Notes of lesson
History of Microbiology
Ancients felt the world filled with invisible spirits, which would
explain things we couldn't understand.
Death and Disease, Disability (there has to be a reason) We still
struggle with these things ideas today.
Greeks had anthropomorphic gods who interacted with them
and could cause disease. Later Greeks lost faith in their gods
and formulated other ideas. They were noted thinkers.
Hippocrates--disease comes from an imbalance of intrinsic
factors (nutrition) and extrinsic factors--air, exercise, etc.
Aristotle and others believed in abiogenesis. Life came from
inanimate material. Myths among many ancient people talk of the
origin of man, even, from decaying corn. Shakespeare wrote about
crockadiles coming from the mud of the Nile. Van Helmont wrote
recipe for mice---dirty underwear, corn, in a vessel---mice come
out fully formed. Solves another inexplicable problem: The origin
1609 Janssen and Galileo grind lenses to produce low resolution
microscopes---microorganisms below the resolution achieved. But
improvements were to come.
Hooke--Discovers cells with an improved microscope.
• Schleiden and Schwann discover all plants and animals are composed of
• 1650's Leewenhoek--Delft Holland in the textile industry and part time lens
grinder. Got good resolution to allow about 3 or 4 hundred X useful
magnification. He put hay and pepper into water and then looked at it
through his microscope. He saw microbes in an infusion as seen below.
• Leewenhoek saw bacteria, protozoa, yeasts and described all the microbial
forms we now know, except for viruses. Although he is not mentioned in
the science literature as observing his animalcules divide, nevertheless he
believed spontaneous generation was untrue and in his original papers
called the idea a "bad joke" as related by Dr. Moll at the University of
• Francesco Redi, in the 1700's did a simple experiment to show flies needed
parents. Used cheesecloth screening and meat.
Spallanzani, a monk, in the first part of the 18th Century boiled and sealed
broths. When he was careful no microbes developed. His work was criticized
by Needham, a Welchman of the Royal Society in that other factors, excluded
by Spallanzani, were needed for Sp. Gen. Notably air. Needham performed
similar experiments, sloppily executed, in which microbes grew from
• Spallanzani died before he could clearly disprove Needham.
• Louis Pasteur, a noted chemist, took up the challance and utilized broths
allowing air but disallowing microbes. Grew broths at different altitudes
and in a dusty cellar. Used broths with cotton to show the germs
accumulated on cotton. Did the Swan neck tube experiment.
• Ignaz Semmelweis, an eastern european physician working in a Vienna
Hospital, noticed the wards where delivery occurred by midwives had 10X
less Puerperal Fever and deaths than those tended to by doctors. He showed
he could dramatically decrease "Childbed Fever" by having doctors wash
hands in chlorine water after dissection of cadavers and between patients.
Was fired for blaming deaths on doctors who didn't wash hands.
Oliver Wendel Holmes wrote on the Contagiousness of Puerperal Fever".
Author, Physician, and Anatomy Professor. Late 19th Century.
Lister used antiseptics on wounds and during surgery. He showed they healed
much faster with the antiseptic treatment. Was also the first to isolate a pure
culture by serial dilution: Bacterium lactis.
Pasteur wanted to isolate a bacterium in pure culture that caused disease. Began
working with Anthrax.
Robert Koch in Germany did too. He worked with various preparations to
provide solid culture media. A woman working in his lab tried agar,
recommended by a cook who used it to solidify puddings because it stayed
solid at warm temps. Koch was using sterile potato slices for media.
Koch isolated anthrax and formulated his Postulates.
• Pasteur went to work on chicken cholera and discovered one could attenuate
cultures and produce artificial vaccines.
• Pasteur solved the riddle of rancid wines in France's vinyards. Recommended
sterile technique and Pasteurization (applied to milk and became a central
method for controlling TB, Diphtheria, and other diseases).
• Weinogradski and others showed soil bacteria recycle nutrients.
• Chamberland developed a bacterial filter. Resulted in:
• a. discovery of viruses b. discovery of toxins
• Pasteur produced attenuated rabies virus and rabies vaccination procedure.
Tried it on Joseph Meister. It worked.
During the 20th Century we have:
• 1. Development of viral culture techniques and attenuation
• 2. Development of the electron microscope.
• 3. Discovery of antibiotics (Fleming and Dubos)
• 4. Discovery of Prions (Pruissner)
• 5. Bioengineering--removal and replication of genes--
incorporating them into microbes and switching them on.
• 6. DNA vaccines
• 7. Antiviral Compounds--ribivirin, protease inhibitors.
• 8. Translation of the entire genome of some microorganisms e.g. yeast.
• All not rosy---reemergence of infectious diseases a constant problem. St
Louis Encephalitis, West Nile Virus, Lyme disease, AIDS, Hanta virus Sin
• Ebola outbreaks, E. coli, Antibiotic resistant bacterial strains and so on.
Includes the following:
• 1. Nomenclature
• 2. Taxonomy (which relates to Nomenclature)
• 3. Identification
• There has been an evolution, over the past 50 years, in the classification
schemes used for microorganisms.
• Early on we had: Fauna and Flora (or Plants and Animals)
• Later---1960's we have the introduction of 3 Kingdoms: Plants, Animals,
and Protists. Then Whittaker's 5 Kingdom System: Plant, Animal, Fungi,
Protists, and Prokaryotes (Monera).
• Currently, with the advent of molecular genetics techniques we have three
Domains serving as higher Taxa over Kingdom. Each domain has its
Kingdoms. Three domains = Eucarya, Archaea, and Eubacteria.
The domains arose because work with ribosomal RNA ca. 1500 base long
units, showed clearly the natural evolutionary history of the cell types.
Ribosomal RNA evolves more slowly than DNA because of a slower
mutation rate. So examination of base sequences more clearly shows natural
relationships (evolutionary relationships) between cells.
1. Binomial system of nomenclature.
2. Latin or Greek or latinized names. Bergey's Manual is the authority.
It is constantly changing. Sarcina lutea to Micrococcus luteus and
Streptococcus faecalis into Enterobacter faecalis. Zillions of more examples.
Taxonomic Heirarchy: = Artificial mechanism to classify living things. But it
can be used on any group of related types. For example:
Definition of a species is different in bacteria:
1. Not based on sex---many do not reproduce sexually
2. Some have "out of species" sexual transfer.
We are evolving in our definition of species from highly similar in
characteristics to a genetic definition. Also, strain is a subgroup of species.
Characteristics of bacteria:
2. Chemical Characteristics
3. Metabolic Characteristics
4. Genetic Testing.
Taxonomy--We use the same scheme eg. Class, Order, Family,
Genus, Species. We don't need to go higher than Family for all
• A. Examples of families: Picornaviridae, Poxviridae,
• B. Genera have the suffix "virus" after them. For
• It is the technical field of using microscopes to view samples or objects.
There are three well-known branches of microscopy, Optical,Electronand
Scanning Probe Microscope
• Optical and electron microscopy involve the diffraction, reflection, or
refraction of electromagnetic radiation incident upon the subject of study,
and the subsequent collection of this scattered radiation in order to build up
an image. This process may be carried out by wide field irradiation of the
sample (for example standard light microscopy and transmission electron
microscopy) or by scanning of a fine beam over the sample (for example
confocal microscopy and scanning electron microscopy).
• Scanning probe microscopy involves the interaction of a scanning probe
with the surface or object of interest. The development of microscopy
revolutionized biology and remains an essential tool in that science, along
with many others.
• Resolving Power
• measures the ability to distinguish small objects close together
• r.p. = 0.61 (lambda)
Where lambda = wavelength of illuminating light.
• R.P. is smallest for violet light, but because human eye is more sensitive to
blue, optimal R.P. is achieved with blue light (~450 nm). Use filters to
remove other light in best microscopes
• n sinØ is called numerical aperture. It measures how much light cone
spreads out between condenser & specimen. More spread = better
resolution. Ø = angle of light cone; maximum value is 1.0
• n = refractive index. n = 1.0 in air. Can increase with certain oils (up to
1.4), called immersion oil. N.A. is property of lens. Look on side of lens to
• Theoretical limit of R.P. for light scope is 0.2 micrometers.
Optical Resolving Power RP in Angstroms
Human eye 0.2 millimeters 2,000,000 A
Light microscope 0.20 micrometers 2000A
Scanning electron 5-10 nanometers 50-100A
microscope (SEM) (nm)
Transmission 0.5 nanometers 5A
electron microscope (nm)
Bright Field Microscopy
• With a conventional bright field microscope, light from
an incandescent source is aimed toward a lens beneath
the stage called the condenser, through the specimen,
through an objective lens, and to the eye through a
second magnifying lens, the ocular or eyepiece. We see
objects in the light path because natural pigmentation or
stains absorb light differentially, or because they are
thick enough to absorb a significant amount of light
despite being colorless. A Paramecium should show up
fairly well in a bright field microscope, although it will
not be easy to see cilia or most organelles. Living
bacteria won't show up at all unless the viewer hits the
focal plane and distorts the image by using maximum
Bright Field Microscopy
• Physicists discovered electrons have wave properties. Can use
magnetic coils like lenses to focus beams of electrons. Basic
design of EM similar to light scope
• But: electrons don't scatter from H, C, O, N: must add heavy
atoms (e.g. Pb, Ur, Os, Gold) as stains.
• Also, electrons are scattered by air molecules. So must remove
air from microscope with vaccum pump. But water in specimen
will evaporate, so must be removed by dehydration after
fixation. Cannot view living specimens.
Transmission Electron Microscope (TEM)
• See slide. R.P. approx. 1000x better than light; 0.2 nm, instead of 0.2
• Excellent for seeing internal detail. But cannot use with large/thick
• Specimen Preparation: specimen must be thin. Use grids with thin film
supports. Prepare thick materials by sectioning with glass knives sections
about 20-100 nm thick. Prepare small preparations (viruses, or subcellular
particles) by negative staining.
Transmission Electron Microscope (TEM)
Scanning Electron Microscope (SEM)
• Same principle as TV screen, except reflected (secondary) electrons used
to produce magnified image.
• complementary to TEM. Only see surface view --no internal detail visible.
Infinite depth of focus, in contrast to light scopes.
• R.P. around 2 nm at best, usually a bit poorer. (100x better than light scope,
not as powerful as TEM)
• Specimen Preparation: fix & dry specimen. Shadow with thin metal film
(e.g. gold). Mount on block and scan. (Note: sometimes possible to use
ordinary air-dried material; but charge builds up on surface, distorts image).
Scanning Electron Microscope (SEM)
• Staining is a biochemical technique of adding a class-specific
(DNA, proteins, lipids, carbohydrates) dye to a substrate to
qualify or quantify the presence of a specific compound. It is
similar to fluorescent tagging.
• Stains and dyes are frequently used in biology and medicine to
highlight structures in biological tissues for viewing, often
with the aid of different microscopes. Stains may be used to
define and examine bulk tissues (highlighting, for example,
muscle fibers or connective tissue), cell populations
(classifying different blood cells, for instance), or organelles
within individual cells.
• Biological staining is also used to mark cells in flow
cytometry, and to flag proteins or nucleic acids in gel
• Both Gram-positive (Gm+) and Gram-negative (Gm) organisms form a
complex of crystal violet and iodine within the bacterial cell during the
Gram-staining procedure. Gm+ organisms are thought to resist
decolorization by alcohol or acetone because cell wall permeability is
markedly decreased when it is dehydrated by these solvents. Thus, the dye
complex is entrapped within the cell, resist being washed out by the
solvents, and Gm+ bacteria remain purple following this differential stain.
• In contrast, cell wall permeability of Gm- organisms is increased by ethyl
alcohol washing because it removes the outer membrane from the Gram-
negative cell wall. This allows the removal of the crystal violet-iodine
complex from within the cell. The decolorized Gm- cell can then be
rendered visible with a suitable counterstain, in this case Safranin O, which
stains them pink. Pink which adheres to the Gm+ bacteria is masked by the
purple of the crystal violet.
REAGENTS FOR THE GRAM STAIN:
Crystal violet (Hucker's Stain)
Gram's iodine: Dissolve 0.33 g of iodine and 0.66 g of potassium
iodide in 100 mL of distilled water.
Alternately, dilute 0.1 N iodine 1:4. (Gram's Iodine
solution should be fresh. If it has weakened and
appears tan it will not work.)
Ethyl alcohol (95%)
Counterstain stock solution : Dissolve 2.5 g of certified safranin 0 in
100 mL of 95% ethyl alcohol.
Counterstain working solution: Dilute stock solution 1:10 with
• After the smear has been dried, heat-fixed, and cooled off, proceed as follows:
• Place slide on staining rack and cover specimen with crystal violet. Let stand
for 1 minute.
• Wash briefly in tap water and shake off excess.
• Cover specimen with iodine solution and let stand for 1 minute.
• Wash with water and shake off excess.
• Tilt slide at 45° angle and decolorize with the acetone-alcohol solution until the
purple color stops running. Wash immediately with water and shake off excess.
• Cover specimen with safranine and let stand for 30 seconds to 1 minute.
• Wash with water, shake off excess, and gently blot dry. The smear is now ready
to be read. (Use oil immersion lens.)
Acid fast staining
• Purpose: To differentiate between acid-fast and non acid-fast bacteria.
• Principle: Some bacteria contain a waxy lipid, mycolic acid, in there cell
wall. This lipid makes the cells more durable and is commonly associated
with pathogens. Acid fast cell walls are so durable that the stain (carbol
fuschin) must be driven into the cells with heat. The cells are then
decolorized with acid-alcohol, all other cells will decolorize with this
strong solvent, but acid fast bacteria will not. Other cells are then
counterstained with methylene blue.
• 1. Deparaffinize and hydrate to distilled water.
• 2. Carbol-fuchsin solution, microwave 80 power, 45 seconds, allow slides
to stand in hot solution for 5 minutes. Filter solution once a week.
• 3. Wash in running tap water.
• 4. 1% Acid alcohol until light pink and color stops running.
• 5. Wash in running tap water for 5 minutes.
• 6. Rinse in distilled water.
• 7. Working methylene blue for 30 seconds.
• 8. Rinse in water. Dehydrate, clear, and coverslip.
Acid fast staining
• 1.With a wax pencil, label the left-hand comer of a clean glass slide with
the name of the bacterium that will be stained.
• 2. As shown in figure 15.3, aseptically transfer a loopful of culture with an
inoculating loop to the slide. Allow the slide to air dry. Do not heat-fix!
• 3. Place the slide on a staining rack. Flood the slide with crystal violet and
let stand for 4 to 7 minutes.
• 4. Rinse the slide thoroughly with 20% copper sulfate.
• 5. Blot dry with bibulous paper.
• 6. Examine under oil immersion (a coverslip is not necessary) and draw the
respective bacteria in the report for exercise 12. Capsules appear as faint
blue halos around dark blue to purple cells.
• 1. With a wax pencil, mark the left-hand comer of a clean glass slide
with the name of the bacterium.
• 2. Aseptically transfer the bacterium with an inoculating loop from the
turbid liquid at the bottom of the slant to 3 small drops of distilled water
in the center of a clean slide that has been carefully wiped off with clean
lens paper. Gently spread the diluted bacterial suspension over a 3 cm
area using the inoculating needle .
• 3. Let the slide air dry for 15 minutes.
• 4. Cover the dry smear with solution A (the mordant) for 4 minutes.
• 5. Rinse thoroughly with distilled water.
• 6. Place a piece of paper toweling on the smear and soak it with solution B
(the stain). Heat the slide in a boiling water bath for 5 minutes in an exhaust
hood with the fan on. Add more stain to keep the slide from drying out.
• 7. Remove the toweling and rinse off excess solution B with distilled water.
Flood the slide with distilled water and allow it to sit for 1 minute while
more silver nitrate residue floats to the surface.
• 8. Then, rinse gently with water once more and carefully shake excess
water off the slide.
• 9. Allow the slide to air dry at room temperature.
• 10. Examine the slide with the oil immersion objective. The best
specimens will probably be seen at the edge of the smear where bacteria
are less dense.
GROWTH OF BACTERIA
Nutritional Requirements of Cells
• Every organism must find in its environment all of the
substances required for energy generation and cellular
biosynthesis. The chemicals and elements of this environment
that are utilized for bacterial growth are referred to as
nutrients or nutritional requirements. Many bacteria can be
grown the laboratory in culture media which are designed to
provide all the essential nutrients in solution for bacterial
• Many bacteria can be identified in the environment by
inspection or using genetic techniques, but attempts to isolate
and grow them in artificial culture has been unsuccessful. This,
in part, is the basis of the estimate that we may know less than
one percent of all procaryotes that exist.
Major elements, their sources and functions in
Element % of dry Source Function;
Carbon 50 organic compounds or CO2, Main constituent of cellular material
Oxygen 20 H2O,organic compounds, Constituent of cell material and cell water O2 is electron acceptor in aerobic
CO2, and O2 respiration
Nitrogen 14 NH3 , NO3, organic Constituent of amino acids, nucleic acids nucleotides, and coenzymes Main
Hydrogen 8 H2O, organic compounds, H2 constituent of organic compounds and cell water
Phosphorus 3 inorganic phosphates (PO4) Constituent of nucleic acids, nucleotides, phospholipids, LPS, teichoic acids
Sulfur 1 SO4, H2S, So, organic sulfur Constituent of cysteine, methionine, glutathione, several coenzymes
Potassium 1 Potassium salts Main cellular inorganic cation and cofactor for certain enzymes
Magnesium 0.5 Magnesium salts Inorganic cellular cation, cofactor for certain enzymatic reactions
Calcium 0.5 Calcium salts Inorganic cellular cation, cofactor for certain enzymes and a component of
Iron 0.2 iron salts Component of cytochromes and certain nonheme iron-proteins and a
cofactor for some enzymatic reactions
are metal ions required by certain cells in such small
amounts that it is difficult to detect (measure) them, and it is not
necessary to add them to culture media as nutrients.
Trace elements are required in such small amounts that they are
present as "contaminants" of the water or other media components.
As metal ions, the trace elements usually act as cofactors for
essential enzymatic reactions in the cell.
One organism's trace element may be another's required element
and vice-versa, but the usual cations that qualify as trace elements
in bacterial nutrition are Mn, Co, Zn, Cu, and Mo.
Major nutritional types of procaryotes
Nutritional Type Energy Carbon Source Examples
Photoautotrophs Light CO2 Cyanobacteria, some
Purple and Green
Photoheterotrophs Light Organic compounds Some Purple and
Chemoautotrophs or Inorganic CO2 A few Bacteria and
Lithotrophs compounds, many Archaea
(Lithoautotrophs) e.g. H2, NH3,
Chemoheterotrophs Organic Organic compounds Most Bacteria, some
or Heterotrophs compounds Archaea
• This simplified scheme for use of carbon, either organic carbon or
CO2, ignores the possibility that an organism, whether it is an
autotroph or a heterotroph, may require small amounts of certain
organic compounds for growth because they are essential
substances that the organism is unable to synthesize from available
nutrients. Such compounds are called growth factors.
• Growth factors are required in small amounts by cells because
they fulfill specific roles in biosynthesis. The need for a growth factor
results from either a blocked or missing metabolic pathway in the
cells. Growth factors are organized into three categories.
• 1. purines and pyrimidines
• 2. amino acids
• 3. vitamins
Culture Media for the Growth of Bacteria
• The biochemical (nutritional) environment is made available as a
culture medium, and depending upon the special needs of particular
bacteria (as well as particular investigators) a large variety and types
of culture media have been developed with different purposes and
• A chemically-defined (synthetic) medium (Table 4a and 4b) is one
in which the exact chemical composition is known.
• A complex (undefined) medium (Table 5a and 5b) is one in which
the exact chemical constitution of the medium is not known
• A defined medium is a minimal medium (Table 4a) if it provides only
the exact nutrients (including any growth factors) needed by the
organism for growth.
A selective medium is one which has a component's) added to it which
will inhibit or prevent the growth of certain types or species of bacteria
and/or promote the growth of desired species.
A culture medium may also be a differential medium if allows the
investigator to distinguish between different types of bacteria based on
some observable trait in their pattern of growth on the medium.
An enrichment medium (Table 5a and 5b) contains some component
that permits the growth of specific types or species of bacteria, usually
because they alone can utilize the component from their environment.
Physical and Environmental Requirements for
• The Effect of Oxygen
• The Effect of Temperature on Growth
• The Effect of pH on Growth
• Water Availability
Bacterial Growth Curve
• A growth curve in biology generally concerns a measured property
such as population size, body height or biomass. Values for the
measured property can be plotted on a graph as a function of time.
• Bacterial Growth Curve:
The schematic growth curve shown below is
associated with simplistic conditions known as a batch culture.
It refers to a single bacterial culture, introduced into and growing in
a fixed volume with a fixed (limited) amount of nutrient. Industrial
situations involving MIC tend to be much more complex in nature
than such a simplified model.
Bacterial Growth Curve
• Lag Phase:
Bacteria are becoming "acclimated" to the new
environmental conditions to which they have been introduced (pH,
temperature, nutrients, etc.). There is no significant increase in
numbers with time.
• Exponential Growth Phase:
The living bacteria population increases rapidly with
time at an exponential growth in numbers, and the growth rate
increasing with time. Conditions are optimal for growth.
• Stationary Phase:
With the exhaustion of nutrients and build-up of
waste and secondary metabolic products, the growth rate has
slowed to the point where the growth rate equals the death rate.
Effectively, there is no net growth in the bacteria population.
• Death Phase:
The living bacteria population decreases with time,
due to a lack of nutrients and toxic metabolic by-products.
Microbes structure and
Organization and Structure of
• Phylogenetic relationships amongst different cell
• Based on ribosomal RNA sequence comparsions
• 3 basic groups or domains established (domains are
a higher order than kingdoms, ie are
• The 3 domain = Bacteria, Archaea and Eucarya
• 3 domains are related to each other; progenote =
hypothetical ancient universal ancestor of all cells.
• Natural relationships amongst cells established
Diagramatic representation of
• Microbes have different shapes and is of advantage
• Cell wall establishes the shape of a microbial cell but
environmenta conditions can change it
• Shapes include:
– Spheres called cocci (greek = berry) can divide once in one
axis to produce diplococci (Neisseria gonnorrhoeae, N.
meningitidis), or more than once to produce a chain
(Streptococcus pyogenes), divides regularly in two planes
at right angles to produce a regular cuboidal packet of cells
(xxx) or in two planes at different angles to produce a
cluster of cells (Staphyloccus aureus)
– Cylinders called rods or bacilli (Latin bacillus = walking
– Spiral or spirilli (Greek spirillum = little coil)
• Shape offers an advantage to the cell:
– Cocci: more ressistant to drying than rods
– Rods: More surface area & easily takes in dilute nutrients
from the environment
– Spiral: Corkscrew motion & therefore less ressistant to
– Square: Assists in dealing with extreme salinities
• At the simplest level, the function of the outer shells (CAPSID) of a
virus particle is to protect the fragile nucleic acid genome from:
• Physical damage - Shearing by mechanical forces.
• Chemical damage- UV irradiation (from sunlight) leading to
• Enzymatic damage - Nucleases derived from dead or leaky cells or
deliberately secreted by vertebrates as defence against infection.
• The protein subunits in a virus capsid are multiply redundant, i.e.
present in many copies per particle. Damage to one or more
subunits may render that particular subunit non-functional, but does
not destroy the infectivity of the whole particle. Furthermore, the
outer surface of the virus is responsible for recognition of the host
cell. Initially, this takes the form of binding of a specific virus-
attachment protein to a cellular receptor molecule. However, the
capsid also has a role to play in initiating infection by delivering the
genome from its protective shell in a form in which it can interact
with the host cell.
• General characteristics
Eukaryotic; placed in Kingdom Protista (also frequently
Photosynthetic pigments- four different kinds of chlorophyll
accessory pigments- a variety, including blue, red, brown,
Require moist environments (lack a waxy cuticle found in terrestrial
May be microscopic and float in surface waters (phytoplankton) or
macroscopic and live attached to rocky coasts (seaweeds)
Size ranges from size of bacteria (0.5 um) to over 50 m long (1
um = 1/25,000th inch; 1 m = 39 inches)
Lack vascular (conducting) tissues- no true roots, stems, or leaves
Modes of reproduction
Sexual and asexual -Have single-celled gametangia
(reproductive organs)- no multicellular reproductive
- Life history has 1, 2, or 3 stages (in contrast, plants have
2 stages, gametophyte and sporophyte)
– Red algae (Division Rhodophyta)
• Evolution: Red algae are some of the oldest eukaryotic
organisms on the planet. Fossils of red algae have been
found that are over 2 billion years old.
• Habitat: There are 4000 different species of red algae.
– They are very abundant in tropical and warm waters, although
many are found in cooler waters.
– Red algae are typically found in marine waters attached to
rocks or other plants in the calmer, deeper waters beyond the
– Some red algae are reef builders in tropical seas, as
important or more important than coral animals.
– The red algae act as habitat and food for some animals.
• Yeasts are single-celled but most fungal species are multicellular.
• Multicellular fungi are composed of filaments called hyphae (singular:
• Hyphae may contain internal crosswalls, called septa, that divide the
hyphae into separate cells. Coenocytic hyphae lack septa. The septa of
many species have pores, allowing cytoplasm to flow freely from one cell to
the next. Cytoplasmic movement within the hypha provides a means to
transport of materials.
• The hyphae may be branched. A dense mass of hyphae is called a
• Fungi have cell wall (like plants) but the cell walls are composed of chitin,
which is what arthropod (insects, crayfish, etc.) exoskeleton are composed
of. The cell walls of plants and some protists are composed of cellulose.
• The hyphae of some symbiotic fungi become specialized for penetrating the
cells of the host. These hyphae are called haustoria.
• Most fungi do not haveflagella in any phase of their lifecycle. They move
toward food by growing toward it.
• Fungi are categorized into phyla (divisions) based on the type of
structures produced during sexual reproduction.
• Some fungal species have not been classified into phyla based on
evolutionary relationships because they do not have a sexual phase
or because details regarding their sexual reproduction are unknown.
They are placed in a separate group called Deuteromycota. When
details concerning their evolutionary relationships become available,
they are reclassified into one of the other phyla.
• In general, the life cycle involves the fusion of hyphae from two
individuals, forming a mycelium that contains haploid nuclei of both
individuals. The fusion of hyphae is called plasmogamy. The fused
hyphae containing haploid nuclei from two individuals is
heterokaryotic. In some cases, plasmogamy results in cells with
one nucleus from each individual. This condition is called
dikaryotic. Eventually, two nuclei that originated from different
individuals fuse to form a diploid zygote. Meiosis then produces
either four haploid nuclei or four haploid cells.
LIFE CYCLE OF YEAST
Bacteriophage life cycle
• A lytic or lysogenic cycle:
• Lytic, coming from the same stem word for lysis, means
to cut/split. When a bacteriophage infects a bacterium on
a lytic mission, the host cell machinery is used to
produce bacteriophage components. Once a critical
mass is attained, the bacterial cytosol is filled with
bacteriophages and the host cell is sacrificed as it spills
open and releases phages into the environment.
• Lysogenic cycles are much less barbaric. Here, the
bacteriophage integrates itself into the bacterial genome.
When this occurs, the bacteriophage is called a provirus
and it is replicated with each generation of bacteria. This
can occur indefinitely or be triggered to cause the
bacteriophage to enter a lytic cycle.
• Heat: most important and widely used. For sterilization
one must consider the type of heat, and most
importantly, the time of application and temperature to
ensure destruction of all microorganisms. Endospores of
bacteria are considered the most thermoduric of all cells
so their destruction guarantees sterility.
• Incineration: burns organisms and physically destroys
them. Used for needles, inoculating wires, glassware,
etc. and objects not destroyed in the incineration
• Boiling: 100o for 30 minutes. Kills everything except
some endospores. To kill endospores, and therefore
sterilize a solution, very long (>6 hours) boiling, or
intermittent boiling is required (See Table 1 below).
• Autoclaving (steam under pressure or pressure cooker)
Autoclaving is the most effective and most efficient means of
sterilization. All autoclaves operate on a time/temperature
relationship. These two variables are extremely important. Higher
temperatures ensure more rapid killing. The usual standard
temperature/pressure employed is 121ºC/15 psi for 15 minutes.
Longer times are needed for larger loads, large volumes of liquid,
and more dense materials. Autoclaving is ideal for sterilizing
biohazardous waste, surgical dressings, glassware, many types of
microbiologic media, liquids, and many other things. However,
certain items, such as plastics and certain medical instruments (e.g.
fiber-optic endoscopes), cannot withstand autoclaving and should
be sterilized with chemical or gas sterilants. When proper conditions
and time are employed, no living organisms will survive a trip
through an autoclave.
• Dry heat (hot air oven): basically the cooking oven. The
rules of relating time and temperature apply, but dry heat
is not as effective as moist heat (i.e., higher
temperatures are needed for longer periods of time). For
example 160o/2hours or 170o/1hour is necessary for
sterilization. The dry heat oven is used for glassware,
metal, and objects that won't melt.
• Irradiation: usually destroys or distorts nucleic acids.
Ultraviolet light is commonly used to sterilize the
surfaces of objects, although x-rays, gamma radiation
and electron beam radiation are also used.
Electron beam (e-beam) radiation,
• Filtration involves the physical removal (exclusion) of all
cells in a liquid or gas. It is especially important for
sterilization of solutions which would be denatured by
heat (e.g. antibiotics, injectable drugs, amino acids,
vitamins, etc.). Portable units can be used in the field for
water purification and industrial units can be used to
"pasteurize" beverages. Essentially, solutions or gases
are passed through a filter of sufficient pore diameter
(generally 0.22 micron) to remove the smallest known
Treatment Temperature Effectiveness
Vaporizes organic material on nonflammable surfaces but may destroy
Incineration >500o many substances in the process
30 minutes of boiling kills microbial pathogens and vegetative forms of
Boiling 100o bacteria but may not kill bacterial endospores
Three 30-minute intervals of boiling, followed by periods of cooling
Intermittent boiling 100o kills bacterial endospores
Autoclave and pressure kills all forms of life including bacterial endospores. The substance
cooker (steam under 121o/15 minutes at 15# being sterilized must be maintained at the effective T for the full
pressure) pressure time
For materials that must remain dry and which are not destroyed at T
between 121o and 170o Good for glassware, metal, not plastic or
Dry heat (hot air oven) 160o/2 hours rubber items
Same as above. Note increasing T by 10 degrees shortens the sterilizing
Dry heat (hot air oven) 170o/1 hour time by 50 percent
Pasteurization (batch kills most vegetative bacterial cells including pathogens such as
method) 63o/30 minutes streptococci, staphylococci and Mycobacterium tuberculosis
Effect on bacterial cells similar to batch method; for milk, this method is
Pasteurization (flash more conducive to industry and has fewer undesirable effects on
method) 72o/15 seconds quality or taste
Ultrapasteurization (direct 140 /2 seconds Effect on most bacterial cells is lethal. For milk, this method creates a
method) product with relatively long shelf life at refrigeration temperatures.
• Chemicals used for sterilization include the gases ethylene oxide
and formaldehyde, and liquids such as glutaraldehyde. Ozone,
hydrogen peroxide and peracetic acid are also examples of
chemical sterilization techniques are based on oxidative capabilities
of the chemical.
• Ethylene oxide (ETO) is the most commonly used form of chemical
sterilization. Due to its low boiling point of 10.4ºC at atmospheric
pressure, EtO) behaves as a gas at room temperature. EtO
chemically reacts with amino acids, proteins, and DNA to prevent
microbial reproduction. The sterilization process is carried out in a
specialized gas chamber. After sterilization, products are transferred
to an aeration cell, where they remain until the gas disperses and
the product is safe to handle.
• Ozone sterilization has been recently approved for use in the U.S. It
uses oxygen that is subjected to an intense electrical field that
separates oxygen molecules into atomic oxygen, which then
combines with other oxygen molecules to form ozone.
Control of microbial growth by
• Types of antimicrobial agents
• Antiseptics: microbicidal agents harmless enough to be
applied to the skin and mucous membrane; should not
be taken internally. Examples include alcohols,
mercurials, silver nitrate, iodine solution, alcohols,
• Disinfectants: agents that kill microorganisms, but not
necessarily their spores, but are not safe for application
to living tissues; they are used on inanimate objects such
as tables, floors, utensils, etc. Examples include,
hypochlorites, chlorine compounds, lye, copper sulfate,
quaternary ammonium compounds, formaldehyde and
Chemical Action Uses
Ethanol (50-70%) Denatures proteins and solubilizes lipids Antiseptic used on skin
Isopropanol (50-70%) Denatures proteins and solubilizes lipids Antiseptic used on skin
Formaldehyde (8%) Reacts with NH2, SH and COOH groups Disinfectant, kills endospores
Antiseptic used on skin
Tincture of Iodine (2% I2
in 70% alcohol) Inactivates proteins Disinfection of drinking water
Forms hypochlorous acid (HClO), a strong oxidizing Disinfect drinking water; general
Chlorine (Cl2) gas agent disinfectant
General antiseptic and used in the eyes of
Silver nitrate (AgNO3) Precipitates proteins newborns
Disinfectant, although occasionally used
Mercuric chloride Inactivates proteins by reacting with sulfide groups as an antiseptic on skin
compounds) Disrupts cell membranes Skin antiseptics and disinfectants
(e.g. carbolic acid,
hexylresorcinol, Antiseptics at low concentrations;
hexachlorophene) Denature proteins and disrupt cell membranes disinfectants at high concentrations
Disinfectant used to sterilize heat-
sensitive objects such as rubber and
Ethylene oxide gas Alkylating agent plastics
Ozone Generates lethal oxygen radicals Purification of water, sewage
Chemical class Examples Biological source Spectrum (effective against) Mode of action
Penicillium Inhibits steps in cell wall
Beta-lactams notatum and (peptidoglycan)
(penicillins and Penicillin G, Cephalospori synthesis and
cephalosporins) Cephalothin um species Gram-positive bacteria murein assembly
Inhibits steps in cell wall
Semisynthetic Ampicillin, Gram-positive and Gram- synthesis and
penicillin Amoxycillin negative bacteria murein assembly
acid plus Streptomyces Gram-positive and Gram- Suicide inhibitor of beta-
Clavulanic Acid amoxycillin clavuligerus negative bacteria lactamases
Inhibits steps in cell wall
Chromobacter Gram-positive and Gram- synthesis and
Monobactams Aztreonam violaceum negative bacteria murein assembly
Inhibits steps in cell wall
Streptomyces Gram-positive and Gram- synthesis and
Carboxypenems Imipenem cattleya negative bacteria murein assembly
Streptomyces Gram-positive and Gram- Inhibit translation (protein
Aminoglycosides Streptomycin griseus negative bacteria synthesis)
Gram-positive and Gram-
Micromonospora negative bacteria esp. Inhibit translation (protein
Gentamicin species Pseudomonas synthesis)
Inhibits steps in murein
Streptomyces Gram-positive bacteria, esp. biosynthesis and
Glycopeptides Vancomycin orientales Staphylococcus aureus assembly
Gram-positive and Gram-
Streptomyces negative bacteria esp. Inhibits translation
Lincomycins Clindamycin lincolnensis anaerobic Bacteroides (protein synthesis)
Gram-positive bacteria, Gram-
negative bacteria not
Streptomyces enterics, Neisseria, Inhibits translation
Macrolides Erythromycin erythreus Legionella, Mycoplasma (protein synthesis)
Polypeptides Polymyxin Bacillus polymyxa Gram-negative bacteria membranes
Inhibits steps in murein
Bacitracin Bacillus subtilis Gram-positive bacteria assembly
Streptomyces Inactivate membranes
Polyenes Amphotericin nodosus Fungi containing sterols
Streptomyces Inactivate membranes
Nystatin noursei Fungi (Candida) containing sterols
Gram-positive and Gram-
negative bacteria, Inhibits transcription
Streptomyces Mycobacterium (eubacterial RNA
Rifamycins Rifampicin mediterranei tuberculosis polymerase)
Gram-positive and Gram-
Streptomyces negative bacteria, Inhibit translation (protein
Tetracyclines Tetracycline species Rickettsias synthesis)
Gram-positive and Gram-
Semisynthetic Rickettsias Ehrlichia, Inhibit translation (protein
tetracycline Doxycycline Borrelia synthesis)
Streptomyces Gram-positive and Gram- Inhibits translation
Chloramphenicol Chloramphenicol venezuelae negative bacteria (protein synthesis)
Industrial and Environmental
• Secondary metabolites are organic compounts
that are not directly involved in the normal
growth, development or reproduction of
organisms. Unlike primary metabolites, absence
of secondary metabolities results not in
immediate death, but in long-term impairment of
the organism's survivability/fecundity or
aesthetics, or perhaps in no significant change
at all. Secondary metabolites are often restricted
to a narrow set of species within a phylogenetic
Preservation of food
• A preservative is a natural or synthetic
chemical that is added to products such as
foods, pharmaceuticals, paints, biological
samples, wood, etc. to prevent
decomposition by microbial growth or by
undesirable chemical changes.
• Natural food preservation
• Health concerns
• The major types of fermentation are:
• Homolactic fermentation (simplest pathway; pyruvate goes directly
to lactic acid; no gas is produced; responsible for soured milk, in
production of many dairy products; Lactobacillus, Streptococcus,
• Alcohol fermentation (ethanol and CO2 are produced; yeasts like
• Heterolactic fermentation (ethanol, CO2, and lactic acid are
produced; Leuconostoc and Lactobacillus),
• Mixed acids fermentation (the characteristic tested for in the
methyl red test; E. coli).
• Butanediol fermentation (Acetoin, a precursor for butanediol, is
produced; Enterobacter aerogenes),
• Anaerobic butyric-butyric fermentation (organic solvents
produced including butanol and butyric acid; Clostridium sp.),
• Propionic acid fermentation (makes holes in Swiss cheese;
• Lab Materials:
• SDA culture of Saccharomyces cerevisiae
malt extract broth
hot plate and beaker with water
• Lab Exercise:
• Each group will:
• Inoculate a malt extract broth with Saccharomyces cerevisiae. Label
this tube A. Do not boil.
• Add three raisons to each of two tubes of sterile water. Label these
tubes B and C. Tube B will not be boiled or inoculated.
• Boil tube C for 10 minutes. Let the tube cool to room temperature,
and inoculate with Saccharomyces cerevisiae.
• Incubate all three tubes at room temperature.
• Bioremediation can be defined as any process
that uses microorganisms, fungi, green plants or
their enzymes to return the natural environment
altered by contaminants to its original condition.
Bioremediation may be employed to attack
specific soil contaminants, such as degradation
of chlorinated hydrocarbons by bacteria. An
example of a more general approach is the
cleanup of oil spills by the addition of nitrate
and/or sulfate fertilisers to facilitate the
decomposition of crude oil by indigenous or
• Genetic engineering approaches
• The use of genetic engineering to create
organisms specifically designed for
bioremediation has great potential. The
bacterium Deinococcus radiodurans (the
most radioresistant organism known) has
been modified to consume and digest
toluene and ionic mercury from highly
radioactive nuclear waste.
Leaching of ores by
• Microbiology of ore leaching
• Microbiological investigations revealed that certain bacteria are the
main agent in natural weathering of sulfidic heavy metal minerals.
• The principal bacteria which play the most important role in
solubilizing sulfidic metal minerals at moderate temperatures are
species of the genus Thiobacillus. They are gramnegative rods,
either polarly or nonflagellated. Most species are acidotolerant,
some even extremely acidotolerant and acidophilic. Some grow best
at pH 2 and may grow at pH 1 or even at pH 0.5. Most species are
tolerant against heavy metal toxicity.
• Thiobacilli are chemolithoautotrophs, that means CO2 may be the
only source of carbon and they derive their energy from a chemical
transformation of inorganic matter. All Thiobacilli oxidize sulfur or
sulfur compounds to sulfate or sulfuric acid.
• Simple laboratory experiments can show, that chemical reactions
catalyzed by bacteria are the essential processes which lead to
decay of sulfidic heavy metal minerals and some other minerals and
that abiotic reactions play a negligible role. If sulfidic ores are
percolated with simple water or diluted salt solutions under aeration
in laboratory percolators in parallel sets, one set not sterilized or
inoculated with natural acid mine effluent, another set under sterile
conditions, it can be seen that disintegration of ore and leaching of
metals proceeds in the not sterilized or inoculated percolators very
much quicker than in the sterilized ones, the ratio being about 104 or
higher. In such percolator experiments it is observed that almost all
the bacteria adhere to the pieces of ore and especially to the
surfaces of the sulfidic minerals. Only a small amount of bacteria is
floating free in the medium. So the bacteria are in close contact to
the almost insoluble substrate which they oxidize to yield energy.
This seems to be necessary because we can assume, that
solubilization of the minerals by some direct mechanisms requires
• The rate of dissolution of the metal
minerals is essentially limited by the
accessible surface of the minerals and can
be enhanced by grinding the minerals or
the pieces of ore resp. to smaller grains. If
the sulfidic minerals are not freely
exposed, but are embedded in rock, as is
normally the case with heavy metal ores,
the rate of leaching is limited above all by
the diffusion rates of solutes through
fissures. Oxygen, ferric ions and hydrogen
ions have to diffuse from the outside of the
piece of ore, to the metal minerals inside
and, conversely, metal, sulfate and
hydrogen ions have to diffuse out to the
surrounding medium, regardless of
whether the bacteria are within the
fissures on the sulfidic minerals or on the
outside of the piece of ore.
• Biofertilizers are mixed with little water for
preparing slurry. In that slurry small quantity of
sugar or jaggary or gum is added so that the
inoculant may get energy for their prolonged
survival. The slurry is poured over the seeds
which should be kept in a container. The seed is
mixed well with the slurry by pouring the mixture
into another container. Thus by pouring fourth
and back into both containers the seed is nicely
mixed with inoculant. Now the treated seed
should be dried in cool and dry shady place and
sown immediately in the field.
• Bio fertilizers are
• microbial products containing living cells of different types of micro
• possess the innate ability either to fix or mobilize important nutrient
elements from non-usable forms through biological process.
• needed to be applied to soil to enhance microbial activity in the
• playing a significant role in intigrated plant nutrient systems (IPNS)
• Types of Bio Fertilizers
– Nitrogen fixers
– Phosphate solubilisers
• Nitrogen fixers
• Among N-fixers azospirillum is widely recommended because of its
easy adaptability and limited host specificity.
• A micro aerophilic bacterium
• Associative symbiotic
• Lives inside the cortical cells and xylem vessels of plant roots.
• Also known to secrete growth promoting substances like gibberellic
acid and IAA which enhance root proliferation and growth of crop
• Having ability to fix 25-40 kg N/ha/year.
• Biopesticides are certain types of pesticides
derived from such natural materials as animals,
plants, bacteria, and certain minerals. For
example, canola oil and baking soda have
pesticidal applications and are considered
biopesticides. At the end of 2001, there were
approximately 195 registered biopesticide active
ingredients and 780 products. Biopesticides fall
into three major classes:
Advantages of using
• Biopesticides are usually inherently less toxic than conventional
• Biopesticides generally affect only the target pest and closely related
organisms, in contrast to broad spectrum, conventional pesticides
that may affect organisms as different as birds, insects, and
• Biopesticides often are effective in very small quantities and often
decompose quickly, thereby resulting in lower exposures and largely
avoiding the pollution problems caused by conventional pesticides.
• When used as a component of Integrated Pest Management (IPM)
programs, biopesticides can greatly decrease the use of
conventional pesticides, while crop yields remain high.
• To use biopesticides effectively, however, users need to know a
great deal about managing pests.
Microorganism and pollution control
• It is the process of reducing or eliminating the release of
pollutants into the enviroment. It is regulated by various
environmental agencies which establish pollutant
discharge limits for air, water, and land.
• Air pollution control strategies can be divided into two
categories, the control of particulate emission and the
control of gaseous emissions. There are many kinds of
equipment which can be used to reduce particulate
emissions. Physical separation of the particulate from
the air using settling chambers, cyclone collectors,
impingers, wet scrubbers, electrostatic precipitators, and
filtration devices, are all processes that are typically
• Condensers operate in a manner so as to
condense vapors by either increasing the
pressure or decreasing the temperature of the
gas stream. Surface condensers are usually of
the shell-and-tube type, and contact condensers
provide physical contact between the vapors,
coolant, and condensate inside the unit.
• Flaring and incineration take advantage of the
combustibility of a gaseous pollutant. In general,
excess air is added to these processes to drive
the combustion reaction to completion, forming
carbon dioxide and water.
• Water pollution control methods can be subdivided into physical,
chemical, and biological treatment systems. Most treatment systems
use combinations of any of these three technologies. Additionally,
Water conservation is a beneficial means to reduce the volume of
• Solid pollution control methods that are typically used include
landfilling, composting, and incineration. Sanitary landfills are
operated by spreading the solid waste in compact layers separated
by a thin layer of soil. Aerobic and anaerobic microorganisms help
break down the biodegradable substances in the landfill and
produce carbon dioxide and methane gas, which is typically vented
to the surface. Landfills also generate a strong wastewater called
leachate that must be collected and treated to avoid groundwater
• A biosensor is a device for the detection of an analyte that
combines a biological component with a physicochemical detector
• It consists of 3 parts:
• the sensitive biological element (biological material (eg. tissue,
microorganisms, organelles, cell receptors, enzymes, antibodies,
nucleic acids, etc), a biologically derived material or biomimic) The
sensitive elements can be created by biological engineering
• the transducer or the detector element (works in a physicochemical
way; optical, piezoelectric, electrochemical, etc.) that transforms the
signal resulting from the interaction of the analyte with the biological
element into another signal (i.e., transducers) that can be more
easily measured and quantified;
• associated electronics or signal processors that is primarily
responsible for the display of the results in a user-friendly way.
• There are many potential application of biosensors of various types. The
main requirements for a biosensor approach to be valuable in terms of
research and commercial applications are the identification of a target
molecule, availability of a suitable biological recognition element, and the
potential for disposable portable detection systems to be preferred to
sensitive laboratory-based techniques in some situations. Some examples
are given below:
• Glucose monitoring in diabetes patients <-- historical market driver
• Other medical health related targets
• Environmental applications e.g. the detection of pesticides and river water
• Remote sensing of airborne bacteria e.g. in counter-bioterrorist activities
• Detection of pathogens
• Determining levels of toxic substances before and after bioremediation
• Detection and determining of organophosphate
• Routine analytical measurement of folic acid, biotin, vitamin B12 and
pantothenic acid as an alternative to microbiological assay
• Determination of drug residues in food, such as antibiotics and growth
promoters, particularly meat and honey.
• Drug discovery and evaluation of biological activity of new compounds.
• Detection of toxic methabolites such as mycotoxins .