Practical 3 : FUNGAL SUBCULTURE AND PURIFICATION

TECHNIQUE, FUNGAL SLIDE PREPARATION & LIGHT

MICROSCOPE

Course Name : Forest Protection (Pathology)

Course Code : FL10103

Lecturer’s Name : Dr. Mandy Maid

Demonstrator’s Name : Ms. Nadzirah Mohd Yunus

Date : 24 November 2022

Student’s Name and Matriculation Number :

1. Alliya Ardaa Binti Lukas (BF22110181)

2. Beatrice Kristy Anak Christopher (BF22110095)
3. Lailatul Shafiah Binti Jackson (BF22110054)
INTRODUCTION.

Sub culturing is the method of transferring microorganisms from one

growth container to another, thereby offering them with fresh supply of nutrients
either in a solid or a liquid medium. So, the basic objective of this test is to
prepare subculture of fungus.

OBJECTIVES.

1. To conduct fungal subculture and purification techniques.

2. To use compound or light microscope techniques.

3. To prepare fungal slide, record and describe the observation.

MATERIALS.

1. 70% ethanol
2. Malt extract agar - MEA
3. Potato dextrose agar - PDA
4. Distilled water
5. Petri dishes
6. Fungus in agar media
7. Scalpel
8. Cork borer
9. Fungal loop
10. Beaker
11. Alcohol / spirit lamp
12. Ethyl alcohol or methanol (fuel)
13. Tissue papers
14. Incubator
15. Parafilm
16. Compound and light microscope
17. Slides and cover slips
18. Metaline blue
METHODS.

A. FUNGAL SLIDE PREPARATION.

1. Scrape the edge of the fungal colony with a sterile loop. With observed
under the microscope, extracted the edge of hyphal growth and any
reproductive structure.
2. Placed the extracted fungal portion on the slide with a drop of water on
the glass slide. Using a glass rod to break up the fungal tissue. Place a
drop of metaline blue or other stain on to the glass slide. Covered it with
a cover slip. Wipe around the edges with tissue paper.
3. View the slide under the light microscope using low to high
magnification. Take photographs under different magnifications.
4. The photographs would be labelled and described.

B. Fungal subculture and purification technique.

1. Work under aseptic conditions. Sterilized hand with ethanol 70%.
2. Each student work on two subculture dishes.
3. The parafilm removed from the petri dish cover contained the fungal
isolation sample.
4. Sterilized the cork borer (4-5 mm) by flaming over the spirit lamp, cool,
and cut a portion of the fungal agar from the edge of the fungal colony.
Transfer the fungal agar plug onto the center of a new media. Covered
and sealed using a parafilm, and label with the date and name.
RESULTS.
Fungal growth under a light Observation
microscope
The colony morphology in this fungal
growth under a light microscope using
4mm magnifications lens are hairy,
yellowish-brown bubble and have dark
circle colour.

Diagram 1: 4 mm
The fungal growth under light
microscope magnifications 10mm are
focus on the edges. There’s a pathogen
in Rhizoctonia which has right angle
branching, septations and hypha that is
slightly constricted where branching
occurs.

Diagram 2: 10 mm
This is the region where the cell wall
extends continuously to produce a long
hyphal tube. Grow moderately rapidly to
slowly and have narrow, septate hyphae.

Diagram 3: 40 mm
Growth of hyphae in most fungi takes
place almost exclusively in the apical zone
(i.e., at the very tip). This is the region
where the cell wall extends continuously to
produce a long hyphal tube. The cytoplasm
within the apical zone is filled with
numerous vesicles. These bubblelike
structures are usually too small to be seen
with an ordinary microscope but are clearly
evident under the electron microscope.

Diagram 4: 100 mm
DISCUSSION.

Culture media are mixtures of nutrients or substances used in the laboratory

for cultivation of many microorganisms.Inside the laminar airflow, take the plate
cultures containing fungus. Open it very less as the spores may come out. Then
show the tip of the inoculating loop in the Bunsen burner to sterilize it. Then a
loopful of fungal culture and streak it in the new sterile PDA plate. Then close the
plate and incubate it for 48-72 hours (Miransari, 2009). All media used in culturing
fungi must be sterilized before use. Steam sterilization by autoclaving is the
customary method of sterilizing most culture media, but it cannot be used with heat
labile compounds.

Often preparations seem to contain only spores or mycellium, or structures

that are so unlike any of the illustrations available that they are unidentifiable. Most
of these problems can be overcome with a little practice and will, in time, seem
trivial. Gently flame a scalpel to sterilize, and cut a 5 mm square block of the
medium fro the plate of Potato dextrose agar medium. Pick up the block of agar by
inserting the scalpel and carefully transfer this block aseptically to the centre of the
slide. Inoculate four sides of the agar square with spores or mycelial fragments of
the fungus to be examined. Be sure to flame and cool the loop prior to picking up
spores. Aseptically, place a sterile cover glass on the upper surface of the agar cube.
Place a drop of metaline blue stain and distilled water on a clean microscope slide
and remove the cover glass from the slide culture and discard the block of agar.
Slide cultures must be observed only after a coverslip has been removed from the
agar plug and not while it is in position on the top of the agar plug (Rijal N, 2022).
Fungi are identified mostly by close examination of its morphology and the
characteristics it possess. It is a rapid method of preparing fungal colonies for
examination and identification (Prescott, 2008).

Under favourable environmental conditions, fungal spores germinate and

form hyphae. During this process, the spore absorbs water through its wall, the
cytoplasm becomes activated, nuclear division takes place, and more cytoplasm is
synthesized. The wall initially grows as a spherical structure. Once polarity is
established, a hyphal apex forms, and from the wall of the spore a germ tube bulges
out, enveloped by a wall of its own that is formed as the germ tube grows. The
colony morphology in this fungal growth under a light microscope using 4mm
magnifications lens are hairy, yellowish-brown bubble and have dark circle colour. On
10 mm magnification, our group focus at the edge of fungi colony.The fungal growth
under light microscope magnifications 10mm are focus on the edges. There’s a
pathogen in Rhizoctonia which has right angle branching, septations and hypha that
is slightly constricted where branching occurs. For the 40 mm magnification focus on
the region where the cell wall extends continuously to produce a long hyphal tube.
Growth of hyphae in most fungi takes place almost exclusively in the apical zone
(i.e., at the very tip). This is the region where the cell wall extends continuously to
produce a long hyphal tube. The cytoplasm within the apical zone is filled with
numerous vesicles. These bubblelike structures are usually too small to be seen with
an ordinary microscope but are clearly evident under the light microscope.

In a compound microscope, the sample is illuminated from the bottom to

observe transmitted light, or from the top to observe reflected light. Light from the
sample is collected by an optical system consisting of two main lens groups: the
objective and the eyepiece, whose individual powers multiply to enable much higher
magnifications than those achieved by a simple microscope. The objective collects
light from the sample and typically has a magnification of 40-100 x (Schadler, 2021).

CONCLUSION.

In, conclusion, the objective of this practical has been successful. For the first
objective, we learn how to conduc fungal subculture and purification in the right
way. Next, we know how to use compound or light microscope technique in the lab
to see clearly fungal growth. Lastly, we know how to prepare fungal slides, record
and describe the observation.

REFERENCES

Schadler, K. (2021, September 21). An introduction to the light microscope, light

microscopy techniques and applications. Analysis & Separations from
Technology Networks. Retrieved December 1, 2022, from
https://www.technologynetworks.com/analysis/articles/an-introduction-to-the-light-
microscope-light-microscopy-techniques-and-applications-35192

Prescott, L. M., Harley, J. P., Klein, D. A., Willey, J. M., Sherwood, L. M., &
Woolverton, C. J. (2008). Microbiology. McGraw-Hill.

Miransari M, Smith D. Biotechnolog, 2009. Available at: docsdrive.com.

TeamLabmonk (2018). To study sub culturing of bacteria and fungus. Retrieved from
https://labmonk.com/to-study-sub-culturing-of-bacteria-and-fungus