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Microbiology Concepts of Culture, Growth and Metabolism




Needing help with quetions 2 & 3 on page 11 of attached document. The question is pertaining to polyurethane as a huge polymer and why is it important that the polyurethanse is a secreted enzyme and the net loss of ATP...etc...;Attachment Preview;Elvis Spring 2014 Wendy Kuennen.docx Download Attachment;Case Study/Application Assignment Number One Spring 2014;General Instructions;The Case Study beginning on page 2 will involve concepts you learn in;Chapters 3, 4, 7, 2 and 8. Use those chapters and related references outside;your text to thoroughly and thoughtfully complete each question you are;asked in the case. Read each section carefully. A grading rubric for this case;will be posted by Feb 10th in Shared Files.;Online Students;Print a copy of the case study first to use as a reference and to give yourself a full-picture of;where youre headed as you work your way through it.;As you work your way through this Case, please type all of your answers directly into the;document in the spaces provided. Follow the required word count.;For the poster graphic, mark your suggested changes right on the page.;My expectation is that each student complete their own work. You are;welcome to bounce ideas off one another and work on solving ideas together;but the writing must be yours and yours alone. For online students in;particular, all submitted writing goes automatically through;and is scored for originality (including if the work is original to other students;in the class).;1;ELVIS Meltdown!;Microbiology Concepts of Culture, Growth and Metabolism;Adapted from the;Department of Cell Biology and Molecular Genetics;University of Maryland - College Park, MD;Part One: Return to Sender;Fresh out of college, with your degree in microbiology, you have landed your first real job as a;scientist with DuPunt, a company that specializes in the development and production of;polyurethane derivatives (specialized plastics). You (and your boss) are not quite sure why;DuPunt has a microbiologist on staff, but you are both about to find out why. Your boss has;called you into his office. Read this article! he says, pushing the front page of a national;newspaper across his desk to you.;Stifling your initial reaction to the article, you manage to mumble, What a tragedy.;2;Yes. Yes. And this could take an ugly turn for DuPunt! your boss says. Im not sure what;caused this mess, but I do know a couple of things that didnt make it into that news article: (1);the only plastics showing damage in the ELV were polyurethanes, and (2) our company provided;those polyurethane products to NASA at a cost of $15,000,000. Were in big trouble if we cant;prove that something from that planet is responsible for destroying ELVIS!;He continues, The polyurethane we provided was first-rate. We didnt cut any corners. Products;from the same batches of polyurethane have been sent into outer space before, and returned with;no damage. There must be some explanation other than our incompetence. This is where you;come in. I need you to find that explanation!;Why me? you ask.;Because of the stink! your boss exclaims.;What do mean by the stink? you ask.;Your boss replies. Some of the scientists present at the ELVIS disaster said the smell reminded;them of an old fermenter or an autoclave. Those are microbiology terms, arent they? Those;comments tell me that this whole stinking mess might be caused by microorganisms you know;bacteria, fungi, viruses, germssomething like that. Get right to work on this! You and I will;have to work closely on this, you know. Ill handle all the communications with the press, and;you handle the science. Just make sure that you explain everything clearly to me so that I can;speak about it to the press without making a fool of myself and DuPunt!;Lets Get Started: As a microbiologist for DuPunt you must determine what has happened to the;polyurethane. Here is your hypothesis;3;The degradation of polyurethane products was caused by a microorganism or;microorganisms present in the soil samples collected by ELVIS.;Questions;1. Using the light microscope, you examine all the soil samples and the goo from the;degraded polyurethane. Will this approach allow you to observe all the potential;microorganisms in the sample? Why or why not? If not, what are the limitations of;this approach? (Minimum 300-word response);There are limitations with using a light microscope. In order for a microscope to work to the;best of its ability it needs to have adequate magnification, resolution and clarity of the image.;The light microscope provides a magnification from 40x to 2000x. Magnification is how;much bigger a sample appears under microscope than in real life. The resolution power of the;microscope defines the object. It helps distinguish between two points on an image. The;resolution of an image is limited by wavelength because when objects in the specimen are;smaller they dont interrupt the waves so they go undetected. You can magnify the image all;you want, but if you dont have the resolution you cant see detail and the image will just;appear blurry. Next comes the type of light microscope used. They are described by the;nature of their field. There is the bright-field in which it forms its image as light is;transmitted through the specimen. The specimen will appear darker than the surrounding;illuminated field. A bright-field microscope can be changed to a dark-field by adding a disc;to stop all the light from entering the objective lens. This will illuminated specimens;surrounded by a dark field. The next field microscope is phase-contrast microscope. Phasecontrast microscope contains devices that change the light to make subtle changes as its;passes through the specimen. This is used to detected internal cellular detail. The final light;microscope to mention is the differential interference. The DIC provides a detailed view of;unstained, live specimens. There are bacteria that are too small to be detected and those;require electron microscopy. There are bacteria that are distorted and not be stained by;normal methods. All these factors need to play into finding the right approach to find the;microorganism that caused the degradation of polyurethane.;2. You next use phase contrast microscopy to observe a wet mount of a soil sample (see;picture below) and a goo sample (see picture below) from the ELVIS. Based only;on the pictures below, in what ways are the samples you see in both the soil and;goo similar to microbes previously characterized on Earth? In what ways are they;different? (Minimum 350-word response);Lets start by describing the make-up of soil. Soil is made up of minerals, organic;matter, water, air and soil organisms. Soil is a big ecosystem that supports complex;relationships between geologic, chemical and biological factors. Organic matter, in soil;is plant and animal residue at various stages of decomposition, cells and tissues of soil;organisms, and other substances that are synthesized by microorganisms. There are;many organisms that find their home in the soil such as fungi and bacteria. When;comparing the slides there are many things to examine. Microbial sizes, shapes and;arrangements are all things that help classify the sample out into categories as;microbes-bacteria, archae, fungi, algae, protozoa, and viruses come in all different sizes;and shapes. When we are comparing the slides we have to remember that the images;are under a light microscope. Viruses are the smallest of microbes and not usually;4;detectable with a light microscope. Archae are single-celled simple organisms. They;contain many bacterial characteristics but their ribosomal RNA contains unique;signature sequences. Fungi are very popular in soil. They are single celled or complex;multicellular. Their role is beneficial in cycling carbon and other elements. Algae can;also be single celled or multicellular. They are the eukaryotic protists that;photosynthesize with chlorophyllia. Protozoa are unicellular eukaryotic protists. This;now leads us to bacteria. Bacteria have no nucleus. Their genetic information is;contained in a single loop of DNA. Bacteria are classified into five groups based on;shape. Sperical (cocci), Rod (bacilli), spiral (spiralla), comma (vibrios) or corkscrews;(spirochaetes) are all shapes of bacteria. Bacteria can be then in single, pairs, chains or;clusters. Lets go back to the samples, the images of the Earth samples have flagella;where the goo does not. The goo sample is actually much simpler as it has much less;organelles. Distant microbes were less complex and did not have flagella to move to new;locations and find nutritional sources. One thing to consider is the complaint of smell;when they returned. This would imply that the microorganisms underwent;fermentation versus respiration.;Figure 1 Soil Sample;Figure 2 Goo Sample;Your boss has done a little reading about microorganisms but he finds it all pretty;complicated. Its like a foreign language! he complains. I have to face the press to explain;our idea that microbes might be responsible for all the damage to ELVIS. Clearly, Im going;to need some visual aids or the press wont have a clue as to what I am saying. I think Ill;need to explain what a bacterium looks like and how it might be possible that they can;degrade polyurethane. I really cant waste time showing them all the potential types of;bacteria (archea, mycoplasma, etc), so I think Ill just show them what a gram negative;bacterium looks like as an example. Ive already put together a poster with a diagram of a;typical gram-negative bacterium. Will you take a look at it, to make sure I havent made any;mistakes? I labeled all the features and also indicated the major biochemical composition of;each feature. Im sure this figure will wind up in lots of newspapers and magazines, so it;really needs to be scientifically correct. We wouldnt want to make DuPunt look stupid;would we? Just proofread it and make any necessary corrections, okay? Go ahead and mark;up the poster as needed.(see Figure 3 next page);5;Make any changes you wish to make to the actual graphic. Highlight those changes.;6;Part Two Suspicious Minds;Your direct microscopic observation of microorganisms in the soil samples has sparked your;bosss interest. He is eager to determine what type of microorganism(s) is/are present. Though he;only presented a model of a bacterium for his public press conference, there is still not enough;evidence to determine if the microorganism is a prokaryote, eukaryote, or even some type of;organism never seen before. He asks you to take a sample of the soil to the electron microscope;for further analysis. To be the most objective, he asks you to run a sample through the electron;microscope and decides to do the same himself for comparative analysis.;You are just as interested in the nature of the microorganism(s) as your boss, but instead of;directly analyzing the soil, you use both the soil sample and the goo sample and, using pure;culture techniques, isolate a similar organism from each for testing.;Question;1. How would you go about isolating a pure and matched culture from each of your;samples? How would you know that you have isolated the same organism from both;the soil and the goo sample? (Be specific) (minimum 450-word response);First thing firstdefinition of culture is to cultivate microorganisms. It is a process to take seeds;(microbes) and plant them into an environment (medium) to let them thrive and grow. To do so;one would introduce a sample into a nutrient also known as medium. This provides an;environment in which the sample can grow or multiply. A medium is the foundation of culturing.;As all microbes are different some require a few inorganic compounds for growth versus others;who require a complex list of specific inorganic and organic compounds. Media (plural for;medium) come in over 500 different types. They can be contained in test tubes, flasks, or Petri;dishes. There are three main categories of media. Those categories are based on their properties;physical state, chemical composition and functional type. Physical state of media is the;consistency of the medium. It can be liquid, semisolid, solid that converts to a liquid or solid that;cant be liquefied. The chemical composition is either synthetic or chemically defined or nonsynthetic with is complex. The functional purpose is the purpose of the medium such as general;purpose, enriched, selective, differential, anaerobic growth, specimen transport, assay or;enumeration. Some media can serve more than one function. Isolation techniques are used to;help separate microbes and spread them apart to create isolated colonies that contain a single;microbe. This method helps define making a pure culture. Proper isolation is required that a;small number of cells be inoculated into a large area of the medium. The tools needed to perform;isolation would be a medium as described earlier, Petri dish or place media is controlled and;inoculating tools. Inoculating tools can be loops, needles, pipettes and swabs. For a controlled;sample, sterile technique needs to be used. Sterile technique is when you start with a sterile;medium, sterile inoculating tools and nothing non-sterile contaminates the specimen. Things that;would be considered non-sterile would be room air, fingers non-sterile materials.;So now that we have a background on what we are using lets carry on to that actual process. We;have collected our sample of the goo and of the soil. We placed those samples into a container of;medium that will support the growth. After placing the sample in the media we place it into an;incubator. An incubator is a controlled environment that helps promote growth. After optimal;growth in the medium we use isolation techniques to spread the colonies apart to grow colonies;7;that contain a single microbe. Those isolation techniques could be techniques such as the streak;plate method or pour plate (loop dilution) method. There is also the spread plate technique. All;these methods use various ways to spread the surface promoting individual colonies. Once our;subculture is separated into a pure culture of the bacteria we observe for the macroscopic;appearance and then under the microscope for basic details such as cell type and shape. Those;basic details should be seen in the soil and goo samples. This is reassurance that you dont have a;contaminated culture.;Later, you and your boss compare samples (see table below).;Test;80 S ribosomes;70S ribosomes;Circular DNA;Linear DNA;RNA;Phospholipid membrane;Peptidoglycan;Lipotechoic acid;Flagellar basal body proteins;Cytoskeleton proteins;Mitochondria;Histone proteins;Nuclear pore proteins;Bosss sample;+;+;+;+;+;+;+;+;+;+;+;+;+;Your sample;+;+;+;+;+;+;+;-;Im not sure whats wrong with your sample, but my results prove that we are dealing with a;new kind of life form hereIm calling it the preukaryote because it has components;characteristic of both prokaryotes and eukaryotes. Its time for a press conference! boasts your;boss.;Question;2. If your goal is to isolate this new microorganism, which results are more informative;yours or your bosss? Why? What do your results indicate about the nature of this;microbe? Does its structure closely resemble that of a prokaryote or a eukaryote? Do;you agree with your bosss conclusion that this new microorganism is a prokaryoticeukaryotic hybrid? Why or why not? (Minimum 450-word response).;As you notice the results vary greatly. This goes back to the pure culture or axenic method;that I used. My boss took the sample and viewed it under an electronic microscope and;found that all the elements were viewed. This is due to the fact that soil contains many;organisms and since they werent separated out, he was able to view all sorts of things. He;was viewing a mixture and nothing can be excluded or included as it isnt about a single;microbe. I had gone through the work of isolating the soil and the goo to find the;microorganism that was found and able to grow in both samples. My boss states that the;new organism is a preukaryote due to the fact that the components characterize both;prokaryotes and eukaryotes. Lets work this through. Due to the fact that my bosss sample;8;was in my eyes not pure, I am going to look at my positive viewings to see if his speculation;is true. The first thing is the 70S ribosome. Ribosome is made up of RNA and protein and is;the site of protein synthesis. 70S ribosome means that it is heavier more compact structure;that sediment faster. Prokaryotic ribosome is 70S as it actually composed of two smaller;subunits. The next positive finding is circular DNA. Most bacterias hereditary material is;in the form of circular DNA which is designated as the bacterial chromosome. Moving on to;RNA, this is found in both prokaryotes and eukaryotes. It is a single strand containing;ribose sugar instead of deoxyribose and uracil instead of thymine. Phospholipids are fatty;acids plus glycerol plus phosphate that are found in membranes, both prokaryotic and;eukaryotic cells have a cell membrane. Although those cell membranes are made up;differently and have different thicknesses but are present in both. Peptidoglycan is a special;class of compounds in which glycans are lined to peptide fragments, also known as a short;chain of amino acids. Eukaryotic cells do not contain peptidoglycan as of this point we;could say that my bosss theory has proven false but lets carry on and see what the last;findings show. I couldnt help but notice that within my findings I didnt have a positive;lipotechoic acid. Lipotechoic acid is found on the cell wall of gram-positive cells. Gramnegative cells dont have this acid. Flagellar basal body proteins are that provide motility or;self-propulsion. They are found in some not all prokaryotic cells but also some eukaryotic;cells as well. They help move the bacteria and this mobility helps growth to spread. The last;positive finding is that of cytoskeleton proteins. Cytoskeleton was known to the eukaryotic;cells as an intracellular framework of fibers and tubules that bind and support the cell.;Prokaryotic cells have more recently been found to house a cytoskeleton into the fine;structure of certain rod and spiral shaped bacteria. Upon reviewing my findings compared;to my bosss I would still have to say that I disagree with his theory as the findings are more;closely related to a prokaryotic cell.;Later on, as you are getting ready to head home, you hear your boss bellow, What the H-E;double hockey sticks is going on here!;You ask him what happened.;This morning I put a few thousand cells from your pure culture onto two slides in water.;Because I had to leave for the press conference and didnt want them to dry out, I sealed the;coverslips. When I left, they ere clearly distributed evenly on the slides. Now look! On this slide;I used a rubber gasket to make the seal. On this slide, I used a Lycra gasket. Now look at the cell;distribution! On the rubber-sealed slide, the cells are evenly distributed, but on the Lycra-sealed;slide all the cells are congregated around the edge of the coverslip. Lookthey are all over the;edges, none are left in the middle part of the slide.;Question;3. Come up with at least two possible explanations for the amazing redistribution of the;new microorganism on the Lycra-sealed slide. (Minimum 400-word response);There are a couple possibilities on the explanation of why the redistribution of the unknown;bacteria on the Lycra-sealed slide. Two of those explanations would be motility and taxis.;9;Motility means the movement of the bacteria by the flagella that are driven by a proton gradient.;Taxis mean a motile response to an environmental stimulus.;Bacteria under the microscope often have appendages noted from their surface. Appendages are;divided into two groups, motility and attachments/channels. If the pure culture was put onto two;slides of water and evenly distributed and sealed and movement happened, motility has occurred.;There can be movement of some cells without it actually making progress. That is why electronic;microscopic examination and sometimes staining of the specimen is necessary to see if the;species is motile. Flagella provide the power of motility or propulsion within bacteria cells.;Flagellum is an appendage that allows a cell to swim freely though an aqueous habitat. There are;three parts to the flagellum, the filament, hook, and basal body. In prokaryotic cells the hook and;filament are free to rotate 360 degrees. This is a differential when comparing eukarotic cells and;prokaryotic cells. The flagella are arranged by two patterns, the polar arrangement and the;peritrichous arrangement. In a polar arrangement the flagella are attached at one or both ends of;the cell. In a pertrichous arrangement the flagella are randomly placed over the cell surface.;There is growth spread though out the entire medium that is an indication of motility as it is;widespread and didnt just stay in the same spot. The flagella can guide the bacteria in the;wanted direction because of the system for detecting chemicals is linked to the drive of the;flagellum. How this works is on the cell surface molecules that bind specifically with other;molecules, receptors, bind specific molecules from the environment. The attachment of sufficient;numbers of these molecules transmits signals to the flagellum which in return set the flagellum;into a rotary motion. As the flagellum rotate counterclockwise the cell itself moves in a linear;motion called a run. Runs are interrupted by tumbles. Tumbles are when the flagellum reverses;direction and rotates clockwise causing a tumble. When placed in a medium a cell moves;randomly in short runs and tumbles until it gets closer to an attractant and then it spends more;time in runs.;So what causes a cell to run and tumble toward or away from something? That would be called;chemotaxis. Chemotaxis is the tendency of organisms to move in response to a chemical;gradient, either towards or away from a chemical stimulus. The chemical stimulus is usually;known as a nutrient or away from a potentially harmful compound. There are other taxis such as;phototaxis which the movement is in response to light rather than chemicals.;Part Three All Shook Up;You have found media that support growth of the pure cultures you isolated (now named by your;boss as the Extraterrestrial Polyurethane-Degrading Microbe (EPTUM)). The recipes for these;media are shown below;Medium 1;10;Medium 2;5 g yeast extract;20 g tryptone extract;0.5 g NaCl;3.6 g glucose;1 liter H2O;Growth;EPTUM Growth - aerobic;EPTUM Growth - anaerobic;E. coli - aerobic;E. coli - anaerobic;10.5 g K2HPO4;4.5 g KH2PO4;1 g MgSO4;10 g polyurethane;1 liter H2O;Medium 1;+;+;+;+;Medium 2;+;-;You are excited because, in Medium 2, EPTUM utilizes polyurethane as its energy source and its;sole source of carbon and nitrogen, a finding that raises the possibility that EPTUM could be a;useful tool for bioremediation of polyurethane-containing wastes (in landfills etc.). You have also;made some progress in characterizing the central metabolic pathways and related biochemical;properties of EPTUM. In particular, you have discovered that;EPTUM secretes an enzyme (polyurethanase) that catalyzes the degradation of;polyurethane and generates citric acid (citrate) as a product.;The cytoplasmic membrane of EPTUM contains a transport system capable of;transporting citrate across the membrane and into the cell at the expense of 4 ATP;molecules (hydrolyzed to form ADP and phosphate) per molecule of citrate transported.;The cytoplasm of EPTUM contains all the enzymes necessary for glycolysis and the;citric acid (Krebs) cycle.;The cytoplasmic membrane of EPTUM contains proteins that form a functional electrontransport system that utilizes oxygen as the final electron acceptor.;Questions;1. Which medium would you consider to be non-selective and which selective? Is;it possible that one of the media is differential? (Minimum 350-word response);Non-selective and selective are terms used when describing medium. Microbiologists can;mix and match agents to fine-tune a medium for any purpose. Selective medium is described;as a media that contains one or more agents that hinder the growth of certain microbe or;microbes. Selective media allows a certain microbe to grow by itself. This method subdues;the unwanted organisms and allows for growth of the desired ones. A selective medium is;11;one that all the chemicals used are known and no yeast, animal or plant tissue is present such;as in Medium 2. Some selective media contain strong inhibitory agents to favor the growth of;a pathogen that would otherwise be overlooked because of its low numbers in a specimen.;Examples of selective media are mannitol salt agar with is used for the isolation of;staphylococcus aureus from infected material. Phenylethanol agar which is used for isolation;of staphylococci and streptococci. MacConkey agar (MAC) is used to isolate gram-negative;enteric. Sabourads agar (SAB) is media that has an acid that inhibits bacteria so it is used to;isolate fungi. The list goes on and on of the medium, selective agent and what they are used;for.;Non-selective media is a medium in which all species will grow. It is a medium that will;contain water, various salts, carbon source and a source of amino acids and nitrogen such as;yeast extract as noted in Medium 1.;Differential media grow several types of microorganisms but are designed to bring out visible;differences amongst them. The differences that may appear in the differential media are such;things as colony size or color, formation of gas bubbles and precipitates. The changes can;also appear as changes in media colors too. The changes come from the types of chemicals;contained in the media and the way the microbes react to them. Dyes are effective differential;agents because of the pH indicators that change color in response to the production of an acid;or base.;So since not all bacteria will grow on the same media, the question is what bacteria grow;where? Medium 2 was an environment that grew EPTUM in an aerobic situation. This;would make Medium 2 a selective media.;2. Given that polyurethane is a huge polymer, why is it important that the;polyurethanase is a secreted enzyme? If we assume that polyurethane is the source;of energy for the organism, how can carbon atoms from the polyurethane find their;way into the central metabolic pathways of the microbe? What is the entry point?;(Minimum 400-word response);3. Why does the growth of EPTUM in Medium 2 require oxygen? Address each of the;following questions in your answer: (at least 3 full sentences per question);Would there be a net gain or loss of ATP in the transport of the citrate across;the EPTUM membrane? Explain.;According to the growthr observations, will glycolysis be useful for;generating any ATP during growth of EPTUM on medium 2? Why or why not?;Could glycolysis be useful for generating any ATP during growth of EPTUM;on medium 1? Why or why not?;How many ATPs can be generated during the citric acid cycle? Where would;the citrate generated in the breakdown of polyurethane on Medium 2 enter the;cycle?;12;What is the relationship between the citric acid cycle and substrate-level;phosphorylation (or the electron transport system)? Can EPTUM generate any;ATP via this cycle when grown on Medium 2? If it can, how many overall ATP is;EPTUM capable of generating when grown on Medium 2? If it cant, explain;why.;How many overall ATP is EPTUM capable of generating when grown on;Medium 1?;What is the importance of oxygen as it relates to the ATP tally for EPTUM on;either medium 1 or medium 2?;Part Four A Little Less Conversation;At a press conference announcing your companys isolation and characterization of EPTUM, a;reporter raises an important question: How do you know that this microbe actually came from;the Nearby Previously Invisible Planet (NPIP) and not from Earth? Could this be a microbe be an;Earth microbe that was present in/on ELVIS before it was launched into space?;Question;1. What kinds of experiments would need to be done to determine an answer to the;reporters question prior to a new attempt to launch ELVIS into outer space? Briefly;suggest a plan of action. (Minimum 450-word response);Why that is a very good question, how do we know that the microbe actually came from the;Nearby Previously Invisible Planet and not from Earth? There are many things that would need;to happen before we can attempt a new launch of ELVIS into outer space. We would first have to;find a process of decontamination of the structure prior to launch. That is, if there is such a thing.;The method of microbial control is called decontamination. There are many methods to;decontamination such as heat or radiation, chemical agents like disinfectants and antiseptics.;There are some microbial forms that constantly present in the external environment. So what is;the difference in disinfection versus sterilization versus antisepsis? Disinfection is the destruction;or removal of vegetative pathogens but no endospores. Sterilization is the complete removal and;destruction of all viable microorganisms and antisepsis is chemicals that are applied to body;13;surfaces to destroy or inhibit vegetative pathogens. To achieve microbial death the cell is;exposed to an agent that promotes cell structures to break down and the entire cell sustains;irreversible damage. The permanent loss of reproductive capability even under optimum growth;conditions is the definition of cell death.;The plan of action to determine this would be to go back to the beginning and sample the areas;of interest. In the specific case those two areas would be Earth and the NPIP. We would have to;inoculate the sample into a container of medium. The inoculated media is then placed in a;controlled environment to grow. After the growth, the known bacteria will be isolated out to;obtain a pure culture. Then information will be gathered to see about finding a suitable way to;promote cell death that is feasible and physically able.;14


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