Elder Microbiology Notes
Elder Microbiology Notes BIOL 3200
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Date Created: 07/24/16
Exam 1 History of Microbiology Syphilis, Tuberculosis have been known about since around 1550 CE Francesco Redi 1688-Spontaneous generation; Chemists in this time believed that organisms could arise without parental organisms, although priests felt this was no biblical. Redi showed that maggots in decaying meat were the offspring of flies. Doyle- was a physician and want to see Koch do a presentation on tuberculosis but when Doyle go to the lecture he wasn’t able to get into the room so he went to his lab instead and snooped and found out Koch was a fraud and published it. Nightingale- nurse, dealt with the idea that if you went to the hospital you were more likely to die because of all the people rather then having a doctor come and see you at your home. Also did a study on the armies and showed the British government that they needed to improve the British armies living conditions. Pasteur- fermentation and pasteurization of milk; showing that fermentation is caused by living yeast, a single-celled fungus, and in the absence of oxygen yeast produces alcohol as a terminal waste product. Fleming- discovered Penicillin; he was culturing Staphylococcus and he found one of his samples was contaminated with mold, Penicillium notatum, which created a clear ring around bacteria. He concluded that mold produced a substance that killed bacteria and it lead to a leading drug, Penicillin. Krebs- Tricarboxylic acid cycle; products of sugar digestion are converted to carbon dioxide. The TCA cycle provides energy for many bacteria and for the mitochondria pf eukaryotes. Watson and Crick- known for pairing the DNA bases found within the double helix of DNA. Thomas Brock-did a lot of work with isolation of organisms within Yellow Stone National Park. He went out to the hot springs collecting organisms. Now being discovered: The DNA of these organisms is what allows them to live in extreme temperatures; ones that tolerate higher temperatures tend to have more Guanine and Cytosine in their DNA because they have triple bonds which are more stable. McDermot was also involved in this study. Lynn Margulis- involved in looking at Eukaryotic cells and how they have the organelles they have; mitochondria, chloroplasts, and membranous organelles. What are now mitochondria came from Prokaryotic organisms along time ago. Costerton- One of the early people involved in looking at biofilms; looking at organisms attached to surfaces. Karl Stetter1982 - Came up with Prokaryotic organisms that are not traditional bacteria, coming up with the classification called Archaea; ones living in extreme environments. They Archaea resemble bacteria in their relatively simple cell structure, their lack of nucleus, and ability to grow in a wide range of environments. They can live in a negative pH, extremely acidic. Glenda Michaels- isolated bacteria from elk feces. She found bacteria in organisms that were resistant to many antibiotics because of a tiny circular piece of DNA in their cells; plasmid. She found that when she moved the plasmid in the cell so did the drug resistance proving it was in there. Review of Chemistry Matter Elements- major as well as trace Atoms-protons, neutrons, electrons, orbits, isotopes, ions, cations, anions Ionic Bonds Covalent bonds/polar covalent bonds/nonpolar covalent bonds Hydrogen bonds Van der Waals forces Molecules/compounds-bonds, isomers Functional groups and interactions Molarity Hydrophobic/hydrophilic pH Water Very important molecule, major component of cytoplasm in cells and between cells For microbes its important because helps to dissolve nutrients, sometimes the cell will secrete enzymes to help break down the molecule for the nutrients Water helps with transporting nutrients across the cell membrane Evaporation Dehydrogenation reactions and Hydrolytic reactions; water breaking down bonds in salts and sugars Establishing a pH; microbes are prone to problems with pH changes (Archaea; live in negative pH) Solid form is lighter than liquid form 2 Ocular Lens High specific heat; takes quite a bit a heat to heat water but it will hold the heat Focusing Knob Molecules are cohesive; trees would die if this wasn’t true because the water from the Arm Nose Piece (Torret) soil goes up the tree and as the water travels up the tree Objective Lens it brings other water Stage molecules behind it. Condenser System Water keeps you cool with Iris Diaphragm sweat and evaporation Microscope Light Source/ Mirror Ocular lens Base • Only one so its called monocular • Called Ocular because its closest to your eye • Mostly 10x magnification Nose piece (torret) • Platform that can turn that has the objective lenses attached Objective Lenses • Called Objective Lenses because they are closest to the object • You can have 3 or 4 Objective Lenses present • Scanning Lens- Short stubby, 4x magnification • Low Dry Lens-10x lens, higher magnification but smaller diameter of viewing area. Called low because low power and dry because you do not have to put any oil on it. • High Dry Lens-40x, high because high magnification and dry because you do not have to put any oil on it. • Oil Immersion Lens- 100x, higher magnification, have to put oil on it. The oil helps retain the light which gives a clearer image. Stage • Holds the slide in place Condenser System • Focuses the light in the right direction • Iris Diaphragm- round ring, it contracts and expands to allow control of light allowed into the system Light source or Mirror • On this drawing it is shown as a mirror that reflects light into the system Base • Platform in which the microscope sits 3 Arm/Body • Holds all the parts together Focusing knob • Allow you to adjust the distance Working Distance- space between the objective lens and the slide when the slide is in focus. On the 100x lens the working distance is almost nonexistent vs. the 4x lens. The longer the lens the shorter the working distance. Resolution- the ability to have two objects together and still be able to tell they are two different objects. Dependent on how good the lenses are and they type of microscope you are using. Parfocal- If you have something in focus on a lower power lens don’t change the position, just go to the next power lens and tweak it. It means you can change from one power to the next without changing much. Total Magnification- Calculating it; ocular magnification x body magnification x objective magnification; most microscopes that we use don’t have body magnification. Prokaryotic Organisms • Eubacteria- refer to as bacteria, live in you and on you, found in soil and water. They can cause illness. • Archaea- organisms that are found in more extreme environments, neg. pH • Shapes and Arrangements: • The arrangements are good for ID Tools and what accounts for the arrangements is the type of organism you are working with and its genetics. • The planes- x, y, and z plane Spherical • Looks like golf balls • Cocci or Coccus • Most Common: • Can be single; ex. Leptospira • Least Common: • Can be in pairs, stuck together side by side, divides in 1 plane; ex. Neisseria, Diplococci • Can for chains, divides in 1 plane, referred to as streptococci (group); ex. Streptococcus • Can be in groups of 4, divides in 2 planes, referred to as tetracocci; ex. Micrococcus • Very Rare: 4 • Can form cubes, divides in 3 planes, with 4 on the backside and 4 on the front side; ex. Sporosarcinae- usually form tubular structure • Can form clusters, like grapes; ex. Genus- Staphylococcus, Group- Staphylococci Cylinders • Bacillus (bacilli plural) • Look like chalk • Common term is rod shaped • Common: Not good Identification tools o Singles, pairs and chains can be in genus Bacillus; capital letter and underline refers to genus and not the shape Singles Pairs- diplobacilli Chains- streptobacilli • Rare • ex. Corynebacterium can show up as stacks or Chinese letters Stacks- one on top of the other; ex. Palisade Chinese Letters- rods that are at unusual angles to each other and look like Chinese letters Spiral • Spirilli/Spirilla/Spirillum • Do not make arrangements • They are all singles, can vary from the number of curls, tend to be long and thin, and tend to move quickly • Treponemma; Spiral shaped organism, could see movement of blood cells because of this movement • Problems in pigs • Problems in humans because it causes syphilis Curved rod- looks like a C; ex. Vibria Pleomorphic- an organism that can change shape; generally, from rod to coccus. As they get older they collapse on themselves, result of enzymes altering components of cells (autolysis) Sizes Microbes are small 6 They are measured in micrometers (um) 10 5 Rods; 1.0-1.5 micrometer Cocci; 1-2 micrometer across, 1-5 micrometers in length Spirilli; 0.25-.5 across, 20 micrometers long Rickettsia: small bacterium; intracellular bacterium-9hat causes tick- born diseases; measured in nanometers (nm), 10 m; Same range (size) you look for with viruses The size that they can go to is limited by the ability to get nutrients into the cell by diffusion, it’s a transport limitation Surface Area per unit of volume (surface area/unit volume) The larger the amount of surface area per unit of volumethe faster it can get in nutrients increases metabolic rate increases division (reproduction) increase the chance of survival Epulopiscium; a large organism, endosymbiotic organism that lives in the gut of certain fish and can be seen without a microscope Thiomargarita namibiensis; large bacterium, green organism and is a sulfur utilizing organism Looking at Parts of the Cell To look at parts of the cell you have to lyse them (open them up) Mechanically apply pressure to the organism and make them break them Use a French press to do this Mix them up with tiny little beads and the beads will put pressure on them Use enzymes to break them down, an ex. To use would be lysozyme to break down the components of the cell then use osmotic shock Sonication- using sound to break them apart Detergent- helps dissolve membranes but doesn’t mess up proteins Prokaryotic Cells Cytoplasm- gel-like matrix that is present to help support cells, helps to transport in reactions Major component in cytoplasm is water Can have wastes that haven't gotten carried out of cell yet Can have nutrients that haven't been used yet Can have precursors- parts of more external structures that haven't been moved to permanent locations Commonly have many enzymes Enzymes- bacteria often have enzymes floating in cytoplasm and this is important because bacteria usually don’t have all the organelles of a normal cell so enzymes take over the functions Inclusions (granules)- areas where sulfur is stored, many would 6 like to store iron because it limits where it can grow, not membrane bound organelles but little collections of nutrients that are present Plasmid- circular exocromisomal piece of DNA; can have multiple ones per cell Its important because they are mechanism by which cells can get new genetic material in Bacteria can divide by binary fission; one cells become 2, 2 becomes 4, 4 becomes 8, its an exact split of cell into 2 parts so they’re all the same without much variation Plasmids can bring new genetic material to cells which can help them adapt Also gives them antibiotic resistance They may not be required Phage- (bacteriophage) Virus that has bacterium as a host Can pick up genetic material and being it into the cell; method of transport They can help bacteria become resistant to antibiotics It may or may not be there Ribosome- Made up of two spherical of eukaryotic cells attached together These are smaller than ribosomes of eukaryotic cells and closer in size to ones you find inside mitochondria but still involved in protein synthesis “Cytoskeleton” Not as organized as what we have in our cells Actin fibers/filaments that are support mechanism for cells Nuclear Region (Nucleoid) Not membrane bound structure so not a nucleus, but is area of cell where DNA and RNA is present Control center of the cell; metabolism, division Gas Vesicle Little sac that has membrane type structure around it and only shows up in bacteria that live in aquatic environments where they store gas Helps in flotation to help keep organism at a certain level in their environment It may or may not be present Cell Membrane Virtually identical to cell membrane of our cells Important in terms of size, transport, survival Important in helping spilt the DNA as cell divides Often a connection between nucleus and cell membrane but 7 there isn’t anything like microtubules in there so it can’t help divide Mesosomes Folds or kinks in the membrane It increases the’ surface area and helps the bacteria in transport of nutrients Can have Enzymes If there are enzymes that are localized makes it more efficient for the cell Cell Wall Important in binary vision Protects the cell again changes in osmotic pressure Glycocalyx Outside the cell Capsule, if it is a capsule it is an organized layer of sugars put in a pattern and are a certain type based on the genetics of an organism Slime layer; sugar that is not organized, just sugars dumped on the outside Regularly structured layer; set pattern of proteins, organized lumping of proteins May or may not be there Pilus Protein type appendage that helps bacterial cell attach, can have many on cell, solid that helps with attachment May or may not be there Sometimes referred to as fimbri Sex Pilus Hollow version of pilus, involved in exchange of genetic material May or may not be there Flagellum Longer and much more elaborate Fewer in number that pilli or fimbri Used in mobility so organism that have them can swim Equivalent to us running 60 mph when they’re in use May or may not be there Endospore Dense, heavy, thick, layer that sections off necessary elements Medically and environmentally dependent pH changing Stress from environment Genetics cause cell to wall off essentials of that bacterial cell Can remain “dormant” in structure for centuries 8 Activated when bacteria are in right environment Protective mechanism, not reproductive when it germinates May or may not be there Phospholipids- Importance in terms of transport and function of the membrane The difference in the fatty acid ends; glycerol Archaea can live in extreme temp due to differences in lipids Cell Wall- Peptidoglycan Made up of different types of sugars – N-acetylglucosamine (G) – N-actylmuramic acid (M) Beta 1-4 linkages (-) between 1 and 4 carbons in the sugars that we are dealing with Peptide linkages – There is a major difference between the types of linkages there are based on what bacteria they are – There is a string of 5 amino acids on the precursors that are being put in the cell wall – As the bind together the 5 amino acid of both sides falls off and then the third on one strand binds to fourth bind together (don’t know why) • Direct Bind • Complex bind- repeating units of amino acids Gram (+) – Lattice set up, chains of sugars – Very thick layer of alternating sugars G-M G-M-G-M-G-M... (found outside relative to membrane) – Peptidoglycan is found in true bacteria but not Archaea – Held together by 1-4 linkages – Not stable without peptide bonds- but the peptide linkages go in random not connecting; only between M – Heavy red line- teichoic acids sometimes referred to as wall teichoic acids and the lower ones sometimes referred to membrane teichoic acids because they are anchor the peptidoglycan layer to the membrane. They are only made in the Gram (+) organisms. – There is an S layer on top of the Peptidoglycan layer that is proteins followed by glycosyl chains – There is a question whether there is a gap between Peptidoglycan and the membrane called periplasmic space (it was initially found in the Gram (-)) 9 Gram (-) – Cell wall (Cell envelope) of Gram (-) – The Peptidoglycan chain, this layer is much thinner – Peptide bonds; different between Gram (+) and organisms that fall under Gram (-) – Lipoproteins; help anchor outer membrane to peptidoglycan layer – Followed by a layer that looks like the fluid mosaic model; there is proteins called porin and phosolipids and lipidpolysacciri that is phospholipids connected with a sugar – There is a space between the Peptidoglycan chain up into the phospholipid layer called the periplasmic layer; it helps break down and bring nutrients into the cell Gram Stain Founded by Christopher Gram 2 types- Gram (+) & Gram (-) Gram stain you would use different reagents to show the difference between Gram (+) and Gram (-); in between reagents you use a wash Use a heat fixed smear Distinguishes Gram positive from Gram negative bacteria REAGENT TIME GRAM + RESULT GRAM - RESULT CRYSTAL VIOLET 1 min Purple Purple Primary stain; rinse IODINE 1 min Purple Purple Mordant; rinse ALCOHOL 15 sec Purple Clear Decolorizer; rinse SAFRANIN 2 min Purple Red Counterstain; rinse; (May vary) dry *There is a rinse between each reagent If you are fighting an infection with a Gram (+) its making peptide bonds for stability as long as its growing; penicillin will work to block the formation of peptide bonds. This doesn’t work for Gram (-) because there aren’t as many peptide bonds. For a Gram (-) you would need a drug to travel up the porin and stop protein synthesis Acid fast organisms REAGENT TIME ACID-FAST NON CARBOL FUCHSIN 3 min Fuchsia Fuchsia Mycolic acid stain; 10 steam; rinse ACID ALCOHOL <30 sec Fuchsia Clear Decolorizer; rinse METHYLENE BLUE 1 min Fuchsia Blue Counterstain; rinse (Red if use safranin) (Can use safranin) To stain you have to heat it, it looks a little more like Gram (+) Cover with paper towel for carbol fuchsin only Distinguishes mycolic acid containing bacteria from those without mycolic acid Acid fast means it does respond to the acid in alcohol Microbacteria in Tuberculosis or Leprosy that causes these diseases The differences will cause different antibiotics to treat them and now some tuberculosis cases are resistant to the antibiotics People with AIDS that have a bad immune system and are susceptible to tuberculosis Smear preparation • Use aseptic technique • Apply loop filled liquid culture to slide (loop of water then loop of colony from agar) • Air dry • Heat fix Simple stain • Used for shape, size, general observation • Flood prepared smear with stain • Allow contact for desired time (generally 1 minute for methylene blue, crystal violet, or safranin) • Rinse gently • Blot gently with bibulous paper Differential stains • Used to distinguish different types of bacterial cells – Gram stain – Acid-fast stain – Endospore – Capsule stain Flagellum Filament Long hair like 11 There is a filament in both Gram (+) and Gram (-) Made up of protein called flagellin; sometimes there are there and sometimes they are Hook; stabilizing unit Rod like anchor below hook Works as anchor Along the rod there are protein rings that energized by ATP; their movement is what makes flagellum move Gram (-) basal body is made up of those rods and those rings is elaborate; M ring, S ring, L ring, P ring, and they line up with the cell membrane, peptidoglycan layer and the outer membrane Gram (+) fewer rings; there is one in plasma membrane and one toward base of Peptidoglycan setup. They both rely on ATP to power then and are very energy intensive set ups, they make the cell move. Sometimes they are there and sometimes they are not. Atrikus- if they aren’t there Monotrikus- if there is 1 cell Peritricus- If it has many, (Peri means around, in many directions) Localtricus and Antricus if it has tufts of these, tuft on one end of the cell or on both ends of the cell Cell Wall Kind of a middle between Gram (+) and Gram (-) in terms of the Peptidoglycan layer Mycolic acids More sugar layer Gylocalyx that is a capsule Endospore- difficult to stain Stains – Malachite Green; primary stain, – Safranin; counter stain If the malachite stain is picked up and it ends up in a spore and the safranin dies the rest of the cell red with a green dot in the middle; sometimes you get a black spot Used a heat fixed smear Cover smear with paper towel for malachite green only Distinguishes endospore former from non-spore former REAGENT TIME W/ SPORE NO SPORE MALACHITE GREEN 5 min Green dot in cell No green dot Spore stain; steam during application; rinse 12 SAFRANIN 1 min Red cell around Red cell without Vegetative cell stain; green dot green dot rinse; dry Glycocalx; uses capsule stain but it’s a different process Distinguishes cells with capsule from those without capsules Use two clean slides Apply drop of Nigrosine or India ink near one end of slide Using aseptic technique introduce bacteria from culture Use second slide to smear fluid across slide (See lab manual for picture) Do not rinse, air dry, do not heat fix, observe If there is a clear ring around then a ring of India ink then it has a capsule around the cell. Differences between True Bacteria and Archaea: True Bacteria No true peptidoglycan but instead they have pseudopeptidoglycan (fake peptidoglycan) Lacks in N-acetylmuromic acid but replaced by N- acetyltalosaminuronic acid (similar sugar but not exactly the same) Lacks Beta 1-4 linkages but instead has Beta 1-3 linkages (different sugars are connected) Like the Gram + you will see an S layer Archaea- extreme environments How the membrane and wall are involved in transport- Rate of transport will depend on lots of factors including: Surface area of membrane 13 – The more lipids present in bilayer- the more you can get materials to move through Amount of lipids in membrane – How much surface area- mesosomes increases the surface area, integral proteins can be used in increasing transport Strength of concentration gradient – A presence and stronger concentration gradient allows to push molecules through Size and/or change of ions/molecules compounds being transported – Larger molecule- transported slowly vs. smaller molecules – If there is a charge it can slow and even stop a molecule Transport proteins present if needed Presence of extracellular enzymes which change material being transported – There could be enzymes associated with mesosomes can have a role in transport Options for Passive Transport: 1. Simple Diffusion Nothing needed to transport molecules – Smell of mouth wash spreading throughout your apartment or blood h – Tissues in your body; carbon dioxide and oxygen in blood… if blood is heading out of lungs and into tissues you would want more oxygen, the transport of oxygen and carbon dioxide in tissues or lungs is an example of simple diffusion 2. Facilitated Diffusion Needs something to help to transport molecules, uses semipermeable membrane. – Glycerol going through a transporter and it attaches to one end of transport set up (ex. Integral protein), it enters the channel and it causes the channel to change configuration and it will go through. – It will be facilitated because it is brought about by protein and it changing shape to allow molecule to pass through, passive because higher lower and uses membrane 3. Osmosis- Hypotonic -If the concentration of the solution is greater than the concentration in the cell then by simple diffusion it will move into the cell and will swell and hit equilibrium or burst When your sick you want something that is hypotonic to move the water from your stomach into 14 your tissues so you do not dehydrate, like diluted Gatorade Isotonic-If the concentration is equal, already equilibrium and nothing happens Saline Solution for contact lenses Hypertonic- more concentrated in the cell and lower concentration in the solution, water moved out and either equilibrium or the cell shrinks Soaking a swollen foot in Epsom salt Options for Active Transport 1. Phagocytosis- active transport; requires energy, low to high, selectively permeable, membrane, can transport foods 2. Pinocytosis- active transport; requires energy, goes against gradient, but something sinks in instead of going out and grabbing it, takes in liquid 3. Na/K pump; active transport 4. Simport One molecule is moved and releases energy and that energy is used to move another molecule Advantage; two things being transported at one time Going in the same direction at the same time Uses selectively permeable membrane 5. Antiport One molecule going one way and the other going another directions Requires energy to go against concentration gradient 6. Group translocation; having a compound such as glucose that is outside the cell, normally there is more outside than inside. A transport system picks up glucose and brings it and picks up a phosphate on the way and it ends up bring glucose-6-phosphate and puts it on a sugar It requires energy, goes against gradient, used semipermeable membrane The sugar molecule is changed in the process 7. Siderophores Made by bacteria Compounds that pick up and hang on to iron, ones it dies it releases the iron then it puts it back in the red blood cells, in order for bacteria to live inside you they need something that will pick up the iron quickly How organisms might attach to surfaces Set up through biofilms 15 Monocultures, Multi-organism cultures Solid surface where organisms attach and they will frequently lose their flagellum, they form colonies and then expand and produce polysaccharides (slime layer or capsule)… Form sensing; then they will begin to move out as they sense there are more organisms around them, and will go through the process again An advantage to attaching to a surface Transport What they need is attached to a cell They wont be picked up phagocytic cells – It could be a disadvantage Competing for nutrients Waste will get into the biofilms If you have a pacemaker they can attach to the pacemaker and cause it to have to be replaced (some are silver because they have antimicrobial properties which cause them not to attach). How to grow these organisms Binary vision is the major mechanism for bacterial reproduction Splitting cells, some go through every 10 minutes 1248 Advantage; speed, large numbers very quickly In Eukaryotic cells; it does not occur as fast Disadvantage; clones, can be a problem for the cells Calculating the population Formula (pop. At a given time) N = (1op. At T 0, original popu0ation (number of generations) Growth Curve • Across the bottom; time, log of the number of cells present • Lag phase; where the organism is adapting to the environment and developing precursors to make new cells, no reproductions • Log phase; also called exponential phase, where you will get reproduction and making cells as quickly as possible • Stationary phase; every cell being reproduced, there is one dying • Death phase; rapid, more cells dying than reproducing • Then prolonged decline • Problems with the growth curve; no particular organism that you are taking about and no specific numbers, also do not 16 know what conditions it is growing under. This is assuming that it is perfect conditions. You also need a closed population; a bad example is deer. Organism need moisture like water to grow; to dissolve nutrients to bring into the cell Hydrolysis; using water to break down compounds, if they get stressed over losing moisture they can use Glycocalyx, a capsule or produce an Endospore as a protective mechanism to keep in moisture pH of the surrounding environment; most bacteria need a pH of 6.8-7.2 to grow successfully. Some will grow at different pH’s. Water can help with pH in lab you can control the pH with buffers. • Helicobacter pylori (True bacteria)- it can live in stomach (not neutral pH) and can cause ulcers • Archaea- live in neg. pH and these can fit into extremophiles for pH and temp. • Neutrophils/Neutrophilic- living in neutral pH • Alkalophiles- higher pH • Acidophiles- lower pH • Most fungi like a pH of 6 more acidic. Nutrients will they need; Carbon Energy Auto-self feeding Photoautotroph; uses inorganic Carbon sources like CO 2nd use light for energy energy Chemoautotroph (Chemolithoautotroph); inorganic Carbon sources like CO ,2and inorganic Carbon for energy Heterotroph- get their food from somewhere Photoheterotroph; use organic Carbon sources and light for energy Chemoorganichetertroph; use chemical compounds for light energy, sources of Carbon for energy Fastidious heterotrophs- picky eaters, need something that is above the normal requirements for them to grow Legionella pneumophila- needed an amino acid for it to grow and activated charcoal which absorbs waste products for it to grow readily, it grows at weird pH (ranges from 9 to 3 to 7), it grows on a black agar and looks like small 17 black dots vs an off white color, lives in water or soil Classifying medium Physical properties • Solid; ex. Potatoes (would be a solid medium for it to grow on) • Broth- making a beef broth with left over beef and bones, probably used brave heart diffusion • Agar- TSA plates in lab, if you didn’t have agar you could use jello- gives proteins and sugar and solid surface to grow Chemical makeup • Complex medium- complex meaning it has a mixture of a lot of things in there; salts, sugars, and other compounds. They are put together for a specific type of medium. • Chemically defined medium- you know how much of every chemical in it (how much sodium or chloride) Application • Optimal medium- give the best growth conditions for a known organism • Minimal medium- the minimum conditions for the known organism to grow; concentrations and ranges are smaller so reproduction is slower Selective • Get Water from aquifer plate it out on nutrient agar and add NaCl (Salt lovers; like ones from ocean) You would grow it out and look for colonies. The salt lovers would not be able to grow in an agar without salt • Eosinmethylene blue agar- if you grow organisms on there it will die the colonies and it will be selective towards versus a Gram (-) bacteria Differential; allow you to tell one organism from the other • Blood agar; base + sheep red blood cells. You will get a pretty red plate; some colonies have no agar, some a green zone and some a clear zone. The clear zone means the organism in the colonies have secreted enzymes in there and they break down the red blood cells… this is referred to Beta hemolysis, Green zone is Alpha hemolysis and no agar is considered to gamma hemolysis. You are trying to get elements such as C, H, O, P, K, Ca, or Se, Mg, Mn, Zn (trace elements; used as cofactors to getting enzymes to work as they should) if you didn’t have Se your enzymes and immune system wouldn’t work but if you had too much it would let your hair fall out and shut down your immune system. 18 For a Eukaryotic cell; mitosis occurs average 60 minutes. G can ho0d up process of mitosis. Sulfur metabolizing organisms split slowly. Limiting nutrients- nutrients that aren’t abundant in an environment and can be a problem Oligotrophic environment • The environment has very little nutrients that an organism needs • Iron is a big nutrient that is limited, organism that live in humans especially have a problem with lacking Iron. When a red blood cells break down the iron is put back into another red blood cells which makes the number organisms that can grow in someone limited. • Siderophores are compounds that can sequester iron very quickly and helps get around the limiting factor Eutrophic environment • An environment that has a high amount of nutrients *These terms are often applied to aquatic environment Pressure • Atmospheric pressure • Ocean has a lot of pressure, this lady went into the ocean and got samples but when they brought them back up and tested them on agars, they didn’t have anything grow correctly because the bacteria blew up from the pressure change. • Barophilic- love high pressure • Barotolerant- can live with high pressure • Osmotic Pressure- • Osmophilic-love the osmotic pressure • Osmotolerant-tolerate the osmotic pressure • Halo-Sale • Halophilic-love the salt • Halotolerant- can stand the salt Temperature • Cardinal Temp • Minimum-lowest temperature at which an organism will survive • Optimum- preferable temperature • Maximum- highest temperature at which an organism will survive 19 • Psychrophilic- lower temperatures 5-15 degrees Celsius ,can lower than that and it has been seen at the bottom of glaciers • Psychrotroph-between Psychrophilic & Mesophilic • Mesophilic- mid range temperatures 25 -45 degrees Celsius • Mesotroph- between Mesophilic & Thermophilic • Thermophilic- 50-75 degrees Celsius • Extremophilic- Archaea rather than true bacteria Gases • Aerobes (Aerobic) deals with free molecular oxygen • Microaerobes- small amounts of free oxygen • Obligate Aerobes; absolutely have to have it • Facultative Aerobes; grow and reproduces better with molecular oxygen but can live without it • Anaerobes- No free molecule oxygen • Obligate Anaerobes- do not do well with free oxygen, if you have a truly obligate anaerobe and it is exposed to air it can have a life expectancy of about 3 sec. • Facultative Anaerobes- wont die immediately without oxygen, grow and reproduce without free oxygen • Aerotolerant- organisms that are indifferent to free oxygen • Capnophiles- require elevated CO to rep2oduce Counting the organisms • Spread plates- count the colonies, look at the characteristics of the colonies • Streak plates- Dip loop into the culture and streak it, it can’t really work as a counting method because you don’t know how much was picked up by the loop. It is more qualitative rather than qualitative. It can be quantitative if you have a calibrated loop. • Each colony came from 1 organism- colony forming unit (CFU) CFU/Ml • Pour plate- Take organism and mix into the Agar while it is still liquid then pour into petri dish and wait for it to solidify. You can see the differences in the organism by looking at the set up. Filtration • There is a filter set up, and there is a funnel shape thing and on top is a platform and you put filter paper on top (these are all sterilized). You take a vacuum set up and attach it to a spicket and pour the liquid through and turn on the vacuum, on the filter paper you have collected all the organisms that are present. You can alter the pour size to collect different things; like viruses, etc. 20 Hemocytrometer/Petrof-Hauser Counter • From the side, it is a slide that has an indentation the center of it. You put a cover slip over it, and put known volume in the small space. Its an old fashion technique for counting blood cells. Flow cytometry • Mechanism that counts cells as they go through beam of light Spectrophotometry • Have source of light & meter; sample goes between the two; measures amount of light that passes through to meter; disadvantage- dead cells reflect same as live cells Broth with Sugar • Goes red & stays red means no acid; red goes to yellow means acid is being produced; look for bubble in top of tube to see if it has produced gas • Broad groups of compounds; haven't been talking about anything dealing with more specific control- antibiotics, etc. Criteria for antimicrobial development/application • Broad spectrum but not kill normal flora • Soluble in body fluids but not easily removed from body • Selective toxicity- problem for eukaryotic pathogens (fungi, protozoans) • Not cause allergic reactions • Not have resistance develop • Inexpensive to manufacture • Reasonable shelf-life without refrigeration: be able to be shipped wherever Mechanisms of resistance 1. Enzymes that inactivate drugs- destroy drugs before enter cells • Beta lactamases: degrade beta lactam rings of penicillin sans cephalosporin- large number of gram positive bacterial have naturally occurring ones • Penicllinases: produced by staphylococcus aureus & Neisseria gonorrheoeae 2. Decrease cell permeability & uptake of drug • Outer membrane of gram negative blocks penicillin • Multidrug resistant pumps & transport proteins- work against broad spectrum antimicrobials such as tetracyclines- can be coded for by genes in plasmid- occur in gram positive cells such as staphylococcus & streptococcus- occur in gram negative org such as pseudomonas & Escherichia coli 3. Add modifying groups that inactivate antibiotic- occurs with 21 aminoglycosides 4. Changes in drug receptors 5. Changes in essential metabolic pathways 6. Lapse into dormancy Convert to cell-wall-deficient form (L form) • Lungs can wall off organism like TB; makes it hard to get drugs where TB is; known way to get rid of it is to go in & cut out portions of lung; went from that TB to multiple drug resistant TB- had to be on 8 or 9 antibiotics for years; TB then became very drug resistant- had to add more drugs for longer periods of time; now have totally drug resistant TB- will kill you if you get it unless you have part of lung cut out • Enzymes are specific in what they do • Semisynthetic; naturally produced but tweaked in lab • You want to have several drugs working together • INH (weaken outer structure of bacteria) in cell wall synthesis and Rifampin both work against Tubercle bacilli. • Cycloserine (talked about during cell wall synthesis) • A drug produced by an organism called Streptomyces garyphalus • Used most against a Tubercle bacillus Ex. Mycobacterium tuberculosis; (what’s different about the mycobacterium is that it is an acid fast organism, meaning they don’t have pseudopeptidoglycan, it makes it difficult to control) This particular one blocks a particular bonding; Amino acid that is D Alene bonds to another D Alene bonded by dipeptide. The drug comes in and inhibits the bonding between the D Alene’s. If you don’t have the bonding you won’t have the pseudopeptidoglycan chain and it makes it unstable and the walls wont hold up and the organism can explode and die. It has to be an actively growing population because the enzyme has to be constantly making the D Alene-D Alene bond to make a strong peptidoglycan chain. • ActoMyosin D • Fits into DNA • It binds to DNA from any source. • You don’t want to take this. 22 Enzymes Composed of proteins Domain- structural functional part within the enzyme itself Subject to denaturation- change in configuration, caused by higher temp, pH, exposure to salt Specific in function Wont be used up in a reaction/or changed Single can be controlled through- con. of enzymes, con. of substrate, pH of rxn, conc. of cofactor Decreases the activation energy Capable of catabolic & anabolic reactions Work singly- 1 enzyme involved in taking care of reaction pathways May require a cofactor-generally inorganic element that helps the enzyme to work efficiently and effectively (Ex. Cr, Mg, Zinc, Mn, Trace elements, Selenium) May require a coenzyme- organic molecules, vitamin B derivatives, electron carriers Coenzyme Vitamin From Substance Example of Use Which It Is Transferred Derived Nicotinamide adenine Niacin (B3) Hydride ions (2 Carrier of reducing dinucleotide (NAD ) electrons and 1 power proton) Flavin adenine Riboflavin (B2) Hydrogen atoms (2 Carrier of reducing dinucleotide (FAD) electrons and 2 power protons) Coenzyme A Pantothenic acid Acyl groups Carries the acetyl Krebs cycle (B5) group that enters the TCA cycle Thiamin pyrophosphate Thiamine (B ) Aldehydes Helps remove CO 1 2 Krebs cycle glycolysis from pyruvate in the transition step Pyridoxal phosphate Pyridoxine (B6) Amino groups Transfers amino groups in amino acid synthesis Tetrahydrofolate Folic acid (9 ) 1-carbon molecules 1-carbon donor in nucleotide synthesis Function on a substrate- molecule upon which enzyme will be active, what its using to build up or break down has to interact directly with enzyme Activate site(s) Portions of the enzyme where it attaches Specific attachment- substrate has to fit in active site Competitive inhibition- Increase in use of glucose; balances off, increase in use of lactose, balances off-way of showing that there is limits to how fast and how long reactions can go on Stationary periods- Glucose is running out (con. of substrate), con of coenzyme, change in pH, lack of substrate (major one) etc. Bacteria tried to turn PABA substrate into Sulfa, if there's enough of the sulfa compound present, it could out compete the substrate and get into active site first, if it happens bacteria can use PABA to create folic acid to survive, can be reversible or irreversible, depends on interaction between competitor and active site whether it stays or not. Enzyme has allosteric site; inhibitor binds to enzyme, it alters the configuration of active site so substrate can’t fit & enzyme doesn’t do its reaction uses separate site from active site. Binds and goes away-reversed depending on how inhibitor binds to allosteric site Allosteric activation- change active site to make it where substrate will fit, could turn off-activation rather than inhibition Optimum in pH- fungi- lower, bacterial- neutral. Neutrophilia- pH 7 Enzyme control Competitive inhibition Inhibitor binds to the active site of the enzyme, blocking access of substrate to that site. Competitive inhibitors such as sulfa drugs are used as antibacterial medications. The molecule that can mimic substrate of enzyme & compete with it. Non-Competitive Inhibitor changes the shape of the enzyme, so that the inhibition (by substrate can no longer bind to the active site. This is a regulatory molecules) reversible action that provides cells with a means to control activity of allosteric enzymes. Non-Competitive Inhibitor permanently changes the shape of the inhibition (by enzyme enzyme, making the enzyme non-functional. Enzyme poisons) poisons such as mercury are used in certain antimicrobial compounds . Metabolic Pathway- Catabolic or anabolic Reciprocal- start with compound and it picks something from that was left over. Each step is brought on by a different enzyme. Has to have stuff that is left over to keep cycle going. Krebs cycle Product inhibition/negative feedback-When you get an increase in the conc. of products can interact with Enzyme 1. It decreases activity of Enzyme 1 & pathway is inhibited, Enzyme 1 becomes rate limiting enzyme. Though out negative feedback the end product of a biosynthetic pathway frequently acts as an allosteric inhibitor of the first enzyme of that pathway. Precursor activation/positive feedback- Increase in concentration of precursor interacting with Enzyme 3 which increases the activity of Enzyme 3. The rate limiting enzyme is Enzyme 3. It allows you to maintain homeostasis. One could be turn on while the other is off Enzyme pathways Monosaccharides can be converted into simple sugars Beta oxidation- breaking down of fatty acids, can lead to the formation of Acetyl-coA Deamination Proteins can be broken down into amino acids, feeding into several layers Reactions are reversible Central of sugars can lead to formation of fats, proteins Proteins- strings of amino acids arbohydrates Polysaccharides- many sugars present Allogosaccharides- 10 or less Disaccharides- 2 Monosaccharides- 1 Central core of using sugars, number of reactions are reversible, core of sugars important can lead to formation of ribosome-makes RNA, converted to make DNA. Formation of purines or pyrimidines Interactions of pathways- fats, proteins, carbohydrates can be converted into core metabolic pathway Glycolysis Oxidizes glucose to pyruvate Cytoplasm of both Prokaryotes & Eukaryotes Can occur under aerobic & anaerobic conditions Good under allosteric control You need- Glucose, ATP's, ADP, NAD, Enzymes, Phosphate, & enzymes What comes out- enzymes, ADP, ATP, NADH, more enzymes, water, & pyruvate Important for- ATP generation, NADH generation & production of intermediate sugars Transition step (GlycolysisKrebs cycle) • Pyruvate is converted into Acetyl-CoA (needed for Krebs cycle) • Can occur in the cytoplasm of Prokaryotic /Eukaryotic • Under Aerobic, sometime Anaerobic - very uncommon • Need- Pyruvate, CoA, NAD, Enzymes • What comes out- Acetyl-CoA, NADH, CO2, H, Enzymes • Can get Acetyl- CoA from sugar (turns into Pyruvate), lipids (turns into fatty acid), peptides (turns into amino acids), lignin (turns into phenolic) Krebs cycle • Prokaryotic- cytoplasm & eukaryotic- mitochondria • Aerobic process ONLY • Have to have- Acetyl-CoA, oxaloacetate, NAD, ATP, FAD, water, CoA molecule, all enzymes, ADP & phosphate • What comes out- oxaloacetate, NADH, H, CO2, ATP, FADH2, Enzymes, CoA, water • Important for- making intermediates, converting energy, and formation of NADH and FADH that will be used in electron transport and it will stay efficient, if not the pyruvate will go into another process and become lactic acid & you can still get some energy but not as much Glyoxylate Process • Is anaerobic • In this system it bypasses some steps and you end up going to succinate or to malate. The bypass could occur because it could be lacking the genes to make the enzymes to make the steps which enables it to follow Krebs cycle. • Don’t get as much energy but it is functional, and still get a chunk of intermediates that could be used for other things. • It does occur in some organisms; microbacterium TB. It goes on when they are picked up by macrophages, they are living in the cells of the body and transported as they are producing the compounds. • There are some variations of the pathway, it can use fatty acids. It could be an advantage to Microbacterium TB because it could grow in macrophages nicely, and it could be taken to all over the body. TB shows up in lungs but can be all in body because of the pathways available. • Can be used if lacking sugars, pull in compounds that are lacking sugars • Uses extra acetyl CoA Entner-Doudoroff pathway- EMP- glycolysis ED- different pathway, it ends up with different compounds in the middle, they can be interlinked on feeding intermediates back and forth. Disadvantage- less energy, less NADH Advantage- do get NADPH that can feed into electron transport Glucose glucose 6-phsophate End up with different intermediate sugars, the outcome is pyruvate Allows for different sugars and different sugar interactions, allows for variety for organisms that don’t have some of the enzymes for other processes Pentose Phosphate Pathway- glucose ribulose 5 phosphate End up with different compounds & sugars that feed into biosynthetic pathways and still get energy Dependent on enzymes being present, and on cell with particular types of energy End up making ribose-5-phosphate (5 carbon sugar)- used in making DNA and RNA Production of- ribose, glycerol, nucleotides & amino acids All pathways have different outcomes & different sugars for different biosynthetic pathways Fermentation Pathways Pyruvic acidAcetaldyhedyeEthanol Produces products that are of industrial use/food use/ethanol products for energy, options that are alternatives dependent on the enzymes present Lactic acid- going on to make yogurt and cheese Most process occur in prokaryotic organisms (some Eukaryotic) in cytoplasm Anaerobic process ONLY Electron Transport Prokaryotic- membranes Eukaryotic-intermembrane of mitochondria membrane What you need- Redox compounds, NADH, FADH2, O2, Enzymes What comes out- reduced redox compounds, NAD, FAD H, enzymes &2 water Eukaryotic- long, some Most electronegative least electronegative As the electrons and hydrogens are moved, ATP is released for 1 NADH you get 3 ATP's Dependent on having ATP synthase E.Coli- has own electron transport system, its short Makes energy for flagella work In gut- doesn’t need a lot of energy because diet is feeding it continuously so it doesn’t need to generate a lot of ATP but it doesn't get a lot because its efficient Anabolic processes Photosynthesis Cyclical abolic process volves the electrons released form Bacteriochlorophyll being sent to bacteriophenophytin followed by cycling through electron carriers and back to bacteriochlorophyll kes energy for carbon fixation reactions of photosynthesis ght reactions in bacterial photosynthesis can involve use of ATP to transfer reducing power to NADH okaryotic- occurs in mesosomes of cell membrane ve to have- photo pigments, electron acceptor, redox compounds, ADP, inorganic phosphate at comes out- photosystem pigments, redox compounds, ATP okaryotic photosynthetic pigments- chlorophyll A, carotenoids, phycobilins, Bacteriochlorophylls Light reactions of photosynthesis Non-cyclical Energy fixing reactions it isn’t cycle it goes through photosystem 2 then 1, need water breaking down to regenerate an
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