I. INTRODUCTION TO MICROBIOLOGY

A. Nature of the subject

l. Definition - The science which deals with organisms which require a microscope to be seen.

a. Considered by many experts to be limited primarily to the study of bacteria, viruses, protozoa, and fungi.

b. Microbiology includes not only the organisms themselves, but other aspects such as body defense (immunology), and control of the organisms (sterilization and disinfection).

2. Importance of microorganisms

a. Health related - Most diseases of human, other animals, and plants are due to microorganisms. The increase in life span which we have enjoyed in this century is largely due to our increased knowledge of microorganisms.

b. Agents of decay - The breakdown of organic matter into simpler substances is brought about by the action of microorganisms. This process is of significance in several different areas.

(l) Natural cycles - These are also known as biogeochemical cycles because elements are constantly being cycled between the living (biological) and nonliving (geochemical) parts of the earth. Green plants remove nitrogen, carbon, phosphorous, sulfur, and other elements from the environment. Some of these elements are incorporated into animals that feed upon the plants. When the plants or animals die, microorganisms decompose the organic matter which contains these elements and they are then released for use again. In this way environmental fertility is maintained for future generations.

(2) Food microbiology - Food is organic matter and is therefore subject to decomposition by microorganisms.

Prevention of food spoilage is the subject of much research and the expenditure of many dollars.

(3) Sewage treatment - The decay promoting activities of microorganisms is utilized to promote the breakdown of sewage into harmless inorganic salts. Sewage treatment is basically intensified management of natural cycles.

c. Industrial microbiology - The ability of microorganisms to transform materials is utilized for the production of many desirable substances. Wine and brewing, cheese and other food products, antibiotics, and other specialized chemicals are all dependent upon microbial activity for their production and or processing.

d. Basic research - Because of their relative simplicity and ease of cultivation, microorganisms have become the premier research organisms for modern biologists. Most of the concepts of modern genetics, molecular biology, biochemistry, and genetic engineering have been developed utilizing microorganisms.

e. Genetic engineering - Out of the basic research conducted on microorganisms has come the knowledge and ability to manipulate the genetic material thereby altering the traits of the organism. We are now custom designing organisms that will produce products and/or carry out processes which we desire. This is currently having great impact in all areas of micribiology.



B. Historical development

l. Discovery

a. Microscope - Microorganisms could not be discovered until the invention of the microscope. This occurred around l590 and the discoverer was a Dutchman named Zacharias Janssen.

b. Anton van Leeuwenhoek was another Dutchman who made the first real use of the microscope. During his lifetime he built some two to three hundred simple microscopes, the best of which could magnify about 300 diameters. Leeuwenhoek had an insatiable curiosity. He examined everything that he could put under the microscope and recorded his observations in great detail. He reported his observations in a series of l25 letters to the Royal Society of London and the society published these letters in its journal. In this manner, the world was first made aware of the existence of microorganisms.

2. Spontaneous generation - After their discovery by Leeuwenhoek, microorganisms remained simply a curiosity. The controversy over the doctrine of spontaneous generation (the generation of living things from non-living matter) sparked the first serious study of microbes.

a. Francesco Redi - Seventeenth century scientist who demonstrated that meat protected from flies did not generate maggots. His experiments convinced most people that large organisms did not spontaneously appear from inanimate matter. The discovery of microbes rekindled the argument.

b. John Needham - In l745 he experimented with meat broths exposed to heat and found that microorganisms appeared in them. He concluded that they had been spontaneously generated as the original heat would have destroyed any living thing.

c. Spallanzani - Working at the same time as Needham he demonstrated that beef broth boiled for an hour and sealed in a flask, did not develop microbes. Needham countered that spontaneous generation required the presence of air.

d. Theodor Schwann - In the early l9th century he repeated

Spallazani"s experiments but permitted air to enter the flask through red hot tubes. No growth occurred, but the critics said that the heating of the air had destroyed its ability to aid spontaneous generation.

e. Schroder and von Dusch - Performed in l850 a similar experiment but sealed the flask with sterile cotton through which air could pass and found no growth.

f. Louis Pasteur - In l864 laid the doctrine of spontaneous

generation to rest by means of a series of elegant experiments. He made special flasks with long curving necks. He then heated a meat broth in these flasks and let it stand. The long curved necks would permit air to move in and out freely but would trap any dust particles in the air. Nothing grew in the flasks. To further prove his point, Pasteur then turned the flasks upside down, thereby permitting the broth to come in contact with the trapped dust. Organisms then grew in the broth.

g. This running feud over the spontaneous generation focused

attention on microorganisms and a great deal was learned about them as well as the techniques for growing and handling them.

3. Development - The controversy over spontaneous generation made the existence of microorganisms known to a wider audience but they still seemed to be of little significance. Two monumental discoveries changed that misconception.

a. Fermentation - The process of fermentation for the making of wine and beer had been known for centuries and was widely believed to be a purely chemical phenomena. The wine industry in France had been suffering from batches of ferment that went sour. Being it was thought to be a chemical process, the winery owners hired a chemist,

Louis Pasteur to help them out. During his studies, Pasteur demonstrated that the fermentation process was actually carried out by "good" microbes, and that the souring was caused by "bad" microbes. He found that if you held the temperature of the juice at 62.8 C. for one half hour that the "bad" organisms would be destroyed and the "good" ones left unharmed. This process became known as Pasteurization and is still used today in the brewing industry as well as in the dairy industry. Pasteur's studies of fermentation showed that microbes were very important industrially.

b. Germ theory of disease - This is the concept that disease is caused by infectious microorganisms.

(l) Girolamo Fracastoro (l483 - l553) - Postulated that

disease was caused by invisible living seeds.

(2) Ignaz Semmelweis (l8l8 - l865) - Introduced antisepsis into hospitals because he thought that microorganisms were transmitting disease to patients.

(3) Louis Pasteur - Considered along with Robert Koch to be the co-founder of the Germ theory. Following his discovery of the role of microbes in fermentation, Pasteur worked with the silk industry in France trying to discover the cause of a disease that affected the silk worms. He never discovered the cause, but became convinced that a microbe was the reason. Pasteur then examined other animal diseases and eventually moved into human diseases. His researches clearly demonstrated the fact that most diseases were caused by microorganisms.

(4) Robert Koch - The other co-founder of this theory was a country physician who was the first person to isolate and demonstrate a microbial cause for a specific disease. This disease was anthrax. Based upon his pioneering experiments Koch developed a series of rules that guide medical microbiologists to this day. These rules are known as Koch's postulates and are as follows.

(a) A specific organism must always be found associated with a given disease.

(b) The organism must be isolated and grown in pure culture in the laboratory.

(c) The pure culture will produce the disease when inoculated into a susceptible animal.

(d) The organism can be recovered in pure culture from the infected animal.

(5) The work of Pasteur and Koch firmly established the concept that disease was caused by microorganisms. Once this was accepted then people reasoned that knowledge of microorganisms might be used to both treat and prevent disease. Thus the study of microorganisms took on a new urgency.

c. Pure cultures - Without the development of techniques for the isolation and growth of pure cultures, Koch's postulates would have not been possible nor would the science of microbiology developed. The first pure culture was obtained by Joseph Lister, a British physician, who is more famous for his development of antiseptic surgery. Lister obtained his culture by a laborious dilution technique. The wife (Fanny Hesse), of one of Koch's students suggested that a solid medium might be prepared by adding a small amount of a gravy thickener known as agar to the liquid mediums. It was tried and it worked. With solid media isolation could be obtained by simply spreading a small amount of a mixed culture thinly on the surface and permitting the organisms to grow. Separated organisms would form pure colonies that could be transferred to fresh sterile media.

4. A new science - The discoveries of Pasteur and Koch ushered in a new science that grew rapidly in several different directions.

a. Immunization - Jenner introduced immunization against

smallpox in l798, but Pasteur was the first to understand

the theory behind immunization and to apply it scientifically. Pasteur discovered this phenomena by accident. He had been working with a batch of fowl cholera culture which he had let sit in the laboratory for 2 weeks. Upon injecting chickens with the old culture he found that nothing happened. He then prepared a fresh culture which he injected the same chickens with plus some new chickens. All of the new chickens became sick and died while the birds which had been injected with the old culture remained perfectly healthy. Pasteur realized that the old culture had become attenuated, it had lost its ability to cause disease,but not its ability to stimulate the animal's immune system. Pasteur next deliberately prepared an attenuated culture of anthrax which he used to protect domestic animals. He named the process of attenuation and injection, vaccination in honor of Jenner (Vaca means cow in latin, which is where Jenner obtained his smallpox serum).

Pasteur now began to devote all of his energy towardsproducing vaccines. His most famous would be the rabies vaccine. During the development of this vaccine Pasteur was visited one night by a Mrs. Meister whose son, Joseph, had been bitten by a rabid dog. She begged Pasteur to vaccinate Joseph with his experimental vaccine and he reluctantly agreed. Joseph did not contract rabies and the news was carried by the papers all over Europe. A few months later l9 Russian peasants from Smolensk showed up at Pasteur's lab. They had been bitten by a rabid wolf and the Czar had sent them by train to Paris for treatment. Pasteur's vaccine saved all but three. The Czar was so pleased that he sent Pasteur a diamond cross and l00 thousand gold franks to establish a center for Vaccine research and production. This money was used to establish the Pasteur Institute which still exists today as one of the premier research and development centers in the world.

b. Applied microbiology - The success of Pasteur and Koch drew students to their laboratories from all over the world. Many of these students then returned to their home countries to establish schools, laboratories, and research centers. This resulted in a vast explosion of knowledge which has frequently been termed the "golden age" of microbiology, a period of time beginning in 1857 and ending with the outbreak of WWI in 1914. Some of the major figures and their contributions are as follows.

(l) Metchnikoff (l884) - First pointed out the importance of phagocytic cells in disease prevention and established the concept of cell mediated immunity.

(2) Nuttal and Bordet - Working at the same time as Metchnikoff demonstrated the bacteriocidal action of serum from immune persons and established the concept of humoral mediated immunity.

(3) Hans Christian Gram (1884) - Developed the Gram stain which permitted the division of bacteria into two major groups.



(4) Klebs and Loeffler (1884) - Isolated the diphtheria bacterium and demonstrated the toxin which it produces.

(5) Von Behring and Kitasato (1890) - Developed antitoxins by injecting toxins from the diphtheria and tetanus causing bacteria into animals and then harvesting from the animal sera the antitoxins produced. These could then be injected into humans to treat the respective diseases.

(6) Iwanowsky (l892) - Isolated the first virus.

(7) Theobald Smith (l893) - Demonstrated that Texas cattle fever was transmitted by the bite of a tick.

(8) Ronald Ross (l895) - Demonstrated that malaria was contracted from the bite of a mosquito.

(9) Walter Reed (l900) - Demonstrated the mosquito spread mechanism of yellow fever. The work of Smith, Ross, and Reed established the importance of arthropods as vectors of disease and established the science known as medical entomology.

(10) Paul Erlich (l909 - Discovered number 606, the first chemotherapeutic agent against syphilis (salvarsan).

(11) John Enders (l949) - Developed the tissue culture technique for growing the polio virus thereby making possible the development of the polio vaccine. Enders developed many other vaccines against viruses, including measles and yellow fever vaccines.

c. Agricultural, industrial, and food microbiology - During the same time period as the discoveries in medical microbiology were being made, scientists in the areas of agriculture, industry, and food processing were also discovering the importance of microorganisms to their various areas. These discoveries further widened and developed the scope of microbiology.

In the latter years of the l9th century and the first years of the 20th century, the discoveries indicated above transformed microbiology from the realm of curiosity to a full fledged science.



5. Modern microbiology - Many of the themes establish in the past are still being pursued at this time. However, two new areas, unique to this century, have further advanced the science of microbiology.

a. Molecular biology - The advances in the studies of the chemistry of life which have been made in this century have been made largely through the use of microbes. Scientists quickly learned that the relative simplicity of microorganisms coupled with their ease of cultivation made them ideal experimental organisms for biochemical studies. Consequently most of what we know about modern biochemistry and molecular genetics was first learned from microorganisms.

b. Genetic engineering - As an outgrowth of the research in molecular biology it was discovered in the seventies that it was quite feasible to take apart the genetic material of microorganisms and splice in new information which coded for new products. This technology is termed genetic engineering, gene splicing, or recombinant DNA. It permits the insertions of characteristics from one organism (including humans) into another organism, usually a microorganism. Such endeavors have yielded bacteria that can produced human insulin as well as other valuable products. The outlook for such production is almost limitless. Gene splicing will be discussed in more detail later in the course.

C. Cellular basis of microorganisms - Two fundamental types of cells appear to have evolved on earth. Procaryotic cells are the simpler and more ancient, with fossil remains going back in time nearly 3.5 billion years. Eucaryotic cells are structurally more complex and go back in the fossil record l.4 billion years, although biochemical evidence suggests that they may be much older.. The best way to understand the differences between these two different cells is to compare them on a feature by feature basis.

Feature Procaryote Eucaryote

Size < 5 microns > 5 microns

Nucleus absent present

Chromosome single, circular few to many, linear histones absent histones present

Sex true sex absent true sex

Pinocytosis absent present

Ribosomes 70s 80s in cytoplasm,

70s in mitochondria and chloroplasts

Membrane bound absent present

organelles



Glycocalyx present present in some cells that lack

a cell wall

Cell wall peptidoglycan is the peptidoglycan major component absent



Cytoskeleton absent present

Flagella simple fibril microtubules, 9 + 2 arrangement.

This table and the ensuing discussion of cellular anatomy demonstrate the structural simplicity of procaryotes compared to eucaryotes. Only the bacteria exhibit the procaryotic cell type, all other organisms are made up of eucaryotic cells.

1. Anatomy of a procaryotic cell

a. Glycocalyx - This is a sticky layer found on the outside of procaryotes. If it is firmly attached and highly organized it is termed a capsule. If it is loosely attached and organized it is termed a slime layer. The glycocalyx has the following functions.



(1) The capsule makes phagocytosis of bacteria more difficult for body defense cells and thereby increases their virulence. Capsules are therefore medically significant.



(2) Attachment to surfaces.



(3) Nutritional reserve.



(4) Protection against dehydration.



b. Cell wall - This is a rigid covering found around most procaryotes. It provides support and protects against osmotic shock. It is composed largely of peptidoglycan, a complex substance with both sugar and amino acid components.

(l) Gram characteristics - Based upon wall differences, most bacteria can be divided up into two great groups using the Gram staining technique. Gram positive organisms retain the Gram stain (crystal violet) after decolorizing with alcohol. Gram negative organisms will not hold the Gram stain and are counter stained pink. Chemical differences in the structure of Gram negative and positive cell walls are responsible for the Gram reaction. The Gram stain is the most important diagnostic feature in bacteriology. Some of the major differences between the two cell types are as follows.



(a) Gram positive bacteria have much more peptidoglycan (several layers) than do Gram negative bacteria.



(b) Gram positive bacteria usually have teichoic acids bonded either to the peptidoglycan or plasma membrane. There are two classes, lipoteichoic acid, which spans the peptidoglycan layer and attached to the plasma membrane, and wall teichoic acid which is linked to the peptidoglycan layer. These teichoic acids serve as important serological (antigenic) markers for bacterial identification.



(c) The wall of Gram negative bacteria has much less peptidoglycan and no teichoic acids. The peptidoglycan is located in the periplasmic space which separates the plasma membrane from a second outer membrane. The space between the two membranes (periplasm) contains in addition to the peptidoglycan, numerous enzymes. The outer membrane is rich in lipoproteins and lipopolysaccharides. One such lipopolysaccharide is known as endotoxin, and is responsible for many disease processes.

(d) Overall, the Gram positive wall is thicker and mechanically stronger than the Gram negative wall. The Gram negative wall is chemically more complex, and more difficult for defense cells and systems of the body to deal with.



(e) The differences in cell wall composition effect not only the Gram stain, but many antibiotics as well. More will be said about this later.

c. Plasma membrane - This is a thin membrane that lies just inside of the cell wall. In procaryotes it is composed largely of phospholipids. The cell membrane plasma membrane) regulates the entry and exit of materials to and from the cell.

d. Flagella - Thin hair-like appendages that protrude through the cell wall and originate just beneath the cell membrane. Not all bacteria possess flagella. They may be few or numerous, but their function is motility.

e. Pili (fimbriae) - These are appendages which are smaller, shorter, and more numerous than flagella. They do not function in motility. There are several different kinds and they function in:

(l) Transfer of genetic information from one cell to another (F pilus or sex pilus).

(2) Mechanisms for adherence to surfaces.

f. Ribosomes - These bodies are composed of RNA and protein. They function as the sites of protein synthesis. Procaryotic ribosomes are the 70s size variety. The s stands for the Svedberg unit and refers to where they settle in an ultracentrifuge.

g. Nuclei - procaryotes have no organized nucleus set off by a membrane, but they do contain genetic centers termed nucleoids. There are usually two nucleoids which attach to the membrane prior to cell division.

h. Granules - These are various inclusions which may be seen in bacterial cytoplasm. Some serve as food storage products and others in reproduction. One common form is volutin which is a stored form of inorganic phosphorous. Volutin granules are also termed metachromatic granules because they stain different colors in the cell.

i. Pigments - Many species form pigments of different colors. Their function is bacteria is usually not known, but they make excellent characteristics for identifying various procaryotic organisms.

j. Endospores - These are small, hard, heat resistant spores that form inside of certain bacterial cells. They can withstand unfavorable growing conditions for extended periods and will begin to grow when conditions become favorable.

k. Plasmids - These are small, extrachromosomal, circular strands of DNA. There function will be discussed in detail later.

m. Gas vacuoles - These are composed of gas vesicles. They are found in many aquatic forms where they promote buoyancy.

2. Morphology - This includes the shapes and arrangements of procaryotic cells.



a. Spherical or ellipsoid - Coccus forms.

(l) diplococcus - pairs.

(2) streptococcus - chains

(3) staphylococcus - irregular clusters.

(4) sarcina - cubical packets of 8 cells.

b. Rod or cylindrical shaped - usually elongate, like a

cigarette. This shape is sometimes referred to as the

bacillus shape.

c. Spiral or helical shaped.

(l) vibrio and spirillum - vibrio look like commas, while

spirillum look like corkscrews.

(2) spirochete - corkscrew shaped, but flexible.



d. While the above morphologies are the most common, some groups of bacterial have a more complex shape, some being club shaped, branching, or even filamentous.



3. Anatomy of the eucaryotic cell

a. Cell wall - Essentially the same function as in procaryotes, but the composition is different. Usually they are composed of cellulose or chitin, but never peptidoglycan.

b. Plasma membrane - Essentially the same function as in procaryotes but in addition to phospholipids, sterols and carbohydrates are also found. and exit.

c. Cytoplasmic skeleton - This is composed of protein rods and tubules known as microfilaments, intermediate filaments, and microtubules. The skeleton gives the cytoplasm form and support. It also permits movement which leads to the constant cycling of the cytoplasm, a phenomenon known as cytoplasmic streaming. This aids greatly in the distribution of materials. Procaryotes lack this cytoskeleton and are therefore incapable of cytoplasmic streaming.



d. Endoplasmic reticulum - This consists of a series of double membranes which ramify throughout the cell. It is continuous with the plasma membrane and the nuclear membrane. It functions as a rapid transit system and a site of biochemical synthesis.

e. Ribosomes - These again function as the sites of protein

synthesis. Most are attached to the endoplasmic reticulum forming what is known as rough ER. Those found in the cytoplasm and on the ER are 80s size. Those inside of the mitochondria and chloroplasts are 70s and therefore identical to the procaryotic types.

f. Mitochondria - These organelles are made up of double membranes and function as the sites of energy production.

g. Lysosomes - These are membrane bound sacs which contain powerful digestive enzymes. When the sac ruptures the enzymes are released and break down organic molecules in the area. Lysosomes function in repair and recycling as well as in destruction of diseased cells.

h. Peroxisomes - These are membranous sacs, similar to lysosomes, contain the enzyme catalase. This enzyme breaks down hydrogen peroxide, a highly toxic substance that is produced as a by product of oxygen metabolism.

i. Golgi body - This a series of membranous sacs that resemble a stack of coins. It functions in the packaging of cell secretions.

j. Chloroplasts - Double membraned organelles similar to

mitochondria in structure. They contain the green pigment chlorophyll which functions in photosynthesis, the conversion of radiant energy into chemical energy.

k. Nucleus - This is the largest organelle in the cell. It is surrounded by a double layered membrane. Inside are the chromosomes, composed of DNA, RNA, and protein. These chromosomes control all of the cells activities and are responsible for the passage of the genetic information from generation to generation. Overall then, the nucleus exercises command and control over all of the cell's activities.

l. Nucleolus - This is a dark staining body found inside of the nucleus. It is the site of ribosome synthesis.

m. Centrioles - These are paired bodies which function in nuclear division (mitosis).

n. Locomotor organelles - These are organelles that propel the eucaryotic cell through aqueous mediums. There are two basic types.

(l) Flagella - These are long and whip like. They are usually arranged one to a cell or at the most, a few per cell.

(2) Cilia - These are hair like projections that cover most of the cell's surface. They beat in a coordinated.fashion and the cell "rows" itself along.

Cilia and flagella have a common structure. Both are composed of a circle of 9 double microtubules with two additional microtubules located in the center. Each flagellum or cilium is connected to a structure inside of the cell membrane termed a kinetosome. The kinetosome is similar in structure to the cilia and flagella. It functions to anchor and move the structures.

D. Microbial reproduction - All microorganisms reproduce or make copies of themselves. Reproduction can be asexual in which the daughter cells are identical copies of the mother cell, or sexual, in which the offspring show genetic variation. Procaryotes have no true sex.

l. Procaryotic pattern- All procaryotes reproduce by means of binary fission, or cell splitting. There is but a single chromosome which is reproduced during the process. The details are as follows.

a. The chromosome duplicates itself so that two identical chromosomes exist.

b. At about midway in the cell, the plasma membrane starts to invaginate. Each chromosome attaches to one side of the invagination.

c. The invagination eventually reaches the other side of the cell, meeting and fusing with the opposite membrane. This separates the mother cell into two compartments, each with its own chromosome.

d. Along the membrane partition a new cell wall begins to form, and eventually, two new cells are formed.

2. Eucaryotic pattern - Eucaryotes exhibit both sexual and asexual reproduction. Frequently eucaryotic microorganisms exhibit an alternation of generations, whereby an asexual phase of reproduction alternates with a sexual phase.

a. Asexual reproduction - All asexual reproduction is based upon mitotic cell division. Here an elaborate series of cellular events insure that each daughter cell receives a full set of chromosomes and thus a complete set of genetic instructions. There are several different ways in which asexual reproduction can occur.

(l) Binary fission - The simplest reproductive mode. Following mitotic duplication of the nucleus, the cell splits either transversely or longitudinally to form two identical daughter cells.

(2) Multiple fission (schizogony) - The mother cell divides to form many daughter cells. It is preceded by multiple mitotic divisions of the mother cell nucleus. Each nucleus is then enclosed by cytoplasm and a membrane and the mother cell "disintegrates" to form a large number of small daughter cells.

(3) Budding - This is the formation of one or more smaller individuals from the mother cell. It is similar to an unequal binary fission.

b. Sexual reproduction

(l) Syngamy - Two compatible (male and female, etc) sex cells are brought together and fuse. This fusion forms a zygote which has twice the chromosome number of the fusing cells. Such a cell is said to be diploid or 2n. The sex cells are haploid or n. Once the diploid zygote has formed it undergoes a special kind of division termed meiosis. This meiotic division results in a reduction of the chromosome number in half, so that the offspring each have the haploid number. The great advantage of sexual reproduction is that the fusion and subsequent meiotic division produces offspring which show genetic variability which may provide increased survivability.

Note: Most microorganisms spend most of their life cycle in the haploid phase whereas most of the more advanced multicellular plants and animals spend most of their life cycle in the diploid phase.



E. Classification of microorganisms.

l. Taxonomy - This is the science of the systematic arrangement of organisms into groups or categories. Organisms are grouped together based upon similarities and differences. A series of taxonomic categories or taxa are arranged into a hierarchial system. The most inclusive category is located at the top of the hierarchy while the least inclusive is located at the bottom. Once a taxonomic system has been established it not only permits identification of organisms, but based upon the position the organism occupies in the system, tells something about the biology of the organism. Ideally, the taxonomic system should also reflect the phylogenetic (evolutionary) relationships of the various organisms to one another.



2. Taxonomic categories - In most taxonomic systems there are at least 7 major taxons. Generally one begins with the most inclusive category and works downward into the more exclusive or specific categories until the organism in question has been identified. The major categories are as follows.

a. Kingdom - A group of related phyla (divisions.

b. Phylum (Division) - A group of related classes.

c. Class - A group of related orders.

d. Order - A group of related families.

e. Family - A group of related genera.

f. Genus - A group of related species.

g. Species - Organisms of one and the same kind.

Using this system, let us classify human beings.

a. kingdom - Animalia (multicelled organisms which ingest food)

b. Phylum - Chordata (animals possessing a dorsal nerve cord)

c. Class - Mammalia (warm blooded with hair, young born alive)

d. Order - Primate (stereoscopic vision, other common traits)



e. Family - Homidae (the "human-like" primates)

f. Genus - Homo (modern and extinct people)

g. Species -sapiens (modern extant human beings)

3. Binomial system - This is the system of nomenclature which was

developed by Linnaeus in the 18th century. In this system each species of organism is given two latin names, the Genus or generic name, and the species or trivial name. This is system is universally accepted around the world. The generic name may be shared by a number of related kinds of organisms and the specific name specifies exactly which one we are talking about. For example, the genus Homo contains only one living species, sapiens but there are a couple of extinct species, habilis and erectus By convention, the genus name is always capitalized while the species (trivial) name is in lower case completely. Genus and species names are always italicized, or if italics are not available, then they are underlined.

4. Major characteristics used to classify microorganisms - In order to classify any organism it is necessary to know as many things about it as possible. The major characteristics used to classify microorganisms are as follows.

a. Morphological characteristics - This is how it looks, size, shape, color, cell arrangement, etc. Staining is frequently used to determine this.

b. Cultural characteristics - Required nutrients, physical conditions necessary for growth (pH, temperature, pressure, etc).

c. Metabolic characteristics - The manner in which the organism carries out the chemical processes of life. Does it utilize glucose as a carbon source? What gases does it produce with sucrose as its carbon source? There are a number of biochemical tests that can be utilized to determine this information.

d. Chemical composition - What are the major chemical constituents of the cell? Differential staining can frequently determine some of this information, for example, Gram negative or positive and acid fast properties are the result of cell wall composition and can be determined with differential stains. Determination of fatty acid composition is another chemical test. Fatty acids tend to be constant for a given species. Tests have been designed to separate fatty acids from unknown forms and compare them to fatty acid profiles for known forms.

e. Antigenic characteristics (serology) - All cells have on their surfaces certain kinds of large molecules. These large molecules are termed antigens because inside of our body (or some other animal) they will stimulate production of antibodies which are specific for them. These antibodies can be used to identify the antigens which they are specific for and cells can thus be identified based upon the types of antigens which they have on their surfaces. For example, type A blood can be detected because there is the A antigen on its surface. Likewise, microorganisms can be "typed" in a similar manner. Frequently antigenic types are referred to as "serotypes, serovars, or biovars." Two test procedures that are clinically important are the ELISA (enzyme linked immunosorbent assay), and the Western blotting test.

f. Phage typing - A phage is a virus that infects bacteria. Many phage are very specific, that is, they will infect only one species of a bacterium or even a serotype. Therefore, using known species of phage is much like using antibodies in serotyping.

g. Flow cytometry - This permits identification of certain bacteria without culturing. A moving fluid containing bacteria is forced through a small opening. Electrical conductivity, scattering of a laser beam of light provide information about cell size, shape, density, and surface features. This is analyzed by a computer that provided tentative identifications.



g. Genetic characteristics - Nucleic acid analysis. This can be determined by examining two different types of molecules, DNA and RNA.



These are the nucleic acids, long chains made up of repeating subunits known as nucleotides. There are four nucleotides in DNA, adenine (A), thymine(T), cytosine(C), and guanine(G). DNA consists of a double chain which are held together by bonds between A and T, and C and G. RNA contains A, C, and G, but T is replaced by uracil (U).



DNA contains the genetic instructions and RNA (which represents a copy of instructions contained in DNA) implements the instructions contained in DNA. The nucleic acids will be considered in detail later.





(2) DNA - This is the genetic material, and the more similar the nucleotide sequence between two organisms, the more closely related they are. DNA similarities are determined by several methods.



(a) Base composition - This is usually expresed as the percentage of the DNA that is composed of G + C. The GC composition for one species is constant and therefore, if two organisms have very similar GC content, they probably share a number of common genes and are therefore closely related. While it is true that two organisms which have widely different GC content, (40% versus 65%) are not closely related, a very close GC content does not prove that they are closely related, only that they may be.



(b) DNA hybridization - In this technique the strands from two separate organisms are mixed together and allowed to react. The more similar the strands are, the greater will be the percentage of reaction, or hybridization.



/1/ Probes - These are fragments of DNA that can be used to rapidly identify a particular organism. Probes are prepared by breaking the DNA of a particular species of organism into a number of small fragments. Fragments are selected which will hybridize completely with all strains of that species, but not with closely related species. The probes are then tagged with fluorescent dyes or radioactive labels. If the probes hybridize with the DNA from an unknown bacterium then that organism is immediately identified as belonging to the species from which the probe was extracted. Probes are therefore analogous to the use of antibodies with antigens except they are specific for genes.



/2/ DNA chips - This is a high tech application in which are large number of probes are attached to a small chip. DNA from an unknown organism is labeled with a fluorescent dye and applied to the chip. Hybridization is detected by fluorescence. This technology will eventually permit rapid identification of unknown organisms .



(c) DNA sequencing - Here the sequences of nucleotides are determined and compared. This provides a complete genetic map (Genome) of the organism. There are currently several microorganisms that have had their genomes determined as well as several multicellular organisms (including humans).



/1/ DNA fingerprinting - Complete genome sequencing is a long, tedious, and expensive process. A technique using DNA fragments offers almost as accurate comparison as sequencing but much more rapidly and cheaply. DNA is cut into pieces by special enzymes known as restriction enzymes. Each enzyme always cuts at a particular nucloetide sequence, i.e. AATTAA. Consequently, two identical DNA molecules would be cut into identical fragments. In practice, two organisms have their DNA cut up using the same restriction enzymes. The fragments are then compared by separating them in a gel by means of an electric current, a technique known as electrophoresis. The more similar the fragments are, the more closely related the organism are.



/2/ PCR - The above DNA techniques require a substantial quantity of DNA. If only one, or a few cells are present, it is possible to amplify the DNA present through a technique known as the Polymerase Chain Reaction (PCR). The PCR and the other molecular techniques will be discussed in more detail later.



(3) Ribosomal RNA sequencing - This has become an especially important method of determining relationships among organisms, especially the bacteria. All cells have ribosomes and part of those are a special kind of RNA known as rRNA. Two closely related organisms will have similar RNA sequences. Over time RNA genes have remained stable so that there are not vast differences in nucleotide sequences that might give false relationship. It is also valuable in that culturing of organisms is not required. The DNA can be amplified using PCR, the rRNA gene identified using a probe, and then the gene can be sequenced to show the differences.



5. Taxonomic systems - Historically a number of different taxonomic schemes have been used. Originally all living things were classified as being either plants or animals, a two kingdom view.



a. The most popular system currently in use in the 5 kingdom system originally proposed by Whittaker in l969. This system is based upon cellular organization and nutritional modes, photosynthesis, absorption of organic molecules, or ingestion of particulate organic matter. The 5 kingdoms of the Whittaker system as follows.

(1) Kingdom Monera - This is the procaryotic kingdom and consists of the bacteria and blue-green algae, a specialized form of bacteria.

The remaining four kingdoms are composed of eucaryotic cells.

(2) Kingdom Protista - Composed of unicellular eucaryotic organisms. All three types of nutrition are found in this kingdom.

(3) Kingdom Fungi - Multicellular and multinucleated organisms which utilize absorption as the mode of nutrition.

(4) Kingdom Plantae - Green plants and multicellular green "algae." Nutrition is by means of photosynthesis.

(5) Kingdom Animalia - Multicellular organisms which utilize ingestion as the mode of nutrition.



b. More recently (1978) a three domain system has been proposed by Carl Woese. Woese and associates utilized modern biochemical techniques, especially nucleic acid sequencing and demonstrated that cells fall into three distinctly different groups. Two of these domains, the bacteria and archaea, are procaryotic in organization, while the third domain are all eucaryotes. While the bacteria and archaea appear similar in morphology there are major differences in their chemistry.



(1) bacterial cell walls contain peptidoglycan, archaea do not.



(2) membrane lipids are very different.



(3) bacteria are sensitive to antibiotics, archaea are not.



(4) rRNA differs considerably.



It was originally thought that eucaryotes were derived from the eubacteria which seem to be more advanced. Recent biochemical evidence suggests that not only are eucaryotes much older than originally thought, but they seem to be most closely related to the archaebacteria.



In this course we will use the three domain concept with the eucaryotic domain being composed of the four Whitaker eucaryotic kingdoms.

F. Procaryotic domains The classification of the procaryotes is found in a special book, Bergey's Manual of Systematic Bacteriology,edition 2. This is a proposed 5 volume work, volume 1, contains the Archae and members of the domain bacteria. It was published in 2001. Subsequent volumes are still in press. An outline of the classification of bacteria and archaea is contained in Appendix A of your textbook. This work represents a profound revision of the first edition (1984) based upon recent biochemical studies, particularly GC ratios and rRNA sequences. While systematic bacteriology presents an evolutionary classification another Bergey's manual is used for practical laboratory identification. This is Bergey's Manual of Determinative Bacteriology (9th ed., 1994). This manual provides a series of criteria for rapid identification of bacteria, but does not present an evolutionary classification scheme.

A brief survey of Bergey's systematic manual will be undertaken concentrating on phyla that contain either ecologically or medically significant genera.



Domain Archaea



A. These are contained in Volume 1 of Bergeys systematic bacteriology. They typically live in extreme environments. Morphologically they consist of rods, cocci, and helixes, plus some unusual forms. They are both Gram + and Gram -. Some divide by binary fission while others by fragmentation or budding. While the formal taxonomy divides them into two phyla and several classes, ecologically they fall into three major lines.

1. Methanogens - These are strict anaerobes that produce methane as a byproduct of metabolism. They are found in anaerobic environments such as swamps, marshes, and the rumen of cattle. They are used in sewage treatment plants to convert sewage into methane. Experiments are underway to adapt them to the production of natural gas (methane).



2. Extreme halophtytes - These organisms require high levels of salt in their environment. They are found in the great salt lake and other areas of high salinity.



3. Thermoacidophils - Grow under high temperature and acidity conditions. Found in hot acidic springs. The genus Sulfolobus grows best at the highly acid pH value of 2.0 and a temperature of 700C. Other forms are found around oceanic volcanic (hydrothermal) vents where water temperature may reach 110 degrees C.



Domain - Bacteria



A. Phylum XII - Proteobacteria - Volume 2 - This is the largest taxonomic group of bacteria. Most of the Gram negative bacteria are found here. Most are chemoheterotrophic meaning they obtain nutrients by absorbing organic molecules from their environment. They are thought to have evolved from a photosynthetic ancestor. There are five classes.



1. Alpha Proteobacteria - Includes most of the proteobacteria that can grow at very low nutrient levels. Several can fix nitrogen, convert atmospheric nitrogen into nitrate (N04). Nitrate is the form of nitrogen that can be used by plants as their nitrogen source. Consequently these play a major role in the nitrogen cycle. This group also includes significant pathogens.



a. Nitrogen fixing genera - Azospirillum, Rhizobium, Nitrobacter.



b. Medically significant genera.



(1) Rickettsia - Minute obligate intracellular parasites. Most are transmitted to humans by insect bites. Cause Typhus and Rocky mountain spotted fever.



(2) Bartonella - Causes cat scratch fever.



(3) Brucella - relapsing fever.



2. Class Beta Proteobacteria - These overlap with the alpha forms. Nitrifying bacteria and forms which promote anaerobic decomposition of organic matter here. There are also several pathogens.



a. Ecologically significant genera



(1) Thiobacillus - Important in sulfur cycle. Convert various sulfur containing compounds into sulfates used by plants.



(2) Zoogloea - Important in aerobic sewage treatment.

b. Medically significant genera.



(1) Bordetella - Causes whooping cough.



(2) Neisseria - Cause gonorrhea and meningitis.



3. Class - Gamma Porteobacteria - This is the largest class of the proteobacteria. Industrial, ecological, and medical importance.



a. Ecologically significant genera



(1) Azotoabacter, Azomonas - Nitrogen fixers.



(2) Pseudomonas - Denitrifiers. Convert nitrate to nitrogen.



b. Medically significant genera.



(1) Francisella - Causes tularemia (Rabbit fever).

(2) Pseudomonas - Numerous types of infections. Multiple antibiotic resistance forms.



(3) Legionella - Causes legionellosis, a type of pneumonia.



(4) Vibrio - Causes cholera.



(5) Escherichia - Common colon form. Strains can cause enteric infections ranging from unpleasant to fatal.



(6) Salmonella - Typhoid fever and a number of food and water borne intestinal infections.



(7) Shigella - Causes bacterial dysentery.



(8) Klebsiella - Mostly beneficially nitrogen fixers but can cause a form of pneumonia.



(9) Yersinia - Causes bubonic plague.



(10) Haemophilus - Meningitis in young children and ear aches.



4. Class - delta Proteobacteria - These include bacteria that are predatory on other bacteria and others which are important in the sulfur cycle.



5. Class - epsilon Proteobacteria- These are slender gram negative rods that are helical or vibrioid.



a. Medially significant genera



(1) Campylobacter - A major cause of foodborne intestinal disease.



(2) Helicobacter - This is the most frequent cause of peptic ulcers.



B. Phylum 10 - Cyanobacteria - These are referred to as the blue green algae. They are photosynthetic and oxygenic, meaning they produce oxygen as a byproduct. Through the process of photosynthesis they combine hydrogen from water with carbon dioxide to form sugars. They are gram negative. Some forms can also fix nitrogen. They are ecologically important in aquatic environments.



C. Phylum 11 - Chlorobium - These are the green sulfur bacteria. They along with several other phyla contain photosynthetic forms which derive the hydrogen needed to combine with carbon dioxide from sources other than water such as H2S and consequently release Sulfur as a byproduct instead of oxygen. For this reason they are referred to as being anoxygenic photosenthesizers.



D. Phylum 13 - Firmicutes - Volume 3 - These are the Gram positive bacteria with low G+C ratios (20-55%). These include both medically and industrially important genera.



1. Industrially important genera.



a. Lactobacillus - This is found in humans in the mouth, vagina, and intestinal tract. They produce lactic acid and are important in the production of sauerkraut, pickles, buttermilk, and yogurt.



2. Medically significant genera.



a. Clostridium - Obligate anaerobes that produce endospores. Cause tetanus (lockjaw), botulism, and gas gangrene.



b. Bacillus - Endospore forming rods. Cause anthrax and food poisoning. One species, B. thuringiensis, is used in the biological control of insects.



c. Staphylococcus - Wound infections, toxic shock syndrome, and food poisoning. Multiple antibiotic resistant forms.



d. Streptococcus - Cause a greater variety of illnesses than any other group of bacterial. Scarlet fever, pneumonia, sore throat, impetigo, dental caries.



e. Enterococcus - Surgical wounds and urinary tract infections.



f. Listeria - Food contaminant, especially dairy. Can cause various illnesses. Especially dangerous in pregnant women where it causes still birth and fetal damage.



g. Mycoplasma - Lack cell walls. May grow as filaments. Extremely small. Cause a form of pneumonia.



E. Phylum 14 - Actinobacteria - Volume 4 - These are high G+C (50-75%). Many have complex morphologies, club shaped, branching, and filamentous. Many medically and industrially significant genera.



1. Medically and industrially significant genera



a. Propionibacterium - Produces propionic acid important in cheese production. Also causes acne.



b. Streptomyces - Common soil organisms with complex mold like branching morphology. Source of most antibiotics. 500 named species.



2. Medically significant genera



a. Mycobacterium - Cause tuberculosis and leprosy.



b. Corynebacterium - Causes diphtheria.



c. Gardnerella - Causes vaginitis.



F. Phylum 16 - Chlamydia - Do not contain peptidoglycan in the cell walls. Obligate, intracellular parasites that were once classified with the rickettsias.



1. Medically significant genera



a. Chlamydia - Gram negative. Cause blindness, ornithosis (parrot fever), and NGU (nongonococcal urethritis), the most common sexually transmitted disease in the world.



The following phyla are contained in Volume 5 of Bergeys Manual.





G. Phylum 16 - Spirochetes - Coiled morphologh. All motile by means of endoflagella, two or more axial filaments found in the periplasmic space. Rotating these filaments causes the cell to move like a corkscrew though the medium. Many are found living in the oral cavity.



1. Medically significant genera



a. Treponema - Causes syphilis.



b. Borrelia - Causes relapsing fever.



c. Leptospira - Causes leptospirosis, a disease spread to humans from water contaminated with domestic animal urine.



H. Phylum 20 - Bacteroidetes - Anaerobic bacteria found in the intestinal tract and in soil



1. Ecologically significant genera



a. Cytophaga - Breaksdown cellulose (wood) and chitin in the soil.



2. Medically significant genera



a. Bacteroides - Causes infections of puncture wounds and peritonitis from bowel punctures. Live in human colon in numbers approaching one billion per gram of feces.



I. Phylum 21 - Fusobacteria - These are anaerobic which are usually spindle shaped rods but are often pleomorphic. The genus Fusobacterium is found in the gingival crevice of the gums and may be responsible for dental abscesses.





Domain Eucarya





A. Kingdom - Eumycota (Fungi) - These were at one time classified as non-green plants. Modern genetic studies show that they not only are not plants, but are more closely related to the animal kingdom than the plant kingdom. They obtain their nutrition by absorption of organic molecules from their environmnets. There are currently 70,000 known species but experts think there may be as many and 1.5 million

1. Distinguishing features - These are eucaryotic organisms that obtain their nutrition from the absorption of organic matter, either living or dead.

2. Morphology - Fungi exhibit two morphologies, a single celled form known as yeast, and the mould, which has a thallus (body) composed of filaments (hyphae).

3. Mould morphology - The thallus of a mould is composed of two parts.



a. Spores - Resistant reproductive structures.

b. Mycelium - This is a complex of filaments termed hyphae.

(l) Hypha structure - A hypha is a tube of protoplasm. The walls are composed of either chitin or hemicellulose.

(2) Septate versus non-septate - Some hyphae contain no cross walls (septa) and thus the hypha is a continuous tube of multinucleated protoplasm. This multinucleated condition is termed coenocytic. Others possess cross walls (though incomplete) and are termed septate.

(3) Hyphae form branching, ramifying masses which collectively form the mycelium. The mycelium is the vegetative (nutrient absorbing) part of the fungus.

c. Dimorphism - Certain fungi can exist as either a mould or a yeast and are thus termed dimorphic. Many pathogenic species fall into this category.

4. Reproduction - This can be either asexual or sexual.

a. Asexual - Fungi reproduce asexually by the following methods.

(l) Budding.

(2) Fragmentation of hyphal strands.

(3) Spores - Minute resting cells. There are many kinds of asexual spores. These include the following types.

(a) Sporangiospores - These are produced inside of sacs termed sporangia which are located at the end of special hypha termed sporangiophores.

(b) Conidiospores - Formed naked (not enclosed) at the tip of a special hypha termed a conidiophore.

(c) Arthrospores (oidia) - Formed by disjointing and

transformation of hyphal cells.

(d) Chlamydospores - Highly resistant, thick walled spores which are formed from cells of the vegetative hyphae.

(e) Blastospores - Spores formed by budding from a parent cell. This is found in certain yeasts.

b. Sexual - Sexual processes in fungi are many and diverse, but all have three basic features.

(l) Plasmogamy - Fusion of the protoplasts of two cells.

(2) Karyogamy - Fusion of haploid nuclei to form a diploid nucleus.

(3) Meiosis - Formation of haploid spores.

c. Sexual spores - These are the spores that are formed as a result of sexual processes described above.

(l) Zygospores - Large, thick walled spores formed when the tips of two sexually compatible hyphae fuse together.

(2) Ascospores - Produced inside of a sac termed an ascus. There are usually eight ascospores per ascus.

(3) Basdidiospores - Single celled spores formed free on a club shaped structure termed a basidium.

5. Classification - The kingdom is divided into four phyla.

a. Phylum - Chytridiomycota - These are the water molds, flagellated. The tropical fish parasite, Saprolegnia, is an example.



b. Zygomycota - Sexual reproduction by means of

zygospores. Asexual reproduction is by means of sporangiospores. Major example is the common bread mould, Rhizopus.



c. Phylum Ascomycota - (sac fungi) - Sexual spores are ascospores produced in a sac called the ascus. Asexual spores are conidia. This class includes most of the yeasts including Saccharomyces cerevisiae (brewer's yeast), truffles, and the pink bread mould Neurospora.



d. Phylum - Basidiomycote - Sexual spores are basidiospores. The basidia which support the spores are formed on a well differentiated fruiting body. Mushrooms, toad stools, smuts, jelly fungi, and bracket fungi.

e. Deuteromycota - This is a holding group in which sex is not present. It one time was given phylum status, but modern molecular studies show that most of these are ascomycetes and a few are badiomycetes. Most have conidiospores.



(1) Teleomorphs - These are fungi that have both sexual and asexual sports. This includes the four phyla of fungi.



(2) Anamorphs - These are fungi which have only asexual reproduction. These were formerly members of the deuteromyctoa. An important anamorph (deuteromycete) is the mould Penicillium, which is famous for the antibiotic that it produces.

6. Occurrence and significance of the fungi - Fungi are ubiquitous. They grow virtually anyplace there is a minimum amount of moisture. They can grow over a wide range of temperature, salinity, and moisture ranges. They can utilize a wide range of organic nutrients and in turn produce a wide range of organic byproducts. Some of the most important roles they play include the following.

a. Decomposers (good) - They break down organic matter into simpler elements which can then be returned to the environment. As such they are very important in maintaining soil fertility.

b. Decomposers (bad) - They breakdown many valuable products such as wood (dry rot), food, and other commodities.

c. Symbiotic associations - These are intimate associations of two different organisms.

(l) Lichens - Fungi unite with algae to form these curious organisms which pioneer inhospitable environments. They play a major role in the initial formation of soil from rock.

(2) Mycorrhiza - These are soil fungi which form associations with plant roots. They enhance mineral absorption by the plant. Truffles are the result of such an arrangement.

d. Industrial microbiology - Many fungi are used to produce valuable products. Alcohol, citric acid, antibiotics, cheese flavoring, and nutrient supplements are but a few examples.

e. Pathogens - A certain number of fungi derive their nourishment from living hosts and cause disease. In humans, ringworm. thrush, and desert fever are common examples. In plants the number of fungal pathogens are more numerous and the damage they cause much more extensive. Millions of dollars of crop damage are done each year by fungal plant pathogens.

B. Kingdom Protista - This kingdom is characterized by unicellular eucaryotic organisms which utilize absorption, ingestion, and photosynthesis as modes of nutrition.

l. Photosynthetic protists - algae - These organisms contain chlorophyll and use it to trap light energy for photosynthesis. Some are motile by flagella while others are not. They are grouped together in several different phyla based upon the types of pigments which they contain and the food storage product the utilize. There are a few pathogens. They are usually considered to be in the realm of the botanist and not the microbiologist.

2. Non-photosynthetic protists - Protozoa - These are unicellular, eucaryotes that obtain their nutrition by ingestion of particulate matter (exception - pathogens which absorb). The feeding and growing form is termed a trophozoite. Many forms can form a protective capsule called a cyst. This allows survival during adverse conditions.



Traditionally they have been divided into four phyla based upon the type of motility which they possess. More recent biochemical studies have completely revamped the classification system, and it is still in flux.

a. Phylum - Archaezoa - These lack mitochondria. They are flagellated and many are found as symbionts in the digestive tract of animals. Two pathogenic species that are found here are Trichomonas vaginalis (vaginal infections), and Girdia lamblia (intestinal upsets).



b. Phylum - Microspora - These also lack mitochondria. They are obligate intracellular parasites. They are associated with a number of human diseases include diarrhea and inflamation of the eyes (conjunctivitis).



c. Phylum - Rhizopoda - These organisms move by means of cytoplasmic extensions known as pseudopods. Asexual reproduction is by means of fission and sexual reproduction (when present) is by means of syngamy. This phylum includes the amoebas, some of which are pathogenic such as Entameba histolytica the cause of amoebic dysentery.

d. Phylum - Apicomplexa - These are nonmotile and are obligate intracellular parasites. They have a complex life cycle involving sexual and asexual phases in different host animals. They include the genus Plasmodium which is the cause of malaria, one of the most devastating diseases known. Another form can be contracted from cats. This is Toxoplama gondii, which causes toxoplasmosis.



e. Phylum - Ciliophora - Motility in this group is by means of cilia. Typically two types of nuclei are found in the cells: a macronucleus that controls the cells activities, and a micronucleus which is involved in a special kind of sexual reproduction known as conjugation. Asexual reproduction is by means of binary fission. These are the structurally most complex of the protozoa. There is only one known human pathogen, Balantidium coli, the cause of a rare form of dysentery.



f. Phylum Euglenozoa - These are flagellated forms which include photosynthetic members such as Euglena. They also include non-photosynthetic members such as Trypanosoma which causes African sleeping sickness.

g. Phylum - Myxomycetes - These are slime molds, organisms which ingest particulate nutrients and which lack cell walls in vegetative state. They were once included with the fungi, but modern studies show them closer to the protozoan phyla. The slime molds consist either of amoeboid swarm cells or a multinucleated protoplasmic mass termed a plasmodium ("the blob". At reproduction time the swarm cells congregate and form a mass which then differentiates into a spore producing structure similar to a small mushroom. The plasmodium form converts directly into a mushroom like structure which then produces spores.

3. Significance of protists

a. Photosynthetic protists form most of the free oxygen and are the foundation of aquatic and marine food chains.

b. Protozoa are important in the following areas.

(l) They are links in the food chain, feeding directly upon algae and being fed upon in turn by larger organisms.

(2) They maintain the ecology of wetlands by feeding upon

bacteria.

(3) They degrade sewage.

(4) They cause disease in humans and other animals.

Non-Domain Agents



These are infectious agents which are acellular and do not fit into any traditional taxonomic system.



A. Viruses - These structures do not fit into any classification scheme because they are acellular. Rather they consist of a complex aggregation of molecules.

l. General characteristics

a. Each virus is called a virion. It consists of a nucleic acid core (DNA or RNA, but never both) which is surrounded by a protein coat (capsid).

b. The nucleic acid may be either double stranded or single stranded.

c. Viruses lack all machinery for energy generation or protein synthesis.

d. Because of their simplicity, all viruses are obligate intracellular parasites and cannot reproduce outside of living cells.

e. Viruses are extremely small. The largest are just visible in the light microscope and approach the size of the rickettsias. The smallest require electron microscopes to be seen.

2. Morphology - Viruses are of various shapes, frequently highly symmetrical. They are usually helical, polyhedral, enveloped, or complex (complicated)

a. Symmetry is due to the capsid. Capsid is composed of subunits termed capsomere. Each capsomere is in turn made up of protein molecules.

b. Capsid functions to protect the nucleic acid core and to serve for attachment to the host cell.

c. Many animal viruses also possess a membranous envelope which surrounds the capsid. Some of these membranous envelopes are derived from a host cell and others are determined by the virus nucleic acid. Some envelopes contain projections known as spikes. These seem to aid in the attachment of the virus to the host cell.

3. Replication - The multiplication of viruses is a complex process. It is divided into five phases.

a. Attachment to host cell and penetration of the virus into the host cell.

b. Synthesis of enzymes needed for replication of virus nucleic acids.

c. Intracellular synthesis of virus components..

d. Assembly of new virus particles.

e. Liberation of progeny virus.

The instructions for all of these events are encoded in the virus nucleic acid. Once it is in the cell it takes over all of the metabolic machinery of the cell and utilizes it to synthesize the necessary components for the viral progeny. Liberation of progeny viroids can result in destruction of the cell (lysis) or by penetration of the cell membrane in which case they are not destroyed.

4. Cultivation of viruses - Viruses can be cultured in living cells only. Three major methods are used.

a. Experimental plants, animals, or microorganisms.

b. Fertile chicken eggs.

c. Cell (Tissue) culture - These are usually of three types.



(1) Primary cell line - These are derived from slices of mature tissues. They tend to die out after a few generations.



(2) Diploid cell lines - These are derived from human embryonic tissue and can survive for around 100 generations.



(3) Continuous cell lines - These are cancerous or transformed cells that will divide indefinitely. One very famous line is the HeLa cells which were derived from a cancer in 1951.

5. Classification of viruses - Because viruses are acellular, they do not fall into any traditional scheme. They have been classified by various methods including:

a. disease caused (yellow fever).

b. tissue attacked (neurotropic - nervous tissue).

c. mode of transmission (arbovirus - arthropod borne).

Most modern systems rely heavily on nucleic acid content.

a. DNA containing.

(l) Single stranded (Dl).

(2) Double stranded (D2).

b. RNA containing.

(l) Single stranded (Rl).

(2) Double stranded (R2).

After nucleic acid content is established, then size, shape, mode of transmission, and other factors are utilized.

6. Bacteriophage - These are viruses which attack bacteria. Because of ease of their cultivation they have been intensely studied and much of what we know about viruses was learned from the phage.

The typical phage looks something like a tadpoles with a head that contains the nucleic acid, and a hollow tail. It attaches tail first to the bacterial cell and injects its nucleic acid into the cell through the tail, much like a hypodermic syringe. The capsid remains on the outside of the cell as a ghost. Once the nucleic acid is inside of the cell, one of two things that happen.

a. Replication of the virus leading to the lysis of the bacterial cell and liberation of progeny viroids.

b. Lysogeny - This is where the viral nucleic acid joins the bacterial chromosome and becomes part of the bacterium's

genetics. The viral genes are therefore replicated along

with the bacterial genes and passed on to future bacterial generations. This can be viewed as a second reproductive

strategy, reproducing with the cell.

(l) Frequently the nucleic acid of the virus are expressed by the bacterium thereby giving new traits to the host cell. Some of these new traits, such as toxin production, are medically significant. Diphtheria and botulism are two examples.

(2) After a number of generations of reproducing with the

bacterium, the provirus may detach, replicate, and

liberate, infecting additional cells.

(3) It is now known that plant and animal viruses can also follow this reproductive strategy, replicate and break out, or incorporate in the host chromosomes and reproduce with them.

7. Animal viruses - These are viruses that parasitize animal cells.

a. Lipoprotein envelope - Most of the larger viruses have a

membranous envelope surrounding the capsid. This envelope may be derived from a previous host cell or coded for by the virus itself. It appears that the envelope makes it easier for the virus to be adsorbed by the host cell.

b. Life cycle of animal viruses

(l) Absorption (Attachment and penetration) - Animal viruses attach to many of the host cell's normal molecular receptors (glycoproteins). Once attached, the virus then penetrate the cell membrane and enter the cell. Penetration appears to occur by two mechanisms.

(a) All naked viruses and probably most enveloped viruses enter by means of endocytosis. Here the virus attaches to receptors on the cell membrane and is drawn into the cell.

(b) Enveloped viruses may also fuse their envelopes with host cell membrane releasing the capsid and nucleic acid inside of the cell.

(2) Uncoating - This is the separation of the capsid from the nucleic acid. It occurs only in animal viruses. The process is not well understood. It appears that at least some viruses utilize the hydrolytic enzymes of the host cell lysosome to carry out this process.

(3) Replication - This may involve either the immediate replication and escape of the offspring or incorporation into the host chromosome.



(a) DNA viruses - The strand of DNA is copied into messenger RNA. This messenger strand then directs the synthesis of the necessary enzymes for viral replication. These in turn initiate the copying of the viral DNA.

(b) Normal RNA viruses - The RNA strand directs the synthesis of enzymes which in turn direct the synthesis of viral RNA. This type of virus cannot become a provirus.

(c) Retroviruses - These RNA viruses utilize the enzyme reverse transcriptase to synthesize a DNA strand that matches the viral RNA. The new viral DNA is again copied to form a double stranded DNA. This viral DNA now incorporates itself into the host cell's chromosome. The virus is now a provirus. Unlike a prophage it will never leave the host cell chromosome. The provirus is transcribed into RNA which then forms new virions that are released. Usually only a few virions are produced. The provirus may also remain latent and reproduce only when the host cell chromosome does.

(4) Liberation - Progeny may escape by destroying the cell. This is the process followed by naked viruses. Most enveloped viruses bud off by pushing out of the cell membrane, a process which may not kill the cell although it is weakened and may die in the future..



8. Viruses and cancer - In addition to their ability to cause infectious disease, a growing body of evidence indicates that many viruses may cause cancer. It is now thought that probably 20% of all cancers are due to viruses.



a. Oncogenes - These are now considered to be the cause of virtually all cancers. Oncogenes are derived from proto-oncogenes, normal genes found in all cells. These normal genes usually control some aspect of cell growth and division. Once converted into oncogenes they cause the cells to divide in an uncontrolled manner, forming a tumor. Proto-oncogenes can be converted into oncogenes by chemicals, radiation, and viruses. Viruses which cause cancer are referred to as oncogenic viruses.



b. Viruses which have been implicated in human cancers include the:



(1) Epstein-Barr (EB) virus - Burkitt's lymphoma

(2) Hepatitis B virus HBV - Liver cancer

(3) Human T Cell Leukemia virus (HTLV 1 and 2)



B. Prions - These are sometimes referred to as "unconventional agents" because they do not fit any category. Prions appear to be infectious proteins. Prions are derived from normal proteins that have been converted to infectious forms. Once converted, they can then convert other porteins. Prions are associated with neurological diseases such as mad cow disease (Bovine spongiform enchephalitis, BSE) Creutzfeldt-Jakob disease and Kuru.