Part A | 2.5
20 Part A Marine Flora and Fauna
racks in a hot water bath (4555
ı C). Instead of this,
the medium may be placed in agar storage bottles and
kept in the refrigerator or on the laboratory shelf. If the
latter technique is used, the medium should be remelted
just before use and pipetted onto the plate with 10 ml
wide-mouth pipettes.
2.5.3 Isolation of Fungi
For the best results in the reading of plates, each plate
should contain 40 to 60 colonies of fungi. To accomplish this, the sample must be diluted before it is plated.
In general, liquids may be diluted only 1 W 10, a fairly
rich sewage may be diluted 1 W 100, sludge containing about 46% dry matter and relatively poor clayey
soils 1 W 1000, and richer, fairly dry materials (dry matter 3060%), as much as 1 W 10 000. When the sample
reaches the laboratory, a series of dilution flasks is
prepared. The samples are then serially diluted and inoculated in the medium, and plating is done by various
methods as per convenience. Different culture media
are available to culture the yeast and other groups of
fungi.
Incubation and Subculture of Isolated Fungi
Commonly, fungi can be grown at room temperature.
25
ı C is a standard temperature for the growth of fungi
and the temperature can be varied in the case of thermophilic fungi when it requires more than 50
ı C to
grow. For subcultures of fungi, a sterile cork-borer is
used. The media block is taken out and inoculated into
an another freshly prepared medium, and incubated at
a suitable temperature.
2.5.4 Identification of Fungal Isolates
Several techniques are available for the identification of
fungi. Because the nature of spore production apparatus
and the spores associated with it is fragile, there are two
techniques that are usually used. These are described
below.
Direct Mounting Technique or LCB Mount
A small portion of mycelium is taken from the medium
with a help of teasing needle. The needle must be incinerated and allowed to cool in laboratory conditions.
Care must be taken when the fungus is taken out from
the medium for mounting because the spores will be
diffused into the environment and this may affect the
individual dealing with the fungus; the laminar airflow
must be in off mode. A drop of lactophenol cotton blue
(LCB) solution is placed on the center part of the microscopic slide. The fungal mycelia should be mounted
over the LCB. The mycelia should be gently handled as
can break into pieces, which results in difficulties during the identification. Later a cover slip is placed over
the mount without air bubbles. The slide is placed under
a high power objective and the structure of the fungi is
observed and identified.
Agar Block Technique
Fungal media are prepared and cut into 1 1 cm blocks
with a sterile scalpel blade and placed over a slide.
A small piece of mycelia is transferred to the agar
block over the slide; a cover slip should be placed. The
whole setup is placed inside the petri dish and small
cotton soaked in water is also placed inside the dish for
maintaining the moisture. The Petri dish is incubated
at 25
ı C for a certain period of time. Once the spores
and mycelia have developed, the cover slip is removed
aseptically from the agar block placed over the slide
mounted with LCB, and observed under a high power
objective. Identification is carried out with the help of
a standard atlas and monograph using key characters
(Table 2.1).
Macroscopic Morphology of Fungi
1. Rate of growth: slow (714 days)
Rapid (27 days)
2. Topography: flat, regularly-folded, tangled
3. Texture: creamy, powdery cottony, mucoid, waxy
4. Color: front color of the colony, back color of the
colony.
Microscopic Examination of Slide Cultures
This will be done for the following characteristics:
A. Hyphae-pigmentation of hyphal elements, shapes
B. Asexual spores – simple or specialized asexual
spores
C. Conidiophores, conidia, macroconidia, and microconidia
D. Sporangiospores
E. Blastospores or chlamydospores
F. Spore size, shape, attachment to the mycelium, unicellular or multicellular, number of compartments
G. Rhizoids, columella.
2.5.5 Molecular Taxonomical Identification
Assembling taxa based primarily on morphological
similarities does not necessarily reflect phylogenetic re-
20 Part A Marine Flora and Fauna
racks in a hot water bath (4555
ı C). Instead of this,
the medium may be placed in agar storage bottles and
kept in the refrigerator or on the laboratory shelf. If the
latter technique is used, the medium should be remelted
just before use and pipetted onto the plate with 10 ml
wide-mouth pipettes.
2.5.3 Isolation of Fungi
For the best results in the reading of plates, each plate
should contain 40 to 60 colonies of fungi. To accomplish this, the sample must be diluted before it is plated.
In general, liquids may be diluted only 1 W 10, a fairly
rich sewage may be diluted 1 W 100, sludge containing about 46% dry matter and relatively poor clayey
soils 1 W 1000, and richer, fairly dry materials (dry matter 3060%), as much as 1 W 10 000. When the sample
reaches the laboratory, a series of dilution flasks is
prepared. The samples are then serially diluted and inoculated in the medium, and plating is done by various
methods as per convenience. Different culture media
are available to culture the yeast and other groups of
fungi.
Incubation and Subculture of Isolated Fungi
Commonly, fungi can be grown at room temperature.
25
ı C is a standard temperature for the growth of fungi
and the temperature can be varied in the case of thermophilic fungi when it requires more than 50
ı C to
grow. For subcultures of fungi, a sterile cork-borer is
used. The media block is taken out and inoculated into
an another freshly prepared medium, and incubated at
a suitable temperature.
2.5.4 Identification of Fungal Isolates
Several techniques are available for the identification of
fungi. Because the nature of spore production apparatus
and the spores associated with it is fragile, there are two
techniques that are usually used. These are described
below.
Direct Mounting Technique or LCB Mount
A small portion of mycelium is taken from the medium
with a help of teasing needle. The needle must be incinerated and allowed to cool in laboratory conditions.
Care must be taken when the fungus is taken out from
the medium for mounting because the spores will be
diffused into the environment and this may affect the
individual dealing with the fungus; the laminar airflow
must be in off mode. A drop of lactophenol cotton blue
(LCB) solution is placed on the center part of the microscopic slide. The fungal mycelia should be mounted
over the LCB. The mycelia should be gently handled as
can break into pieces, which results in difficulties during the identification. Later a cover slip is placed over
the mount without air bubbles. The slide is placed under
a high power objective and the structure of the fungi is
observed and identified.
Agar Block Technique
Fungal media are prepared and cut into 1 1 cm blocks
with a sterile scalpel blade and placed over a slide.
A small piece of mycelia is transferred to the agar
block over the slide; a cover slip should be placed. The
whole setup is placed inside the petri dish and small
cotton soaked in water is also placed inside the dish for
maintaining the moisture. The Petri dish is incubated
at 25
ı C for a certain period of time. Once the spores
and mycelia have developed, the cover slip is removed
aseptically from the agar block placed over the slide
mounted with LCB, and observed under a high power
objective. Identification is carried out with the help of
a standard atlas and monograph using key characters
(Table 2.1).
Macroscopic Morphology of Fungi
1. Rate of growth: slow (714 days)
Rapid (27 days)
2. Topography: flat, regularly-folded, tangled
3. Texture: creamy, powdery cottony, mucoid, waxy
4. Color: front color of the colony, back color of the
colony.
Microscopic Examination of Slide Cultures
This will be done for the following characteristics:
A. Hyphae-pigmentation of hyphal elements, shapes
B. Asexual spores – simple or specialized asexual
spores
C. Conidiophores, conidia, macroconidia, and microconidia
D. Sporangiospores
E. Blastospores or chlamydospores
F. Spore size, shape, attachment to the mycelium, unicellular or multicellular, number of compartments
G. Rhizoids, columella.
2.5.5 Molecular Taxonomical Identification
Assembling taxa based primarily on morphological
similarities does not necessarily reflect phylogenetic re-