Can you distinguish between … the living and the
dead?
Technical & Teaching Notes
This is a developing resource idea, first published in SAPS Osmosis newsletter, Spring
2000. If you'd like to see this become a fully-fledged teaching resource, why not become a
SAPS Associate and apply for funding to develop it further?
Teaching Notes
This relatively simple but elegant staining technique gives an attractive way of detecting and
differentiating between living and non-living cells. It can be used to make students think about cell
membrane structure and function thus providing a useful practical in an otherwise rather 'lean'
part of a teaching programme. It can even provide an introduction to PCD (programmed cell
death or apoptosis).
During the maturation of flowering plant seeds, the endosperm dies long before the seed
germinates. Using maize (Zea mais) seeds, or any other endospermic seeds, it is possible to
demonstrate the presence of living and dead tissue, using metabolic activity and cell membrane
integrity as markers.
Staining with tetrazolium chloride (TTC) is a common method for determining the viability of
seeds. When TTC diffuses into actively respiring tissues it accepts electrons from the
mitochondrial electron transport chain and the stain is reduced to the pink compound formazan.
The accumulation of this pink compound stains the tissues red, and the inensity of the red is
proportional to the rate of respiration in those tissues. To carry out the test, imbibed seeds should
be bisected with a razor or scalpel, and the cut faces placed face down in a watch glass
containing a few drops of 1% TTC solution. In a warm room, actively respiring tissues of the cut
face will become coloured within 10 to 20 minutes. If the respiratory rate is slow, longer periods of
incubation are needed. The staining is permanent and seeds stained in this way can be kept in a
refrigerator until the next lesson, or even dried out.
1. Bisecting the seeds
Science & Plants for Schools: www.saps.org.uk
Can You Distinguish Between the Living and the Dead: p. 1
This document may be photocopied for educational use in any institution taking part in the SAPS programme.
It may not be photocopied for any other purpose. Revised 2010.
2. Staining the seeds
Staining with Evans Blue has been used to reveal cells that have lost their cell membrane
integrity. The stain is excluded from living animal and plant cells - and can, for example, be used
to distinguish living onion skin epidermal cells from dead ones, simply by adding a drop of the
stain to a piece of epidermis on a slide. The protoplast of dead cells is stained blue. To carry out
the test with maize seeds, imbibed seeds are bisected with a razor or scalpel, and the cut faces
placed face down in a watch glass containing a few drops of Evans Blue (0.1% w/v aqueous)
solution. After 1 minute the sections are removed from the solution with forceps and gently rinsed
in distilled water for 10 minutes to remove the excess stain. The staining is permanent and such
seeds can also be kept in a refrigerator until the next lesson, or dried out. It is even possible to
achieve double staining of bisected seeds by using both the TTC and Evans blue techniques on
the same half-seeds!
3. Internal Structure of maize seed and its appearance after staining (shown by shading)
We can now look at this in terms of programmed cell death (PCD or apoptosis). At the cellular
level, animals and plants share many of their fundamental processes. In recent years there has
been much interest in the process of programmed cell death (PCD) in animals and plants, as it is
crucial in many of the processes of life including embryonic development, tissue differentiation,
senescence, and disease. Programmed cell death is distinguished from death by traumatic
causes because the cells seem to die in an organised (programmed) fashion. It is thought that the
PCD mechanism has been conserved amongst eukaryotes - a cascade of biochemical events
leads to the fragmentation of nuclear DNA by nucleases, producing fragments in multiples of 180
base-pairs (representing the lengths of DNA associated with the nucleosomes) and finally a loss
of cell membrane integrity.
As another idea, Evans Blue can be included in solutions being used to plasmolyse plant cells,
such as those of an onion skin. It should immediately be apparent to students examining the cells
under the microscope, that the space between the retreating cell membrane and the cell wall is
being filled by the (blue-coloured) bathing solution. The stain will, incidentally, reveal the
occasional dead cell amongst the living ones in the onion skin.
Safety Notes
Science & Plants for Schools: www.saps.org.uk
Can You Distinguish Between the Living and the Dead: p. 2
This document may be photocopied for educational use in any institution taking part in the SAPS programme.
It may not be photocopied for any other purpose. Revised 2010.
All biological stains should be regarded as potentially hazardous and relevant hazard warnings
are given in the catalogues. With respect to Evans Blue, CLEAPSS has advised that this stain
can be used by students and teachers in schools, provided it is used as a 0.1% solution, under
normal laboratory conditions.
Teachers and technicians should ensure that the employer's risk assessment has been carried
out before attempting any practical work.
Suppliers
Evans Blue is available as a powder from Sigma (tel: 0800 717181).
Tetrazolium chloride (TTC) is available as a powdered sodium salt from Philip Harris Ltd (tel:
0845 1204520).
Acknowledgements
Roger Delpech (Haberdasher's Askes' School, Elstree)
Science & Plants for Schools: www.saps.org.uk
Can You Distinguish Between the Living and the Dead: p. 3
This document may be photocopied for educational use in any institution taking part in the SAPS programme.
It may not be photocopied for any other purpose. Revised 2010.