Seed Science and Technology

LABORATORY EXCERCISE
Tetrazolium Test

Introduction
The tetrazolium test is widely recognized as an accurate means of estimating seed viability.
This method was developed in Germany in the early 1940s by Professor George Lakon who
had been trying to distinguish between live and dead seed by exposing them to selenium
salts. He then tried tetrazolium salts and found them more effective. Today the test is used
throughout the world as a highly regarded method of estimating seed viability and is routine
test in many seed testing laboratories. It is often referred to as a “quick test”, since it can be
completed in only a few hours (as compared to regular germination tests that require as
long as two months for some species). Tetrazolium test results can be extremely valuable
for providing labelling information for immediate shipment of seed lots without waiting for
completion of germination tests. It is also a valuable research technique for estimating seed
viability and determining reasons for poor germination.

Objectives
1. To provide hands-on for student to perform tetrazolium test.
2. To expose students the importance of tetrazolium test and the parameters to be taken in
determining seed viability and enzyme activity.

Materials
Various types of seeds: green beans, black eyed peas, soy beans, and ground nuts; petri
dishes, magnifying glass or microscope, tetrazolium solution.

Methodology
Seeds to be tested were soaked in sterile distilled water for about half an hour. Seeds were
then divided into species before cracked or opened into half and soaked in petri dishes filled
with tetrazolium. The samples were incubated for 30 minutes at 37°C. Seeds were observed
and diagnosed using magnifying glass or microscope.

Results and Discussion

Tetrazolium test is a biochemical test, which differentiates live from dead tissues of seed
embryos on the basis of dehydrogenase enzyme activity (respiration enzymes). Upon seed
hydration, the activity of dehydrogenase enzymes increases, resulting in the release of
hydrogen ions, which reduce the colourless tetrazolium salt solution (2,3,5-triphenyl
tetrazolium chloride) into a chemical red compound called formazan. Formazan stains living
cells with a red colour, while dead cells remain colourless. The viability of seeds is
interpreted according to the staining pattern of the embryo and the intensity of the
colouration. But the interpretation requires considerable skills and experience. Sound
embryo tissues absorbs tetrazolium slowly and tend to develop lighter colour (Figure A)
than embryos that are bruised, aged, frozen, or disturbed in other ways (Figure B). The
staining patterns reveal the live and dead areas of the embryos thus enabling us to
determine if seeds have the capacity to produce normal seedlings. The cell division areas of
the embryos are the most critical during germination, and if they are unstained or
abnormally stained, the seed’s germination potential is weakened.

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Figure A: Soy bean seeds (left) and ground nuts showed relatively good viability after 30
minutes incubation in oven at 37°C. According to Patil and Dadlani (2009), soaking duration
(normally between 15 to 45 minutes) and temperature (between 20°C and 40°C) has no
effect on accuracy of tetrazolium test, but staining performed faster at higher temperature.
It is essential that the tetrazolium solution penetrates into the embryo. In the case of hard
legume seed, the impermeable seed coat should be cut, and in the case of grass seed, a
longitudinal cut through the embryo may be required before placing the seed in tetrazolium
solution. A gradual reduction in colour intensity from the surface to the interior of the seed
indicating slow absorption and penetration of tetrazolium solution.

Figure B: Several seeds of black

eyed peas showed low viability
during tetrazolium test. Almost
entire part of endosperm (bottom)
was stained red, indicating active
enzyme degradation of food
storage and seed deteriorates fast.
Uneven stained regions in
coleoptiles and radicle (labelled
“a”) demonstrating weak viability
of those parts – seed may not
germinate. The unstained dead
tissues of coleoptiles and radicle
(labelled “b”) look flaccid, liquid-
logged, and chalky white in colour.
a Seed will never germinate.

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The rapidity of the test is its most obvious advantage and may justify its usage when speed
is important. Another advantage is its usefulness for dormant seeds, as well as for
nondormant seeds. It is also useful for researching and teaching the biology and physiology
of seed quality and deterioration processes. Like any other seed testing method, the
tetrazolium test requires special training and experience. This may be the reason why this
test has been underutilized in the past. However in recent years, this test becomes more
standardized and more analysts are trained to use them.

Conclusion
From this experiment, we become aware and understood about how to perform tetrazolium
test and determining seed viability and enzyme activity.

References
Copeland, L.O. 2001. Principle of Seed Science and Technology. Kluwer Academic Publishers,
Netherlands.

Hampton, J.G., Ferguson, J.E., Loch, D.S., and Fairey, D.T. 1999. Forage Seed Production:
Tropical and Subtropical Species. CABI Publishing, London, UK.

McDonald, M.B. and Kwong, F.Y. 2005. Flower Seed: Biology and Technology. CAB
International, Oxfordshire, UK.

Patil, V.N. and Dadlani, M. 2009. Tetrazolium Test for Seed Viability and Vigour. Retrieved
from http://dacnet.nic.in/seednet/seeds/Material/Handbook_of_seed_testing
/Chapter%2014.pdf on 051109.

http://www.seedlab.oscs.orst.edu/Page_Technical_Brochures/ValueTZTests.htm (031109)