probable cause. If the onion was washed twice, then the washing step should be conducted only once.
It could also be that the washing times were too long. If the nuclei are stained lightly blue, then this indicates that the staining procedure is in principle working, but that too much of the ink was removed.