Microbiología Grupo 2 Año 2022-1, No 3, Mayo 7 de Año 2022.

Universidad Tecnológica de Pereira 1

Macroscopic and microscopic analysis of the lichen

fungus of the Caracolí tree
Análisis microscópico y macroscópico del hongo proveniente de los líquenes del árbol Caracolí
Manuela García Arango, José Alejandro Grisales Lopez
manuela.garcia2@utp.edu.co - a.grisales@utp.edu.co
Abstract— Lichens are considered as a mutualistic association dilutions are prepared in a 10-1 sequence, until reaching
between fungi and photoautotrophic species (algae, dilutions of 10-7 or greater. These dilutions are seeded in a
cyanobacteria), but they can also host other microbial nutrient plate medium, where the microorganisms develop
communities [7]. To analyze the macroscopic and microscopic forming colonies [3].
characteristics of the fungi contained in the lichens, the serial
dilution technique was performed to decrease the concentration
To carry out serial dilutions, peptone water is used, which is
of the sample, in order to identify and compare the different a non-selective liquid enrichment medium, used mainly as a
structures. Fungal growth was obtained in each of the petri diluent for food samples or other materials. This medium
dishes planted with the dilutions, in addition, by means of contains meat peptone, sodium chloride, and water [4].
staining, the different microscopic forms of each fungus were Therefore, our objective is to isolate the fungus present in
observed. Additionally, an isolation of the fungus of the 10-3 the bark of Anacardium excelsum, using the method of seeding
dilution was made, from which a pure growth was obtained, by dilution in a Sabouraud agar medium. In addition to
without contamination, with which all its morphology could be characterizing the macro and microscopic morphology of the
observed and analyzed. isolated fungus.
Index Terms—fungi isolation, dilution, fungi, lichens,
morphology II. METHODS
A sample was taken from the bark of the Caracolí
Resumen— Los líquenes son considerados como una asociación Anacardium excelsum tree, which is located outside building
mutualista entre hongos y especies fotoautótrofas (algas, 16-C of the Technological University of Pereira. Said sample
cianobacterias), pero también pueden albergar otras was macerated and a homogeneous suspension was created
comunidades microbianas [8]. Para analizar las características that was poured into the test tube with a dilution of 10-0 (fig.
macroscópicas y microscópicas de los hongos que contienen los
1).
líquenes, se realizó la técnica de diluciones seriadas para
disminuir la concentración de la muestra, con el fin de identificar On the other hand, 9 mL of peptone water was added to four
y comparar las diferentes estructuras. Se obtuvo crecimiento de test tubes to seed by dilution in the sample obtained.
hongos en cada una de las cajas de petri sembradas con las
diluciones, además, por medio de la tinción, se observaron las A. Sowing by dilution
diferentes formas microscópicas de cada hongo. Adicionalmente,
With the sterile pipette, 1 mL of the mother sample (10-0)
se realizó un aislamiento del hongo de la dilución 10-3, del cual se
obtuvo un crecimiento puro, sin contaminación, con el que se was extracted. Next, it was added to the test tube which
pudo observar y analizar toda la morfología de este. contains 9 ml of peptone water and stirred until a
homogeneous mixture was obtained with a dilution of 10-1.
Palabras claves—aislamiento de hongos, dilución, hongos, Similarly, with a sterile pipette, 1 mL of the 10-1 dilution
líquenes, morfología was extracted and added to the next test tube with 9 mL of
peptone water, achieving a 10-2 dilution. This process was
I. INTRODUCTION repeated until a 10-14 dilution was reached (fig.1).
Trees often show a blue-green growth on their bark that
appears to be a fungus, these are harmless organisms called
lichens which appear on trees in poor health. Lichens are
organisms made up of an algae and a fungus which work
together for their growth and development, this symbiotic
relationship is abundant and resistant, it can survive in almost
any environment, as well as in trees, shrubs, on the ground, in
the rocks and other materials [1].
In most environments, the density of microorganisms is too
high to carry out a sowing in which numeral microorganisms
can be obtained. This makes dissolution necessary in order to
obtain results in which microorganisms can be better
identified, in addition, solid samples require dissolution to
facilitate handling [2].
When the concentration of the sample is high, serial Fig. 1. Fungus sample diluted in peptone water
2 Microbiología Grupo 2 Año 2022-1, No 3, Mayo 7 de Año 2022. Universidad Tecnológica de Pereira

Next, 1 mL of each dilution was taken and cultured in

Sabouraud dextrose agar and the Petri dishes were
immediately sealed and incubated for 5 days at a temperature
of 30 ºC.

B. Macro and microscopic observation

After 14 days from sowing, the macroscopic characteristics
of the fungus isolated in their respective Petri dishes were
observed.
To microscopically characterize the morphology of the
fungus, first a strip of adhesive tape was taken, which was
pressed firmly on the surface of the fungus contained in the
Petri dish with a 10-0 dilution and a few drops of methylene
blue were added, and then be observed with the different
objectives of the microscope. Process that was repeated for
each sample in dilution (fig. 4).

C. fungus isolation
In order to carry out a pure isolation, the Petri dish with the
10-3 dilution was selected, from which a sample with a size of
1x1 cm was extracted, seeded on the surface of the new
nutrient agar (fig. 2 ) and then incubated. After 7 days, their
morphology was identified macroscopically.

Fig. 2. Extraction area and subsequent inoculum of the sample.

III. RESULTS
A. Macroscopic characterization
After incubation and after 14 days of growth, the Petri
dishes were examined and in all the dilutions a white cottony
mycelium was present.
Small purple dots were identified at 10-0 and 10-1 under the
cottony white micelles. For 10-2 and 10-3 a similar morphology
was observed where the cottony mycelium covers Fig. 3. Macroscopic characterization of the tree bark sample with different
approximately a little more than half of the Petri dish. While dilutions
in sample 10-4 the cottony mycelium covered almost the entire
surface of the agar (fig. 3). B. Macroscopic characterization
The growth and formation of hyphae and blue-stained spores
were observed in all the solutions from the mother sample, as
indicated in figure 4.
Microbiología Grupo 2 Año 2022-1, No 3, Mayo 7 de Año 2022. Universidad Tecnológica de Pereira
Fig. 4. Microscopic characterization under staining of the bark of the tree.
C. Pure culture characterization
A totally pure isolated culture with white cottony mycelium
and radial growth that covers the entire surface of the agar is
observed (fig, 5).
Fig. 5. Macroscopic characterization of the pure isolation of the sample
contained in the Petri dish with dilution 10-3.
IV. ANALYSIS OF RESULTS
Most fungi have vegetative filamentous structures called
3
hyphae, which can be clearly seen in figure 4. These grow in
the form of long arms in all directions, they are usually
uniform and thin with diameters of 1 to 2 mm, the set of
hyphae form what is called mycelium [5].
In sample 10-1, a structure of skeletal hyphae with thick
walls, unbranched and without septa can be observed.
Sabouraud Dextrose Agar is a peptone medium
supplemented with dextrose. Peptones are sources of
nitrogenous growth factors and dextrose provides a source of
energy [6], which allowed a favorable growth of the fungus in
all the isolates that were carried out during the practice.
V. CONCLUSIONS
Serial dilutions help us to analyze the behavior of the
fungus at different concentrations and to recognize its
morphological structure; the staining of these results and the
structural identification of the fungi allows us to diagnose
infectious diseases and allows us to solve problems of
microbial etiology. Stains are elementary, current and
universally used tools that contribute to microbiological
diagnosis [8].
In addition, it was found that Sabouraud dextrose agar is an
optimal in vitro culture medium that favors the growth of
fungi under controlled conditions.
REFERENCES
[1] Alexopoulos C. y Mims C. (1985). Introducción a la micología. Omega,
Barcelona, 660 pp.
[2] Arana, I., Orruño, M., & Barcina, I. (s. f.). CÓMO ABORDAR Y
RESOLVER ASPECTOS PRÁCTICOS DE MICROBIOLOGÍA.
ocw.ehu.eus. https://ocw.ehu.eus/mod/resource/view.php?id=39642
[3] Scheffers D-J. Bacterial Reproduction and Growth. In: eLS [Internet].
Chichester, UK: John Wiley & Sons, Ltd; 2013 [cited 2017 Nov 20].
Available from: http://doi.wiley.com/10.1002/9780470
015902.a0001419.pub2
[4] Gil, M. (2019, 22 marzo). Agua peptonada: fundamento, preparación y
usos. Lifeder. https://www.lifeder.com/agua-peptonada/
[5] Garces, E., Correa, M., Coba, B., Orozco, M., Zapata, A., Anacona, A.,
Sabogal, S. (z.d.). MORFOLOGÍA Y CLASIFICACIÓN DE LOS
HONGOS. ciencias.bogotá.unal.
http://ciencias.bogota.unal.edu.co/fileadmin/Facultad_de_Ciencias/Public
aciones/Imagenes/Portadas_Libros/Biologia/Morfologia_y_Clasificacion
_de_los_Hongos/Morfologia_y_clasificacion_de_los_hongos_libro.pdf
[6] Koneman E. Diagnostico microbiologico Texto y Atlas color. 6º edición.
Editorial Panamerica. 2006. Oxoid manual 9º edición. 2006. BD. BBL
Sabouraud dextrose agar. Rev 10. Diciembre 2012
[7] Grube M, Berg G. 2009. Fungal Biology Reviews 23: 72-85.
[8] López-Jácome LE, Hernández-Durán M, Colín-Castro CA, et al. Las
tinciones básicas en el laboratorio de microbiología. Investigación en
Discapacidad. 2014;3 (1):10-18.