STM 311O

ADVANCED FOOD MICROBIOLOGY

LAB 1
ISOLATION OF FUNGI FROM DIFFERENT SPICES AND
SPOILAGE FRUIT AND VEGETABLE

NAME: LOGAVATHANAA A/P SAMON

MATRIC NUMBER: UK 20826

PROGRAMME: FOOD TECHNOLOGY

LECTURER: DR. MOHD NIZAM LANI

Topic

Isolation of fungi from different spices

Objective

1. Isolate fungi from different spices.

2. To observe the phenotypic characteristic of different fungi grown on the selective agar from
different spices.

Materials

Samples - Ginger, spoiled apple fruit and some prepared slide of fungi.
Media - PCA, PDA, DRBC
Others - Chlorine solution and other common equipment for microbiological works.

Introduction

Fungi can be single celled or very complex multicellular organisms. They are found in just about any
habitat but most live on the land, mainly in soil or on plant material rather than in sea or fresh
water. A group called the decomposers grow in the soil or on dead plant matter where they play an
important role in the cycling of carbon and other elements. Some are parasites of plants causing
diseases such as mildews, rusts, scabs or canker. In crops fungal diseases can lead to significant
monetary loss for the farmer.

Procedure

Procedures are stated in jotter book.

Results

Sample Agar Plate Color

Front Behind Observation

DRBC White, Green, Pinky purple,  Spore formation

Apple Yellow, Pink purple, white, dark  Mycelium and
green hyphae present
PDA White hairy Dark grey, white  Spore at the
structure, greenish surface of the
black, dark green fruit
 Mycelium and
hyphae present
PCA Light brown, white Light yellow, white  Black spore
 Hairy structure
 Present of
mycelium and
hyphae
DRBC Pink, white, light Pink, yellow, white  Spore formation
Ginger yellow  Mycelium and
hyphae
PDA White, yellow Milky white, yellow  Black spore
present
 Mycelium and
hyphae present

PCA White ,yellow, light Pale yellow  No spore

yellow formation
 Mycelium
present
Discussion

The sample of ginger was washed with chlorine 5% solution for 3 times. This step is to
ensure all the impurities which attach in the surface of ginger is taken out. Lab manual mentioned
that we should wash sample for 80 minutes. But for this experiment we only wash it for below than
ten minutes since the sample is in very small volume. For those sample which with greater volume
should be washed for 80 minutes to remove all the impurities. After that the sample is washed with
distilled water for 3 times. Distilled water is used instead of tap water because tap water contains
chlorine, iron and some minerals. It maybe distracts the experiment.

The sample of ginger and apple is isolated by using aseptic technique into the 3 selected
plates. The three agar plates were chosen for this experiment as a medium for fungi to grow. They
are DRBC (Dichloran Rose Bengal Chloramphenicol), PDA (Potato Dextrose Agar) and PCA ( Plate Count
Agar).

DRBC Agar conforms with APHA guidelines for the mycological examination of foods,
containing chloramphenicol rather than chlortetracycline as originally proposed. DRBC Agar is a
selective medium that supports good growth of yeasts and molds. The presence of rose bengal in
the medium suppresses the growth of bacteria and restricts the size and height of colonies of the
more rapidly growing molds. Chloramphenicol is included in this medium to inhibit the growth of
bacteria present in environmental and food samples. Inhibition of growth of bacteria and restriction
of spreading of more-rapidly growing molds aids in the isolation of slow-growing fungi by preventing
their overgrowth by more-rapidly growing species. In addition, rose bengal is taken up by yeast and
mold colonies, which allows these colonies to be easily recognized and enumerated. DRBC agar
shows good result if it incubated at room temperature for 5 days.

Potato Dextrose Agar (PDA) is a general purpose medium for yeasts and molds that can be
supplemented with acid or antibiotics to inhibit bacterial growth. It is recommended for plate count
methods for foods, dairy products and testing cosmetics. PDA can be used for growing clinically
significant yeast and molds. The nutritionally rich base (potato infusion) encourages mold
sporulation and pigment production in some dermatophytes. When we using the medium to test
fungi growth the pH prefer is 3.5 where acidic condition inhibits some bacteria growth. Pda also
supplemented with antibiotics to inhibit bacterial growth. Incubation time and temperature for this
plate is room temperature and for 2 days. Inoculation of Potato Dextrose Broth with pure cultures of
yeasts can assist in their identification.

Plate Count Agar and Standard Methods Agar (Plate Count Agar; Tryptone Glucose Yeast
Agar) are used for obtaining microbial plate counts from milk and dairy products, foods, water and
other materials of sanitary importance. Enzymatic digest of casein provides the amino acids and
other complex nitrogenous substances necessary to support bacterial growth. Yeast extract primarily
supplies the B-complex vitamins, and dextrose is an energy source. TTC is reduced to the insoluble
formazan inside the bacterial cell producing red colored colonies. . Incubate hardened plates for 48 ±
3 hours at 32 ± 1°C (dairy products) or 35 ± 0.5°C (foods) in an aerobic atmosphere.
 By comparing the 3 experimented plates DRBC show a greater growth of fungi. The fungi
from the spoiled apple and ginger are more clearly and largely formed in DRBC plate compare to
PDA and PCA plates. Additionally, some bacteria found in PDA and PCA. This is maybe due to the
properties of agar which is not selective. DRBC agar is selective for fungi growth. On the other hand
PDA and PCA is not a differential medium.

Most scientists performing identifications on fungal samples still use traditional methods of
macroscopic and microscopic examination. The FF MicroPlate and Database provide a simple and
accurate method as an alternative or as a complement to these traditional methods that require a
high degree of skill, training, and judgment. The ‘Biolog FF MicroPlate’ performs 95 discrete tests
Simultaneously and gives a characteristic reaction pattern called a “fingerprint”. These fingerprint
reaction patterns provide a vast amount of information about the metabolic properties of each
fungus tested, along with a species level identification. The FF Database contains over 500 taxa of
fungi from over 120 genera.

“Fungi Identification Kit” is a prepared, self-contained slide culture kit system for the
identification of fungi is presented. The kit consists of a sterile two part, clear plastic container. In
the container is a block of an appropriate agar medium. The agar block is one square centimetre. In
the bottom of the container is affixed a piece of absorbent material. The absorbent material acts as a
sponge when dampened and provides a moist atmosphere during incubation. Over the agar block is
a sterile covering. Prior to inoculation the covering of the agar block is removed. The surface of the
agar block is inoculated with the fungus being identified. A sterile cover slip is placed on the surface
of the agar. The absorbent material is dampened with water, the cover of the container is replaced,
and the kit is incubated at the required temperature. After incubation the cover slip is removed and
examined microscopically.

There are several precautions in mycology studies. Besides caring out the experiment in
normal lab, carry out the experiment in special lab which reserved for mycology studies. For the
DRBC agar plate care should be taken not to expose this medium to light, since photo-degradation of
rose bengal yields compounds that are toxic to fungi. Heating Potato Dextrose Agar after acidifying
hydrolyzes the agar and may destroy the solidifying properties.
Attachment

Rhizopus sporangia zygotes

Mucor sporangia and zygotes

 Mucor sporangia

Sporangium Rhizopus
Sachramyces sp yeast budding
Penicillium blue mold
The apple and ginger samples and observation.