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Lab 7 Objectives
Cultivation of Microorganisms
(part 1):
Since our lab exercises frequently make use of living microbes, it is necessary to understand how they can be successfully grown and
maintained in the laboratory. Microbes are typically grown on or within a variety of culture media.
A culture medium is defined as a nutritional substrate containing
substances required to grow microbes in the laboratory.
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Types of Culture Media:
Culture media used in microbiology come in many different forms. Based
on its formulation, each medium can be initially classified by its 1) physical
state, 2) chemical composition, or 3) functional type. Each of these
categories and their subdivisions are described below. |
Parasitic Worms:
The worms (or helminths) are multicellular organisms belonging
the Kingdom Animalia. Being heterotrophic, many species in this group have
evolved parasitic lifestyles, making them the causative agents of diseases in
most vertebrates, including humans. Although many of these worms are
macroscopic as adults, they exhibit microscopic life stages (e.g., egg, larva)
which make them appropriate subjects in microbiology. The life cycles of
these parasites can
be simple to complex, and may include more than one type of host. The following represent
some of the medically-important helminths known to parasitize humans.
The
Fungi: Yeasts and Molds
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Background and Purpose:
The fungi form a large, diverse group of eucaryotic organisms belonging to the Kingdom
Myceteae. Members include the mushrooms, bracket fungi, puffballs,
yeasts, and molds. Nutritionally, these organisms are heterotrophs.
After secreting exoenzymes into the environment, digested nutrients
are then absorbed. Most fungi exists as saprobes by decomposing
dead organic material, whereas some may also be parasites of living plants and
animals. Several species are true or opportunistic fungal pathogens
of humans.
In this lab we will
then examine the morphological characteristics of yeasts and molds. The
molds consists of filaments called hyphae. Most molds possess
septate hyphae where each filament is structurally composed of
individual cells separated by septa or cross-walls. These septa
possess central openings that permit cytoplasmic streaming between the cells.
Other molds feature
nonseptate hyphae which lack
cross-walls. Hyphae spread across the surface of a food source and
penetrate into it. Besides anchoring the mold to this material, they function to digest and absorb nutrients.
Hyphae which grows above the surface
produce fruiting bodies that form spores. After spores
disseminate to another suitable substrate, they can germinate to initiate the
formation of new colonies.
The molds we will examine include
four species. Penicillium and
Aspergillus possessing septate hyphae and produce conidia
(asexual spores) at the end of aerial hyphae called conidiophores.
These molds are also capable of producing
ascospores (sexual spores)
inside a sac-like structure, the ascus. In comparison, Rhizopus
and Mucor having nonseptate
hyphae and produce asexual sporangiospores within a sac called
the sporangium. The sporangia located at the end of aerial hyphae
are called sporangiophores. The sexual spores of these molds
called zygospores are formed when
the hyphae from two different strains fuse together.
Clinically, all the
mold species we will
study here can cause opportunistic infections in critically-ill or
immunocompromised patients. In addition to histological stains
and serological tests, accurate diagnosis of these and other mycoses are dependent upon
isolating and correctly identifying the fungal pathogens through macroscopic and
microscopic examination of laboratory cultures. When isolated on solid culture media, each mold
grows into an interwoven network of hyphae collectively called the
mycelium. The overall size,
color, texture, and organization of the vegetative and aerial mycelia are useful cultural
characteristics when identifying molds. Equally important, we will
concurrently examine
each mold's microscopic features. These include the type of hyphae
(septate or nonseptate) and spore-producing structures
(fruiting bodies) they exhibit.
We will
also examine three species of yeast. Unlike the molds, these microscopic fungi
do not form hyphae. Instead, yeasts are unicellular,
typically having spherical or oval shape cells. Reproduction is mostly accomplished
asexually through budding. During this process the parent cell
forms an outgrowth or bud that eventually detaches, becoming a new
daughter cell. If buds fail to separate from the parent cells, this may
lead to the formation of short chains of cells called a pseudohyphae.
When grown on a particular medium, some yeasts may exhibit sexual reproduction
and produce sexual spores.
The yeasts species we
will study have been reported to cause various opportunistic infections in
humans. Correct identification requires the examination of both macro- and
microscopic features. These characteristics, in conjunction with other
clinical tests and observations, are needed for accurate diagnoses.
It is worth mentioning
that some fungi may actually have both a mold and yeast life cycle stage.
These species are said to be dimorphic or biphasic. This ability
is largely determined by temperature, but it is also influenced by other factors
such as levels of oxygen and nutrients. True fungal
pathogens to humans are dimorphic. These include Histoplasma capsulatum and
Coccidioides immitis. In their natural habitats, these saprophytic
fungi grow as molds in soils where temperatures are usually lower than 30ºC.
However, when their spores gain entrance into a warm human body (37ºC),
these fungi germinate into a more invasive, parasitic yeast phase.
Structurally and physiologically, yeast cells are better adapted for survival at
the body's warmer temperature and are relatively resistant to phagocytosis.
Some yeasts may actually reproduce within phagocytes.
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General Procedure:
Wet mount slides and streak plates will be prepared from working
slant tube cultures of selected yeast species. Plate cultures will require
3-5 days of incubation at 25ºC. The wet mount
slides will be examined under the microscope to identify the morphological
features of yeast cells, whereas the streak plate cultures will be used to
identify macroscopic features of yeast colonies. Fungal spores from
working slant cultures will be aseptically transferred to prepare plate and
slide cultures of various molds. After a 5-day period of incubation at 25ºC, the morphological characteristics
of these mold cultures will be examined macroscopically and microscopically.
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Materials:
Sabouraud agar slant tube cultures of Saccharomyces,
Candida, Rhodotorula, Penicillium,
Aspergillus, Rhizopus, and Mucor, Sabouraud agar Petri plates, microscope
slides, cover glasses, lactophenol cotton blue, empty Petri plates, cotton balls, glass
support rods, microscope slides, cover glasses, paraffin wax, petroleum jelly, 3
cc disposable syringe, sterile Pasteur pipette and bulb, 400 ml beaker, 250 ml beaker, hot plate, glass stir rod,
50ºC
water bath, tube rack
 | Preparation of Mold Plate Cultures:
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Preparation
of a Mold Slide Culture:
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The Examination of Yeast Cultures:
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The Examination of Mold Cultures:
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View Mold Plate Cultures of Different Ages (24-96
hours):
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For Practice
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