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					Notes of lesson
      Mr. Sridhar
III Semester

                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
of life.

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:
             Taxonomy Distributions








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:
1. Morphological-

2. Chemical Characteristics

3. Metabolic Characteristics

4. Genetic Testing.
• Viruses:
Taxonomy--We use the same scheme eg. Class, Order, Family,
  Genus, Species. We don't need to go higher than Family for all
  common viruses.
• 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)
                (N sinØ)
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
                   Electron Microscope

• 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 technique
                     Staining technique

• 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
                            Gram staining

• 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.

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.)
Gram staining
                          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.
• Procedure:
• 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
                           Capsular 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.
Capsular staining
                        Flagellar staining
•   Procedure:

•   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.
Flagellar staining
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
                                           bacterial cells.
 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
                              compounds, N2

 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
                      Trace Elements

Trace elements
             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
                                                           Green Bacteria

Chemoautotrophs or     Inorganic           CO2            A few Bacteria and
     Lithotrophs      compounds,                            many Archaea
 (Lithoautotrophs)   e.g. H2, NH3,
                       NO2, H2S
Chemoheterotrophs      Organic       Organic compounds   Most Bacteria, some
 or Heterotrophs     compounds                                Archaea
                         Growth Factors

• 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
              Microbial Growth

•   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
  (16S, 23S)
• 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
                   Virus structure
• 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
  chemical modification.
• 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.
                     Algal structure
•      General characteristics
Eukaryotic; placed in Kingdom Protista (also frequently
     termed Protoctista
    Mostly photosynthetic
        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
            tidal zone.
        –   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.
                    Fungal structure
•   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.
      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.
  Control of
• 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.
• Ultraviolet
  Gamma radiation
  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
  bacterial cells.
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
        chemical agents
• 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
  phenolic compounds.
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
Detergents           (e.g.
     compounds)              Disrupts cell membranes                                Skin antiseptics and disinfectants
Phenolic      compounds
     (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
                        Clavamox        is
                             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)

                                                                                     Damages    cytoplasmic
Polypeptides    Polymyxin      Bacillus polymyxa    Gram-negative bacteria               membranes
                                                                                     Inhibits steps in murein
                                                                                           biosynthesis    and
                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-
                                                               negative      bacteria,
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
            Alcohol Production
• 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
  Saccharomyces sp.),
• 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;
  Propionibacterium sp.).
• Lab Materials:
• SDA culture of Saccharomyces cerevisiae
  malt extract broth
  sterile water
  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
  exogenous bacteria.
• Genetic engineering approaches
• The use of genetic engineering to create
  organisms specifically designed for
  bioremediation has great potential.[3] 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.[4]
                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.
• Thiobacilli
• 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
  direct contact.
• 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
  wastewater generated.

• 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.[2]
•   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 .