INTRODUCTION

Fungi are common food-borne microorganisms. The fungi are capable to live within
unfavourable condition as it can derive spores. The major factors that affect fungi growth
are humidity, availability of carbon and nitrogen source, and temperature.
Various types of fungi are known as food-borne. In this experiment, students are exposed
in handling inoculation of fungi from their own rotten food. Spore counting is the method
used to measure the spores produced from cultured fungi. In this practical growth, a
hemocytometer is used to count the spores harvested.

EXPERIMENT 5.1
Inoculation of Fungi from Rotten Food.

OBJECTIVES
1. To observe food-borne fungi on the rotten samples
2. To make an inoculation of fungi from rotten food

MATERIALS
Sample
 Rotten food
Apparatus:
 Slant containing Potato Dextrose Agar (PDA)
 Wire loop
 Face mask
Chemicals:
Potato Dextrose Agar (PDA)
METHOD
1. A face mask was took and it was wore properly before starting the experiment
2. The inoculating loop was placed on the Bunsen burner flamed until it was red hot
3. The loop was allowed to cool and a small piece of fungal sample was scraped from
the rotten food.
4. The sample was placed at the middle of the sterile slant agar (PDA) and it was
closed it by hand tightly.
5. It was incubated for 3 days at room temperature or 35°C in an incubator

EXPERIMENT 5.2
Spore counting

OBJECTIVE
 To count spores from the cultured fungi by using a hemocytometer

MATERIALS
Sample:
 Fungi spores in slant agar
Apparatus:
 Pasteur pipette
 Face mask
 Compound microscope
 Hemocytometer
Chemicals:
 0.001% v/v Tween 80 solution
METHODS:
1. A suspension of spores was made by mixing 10 mL of Tween 80 solution into the
slant agar bottle
2. Clean hemicytometer was obtained. A drop of the spores suspension was
transferred to the center of hemocytometer using a Pasteur pipette
3. A clean coverslip was placed over the slide and allowed the spores suspension to
flow into the chamber of the hemocytometer.
4. The hemocytometer was placed on a compound microscope. 10X magnification
was used to count the spores in the small squars of hemocytometer grid.
5. A small square was observed to determine the number of spores.
6. 30 smallest squares was choose randomly. The number of spores in 1 mL of
undiluted sample was calculated.

EXPERIMENT 5.3
Staining of Fungi

OBJECTIVES
 To make a slide by using spread technique
 To prepare, stain and make a slide of fungal smear
 To illustrate the observation of fungal smear

MATERIALS
Sample
 Mould
Apparatus:
 Compound microscope
 Scalpel
 Sterile Petri Plate with layers of filter paper
 Cork borer
 Wire loop
 Glass coverslip
 Glass slide
Chemicals:
 70% alcohol
 Lactophenol Cotton Blue (LPCB)
 Sterile distilled water

METHOD:
1. A drop of LPCB was placed on a slide
2. The sample of mould was taken using sterile scalpel or needle, and the mycelium
was transferred into the LPCB on the slide. The hyphae was teased out with the
helped of mounted needles.
3. The fungal specimen was covered with a coverslip. Air bubbles were avoided in
the solution.
4. The slide was examined using a microscope with the low and high power
objectives. The result was recorded.
5. Step 1 until 4 were repeated for the wild fungi and the observation of pure culture
fungi with the wild sample were compared.
RESULT:
Table 5.1:
Spore counting using hemocytometer
Square Spore Count
1 13
2 12
3 14
4 12
5 11

Total count spore = 62

62
Average of Spore = = 12.4
5
The number of spores per mL
1 1
Average spores per small square X X X Dilution
0.00025 1000
1 1
= 12.4 X X X Dilution
0.00025 1000
= 49.6 number of spores per mL
 Sample Characteristics
Mucor sp. Filamentous Structure

Sporangiophore
e
Magnification 4x10
Magnification 4x10
Reproductive Structure

Sporangium

Spore

Magnification 4x10
Aspergillus sp. Filamentous Structure

Conidiophore

Magnification 4x10
Magnification 4x10
Reproductive Structure

Conidia

Magnification 4x10
 Sample Characteristics
Penicillium sp. Filamentous Structure

Branch

Septate Hyphae

Magnification 4x10
Magnification 4x10
Reproductive Structure

Conidium

Magnification 4x10
Wild Fungi: Rotten Tomatoes Filamentous Structure

Sporangiophore

Magnificatin 4x10

Magnification 4x10 Reproductive Structure

Spores

Magnification 4x10
Table 5.2: Characteristics of Fungi
POST LAB QUESTIONS
1. List the factor that are favourable for fungi growth
 Temperature
 Light
 Aeration
 pH
 water activity
2. Discuss the precautionary steps involved in spore counting procedure
Prepare a hemacytometer and carefully clean all surface of the hemacytometer
and cover-slip. Ensure that all surface are completely dry using non-linting tissue.
Finally, center the cover slip on the hemacytometer.

3. Explain the purpose of using staining in fungal observation under light microscope
Most cells and their components are colourless in optical microscope. So, staining
enables the specimen to be seen as stains absorb or scatter light. Additionally,
most object in cells are too small to have any significant impact on absorbing light.
Stain are used to distinguish between cell based on their physical properties
DISCUSSION:
A hemocytometer is a device used to count number of cells/spores present in a
given sample solution. It consists of a thick glass microscope slide with a rectangular
indentation that creates a chamber. Each chamber is engraved with a laser-etched grid
of perpendicular lines. The area bounded by the lines and depth of the chamber is known.
It is therefore possible to count the number of cells or particles in a specific volume of
fluid, and thereby calculate the concentration of cells in the fluid overall.
Precaution step when using hemocytometer are to calculate based on type of
counting chamber. There are different types of counting chamber available, with different
grid size. Some counting chamber also have grid of different size. Take care to know the
grid height and size otherwise it will make the calculation error. Next use the provided
glass cover. They are thicker than the standard 0.15mm cover glasses. Therefore they
are less flexible and the surface tension of the fluid is standardized this way.
Penicillium oqueforti is used as a fungal starter culture for the production of a
number of blue-veined cheeses, with both proteolytic and lipolytic enzymes produced by
the fungus involved in cheese ripening and flavour production. Thus the fungus has the
lowest oxygen requirement for growth of any penicillium species.
Hypal extension in mucor is a non-stop septate fungi. It produce tube that can
extend for many milimeters. Three diemensional colonies of these fungi develop by lateral
branching, creating continuous network of cytoplasm. These colonies are multinucleate,
but do not multicellular. Colonies are septate fungi also proliferate through tip growth and
branching.
CONCLUSION:
The food-borne fungi on the rotten samples was observed. Inoculation of fungi from rotten
food were made. Filamentous Structure and reproductive structure for Mucor sp.,
Aspergillus sp., penicillium sp., and wild fungi were determined and labelled.

REFERENCES:
https://www.biotopics.co.uk/microbes/tech1.html
https://www.google.com/amp/s/www.researchgate.net/post/Which_stains_can_be_used
_for_staining_fungi/amp
https://www.rapidmicrobiology.com/news/detection-of-fungi-using-stainsstaining-
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