Microbiology Week 2 Notes - Chapters 1 and 2
Microbiology Week 2 Notes - Chapters 1 and 2
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Date Created: 01/17/16
MICR 3050 – Notes Set 2, 01/11/2016 Dr. Whitehead, Clemson University Chapter 1 Cont’, Intro to Microbiology It is hypothesized that life began with prokaryotic cells that evolved into eukaryotic cells, then to multicellular organisms. It is believed the earth has been around for 4.5 billion years, and life appeared about 3.5 billion years ago. However, there has been some debate due to the discovery of possible microbial fossils, though they are sparse. Most of the evidence we have of early microbes is indirect evidence – For example: it is believed that there was no oxygen in the atmosphere early on, and development of photosynthetic organisms introduced oxygen. Extant: Organisms currently living on earth; the opposite of extinct. – Studying extant organisms can give us insights and allow us to hypothesize about historic microbes. Evolution of Organisms: Variation in larger, multicellular organisms is generally achieved through sexual reproduction. However, microbes cannot rely on that. Instead, microorganisms have a couple of ways variation is introduced into evolutionary lines 1.) Mutation: The main way microbes evolve is through mutation of genetic material, aka mistakes in genetic replication. Usually mutations aren’t beneficial, but occasional an organism gets lucky, and the mutation helps them survive. Leads to new genotypes (genetic characteristics) Often leads to new phenotypes (visible characteristics made from combination of genetics and environment) Allows for natural selection – most beneficial/viable mutations survive, replicate, and spread A common example of mutation is antibiotic resistant bacteria 2.) Horizontal Gene Transfer: Microbes perform both vertical and horizontal gene transfer. Vertical is the most recognized form of passing genetic material where genes are passed from one generation to the next. This is done in microbes by asexual reproduction (also called binary fission) as well as sexual reproduction in larger animals. However, there is also horizontal gene transfer Occurs between two different types of microbes living in the same time/within the same generation Relatively unique capability of microbes to acquire new characteristics Example: MRSA and VRSA: Methicillin/Vancomycinresistant Staphylococcus aureus – A man with an ulcer on his foot started out with MRSA in the wound. Somewhere in/on his body, there were other bacteria that were resistant to Vancomycin, and those microbes passed their resistance VERTICALLY to the MRSA, creating VRSA, making the wound incredibly difficult to treat. Microbial “Species”: The term species is mostly associated with the idea of reproductive isolation, or the ability of a certain organism to only reproduce with like species to produce viable offspring. A good example of this is in horses and donkeys that, when crossed, create a sterile offspring. However, there is not reproductive isolation in microbes, and therefore it causes some debate about using the term “species” for microbes. There are instead some general ideas and rules of microbe species: 1.) Naming: Microbes, like larger animals, are named using binomial nomenclature, or a genus and species. The genus name comes first, and is always capitalized. The species name comes second, and is always lowercase. The entire scientific name is then italicized. When writing about microbes, the first time you mention one, you spell the entire name out. After that, you can shorten the genus (Escherichia coli v E. coli). A strain (see below) is designated with a number after the species name, and is not italicized. 2.) Strains of microbes: A group of microorganisms that are more like each other than anything else. 3.) A microbial species contains a collection of strains. Microbes within a certain group of strains share many stable properties and differ significantly from other groups of strains. Strains within a species differ from each other as well. 4.) To determine a strain, you can take a pure microbial culture (one, single microbe present at the beginning), and all microbes that descend from that culture are a part of the strain 5.) Within a species of microbe, there is a “type strain,” which is a random designated strain within the species. It is usually the first discovered microbe within the strain, and the one we know the most about/it is the most well characterized. It may or may not have typical characteristics of the species. In other words, a type strain isn’t always the most “normal” of a species. 6.) Strains are often characterized with a number after the scientific name. Example Breakdown: Bacillus subtilis: A bacterium commonly found in soil that very rarely causes issues in humans. Bacillus anthracis: The causative agent of anthrax. Both are in the genus Bacillus, but have different species. There can be different strains within each of them (Ex: B. subtilis 23 and B. subtilis W23) History of Microbiology: It took a while for humans to realize that microbes could be a cause of disease. At the time, it was thought illness was caused by supernatural forces, infectious spirits, etc. Sanitary methods did not exist. Once they realized there was a connection, this is where the field of microbiology caught on, and led to study of host defenses, aka immunology. Robert Hooke (1665): One of the first people to look at microorganisms and describe them. Developed and used the microscope, with which he observed and described the fruiting structurs of molds. Antony van Leeuwenhoek (1674 – 1676): His microscope was improved from Hooke’s, and could magnify things from 50x – 300x. He was the first to observe and describe bacteria and protists accurately, including those capable of movement. Famous for referring to them as wee animalcules. His microscope shined light at a 45 degree angle to illuminate a specimen between two pieces of glass – in other words, he developed a lightfield microscope. Around the time of Hooke and Leeuwenhoek (17 century), there was little belief in the existence of anything smaller than what the naked eye could see. Instead, the idea of spontaneous generation was prevalent: the idea that living organisms could develop from nonliving or decomposing matter. This idea came about due to flies and maggots appearing from rotting food. Was believed to require air to work. Francesco Redi (1668): Discredited spontaneous generation for larger animals by experimenting with three jars of meat that were allowed to rot. One was open to the air, one was stoppered, and one had gauze over the top. Maggots and flies appeared on the meat of the open jar, nothing appeared in the stoppered jar, and flies and maggots stayed on the gauze of the third jar, but never entered. This experiment suggested that large organisms do not adhere to spontaneous generation, though small organisms were still thought to use it. Louis Pasteur (1861): Fully disproved spontaneous generation by placing nutrient broth in flasks with long, curved necks. He boiled the solutions to purify them, and left them exposed to the air. No microorganisms grew unless he broke the neck of the flask. The idea was that microorganisms would get stuck in the neck. John Tyndall (1820 – 1893): Final blow to spontaneous generation – suggested that there are living things in the air, and that they were being carried by dust. Claimed that Pasteur’s experiment worked because he kept dust from entering the solution. He also provided some evidence that there are heat resistant microbes, which had caused problems in previous experiments. (Bacillus subtilis and bacillus anthracis both have heat resistant strains). Ferdinand Cohn (18281898): Referred to as the father of bacteriology. Discovered bacterial endospores in very resilient strains of microbes, and classified bacteria by shape. There are 3 shapes bacteria fall into, typically. He also used the term Bacillus for the first time. Joseph Lister (1827 – 1912): Gave first evidence of infectious microbes. He noticed we had large numbers of people dying after surgeries, so he suggested cleaning instruments between procedures. He became known as a surgeon whose patients rarely died after surgeries due to few postoperative infections. However, this wasn’t well understood. Ignaz Semmelweis (1847): While he was working on a hospital ward, he noticed that doctors (who performed autopsies as well) lost 5x more pregnant women to an illness called childbed fever than the midwives (who did not perform autopsies). It is estimated that as many as 25% of women died from this infection, and it is believed that it was a bacterial infection caused by Streptococcus pyogenes, or other strep species. The midwives had no priest go through the ward when women died, but the doctors did, and they also had women birth on their sides, while doctors had them deliver on their back. Semmelweis tried to have doctors eliminate the priest, and have women deliver on their sides, but nothing changed. After taking a vacation, he found a colleague had died of what appeared to be childbed fever even though they were not a pregnant woman, so he suggested that doctors wash hands and instruments with chlorine between autopsies and delivering babies (which, fortunately, is excellent at killing microbes). He believed that “cadaverous particles” were being passed to pregnant women by the unclean hands and instruments. The death rate in the doctor’s ward dropped. However, Semmelweis made a public announcement about hand washing that made doctors and med students look bad, so his suggestions were ignored. He also lost his license, and hand washing fell out of favor for a while after. He died in his forties in an insane asylum. Sidenote: A suggestion for keeping cultures clean in lab Tilt a bottle of broth while handling it to prevent microbes from “falling” into it due to gravity (though good air circulation will negate this), and don’t leave things open to the air. MICR 3050 – Notes Set 4, 01/15/2016 Dr. Whitehead, Clemson University Chapter 2: Microscopes and Staining Procedures Party Fact: Symbiotic relationship of Hawaiian bobtail squid (Euprymna scolopes) and Vibrio fischeri: The squid is very small and nocturnal. They only hunt and birth at night. The monk seal is the most common predator for them, and they sit and wait on the sand under the water while the moon is up. They wait for a shadow to pass over before attacking. As a defense mechanism, these squid have coevolved with a bacteria called Vibrio fischeri, which are bioluminescent. The squid have evolved “light organs” that hold a huge number of only the motile strain of V. fischeri that casts light to trick the seals. Scientists aren’t sure how the bacteria benefit, but they think the squid might provide them with nutrients. Every sunrise the light organ is emptied, and fills throughout the day. Chapter 2: Microscopes and staining procedures. Stuff to view particularly bacteria. Lenses and the bending of light: Terminology: Resolution: How well we are able to distinguish between two objects; if there are 2 really tiny objects next to each other, can we tell they are two distinct things? The number tells you the smallest distance between two objects where you can still tell they are two distinct objects. The smaller the number, the better the resolution. The equation for it is d = (0.5λ)/(nsinΘ), where d is resolution. Focal Point: The point at which a lens concentrates all of the rays of light passing through it – falls some distance away from the lens. Focal Length: The distance from the center of the lens to the focal point. The shorter the focal length, the greater the magnification of the lens. Compound microscope: Microscope with 2 sets of lenses – there is a lens very close to the eye, and one close to the specimen. Total magnification comes from the magnification of both of the lenses combined. Refraction: The bending of light rays through a medium. Refractive Index: A unitless value assigned to represent how light propagates/travels through a certain medium. Every medium has its own refractive index. A measure of how much an object slows the velocity of light. Parfocal: The ability of a microscope to keep an object in focus when switching from one level of magnification to another. Parcentric: The ability of a microscope to keep an object centered when switching from one level of magnification to another. Numerical Aperture: The measure of light gathering ability. NA has two parts to it. In any lens you have, you have an opening on the lens where light can enter. The size of this opening determines how much light can enter the lens. Theta is the measure of half of the “cone of light” from the surface of the specimen to the center of the lens. Equation for NA is nsinΘ, where n is the refractive index. Theta is the measure of half of the “cone of light,” from the surface of the specimen to the center of the lens. Working Distance: The distance from the objective lens to the closest surface of the coverslip when the specimen is in focus. Light Microscopes – Two Types: Brightfield microscopes: Field around the objects is going to be light, and the objects are dark. Compound microscopes, but the upper limit of magnification typically falls around 1500x. The upper limit of resolution is .2 micrometers. Uses several objective lenses closer to the specimen and one ocular lens closer to your eyes. The ocular lens magnifies the power of the objective lens. (So, if the ocular lens is 10x, and an objective lens is 4x, the total magnification is 40x). In a perfect world, brightfield microscopes are parfocal and parcentric. However, they are rarely, truly either. o You can technically use brightfield microscopes for stained and unstained objects. But it is harder to see unstained objects, especially because microbes don’t tend to have natural pigmentation. Darkfield microscopes: The field around the objects is going to be dark, and the objects are light. This is because you’re viewing light focusing on, and then reflecting off of, the object being observed. This is better for seeing living organisms because there is a very big difference in the object you’re looking at compared to field around it. o Especially helpful for looking at unstained objects, identify bacteria, and can be used to observe internal structures in eukaryotic microorganisms. Light Microscope Basics: Light is refracted (bent) when passing from one medium to another. When light hits an object, the speed and directionality of light traveling will change due to the refractive index of the medium it is passing through. Using immersion oil changes the resolution because it has a different refractive index. If light passes through two mediums (such as air and oil), the direction and magnitude of light bending is determined by the refractive indices of the two media forming the interface. Lenses often act as a collection of prisms. They focus light rays at a focal point, or concentrate the rays of light at a single, further location. The distance between the center of the lens and the focal point is the focal length. In other words, how far away is the lens from the object you’re trying to look at? The focal length plays an important role in determining the strength of the lens or the microscope and total magnification. o In the microscopes we used in lab, the 4 lens was the shortest, and the 100 the longest. These are a result of the different focal lengths. The SMALLER the focal length, the GREATER the magnification. A lens with a short focal length is capable of greater magnification, and will be closer to the object. Microscope Resolution: In terms of the strength of a microscope, magnification isn’t enough. If the image isn’t clear, it isn’t useful, so you have to have magnification and resolution. o The wavelength of light used in a microscope is a major factor in resolution: shorter wavelength = better resolution. Resolution is what limits the overall power of a light microscope. This determines if we have to switch to an electron microscope. o Formula for resolution (d): d = (0.5λ)/(nsinΘ). In other words, resolution is equal to ½ the wavelength (λ) of light divided by the numerical apperture . The shorter λ is, the better the resolution, because the smaller resolution (or d) is going to be. It turns out that you can get a wavelength of energy for electrons that is smaller than any visible light, which allows electron microscopes to give us better resolution. Also, the greater the aperture, the better the resolution. This is why immersion oil is important – it creates a greater aperture, and therefore a better resolution. Increasing theta will also increase NA. MICR 3050 – Notes Set 3, 01/13/2016 Dr. Whitehead, Clemson University Chapter 1 Cont’, Intro to Microbiology Best way to study for this class: Review the material you’ve learned by seeing if you can answer the study guide questions at the end of each slide set! History of Microbiology Continued: Direct evidence: Robert Koch (1884): Established the relationship between Bacillus anthracis and anthrax. He used criteria developed by his teacher Jacob Henle (1809 – 1895). Note: Bacillus anthracis can cause a variety of illnesses based on how it enters the body, and is commonly associated with severe animal illnesses. The story goes that Koch originally experimented on his daughter’s wide variety of pets, but due to their high mortality rate, began using mice. He would take sick mice he believed were infected with anthrax, and extract blood and materials from them. He would then inject healthy mice with these materialth and they would get sick and die. He did this 20 different times. After the 20 time, he took the spleen of the dead mouse and put it in a dish with a broth medium. He found large amounts of tiny bacteria grew, of which he took a sample and injected it into a healthy 21 mouse. This mouse fell ill and died of the same sickness as the others. This led to Koch’s postulates, which are still used today to connect an organism to a disease. Koch’s Postulates: 1.) The suspected pathogenic organism should be present in ALL cases of the disease, and absent from healthy animals. 2.) The suspected organism should be grown in pure culture – nothing else should be in the culture with it. 3.) Cells from a pure culture of the suspected organism should cause disease in a healthy animal. You should be able to take bacteria from the culture, inoculate it in healthy host, which should subsequently fall ill of the same disease. 4.) The organism should be reisolated from the individual inoculated from the culture, and show to be the same organism. Note: Within microbiology, there are organisms for which we cannot fulfill all koch’s postulates due to ethical or situational difficulties. However, we are virtually certain that a specific pathogen is what causes a certain disease: Example: small pox. See below. Possible Problems with Koch’s postulates: Isolating and growing things in a pure culture can be very difficult. There are some viruses, like noroviruses that cause gastrointestinal distress (vomiting and diarrhea), that are notoriously difficult to culture in a lab setting. It is not ethical to use Koch’s postulates on humans, so it’s hard to study human specific disease, such as Ebola. You can extract pathogens from ill humans, but cannot reinfect others. Newer problem: A number of pathogens, called opportunistic organisms, live all the time in healthy humans. Then, if the human gets sick, it will take advantage of the lowered immunity and cause illness. A good example is Staph aureus. Therefore, they are found in healthy humans and ill humans, a violation of Koch’s postulates. Koch’s work led to the discovery or development of agar, a culture medium that consists of gelatin made from seaweed (of which there has been a shortage of due to lower seaweed populations). His work also led to the development of the petri dish, and nutrient broth and nutrient agar. These nutrient mediums are very rich, and will grow a lot of different things. His work also led to the idea of isolating microorganisms. Other influential people in the History of microbiology: Edward Jenner (1798): Used a vaccination procedure to protect individuals from smallpox. He noticed that milkmaids that were infected by “cowpox” (which is in the same strain as small pox) were no longer susceptible to smallpox. He performed an experiment with a young boy, infected him with cow pox, and exposed him to smallpox. He stayed healthy. Pasteur and Roux (1880): They discovered that incubation of cultures for long intervals between transfers caused pathogens to lose their ability to cause disease. This is called attenuation. The viruses or infectious agents exposed outside of a host to incubation causes weakening mutations, and makes them less dangerous to vaccinate with. Pasteur and his coworkers (1885): Developed vaccines for chicken cholera, anthrax, and rabies. Developments and influential people in Industrial Microbiology: Microbes are often genetically modified and used to produce a variety of substances such as insulin. Also used to make alcohol. Louis Pasteur (1856): Demonstrated microorganisms carried out fermentations, and then developed the process of pasteurization, or the heating method used to make foods safe to consume, and store them for longer periods of time. Used in milk, chocolate, guiness beer, etc. Not used as much in Europe. Alexander Flemming (1929): Discovered penicillin. He had grown a culture, and noticed a fungal colony had grown in one are. He also noticed that the bacteria weren’t present right around the fungus. He hypothesized that the fungi were excreting something that either inhibited bacterial growth, or killed them. He did NOT isolate this compound, other scientists did in the future to develop antibiotics. Once developed, penicillin was thought to save thousands of lives during WWII. Developments and influential people in Microbial Ecology: Sergei Winogradsky (1856 – 1953) and Martinus Beijerinck (1851 – 1931): Pioneered the use of enrichment cultures and selective media to promote the growth of certain organisms while discouraging the growth of other organisms. Fine tunes what will actually grow on a plate. They were very interested in soil microorganisms, and they discovered numerous interesting metabolic processes (such as nitrogen fixation). Winogradsky discovered chemolithotrophy, which consists of specific organisms that don’t need organic compounds or carbon for energy sources. They use inorganic compounds as energy and electron sources. Will use CO2 as a carbon source. Beijerinck considered the “Father of Virology.” Developments in Molecular Microbiology: Avery, MacLeod, and McCarty (1944): Found that DNA is the genetic material of living things. Arber and Smith (1970): Discovered restriction endonucleases – enzymes that destroy genetic information. These enzymes are often found in bacteria as a defense mechanism against infective, foreign DNA from bacteriophage viruses. Can also be used in cloning and genetic modification. Jackson, Symons, and Berg (1972): Developed the first recombinant molecule – recombination of genetic material. Woese and Sanger (1977): Developed DNA sequencing. Kary Mullis (1983): Awarded a Nobel prize for PCR used in genetic sequencing and evolutionary trends. General overview of Microbiology timeline: The first few centuries of the start of Microbiology (1600s – 1800s) were all about discovery, and then we moved on in the 1940s for the era of molecular biology and general microbiology. It took until 1970s to realize that archea were separate organisms. Since the mid1980s, appreciation of microbiology has really developed. DNA sequencing has been incredibly developed. Study Guide questions/information: 1.) Define “microorganism” and describe the types studied by microbiologists (cellular and acellular): Organisms and acellular entities too small to be clearly seen by the unaided eye generally less than or equal to 1mm in diameter, and often unicellular. Acellular (A meaning without, so without cells) – viruses with a protein coat and genetic material core. Require a host to replicate. Cellular: o Bacteria: All Prokaryotes – Pro means before, karyote means nucleus. Usually singlecelled with a cell wall made of peptidoglycan. o Archaea: Prokaryotes Pro means before, karyote means nucleus. Usually singlecelled with no membrane bound nucleus or membrane bound organelles. No peptidoglycan in their cell wall. Very small, very simple o Eukaryotes: Eu means true, karyote means nucleus. Usually have membrane bound nuclei and organelles – contains protists (usually single celled) and fungi (can be single or multicelled). Also include larger animals, but they are minimal in number compared to microorganisms, and not discussed in microbiology. 2.) Understand the importance of microorganisms (scope and relevance): Found everywhere, all around us. Symbiotic relationships, often. Found in healthy humans. Most are nonpathogenic. Those that are can be very dangerous. Can be used for industrial purposes and studying other scientific ideas. 3.) Compare and contrast prokaryotic and eukaryotic microbial cells: See number 1. 4.) Explain how the Universal Phylogenetic Tree was developed: 3 domains developed by Carl Woes. Based on a comparison of the DNA encoding small subunit ribosomal RNA (SSU RNA) that is found in all organisms. 5.) What is LUCA: Phylogenetic Tree base organism: Last Universal Common Ancestor 6.) Distinguish between the three domains of life and understand their relatedness: See number 1. 7.) Describe the microorganisms of the three domains: See number 1. 8.) How do microbial populations evolve: Mutation Horizontal Gene Transfer 9.) Define the prokaryotic “species” and the bacterial strain: Groups of strains of bacteria, where a strain is a group of bacteria more like each other than anything else. 10.) Explain how microorganisms are named: Binomial Nomenclature: Genus and Species, genus capitalized, species lowercase, all italicized. Strain will be a number that is not italicized following the species name. 11.) Know the contributions of the following scientists discussed to the science of microbiology. Robert Hooke: Developed and used the microscope to first describe largermicroorganisms and the fruiting structures of molds. Antony van Leeuwenhoek: Developed an improved microscope (brightfield) to view and describe bacteria and protists, some of which were mobile, in detail. Francesco Redi: Discredited spontaneous generation for larger organisms with jars of meat. Louis Pasteur: Developed pasteurization to avoid fermentation; developed vaccines for chicken cholera, anthrax, and rabies; developed attenuated vaccines; fully disproved spontaneous generation with his longnecked flask experiment. John Tyndall: Finally put spontaneous generation to rest by suggesting living things traveled on dust; provided evidence of heatresistnat microbes. Ferdinand Cohn: Father of bacteriology; discovered bacterial endospores; classified bacteria by shape. Joseph Lister: Cleaned instruments between surgeries – gave evidence of infectious microbes. Ignaz Semmelweis: Suggested washing hands between autopsies and delivery of babies. Robert Koch: Koch’s postulates. Alexander Flemming: Credited with discovering, but not isolating and developing, penicillin. 12.) Describe the experiments that led to the downfall of the theory of Spontaneous Generation: Redi used three jars of rotting meat, one open, one stoppered, and one covered in gauze, to show that flies and maggots only appeared in the meat in the open jar, and appeared on the gauze of the covered jar. They did not appear in the stoppered jar. Pasteur used long, curved necked flasks with boiled broth to show that microorganisms did not grow unless the neck was broken. Tyndall suggested that dust carried microorganisms, which is why pasteur’s experiments worked. 13.) List Koch’s Postulates and understand how they are used to determine the cause of a disease (including the microbiological techniques employed). 1 The suspected pathogenic organism should be present in ALL cases of the disease, and absent from healthy animals. 2 The suspected organism should be grown in pure culture – nothing else should be in the culture with it. 3 Cells from a pure culture of the suspected organism should cause disease in a healthy animal. You should be able to take bacteria from the culture, inoculate it in healthy host, which should subsequently fall ill of the same disease. 4 The organism should be reisolated from the individual inoculated from the culture, and show to be the same organism. 14.) Why is it not always possible to fulfill these postulates? Some organisms are difficult to culture in a lab setting. Human ethical issues. Opportunistic organisms.
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