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Unit I - Introduction {Quotes on board - discussion} {Attendance} - Global to Local {Articles} - Lyme Disease – endemic - Climate Change & Disease - Bird Flu – potential pandemic - Microbiology, Is It Hard? {*} - Microbes/Discipline are everywhere Infectious Disease - WTBY/World History - Plague - epidemic - history - Syllabus - biology/ecology Lyme Disease - No. #1 vectorborne disease in the USA --------------------------------------------------* VISIT TO MATTATUCK MUSEUM 1st station - What happened to the local Native People? measles (v) typhus (b) small pox (v) 2nd station - Door / Pest House, Dr. Abel Bronson 1784. Of course, we now have had a best selling book, “The Hot Zone” And a number of Hollywood movies, Dustin Hoffman movie and “Medicine Man.” WORLD HISTORY – Population Curve & Disease

____________________________________ | | | | | 8,000 BC 4,000 0 1,000 AD 2,000 TIME (YEARS) NOW USING BP (or BCE) - Gathering & Hunting / life span 20 years - Organized Slave Societies (Egypt, Greece, Rome) / 30 years why avg. life span so low - infant mortality! - ~1200 AD / plague era (we’ll come back to this.) Why does curve, our population curve go almost straight up? - organized production (agriculture & industry)/ modern medicine (sanitation etc.) Now, the plague: 1st some data: DATE





Almost all



Almost all






Almost none

Classic epidemic - explosive in a given area. Historical Background:

Imagine the terror

- Queen Elizabeth (1565) goes to Windsor Castle and orders a gallows be set up outside the gates. Hang anyone who comes from London. And they did it! Survival was class based. - How would you like to be a gravedigger? - good pay. Used to take the bodies out in carts and drag them into ditches with a pole with a hook at the end of it. Did you ever hear, “I wouldn’t touch it with a ten foot pole.” - Culturally, plague is reflected in songs and poems. How about, I remember from Kindergarden, “Ashes, ashes, all fall down.” Let’s look at a parable.

- What’s a parable? A simple story with a lesson. So we will be talking about some daunting stuff in here, but don’t be overwhelmed by it. We also have some exciting stories to tell on how microbes do many good things and how we have won some battles against disease. {Now - rest of syllabus}

Unit I

Introduction continued

Conquistadors & S.A. {Population graph (OH)} biology/ecology 3 kinds of plague AIDS - pandemic - biology/biochemistry - defined - CT - HIV+ - HIV Syllabus Interviews -------------------------------------Conquistadors brought diseases (same as WTBY area) = 50 million Native Peoples died in South America {movie, Medicine Man}. {Pop. graph revisited}

Plague causative agent is Yersinia pestis. It is a small, gram negative rod. Reservoir host = the black rat, Rattus rattus. Vector {carrier}= Fleas. Cause = ecological imbalance. {lyme disease, same} 3 kinds: 1. - Lymphatic System, lymph nodes {demo} swell (buboes), hence the name bubonic plague. symptoms = fever, delirium, swellings {can lead to shock & death}. 2. Circulatory System - septicemia, infection of the blood . Black lesions. Thus the name black death. {virulence due to hiding of bacteria in phagocytic cells} 3. Respiratory System- heavy cough = pneumonic plague. Lungs. heavy cough and Y.p. goes airborne, contagious. Still occurs in USA. Ground squirrels out west. India, turn of the 20th century, 10 million died. Rats are in a diseased state. Can be bitten by rat and get plague. Streptomycin. By the way, epidemiology is the study of the incidence, distribution and transmission of diseases among people . Now Acquired Immune Deficiency Syndrome Pandemic, First described in 1981 Luc {} Montagnier and Robert Gallo (WTBY!). - believe it began as early as 1950s. {contrast to small pox in Egyptian mummies}. source = chimps. {used to think green monkeys} Central Africa to Caribbean to USA & Europe & Asia. Biology/biochemistry. Etiological agent = Human Immunodeficiency Virus (HIV).

Attacks T4 lymphocytes or helper cells. Help in maturation of other WBC that produces antibodies. You know DNA makes RNA makes Protein (Central Dogma) HIV works in reverse. {See diagram on syllabus}. Patient becomes immunodeficient and dies from other infection. Syllabus + annoucements THE INTERVIEW NAME TOWN FAMILY/JOB (optional) Academic goal The last book I read or am reading, is _____/ about______. What I would most like to understand about microbiology is ____ What I worry about most is _____________ This afternoon I will ______________________

Units II & III

taxonomy & history

AIDS CONT. - USA/ CT/ World Scene - How They Die Taxonomy of Microbes - DOMAINS - Kingdoms / Sci Name History of Microbiology - introduction - Fracostoro (new evidence)/ xenophobia

- Jenner - Leeuwenhoek Royal Society ______________________________ - USA/ CT/ World Scene {Text + Students Web Search e.g.CDC} To bring these stats alive: - By 1992, it was the main cause of death for young American men. AIDS is already Africa’s greatest killer. In Botswana, children born today have a life expectancy of 20. Take AIDS out of the stats and it goes to almost 70. HOW THEY DIE - The cause of death in over 50% of AIDS patients = Pneumocystosis infectious agent = Pneumocystis carinii A protist It is a particularly nasty protist, a sporazoan PCP = pneumonia, high fever, breathing difficulties (aveoli destroyed) - 20 to 25 % of patients with HIV develop Kaposi’s sarcoma common in Africa, purple skin, skin cancer. There is no vaccine.


Taxonomy of Microbes - What is Microbiology?

- Kingdoms / Sci Name - What’s in a name? - DOMAINS -----------------------------WHAT IS MICROBIOLOGY? It is the study of cellular and acellular organisms beyond macroscopic vision. {Not just a list. How they effect the environment, how they are affected by the environment, products they make from the nitrogen in the air to the sourgrout on your hotdog if you are still eating them.} WHERE ARE THE MICROBES? FUNGI (Mycology) e.g. penicillium yeasts {British Museum has

PLANTS (Botany) ANIMALS (Zoology) e.g. algae (Parasitology) e.g. ticks nematodes, found in soil

samples of bread from Egypt before 4000 BP} PROTISTS (Protozoology) e.g. Euglena MONERA (Bacteriology) e.g. E. coli A Case Apart - ACELLULAR MICROBES e.g. Viruses (Virology) e.g. Viroids, a “naked” virus, no protein coat. e.g. Prions, just protein, can self-replicate (AIDS?) There are many fields (read careers) in Microbiology e.g microbial ecology. industrial microbiology, medical microbiology, agricultural microbiology, immunology and more. Conclusion - MICROBIOLOGY IS INTERDISCIPLINARY. Now, taxonomy continued, each kingdom can be broken down into phylum, class order, family genus species, subspecies (we usually say strains. As the taxonomy of microbes is in flux, don’t memorize these unless directed by me. {ad lib - we usually work with kingdoms and genus/species}

Carolus Linnaeus developed the genus/species idea in the mid1700s. This name usually tells you something useful about the organism. e.g. Streptococcus pyogenes R. + 1884 chains / spheres

Rosenbach / Gram positive / date

See inside covers of text for pronunciation. This bacterium commonly causes sore throat. Can lead to Scarlet Fever, rheumatic heart disease. One version Strep A. One reason so nasty is it has the M protein that retards phagocytosis. DOMAIN SYSTEM - better represents the biodiversity of microbes. (Carl Woese, 1977) Eubacteria e.g. Cyanobacteria

Archae(a) Eukarya - Halophiles - Protista - Thermophiles - multicellularity Universal Ancestor {OH} Most important - proper taxonomy reflects diversity and research follows e.g. slime molds - noncharismatic species {have flagella and no chitin, so a Protist! No say mycologists.}

MICROBIOLOGY – A HISTORY We have said in different ways: Microorganisms are found in Antarctica, Mt. Everest, in the air, water, on and inside our bodies. They are ubiquitous. Throughout the ages, humans have been aware of the effects of microbial activity

even though they were unaware of microbes as living entities. e.g. Memphis Egypt, 5400 BP – beer Breads /didn’t understand fermentation. e.g. Concerning disease: Ordinances of Manu / India 1300 BCE (same as BC) Bridegrooms were warned about marrying into a family that had a coughing disease (tuberculosis) because the bridegroom might acquire the disease. Tiberius Claudius Neru Caesar (14-37 CE) banned kissing in Rome to avoid cold sores (oral herpes). Did he or they know about viruses? NO. ITALIAN SCIENTIST – Girolamo Fracostoro, published a manuscript in 1530 describing the symptoms of a shepherd named Syphilus. This disease, STD, became know as syphilis. In Italy it was known as that French disease. In France it was known as that English disease.

Syphilis is caused by the bacterium, Treponema pallidum, a spirochete. They are spiral shaped. Also have many flagella Figure: Even the treatment of disease caused by microorganisms, including viruses – acellular microorganisms – was done for centuries without understanding the organisms that were the causative agents. e.g. The Cherokee Indians used the root of the cardinal flower (Lobelia cardialis) for the treatment of syphilis. e.g. Edward Jenner (1749-1823), an English country doctor, observed that milkmaids often caught a mild form of “the pox.” These milkmaids apparently contracted this from cows – cowpox. He noticed they rarely contracted The Pox.

Using these observations Jenner deliberately infected people with the relatively harmless cowpox virus, causing the recipient’s body to develop antibodies against cowpox. These antibodies were also effective against small pox. Jenner was totally ignorant of viruses and antibodies. He just saw “it worked”. Viruses would not be even indirectly observed for more than a century. – Tobacco mosaic virus (TMV). But that’s getting ahead of our progression. NOW, who was the first to describe microorganisms? - Robert Hooke: coined the word cell {ad lib} - Zacharias Jansson: first microscope. • Anton van Leewenhoek (1632 -1723) - He was a Dutch Dry Goods Merchant

Now, people thought ole’ Anton was a bit weird. He did his science work at night in the back of his store. He began to talk about what he was observing under his microscope. / pond / mouth. You have to keep in mind to investigate and observe life as it is could be very dangerous business back then – 350 years ago. e.g. Servetius was burned at the stake for daring to cut up and examine the body of a dead man. e.g. Galileo was shut up by the church for daring to say that the earth turns and orbits the sun. In England, scientists started a society called the invisible college so they could observe and experiment – and they had better be invisible or they would have been hung as heretics had it been known what they were up to. The Invisible College morphs into the Royal Society of England. Leewenhoek writes them and tells of “little beasties.”

They’re astounded! He tells them he finds them in his mouth and can be found in your mouths. He sends drawings that can be found on your syllabus (near the numerical grades). He was a humble man who worked also as the janitor in town hall. He was later to join the town council. {Read quote- syllabus} Interested - read “The Microbe Hunters” by De Grauf. Van Helmont (1577-1644) – He developed the following recipe: dirty underwear/ grain / damp place and presto you get mice, fully formed. Now Van Helmont was an excellent chemist and helped elucidate the photosynthetic process/ Yet he held the idea that life could arise from nonliving matter – a widely held idea at the time and for another 200 years. Spontaneous Generation. The disproving of spontaneous generation and the affirmation of biogenesis (life comes from life) is integral to the history of microbiology, indeed all the life sciences. – can’t separate these.

See in your text Francesco Redi { maggots, the footless larvae of flies} Proponents of SG responded by saying OK but microbes arise spontaneously. This was challenged by Lazzaro Spallanzani (1765)

Figure Once opened to the air: Turbidity

Figure The adherents of SG said well the SG of microbes requires air. So Theodor Schwann (1810-1882)

Figure Now proponents of SG said the heat was destroying the “vital force” necessary for SG. Keep in mind, this was a current idea in the mid 19th century e.g. Mary Shelly. Now, also some who repeated Spallanzani’s and Schwann’s experiments found microbes – now think this was due to spores – hold that thought. The most convincing evidence against the idea of SG and for biogenesis provided by Louis Pasteur. Entering the first and classic Golden Age of Microbiology Mid-1800s to WWI 1914. Louis Pasteur (1822-1895) A French Scientist. Why was he in a prime situation to do microbiology and the many advances he authored?

1. A chemist. 2. He studied crystals. To study crystals he had to use a __________ microscope. {Think of our labs} 1861 Figure: - broth of sugar water or beet juice - heated broth to kill microbes already present. Figure: - unheated air - many days / no microbes 1857 – tragedy strikes Paris, the wine goes sour. Thinking up to this point is that grape juice “breaks down” resulting in wine. No living organisms involved. Pasteur, using his chemistry and microscopy skills, went to work. Normal (grape juice) / Sour - yeast cells

/ yeast & Rod-Shaped cells

------------------------------------------------------------removed {o} no alcohol / removed {rods} no fermentation / no spoilage 3 important conclusions: 1. Microbes have specific metabolisms. 2. Microbes generate products we use. • 3. Microbes can be agents of change. Prevailing thought was that bacteria moved in when there was sickness – an effect – now it seemed that microbes could be on the causative side. Figure:

Pasteur is not a distant observer in all this. He loses 3 daughters to disease. But it was Robert Koch, that other microbe hunter, who published findings that showed microbes cause disease. Competitive period: Pasteur took pathogenic organisms and made them nonvirulent. He would inject them into animals, eventually including humans, and make them immune. He honors Jenner calling them vaccines {vaca = cow} {Joseph Meister story and rabies vaccine} Of course all this suggested strongly that microbes cause disease. Robert Koch (1843-1910) main contribution was the linkage of specific diseases to specific organisms. E.g. Anthrax – Bacillus anthracis

Anthrax was killing large number of cattle in the late 19th C. and people contracted it. Symptoms: skin disease / flu like / endospores enter the skin through a cut or scratch then germinate in the wound. Figure: Remember it’s a competitive period: Pasteur recovers spore from soil and says more cattle are probably infected while grazing so, burn the dead infected animals and bury them in non grazing fields. Spores > blood > germinate there > all throughout the body > lungs > fatal. e.g. veterinarians must be careful, farmers. B.a. exotoxin > CNS (phrenic nerve) > Respiratory failure.

Koch noticed these rod-shaped structures in the blood of sick animals. He grew the bacteria in media > the mice died > mouse Bld > bacteria. Specific microbe connected to a specific disease. His lab also figures out tuberculosis (TB) Mycobacterium tuberculosis. E.g. doc holiday, Henry David Thoreau. Fellow by the name of Petri works in his lab. So Pasteur and Koch stimulated others to study microbes and disease. This time period is called the classic or First Golden Age of Microbiology. Koch’s Postulates (OH) Warning: It is not always applicable e.g. poisons Clostridium tetani; H.D. Thoreau’s brother dies of tetanus. Microbe not present. C. botulinum also. - also some organisms don’t grow well in vitro, in the lab. E.g. viruses. {PASS OUT STUDY GROUP SHEETS}

The Origin of Microbes Unit IV Evolution = Change through time or Descent with modification. 13.8 BYA > The Big Bang. 5.0 BYA > Mother Earth But where is life? First there would have to be organic (Carbon) matter. Oparin/Haldane Hypothesis: (1920s) CH4 = methane NH3= ammonia + ENERGY (lightening, volcanoes, sun) = Organic Molecules H2O= water H2= hydrogen Tested by Stanley Miller, University of Chicago, 1953. {OH} - amino acids! (repeated - found bits and pieces of carbohydrates and nucleic acids). Now, still a far cry from a living organism. so in the lab: H-N-C-C=O / / H OH

+ same >

Dehydration reaction + peptide bond. H2O driven off C-N

- less than fifty amino acids = polypeptide / more = protein. Bonding can lead to > O {Done in lab} / also O split into O O - This is very much like the binary fission of bacteria. 1. These are called Microspheres. May have formed in lakes on primitive earth. About 4.0 BYA. 2. Next thinking is that some contained protein. And those protein molecules functioned as templates to make more protein. Some present day bacteria make polypeptides from protein templates. These are called Progenotes. (Remember no DNA or RNA!) 3. At some point, RNA took over this function.. Now called Eugenotes. {RNA was first!!}

4. Eventually DNA evolved. Now at 3.5 BYA. How do we know this? Science has the microfossils! Rock outcrops Australia and South Africa. Flow Diagram of Microbial Evolution: 5.0 BYA 4.0 BYA

- Formation of the Earth - Microspheres - Progenotes - Eugenotes

3.5 BYA

- Early Prokaryotes

BACTERIAL EVOLUTION These bacteria represent all living organisms. For 2.0 BY they ruled the earth. dinosaurs “ruled” for 200 MY so. . . } Once they appeared on earth, they evolved rapidly. They made use of nutrients that were available in the environment, were affected by the environment and CHANGED the environment. (Sun) e.g Cyanobacteria - H2O + CO2 > O2 + Organic matter {Hold do bacterial evo. first} / / Eubacteria Cyanobacteria e.g. B. subtilis

e.g. Anabena

/ Archaea e.g. Halobacterium

NOTE: Lenski - studied a strain of E. coli over 10,000 generations. He observed punctuated equilibrium. (vs. Darwin’s slow evolution). 1.5 BYA

- 1st Eukaryotes e.g. Protista

- 600 MYA So



- muticellularity Plants



So up to 1.5 BYA, we have these small simple {ad lib} cells, all about 5 to 20 nanometers thick {mm to Um to nm, 9 places to the right of the decimal point.} Starting about 1.5 BYA we see much larger cells; cells more than 100 nm thick up to 600 nm thick. And there are now the appearance of complex structures inside those cells (organelles). Now we have the fossils, we know it happened proceeded from these Pros to Eus. How it occurred is debated. - Need to back up a bit. e.g. Cells in Eus now have a mitochondria and through electron transport {remember?} produce ATPs. - at the very end of the electron transport chain O2 + electrons (H) > H2O - oxygen is reduced (gains electrons) to produce H2O. I. Some scientists say the reason the larger more complex Eu cells arrived on the scene is due to the availability of large amounts of O2.* * products of O2 producing Pros. II. Wait a minute. Other scientists say we are finding rocks billions of years old with rust. e.g. Fe + O2 > Fe2O3 (Iron III Oxide) EVOLUTION OF ORGANELLES There is debate here also: 1. Endosymbiosis Hypothesis - Lynn Margulis - U. Mass. Diagram:

A. Small cells invaded large cells for protection. B. Large cell tolerates the small cells for increased energy from the small cells respiring. {Remember more O2 around}

Also, some adhearing to the endosymbiosis hypothesis say the flagella originated by an ectosymbiosis Diagram:

Lately, some scientists are saying that a structure found in some microbes e.g. Yersinia that use this structure to shoot off toxins, is closely related structure-wise to the flagella. (see microfocus) Diagram:

MEMBRANE PROLIFERATION HYPOTHESIS Say organelles e.g. nuclei, mitochondria, , chloroplasts arose from invagination of the cytoplasmic membrane. Diagram:

Are both possible? We will look closer to the evidence for these hypotheses as we go through the morphology of the prokaryote and eukaryote cell.

Origin of Microbes Cont. Unit IV The Prokaryote and Eukaryote Cell Unit V Review -Timeline ---------------------------HIV & Evolution - HIV-I & II - retroviruses

- microbial genes & us Pros vs. Eus Cells -------------Circle Discussion

HIV & EVOLUTION Every AIDS patient is a monument to the theory of evolution. Darwin thought that evolution was too slow to observe. Wrong. Genes and time have come together on a human scale here. It is descent with modification. DOES THIS MEAN DARWIN WAS WRONG? Is evolution wrong? discussion. - RNA (HIV) has 300 N.A. at its signature point. - HIV-1 = homosexuals, HIV-2 = heterosexuals - HIV tracked to the 1940s - can pick up both HIV-1 and HIV-2 but no hybrids. Do these represent separate species? - HIV-1 origin in chimps (ape), HIV-2 origin in the sooty mangabey (monkey). Could it be that AIDS occurred in Africa in past centuries but general isolation prevented its spread? Discussion.


Unit V

1. Size : Bacteria, as a group, exhibit a wide range of sizes ( 5 - 20 na). They are usually much smaller than Eukaryotic cells. 2. Genetic Material : 1 DNA molecule (what do we have? 46) / Not paired in Pros/ Circular. Also note - no nucleus. 3. Organelles : absent. Eus - mitochondria {OH} - rectangular or rod shaped, - divides by binary fission - circular DNA Eus - Nucleus is now thought connected to the mimivirus (huge, can be seen with a light microscope). {diagram on board}. * ALL THE ABOVE IS MORE EVIDENCE SUPPORTING THE ENDOSYMBIOSIS HYPOTHEISIS.

4. Ribosomes: Smaller than Eus and free in the bacterioplasm. Pros = 70s

Eus = 80s (svedberg unit)

Why else is this significant? Because we have found out that certain antibiotics attack protein synthesis at the 70s and NOT the 80s. e.g. erythromycin, chloramphenicol, streptomycin, tetracycline ****** So no E.R., Golgi Body, Chloroplasts in Pros. 5. Locomotion: Flagella - Many different arrangements in the Pros. Very thin. Mordant necessary to visualize. Rotation is stiff versus Eus that is undulating. 6. Cell Wall : Almost always present (exception mycoplasms). Contain peptidoglycan (murein). No peptidoglycan in plants (cellulose) or fungi (chitin). How about animal cells. NO CELL WALLS! 7. Plasma membrane : Yes. Similar to Eus. Also, contains respiratory enzymes (remember! no mitochondria.). Genome (genetic material) binds at some point to the C.M., so it plays a role in, 8. Reproduction: by binary fission (no mitosis, meiosis). Sometimes the daughter cells remain attached after fission. [Board]

Strepto: Staphylo:

SUMMARY: Prokaryotic and eukaryotic cells are structurally distinct; they carry out some of the same life activities, but do these differently.

Unit V continues with a focus on the outer cell structures of the prokaryotic cell. Surface Structures of the Prokaryote Cell 1. Capsule/Slime Layer - is composed of polysaccharides which, quite often, are glycoproteins. The capsule can vary in, a. chemically b. size c. rigidity Note: Capsules can be removed and the bacteria remains viable. The function of capsules are, i. protection against – dehydration. So pathogens with capsules are associated with virulence (The degree of pathogenicity). - Protection against phagocytes - antiphagocytic. A slimy smooth capsule can protect bacteria e.g. Streptococcus pneumoniae, from phagocytosis. The WBC slips off and the bacteria slips away. ii. attachment, Capsules composed of polysaccharides are sticky and play an important role in attachment. e.g. Streptoccocus mutans, an initiator of dental caries, is a good example. S. mutans takes in sucrose (common table sugar) and produces

dextrans. This latter chemical flows over the capsule and allows S. mutans to attach to the surface of teeth. This chemical also becomes home for more bacteria to attach to the site forming dental plaque. These bacteria commence producing weak organic acids that demineralise the enamel of our teeth. Note: The capsules of bacteria are antigenic. They can be used to I.D. different organisms. There are over 80 strains of S. pneumoniae. 2. Pili a. Hair-like protein 4 – 35 nm wide. Not all cells have them. b. Some have what are considered sex pili. This means that pili connect two cells and nucleic acid material crosses this pili bridge. (OH E.coli with pili) c. Pili are important with pathogens where these fibrous protein threads can play an attachment role. e.g. Neissera gonorrhea (gram -, diplococci) in the urogenital system e.g. E. coli (gram -, rod) that causes infections has pili. *Many gram –s have pili. 3. Flagella -are helical protein threads, 12 – 18 nm wide. They are in three basic components: the filament, hook, and basal body (OH) - The prokaryote flagella is not like those in eukaryota which are fluid e.g. Euglena, human sperm. The prokaryote flagella are rigid and rotate. 4. Cell membrane or cytoplasmic membrane - It is composed of a lipid bilayer that is mostly phospholipid (20%) and protein (80%). It is a fluid mosaic with proteins moving laterally. (OH) - Its main function is to control what gets in and out of cells. While freely permeable to organic substances and H20, it doesn’t readily allow charged substances to penetrate its boundaries.

- Substances enter the plasma membrane by, a. simple diffusion – down a concentration gradient. b. facilitated diffusion – carrier proteins are involved but no energy input is necessary. c. active transport – binding proteins are involved plus energy (ATPs) input. - However the membrane in the prokaryotes have more functions. i. Synthesis of lipids ii. Synthesis of cell wall here. The enzymes are here. iii. Replication of DNA involved here. The DNA is attached here. iv. Electron transport system here. • The cell membranes of the prokaryotes are busy places. 5. Cell Wall - Main function is protection. It maintains the osmotic integrity of the cell. Peptidoglycan, unique to bacteria, dominates while there are other chemicals present. - Most bacteria have wall with honorable exceptions. The Mycoplasma Have no cell wall. Cause throat irritations. Also, the archae e.g. methanogens, halophiles have cell walls but no peptidoglycan. History of the Gram Stain - In 1884, Hans Christian Gram (1853-1935), was searching for a way to stain the background of a slide different from the bacteria. He stumbled on a differential stain that showed there were two fundamental bacterial groups. We now called them gram + and gram -. - It was quickly realized how valuable a discovery he had made. It is universally used in research, clinical and academic microbiology labs to this day. The Gram Stain - first 5 steps same as preparing a simple stain, then,

- 1. Crystal Violet – it reacts with negatively charged groups in the cell with a purple color. - 2. Gram’s Iodine (Lugol’s Solution) – functions as a mordent. It strengthens the attachment of the crystal violet to the cell - 3. Ethanol – functions as a decolorizer. It removes both the crystal violet and iodine from the cells. *But because of the thickness of the wall of gram +’s, it diffuses out more slowing here. Therefore the gram + remain purple while the gram – becomes clear. - 4. Safranin – functions as a counterstain. It also attaches to negatively charged elements in the cell. As there are many more sites now in the gram – cells, it turns these cells pink. The gram +’s remain purple. Teichoic Acid plays a role in the wall of Gram +’s. In the wall it plays a structural role and is called wall teichoic acid. There is a teichoic acid that reached from the wall to the cell membrane. It functions to as a chemical “glue” here. It is called membrane teichoic acid. * Look at peptidoglycan structure (OH) and in text. ************************************************* Unit VI – Bacterial Reproduction and Growth Unit VII – Spore & Antibiotics / Resistance Unit VIII – Microbial Metabolism Unit IX - Microbes and Disease – Study Group Reports MICROBIAL GENETICS

Unit X

Genetic Engineering - why bacteria - how? - products - transposons {natural mutations} - Barbara Mc Clintock DNA - A History - 1860s

- Griffith{Frederick} Second Golden Age Of Microbiology - Avery & Transformation - Rosalind Franklin - Watson & Crick DNA Basics - central dogma - base pairs {OH} Synthesis - replication semiconservative - bidirectional synthesis - rolling circle {conjugation} - transduction ------------------------------------Why are bacteria in the forefont of the Molecular Genetics Revolution? 1. One DNA molecule (circular) {We have 46 and linear} 2. No dominance/recessive {No Punnett Squares} 3. Easy to cultivate {Know this from labs} 4. Reproduce rapidly {eg. E. coli 1 generation/20 min.} How? Key - Usually done with plasmids = small bits of DNA, independent of the genome. Cell #1 = Streptomyces

- gene a makes the antibiotic streptomycin {effective against TB, Plague} But not fast enough. Step #1 So we clip out the section of DNA, with a restriction enzyme, responsible for producing the antibiotic. Step #2 Make many copies of it in PCR

Step #3 Sew a into plasmids using DNA ligase Step #4 Make Cell #2, which has a fast metabolism, competent {in lab use cooling and heating} Step #5 Insert plasmids into Cell #2. They produce the antibiotic fast. OTHER PRODUCTS: THIS CAN BE DONE TO MAKE GROWTH HORMONE / INSULIN. Transposons - Jumping genes. Happens naturally. Plasmid to plasmid or plasmid to chromosome or chromosome to plasmid. CAN CARRY ANTIBIOTIC RESISTENCE. Barbara Mc Clintock won a nobel prize (1983). {More Microfocus 6.6} When we talk of genetic engineering and genomics we’re talking about the 3rd Golden Age Of Microbiology (1970 - to date). DNA - A HISTORY How long have we known about DNA? Since the 1860s! A German scientist talked about a substance in the cell called “nuclein.” We know now that substance is DNA. Most scientists thought, prior to 1944, that chromosomes consisted entirely of protein. After all, everything else seemly of importance was coming up protein e.g. hemoglobin, Insulin. Proteins play an important structural role and regulatory role. But this was wrong. It was DNA. This is getting ahead of the story. Elegant, but simple, {and so many elegant experiments are simple} experiment by Fredrick Griffith, a British Bacteriologist. Streptococcus pneumoniae s- strain = smooth appearance - capsule. r-strain = rough appearance- no capsule. s-strain --------> mice = died r-strain ---------> mice = lived

Then he took live r-strain + dead s-strain ---> mice = died!!! {heat killed} What happened? He looked at the blood of the dead mice and saw live sstrain. Thus, something must have passed from the dead s-strain to the live rstrain that made them pathogenic. What was that substance? {Good diagram on P. 234} It wasn’t until 1944 and Avery isolated that substance. {Added protein digesting enzyme and substance didn’t break down. Added nuclein digesting enzyme and substance was lost.} Still didn’t know the specifics, the structure of this substance of what we now know is

Deoxyribonucleic Acid (DNA) Famous Diagram that is in every biology book except yours. Rosalind Franklin {see Microfocus 6.2}. Died in 1958. As you probably know, Watson and Crick get the recognition and Nobel (Indiana Ed.) (English) Prize (1962). They don’t give Nobel prizes posthumously. They publish the famous “Double Helix.” {Use below or skip to OH}

Twisted Ladder Analogy

s = 5 C sugar - deoxyribose p = phosphate group n = nitrogen base Nitrogen Bases: Adenine (A) Thymine (T) Guanine (G) Cytosine (C) A + G = purines

T + C = pyrimidines Because the bases pair up, if you know one side of the DNA molecule, you can figure out the complimentary strand. e.g.

ACTTG TGAAC DNA Synthesis The genetic material in bacteria is called the genome. Remember that the genome of bacteria is simple in that it is circular and one molecule. But even while saying this, e.g. E.coli has over 4 million base pairs and 4,288 genes. {We – Homo sapiens - have 23,000 genes.} Remember homopolymers e.g. glycogen. Just put some glucose in a beaker with some enzymes and bingo = glycogen. Can we do this with DNA replication. No! It is a heteropolymer. What else is a heteropolymer? Yes/ Proteins. So in these cases you need a template. DNA is the ultimate template. Bidirectional Synthesis:

- Okasaki Fragments {come in chucks of nucleotides forming the new strand. The other side come in as single nucleotides} - Each chromosome contains an old and new strand, thus it is semiconservative. {Fig.6.3}

Rolling Circle: e.g. “conjugation� bacterial chromosome not involved. Plasmids.

Transduction: - invasion of a bacteria by a virus called a phage or bacteriophage. {Think this happens with TSS (toxic shock syndrome), staph infection, makes the bacteria more pathogenic.} {OH} Question: E. coli - DNA replication takes about 40 minutes yet cell division takes about 20 minutes. How? More than one growing point. 3 in fact. Protein Synthesis like rolling circle except M-RNA is made. DNA ------>RNA--------->Protein = Central Dogma transcription


Remember no T (thymine) in RNA. U or Uracil in its place. {OH} then make enzymes or toxins. e.g. Clostridium botulinum which inhibits the release of ACH so no nerve muscle stimulation.

MICROBES & IMMUNOLOGY Unit XI 1. Introduction - who? / Evo microbes - humans 2. History 3. Defined 4. Antigens - allergic reactions 5. Components 6. T-Cells 7. B-Cells 8. Phagocytosis 9. Complement/Opsonins 10. ELISAs 11. Vaccines - AIDS -------------------------------INTRODUCTION - All mammals, including humans, and also birds, have an immune system. Successful microbes and viruses, IN AN EVOLUTIONARY SENSE, are those that have defeated this system with one of two strategies: 1. Antigen Shifting e.g. Trypanosomes {Board} 2. Direct Attack e.g. HIV ad lib. It actually does both! For Reference: Immunology and Evolution of Infectious Disease By Steven Frank, Princeton University Press, 2002. HISTORY - The science of immunology sees its origins with the work of Edward Jenner (1798) with small pox and Pasteur, with vaccines for anthrax, rabies etc. It is of particular interest to the microbiologist - to see how the immune system reacts to a. microbial invasion b. vaccine dev. * The fact that we have an immune system is proof positive that we have coevolved with microbes! Without it, YOU DIE! E.G. AIDS {Coevolution is when two or more organisms show mutual adaptations to one another over time. e.g. Darwin’s Orchid and proboscis or tongue 20 inches long! e.g. bees and flowers.}

DEFINED - Immunology is the study of the cells and their products whose primary function is to protect animals from invading microbes and cancerous growths. ANTIGENS - a chemical substance that elicits a response by the body’s immune system. e.g. surface structure of a microbe - capsule, pili, flagella e.g. bee venom, poison ivy, cosmetics. {read leaflet, allergic Rx} COMPONENTS: Humoral Response - antibodies, B-Cells. Remember, antibodies alone kill nothing! Cellular Response - T-Cells, some are regulators like the famous help Ts or killer cells. Now all these are forms of lymphocytes, the letter indicates where they maturate, the B-Cells in the bone marrow and the T-Cells, while originating in bone marrow, complete their development in the thymus gland. By the way, B-Cells produce 2,000 molecules (antibodies) per second! They are then called plasma cells. Some become memory cells. Phagocytes - are monocytes. In tissues we call them macrophages. {part of the reticuloendothelial system} One Scenario: Go to the lungs / Streptococcus pneumoniae / Fixed Macrophage

{1st Diagram Here. S.p. with slippery capsule slips away!}

What has evolved here? B-Cells take a molecular read of the invading antigen and produce antibodies. These stick to the surface of the bacteria (antigen). There are five classes of antibodies - IgM, IgG, IgA, IgE, and IgD Ig = immunoglobulin IgM, M = Macroglobulin, 1 st to appear with an infection. {Only use if necessary IgG, G = gamma globulin. It is the 2 nd response. Appears 24 to 48 hours after antigen stimulation.} Back to our one scenario: Now, serum proteins called complement ( 11 serum proteins produced by the epithelium of the gastrointestinal area and other cells) also “tag” the antigen. {2nd diagram}

These together are called opsonins (from the Greek, meaning “to eat.”) Process is called enhanced phagocytosis or opsonization. {Show PMN cell engulfing a bacteria now or next time.} NOTE {end} - Antibodies attack toxins, function as an antitoxin. ELISA - {use OH/ also see lab notes } a molecular sandwich How vaccines are made - {use HIV & AIDS OH} *************************END***********************************