1. The Natural Habitat Of Microbes
Microbial cells in nature live as:
Populations: Populations are a group of related cells, generally
derived by successive cell divisions from a single parent.
Habitat: The location in which populations live is called
a habitat (eg hot spings, GI tract, skin etc).
Community: An assemblage of different types of populations
living in the same habitat is called a community. Different populations
in a community interact (beneficial, harmful or neutral interactions occur).
Ecosystem: The interactions that occur between the chemical
& physical properties of the environment with that of the organnisms
is collectively called an ecosystem. Eg, microbes grow in their habitat,
deplete nutrients required for growth and excrete wastes, thus changing
the environment they live in.
2. Laboratory Studies Of Microbes
Knowledge on microbial structures, physiology, biochemistry
and genetics can be better understood from homogenous populations rather
than form mixed populations. Homogenous populations can be obtained in
the laboratory by using microbiological procedures.
A. Microscopes and Microscopy
Microscopes magnify cells too small to see by naked eyes.
Microbial size or the structure to be viewed determines the
type of microscope that is used
Microscopes use visible or ultraviolet light (Brightfield,
Phase contrast and fluroscence microscopes) for low resolution (100 to
200 nm) and electrons (Transmission Electron and Scanning Electron) for
high resolution (1 to 10 nm).
Bright Field Microscope:
To understand microscopes it is important to understand the
properties of light and how the light interacts with the object:
Visible light is a narow range of the spectrum of electromagnetic
radiation with wavelengths of approximately 400 (blue light) to 700nm (red
light) that we are able to see. (The electromagnetic radiation spectrum
includes Gamma rays, X-rays, UV light, Visible light, Infrared and
Light passes through an object (transmission) or is
bounced of an object (refraction). When light passes through a fine
opening (aperture) light is broken into light of differing wavelengths
(diffracted). Numerical aperture (NA) is a property of a
lens that describes the amount of light that can enter it.
Light bends (refracted) as it moves from one medium
to another (air to glass slides to microscope lens). The degree of bending
is called it's refractive index (RI) and the shape of the lens determines
the degree of bending (a convex-convex lens pair bends parallel light rays
so that the light is focussed at one point).
Light can be adsorbed at one wavelength (excitation) and
emitted at another that is at a lower energy level (emmision) and this
phenomenon is called fluorescence. Some molecules emmit fluorescence
naturally (photosynthetic pigments) whereas others can be "made" fluorescent
by dyes (FITC, Rhodoamine).
In compound microscopes, a series of lenses enable magnification
to be achieved (two convex-convex lenses, an ocular lens and
an objective lens). Magnification = magnifying power of occular
x magnifying power of the objective lenses.
Resolution is the degree of detail that is retained
in the magnified image. This is determined by the resolving power
(R) ie the the closest spacing between two points at which the points can
still be clearly seen as individual entities.
Shorter wavelength of illuminating light (blue light, 400nm
as opposed to red light, 700nm), increased value of Numerical Aperture
(NA) and small R all improve resolution.
Oil immersion lenses has short focal length ie the plane
of focus is near the lens and therefore there is a short working distance
between the objective lens and the specimen. Short working distance results
in a very shallow depth of field, ie only a very thin section of
the specimen cen be in focuss at any one time. (The short working distance
and shallow depth results in students have problems focussing bacteria
on slides and and leads to slide breakages). Oil immersion lens also have
an RI in common with glass rather than air which improves resolving power.
are organic compounds that have affinity for specific cellular material
and increase contrast.
Most common laboratory microscope
Cells & structures of a cell absorb
& scatter light in varying degrees. Eucaryotes have enought contrasting
structures and can be viewed without staining ie live cells observed.
Bacteria lack contrast & are stained
prior to observation (rarely used for observing live bacterial cells)
(positively charged): Cell walls are negatively charged and hence positively
charged chromophores (chroma = color) can bind (methylene blue, crystal
violet, safrarnin). Dyes can be simple (cationic or anionic) or differential
types. A smear is prepared air dried, heat fixed and flooded with the dye
for a minute, washed, blotted, dried and observed using an oil immersion
(negatively charged): Negative staining with India Ink or Nigrosine. Negatively
charged dyes are repelled by the negative charges on the cell wall of bacteria
and hence the background is colored but not the cell. Used to demonstrate
Dyes: Gram staining (the thick cell walls of Gram-positive bacteria do
not allow the alcohol to penetrate into the cell and hence are not destained
whereas the thin Gram-negative cells are), flagella staining and spore
staining are examples of this.
Phase Contrast Microscope:
Cells differ in their RI from their surrounding medium and
hence bend some of the light rays that pass throught them.
Unstained bacterial cells can be observed
A fluorescent substance emits light of one color when light
of a different color shines on it.
Specimens are visualised because they contain fluorescent
substance (chlorophyll = brilliant red) or are treated with a fluorescent
dye (FITC, Texas Red, Rhodoamine, TAMARA).
Used in medical microbiology and immunology laboratories.
Used for studying cellular structural details and viruses.
Transmission Electron Microscope (TEM):
A beam of electrons is used (60 to 80 kV accelerating voltage
produces a wavelength of 0.005 nm which is much much shorter than that
of visible light and hence a theoretical resolution of 0.2nm can be achieved).
Cells are too thick to be seen thro' a TEM and thin sections
are prepared (using an ultramicrotome) following a special protocol (cells
are dehydratedfixed The sections are stained with heavy metals such as
Osmium tetraoxide, Uranyl acetate or phosphotungstic acid which scatter
Scanning Electron Micoscope (SEM):
External rather than internal structures are observed
by a Scanning Electron Microscope (SEM). Gold is used as a stain for scattering
B. Culturing Microbes
It is essential to study pure cultures of microorganisms.
A pure culture is a culture consisting of only one type of microorganism.
This pure culture multiplies and cell numbers increase. This process is
known as growth (Growth is primarily an increase in numbers).
A pure culture is obtained by the principle of cloning. A
clone is a collection of cells derived from one cell. A pure culture
can also be called a clone.
Microbes grow rapidly (E. coli doubles every 20 mins)
and contaminants (unwanted microbes) have to kept out of the
When dealing with pathogenic microbes, precautions to prevent
infection must be taken. Manipulation of microbes so as to prevent unwanted
spreading and unwanted contamination of the culture under investigation,
is known as aseptic techniques. (PLEASE READ THE INSTRUCTIONS FOR
SAFE HANDLING OF MICROBES IN THE ASSOCIATED LABORATORY).
Microbes are grown in water containing appropriate nutrients
called culture medium. Culture medium contains:
Energy source: Organic (starch , glucose, proteins,
lipids) -- heterotrophs; inorganic compounds (Fe+ NH4
H2S, So, H2 ) -- lithotrophs; light
(cyanobacteria, green bacteria, algae) -- phototrophs. Lithotrophs
and phototrophs use and grow completely on inorganic medium using CO2
as the sole carbon source. They are also know as autotrophs.
Cell structural components: Metals such as Ca, Mg, Mn, Fe
for enzyme functioning eg metaloenzymes.
Environmental factors: Temperature: grow fastest at the optimum
temperature but span 5 to 110oC (pathogens = 37oC;
soil & water = 25oC), pH (acidic, alakaline, neutral), oxygen
(aerobic, anaerobic, facultative & microaerophilic)
The liquid culture medium can be converted into a solid form
by adding agar (sea weed extract) and heated to boiling in order to disolve
it. The agar containing medium is cooled to 50 - 55oC and dispensed
into flat round dishes (petri dishes)
The culture medium is sterilized (steam under pressure)
to render it free of contaminants prior to use.
Steps to grow microbes in the laboratory from natural samples:
Enrichment cultures: Increase numbers of bacteria in culture
Purification and isolation by streaking agar plates
Pure cultures are preserved by lyophilization or by freezing
for future use. Cultures can also be purchased commercially from ATCC or
Author and HTML'd by: Dr Bharat Patel <B.Patel@griffith.gu.edu.au>
[Created 20 Sept 1995]
[Modified 2 Aug 1997]