MYCOTOXICOLOGY:

ENUMERATION, DETECTION
AND ISOLATION OF FUNGI
TOXICOLOGY AND BIOSENSORS

Freddie K. L. Rakwadi | 19001137 | Biological Sciences and Biotechnology

ABSTRACT
The experiment was carried out to detect toxins produced by fungi that grow either on the
field crop or the grains that grow on the crop. The growth of the fungi depends on many
factors including the amount of exposure to light, temperature, length of time and the
condition of the grains. This was done as the aflatoxins produced by the fungi; Aspergillus
pose a threat to all vertebrates. It is also important as grains are staple foods in many
developing countries and the aflatoxins from the fungi are known to be associated with
multiple diseases. In this experiment, the aflatoxins were to be detected in the cereal crop
(sorghum)

INTRODUCTION
Food toxicology has aimed to detect chemical substances in food that pose a threat to the
homeostatic balance within the human body upon consumption (Dellaforia and
Dall’Asta, 2017). Over the decades, studies in food toxicology have helped discovered
that the toxins (chemical substances) have been found to be either naturally occurring in
the food or foreign toxins that have entered the food as contaminants from other
organisms. These contaminants can enter the food production chain at various levels.
These levels can be accidental or intentional contamination by people or the presence of
microorganisms in food produce that produce toxins. Aflatoxins are known to present
major concern in food and feed safety in agriculture. This is because it has been known
that aflatoxins are known to affect every vertebrate species. They are produced by fungi
from the Aspergillus species. These aflatoxin producing fungi are often characterized into
two groups, field fungi (preharvest fungi) and storage fungi (Coppock et al. 2018).
Storage fungi attack the grains or seeds after harvest and are generally in small quantities
before the crop is harvested. They begin to thrive when the grains are stored in an area
that is warm and humid. The most common storage fungi are the species of Aspergillus
and Penicillium. The fungi are widely spread and can be found anywhere (Sweets 2018).
Field fungi are fungi that invade the seeds before they are harvested. They affect the seed
or grain’s physical appearance and can usually be detected using routine inspections and
they are usually not found in the seeds after harvesting. Most field fungi thrive in
conditions where rainfall is above normal and one of the fungi that can be classified as
field fungi are Fusarium. Aspergillus flavus is well known to produce aflatoxins as
secondary metabolite. Although little is known about the secondary metabolite and its
association with the development of the fungi, its survival and the virulence of the fungi
(Cary et al. 2018). The field fungi and storage fungi are filamentous fungi. Filamentous
fungi are a notorious constraint in food safety (Rodríguez et al. 2015). Filamentous fungi are
usually detected using culturing and mycotoxin production evaluation. The detection of
aflatoxins in the plates are also vital when it comes to agricultural feeds and food
production. The techniques used to detect these aflatoxins are sensitive, specific and easy
to carry out. These techniques are Thin Layer Chromatography (TLC), High Performance

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Liquid Chromatography (HPLC), electrical immunosensor and many more can be used to
detect aflatoxins (Wacoo et al. 2014).

MATERIALS AND METHODS

Week 1

Isolation and Enumeration of fungi

50g of each sample was suspended in 450 ml of 0.1% peptone water to make a 10^-1
dilution. Further dilutions were made in 0.1% peptone water in series up to 10^-4. 0.1 mL
of the diluted samples were then plated in DRBC and DG18 medium plates. A plate was
prepared for each diluted sample. After dilution the DG18 and DRBC plates was
incubated in a cabinet to simulate a dark environment. The incubation was done for 7
days at 25℃. The DPCVA plate was incubated at 25℃ in alternating 12-hr periods of
fluorescent light on a designated area and in the cabinet.

Week 2

Enumeration and Identification of fungi

Colonies that grew on the culture plates from the first week were counted and recorded as
Colony Forming Units per gram of the sample plated. Isolates from each culture plate
were subcultured onto Malt Extract Agar for identification. The MEA plates were then
incubated for 7 days at 25℃.

Extraction and Analysis of aflatoxins from food samples

50g of the sample was mixed with 15- mL of methanol/water (85/15, v/v) solution in a
container. The container was then placed on an orbital shaker for 30 minutes. The
contents of the container were then filtered using the Whatman paper No. 1. Two rounds
of 25 mL hexane were then added to 40 mL of the filtrate. The aqueous layer was
extracted and 40 mL of NaCl was added. The mixture was then partitioned through using
two rounds of 25 mL chloroform. The chloroform layer was filtered through using
anhydrous sodium sulphate. The mixture was then taken to a rotatory evaporator to
evaporate the chloroform for two weeks. The mixture was then redissolved using two
rounds of 1 mL chloroform and transferred to Eppendorf tubes. The solution was then
evaporated in a fume hood. The solution was then redissolved in 250 µL chloroform.

Week 3

Thin Layer Chromatography Analysis

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Samples were taken to screen for aflatoxins on 0.2 mm silica gel TLC plates. The 10 µL
extracts spotted using 2, 5 and 10 µL aflatoxin standards (0.5ng/µL). A solvent of
chloroform: acetone (9:1, v/v) for the development using TLC tanks. After development,
the plates were then dried in fume hoods and observed under short and long wavelength
of UV light (254 and 365 nm).

Identification of fungi

Fungal isolates on the MEA plates were identified. The subcultures of Aspergillus flavus
as well as A. parasiticus were distinguished from the other Aspergillus spp. Subcultures
on AFPA. The isolates were incubated for 7 days at 25℃ and observed under a
microscope. The Fusarium species were then confirmed as they grew in the DPCVA
plates.

Week 4

Identification of fungi

Cultures of A. flavus/parasiticus were recorded. Any outstanding work was then

completed during that time.

RESULTS AND DISCUSSION

Enumeration of fungal colonies

Dilution Value Number of filamentous fungi colonies (CFU/mL)

DCPCVA DG18 DRBC
10-1
10-2
10-3
10-4

Figure 1: The table above displays the number of filamentous fungi colonies with respect
to the dilution value of the sample in each test tube.

CFU

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Figure 2: The images above show DRBC plates after one week of incubation. The images
show fungal colonies (the black colonies) and the many pink colonies that have grown on
the plate. The DRBC were inoculated with various concentrations of the sorghum
mixture.

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Figure 3: The image above shows DCPCVA media after one week of incubation. Very
few colonies of filamentous fungi had grown, and majority of the colonies are orange
with an irregular shape.

Figure 4: The images above show the of the DG18 media plates inoculated by various
concentrations of the sorghum mixture. In the plates, there are no shiny colonies forming
in the media. The plates only contain filamentous fungi colonies.

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Figure 5: The image above shows Aspergillus flavus growing in a Malt Extract Agar
(MEA).

Thin Layer Chromatography

Figure 6: The image above shows the results of the thin layer chromatography. The
chromatography shows SA, B1, B2, G1 and G2. The image above was viewed under
ultraviolet radiation.

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Calculations

Rf value=Distance travelled by sample/distance travelled by solvent

Rf value for B1=51mm/93mm=O.55

Rf value for G2=36mm/93mm=0.39

Rf value for G1=41mm/93mm=0.44

Rf value for B2=43.5mm/93mm=0.48

Rf value for mealie meal sample -32mm/93mm=0.34

During the one-week incubation, the plates were moved from a light source to a cabinet
where there was no light and vice versa every 12 hours. Throughout the first four days of
incubation, the fungi had not grown but the other irregular shaped, shiny colonies had
started growing in the media. This may be because the fungi needed a warm and humid
environment for them to grow (Sweets 2018). This could support the fact that the
temperature of the area where the plates were kept were as low as room temperature. This
could have led to the fungi to grow at a much slower pace. The fungi form the sorghum
grain sample were isolated using different media plates to determine the type of fungi
isolated from the sorghum grains. The media plates used were DRBC (Dichloran Rose-
Bengal Chloramphenicol), DCPCVA (dichloran chloramphenicol Peptone Agar
containing Crystal Violet) and DG18 (dichloran glycerol). After incubating the media
plates, it was found that all the plates had mostly shiny, irregular shaped colonies that
occupied majority of the plates. Only a few plates had contained the desired fungal
colonies. DCPCVA media is a media that is used to isolate the Fusarium species of fungi
and dematiaceous hyphomycetes from cereal grains (Hocking & Andrews, 2009).
Fusarium was alternated between light and dark areas because light is used by the fungi to
synthesize carotenoids which aid in the development of sexual fruity bodies and asexual
spored that it utilizes to colonize an area (AF 2010). This could explain why the Fusarium
plant did not thrive well in the environment as it did not stay in the light too long to
produce fruiting bodies that would have allowed it to spread and colonize the plates. DG18
(Dichloran Glycerol) is a media used to isolate xerophilic molds in foods and is used to
prevent the growth of bacteria. Bacterial growth is inhibited due to the compound,
chloramphenicol and reduces the amount of available water. This is supported by Figure 4
which shows that there are only filamentous fungi that grew in the DG18 media plates.
MEA (Malt Extract Agar) is a medium which is a selective media that is used to isolate
Aspergillus flavus, one of the fungi that produces aflatoxins. This was shown by Figure 5 as
there were two large colonies that grew in the Malt Extract Agar, meaning that Aspergillus
flavus was present in the sorghum sample. The media was developed to only favor the
conditions for fungi to grow and inhibit any bacterial growth. Aspergillus colonies
managed to thrive in the dark and appear as two dark brown colonies with a white

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outline. The Thin Layer Chromatography was used to determine the presence of the
aflatoxins as it is cheap and reliable to use when detecting molecules such as aflatoxins.
The dyes illuminated by the ultraviolet radiation are the molecules that managed to
separate from the other molecules in the mixture. The higher the polarity of the molecule,
the further it moves up the chromatography paper. If the dyes are on the same level, we
can consider the molecules identical or similar when it comes to their polarity. The
distance of the solvent front and the distance travelled by the dye can be expressed as a
ratio known as the Retention factor (R f). From the determined retention factors, the
sample (SA) does not match the retention factors of the standards ( B1, B2, G1 and G2). This
means that there are no aflatoxins that can be found in the sorghum sample.

CONCLUSION

There were no aflatoxins found within the sorghum sample. There were fungi form the
Fusarium and Aspergillus spp but in small quantities. There was no competition for space
in the colonies for the fungi to begin producing aflatoxins to eliminate the other
microorganisms growing in the plates.

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REFERENCES
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Detection of Aflatoxins in Agricultural Food Crops", Journal of Applied
Chemistry, vol. 2014, Article
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Avalos J, Estrada AF. Regulation by light in Fusarium. Fungal Genet Biol. 2010
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Aspergillus flavus secondary metabolites: more than just aflatoxins. Food Safety, 6(1), 7-
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Dellafiora, L., & Dall’Asta, C. (2017). Forthcoming Challenges in Mycotoxins

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