This document discusses various screening methods for evaluating the antimicrobial activity of natural products. It describes diffusion methods like the agar well diffusion method and disc diffusion method. Dilution methods, like the tube assay and agar dilution method, are also covered. Bioautographic methods like contact bioautography, direct bioautography, and immersion bioautography are explained. Plant selection criteria for screening include chemotaxonomy, traditional medicine information, field observations, and random collection. LC-hyphenated techniques are also mentioned as screening methods. The document concludes by stating that diffusion methods are commonly used despite some difficulties and that screening aims to efficiently test large numbers of materials.

1. Screening
methods for
natural
products

2. Group Members
Lucia Mapuranga
Chiedza Kudyarawanza
Forget Dimingu
Yvette Nyandoro
Melrose Chikanya
Ropafadzo Makumbe
Rosemary Nyanhoro
Tafadzwa Muchemwa

3. Introduction
• Because of available antimicrobials failure to treat
infectious diseases, many researchers have focused on the
investigation of natural products as source of new bioactive
molecules.
• A variety of methods are found for this purpose and since
not all of them are based on same principles, results
obtained will also be profoundly influenced not only by the
method selected, but also by the microorganisms used to
carry out the test, and by the degree of solubility of each
test-compound.
• The test systems should ideally be simple, rapid,
reproducible, and inexpensive and maximize high sample
throughput in order to cope with a varied number of
extracts and fractions.

4. Introduction
• The current screening methods for the antimicrobial
activity of natural products are diffusion, dilution and
bioautographic.
• The bioautographic and diffusion methods are known as
qualitative techniques since these methods will only give
an idea of the presence or absence of substances with
antimicrobial activity.
• Dilution methods are considered quantitative assays
since they determine the minimal inhibitory
concentration.

5. Introduction continued
• LC-hyphenated techniques are also used as screening
methods for natural products.
• These techniques are not only limited to screening
antimicrobials, but a variety of things like pesticides,
antifungal, antivirals etc.

6. Plant Selection
• Plant selection is done before screening and several
criteria are used.
• Chemotaxonomy, or the science of classification of plants
as a function of the structures of their chemical
constituents, can introduce useful elements. If a natural
compound or class of natural compounds possess
relevant properties, analogous substances may be found
in parent species.
• Chemotaxonomic information can be obtained before
starting a phytochemical and pharmacological
investigation by a literature or data bank search; it is
therefore possible to gain access to all the previous
research which has been performed on the plant selected
for study.

7. Plant Selection
• Plant selection can be done using information from
traditional medicines.
• Field observations are also done. A plant species growing
in a hostile environment, such as warm and humid
tropical forests, will attempt to protect itself by
synthesising insecticidal, fungicidal, antibacterial, or
virucidal constituents.
• Then, for example, if the leaves of a plant show no signs
of aggression, they may contain defensive compounds
against insects or microorganisms.
• Roots often synthesize antifungal substances because
soil is rich in pathogenic fungi.

8. Plant Selection
• Random collection is also an indispensable method of
finding new drugs or leads. In a random sample,
preference is given to endemic plants that have not been
investigated. If the plant species is distributed very
narrowly, it may produce secondary metabolites specific
to the bio- and ecosystems where it is endemic.
• Endemicity and degree of investigation of the plant is
also important.

9. Diffusion
Methods

10. Agar diffusion well-variant
• It is also called the hole-plate assay method.
• This method depends upon the diffusion of the antibiotic
from a vertical hole through the solidified layer of a
petri dish or plate to such an extent that the growth of
the microorganism is prevented entirely in a circular
area or zone around the hole containing a solution of the
antibiotic.
• The plate or petri dish is then incubated under
anaerobic conditions.
PS: Agar refers to some gelatinous material from red
algae which is used as a culture medium.

11. Agar diffusion disc-variant
• Its essential feature is the placing of filter paper discs
with the antibiotic on the surface of agar immediately
after inoculation with the organism tested.
• Natural products are dissolved and diluted the solvents.
• The discs are allowed to remain at room temperature
until complete diluent evaporation and kept under
refrigeration.
• Discs loaded with natural products are placed onto the
surface of the agar and observations are made.

12. Dilution Methods
Dilution techniques are those which require a
homogenous dispersion of the sample in water. They are
used to determine, principally the minimum
concentration values of an extract, essential oil or pure
substance. They can be used in the preliminary screening
of antimicrobial activity.

13. Tube assay or turbidimetric
method
• It is based on the homogenous diffusion of a sample,
dissolved in purified water, MeOH, water/MeOH
mixtures, phosphate buffers, in the broth culture
required for the organism assayed.
• After incubating 3-4 hours, transmittance or absorbance
is read in a suitable spectrophotometer fitted with a
530nm filter .

14. Agar dilution method
• The sample is dissolved or suspended in an appropriate
solvent (water, MeOH or acetone) and mixed with agar
medium.
• The microbial is then inoculated and incubation is done
under anaerobic conditions.
• The results obtained with this method are equivalent to
those obtained with diffusion and dilution methods.

15. Bioautographic
Methods

16. Contact Bioautography
• It is based on the diffusion of separated compounds by TLC
or PC from sheets or chromatoplaques.
• These are placed on the surface of large nutrient agar
plates inoculated with microorganisms that are sensitive to
the antibiotics being analyzed.
• After 15-30 minutes, the sheets or chromatoplaques are
removed in both instances, antibiotics diffuse into the agar
layer and inhibit the growth of the test microorganism.
• The plates are then incubated at an appropriate
temperature until a thin film of the growing
microorganisms is visible on the surface. Zones of inhibition
are then clearly visible.
• Inhibition zones can be made more conspicuous and visible
earlier by using dehydrogenase activity inhibitors.

17. Direct Bioautrography
• A microorganism suspension in liquid medium is
sprayed on a developed chromatoplaque after removing
the solvents. It is then incubated.
• There is no diffusion and the problems of the contact
method are eliminated.

18. Immersion Bioautography
• The chromatoplaque must be included in the medium.
The developed plaque is covered with fused agar.
• The solidified agar is inoculated with the
microorganisms and the plates are left at 4 degrees
celcius or at room temperature. After four hours the
plate is inoculated and incubated.

19. LC-hyphenated techniques

20. Conclusion
• The purpose of screening is to expose the largest number
of test materials to assess their effect on a biological
activity.
• Screens can either be cell-based or biochemical (cell-
free).
• The diffusion methods are the most often employed in
research in spite of certain difficulties.

21. References
• Burgess, J.G.; Jordan, E.M.; Bregu, M.; Mearns-Spragg, A.;
Boyd, K.G. (1999), Microbial antagonism: a neglected
avenue of natural products research, J. Biotechnol., 70, 27-
32.
• Hadacek, F.; Greger, H. (2000). Testing of antifungal
natural products: methodologies, comparability of results
and assay choice, Phytochem Anal., 11, 137-147.
• Recio, M.C.; Rios, J.L. (1989). A review of some
antimicrobial compounds isolated from medicinal plants
reported in the literature 1978-1988. Phytoter. Res., 3, 117-
125.
• Rios, J.L.; Recio, M.C.; Villar, A. (1988). Screening methods
for natural products with antimicrobial activity: a review of
the literature, J. Ethnopharmac., 23, 127-149.
• Hamburger, M.O.; Cordell, G.A. (1987). A direct
bioautographic TLC assay for compounds possessing
antibacterial activity. J. Nat. Prod., 50,19-22.

22. THANK YOU