Microbiology Test #1
Microbiology Test #1 BIOL 2230
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This 18 page Study Guide was uploaded by Allison Collins on Sunday February 14, 2016. The Study Guide belongs to BIOL 2230 at Middle Tennessee State University taught by Anthony L Newsome in Fall 2015. Since its upload, it has received 115 views. For similar materials see Microbiology in Biology at Middle Tennessee State University.
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Date Created: 02/14/16
MICROBIOLOGY UNIT 1 TEST Highlight = important term Highlight = important concept Highlight = important person or specific organism History and general principles of antibiotics Eukaryotes – humans, plants, protozoa, algae Prokaryotes – bacteria ONLY All cells – contain nucleic acids, lipids, proteins, polysaccharides Viruses – not cells – live inside eukaryotes or prokaryotes • Have no metabolism outside host cell • Referred to as a viral AGENT – not a viral CELL Timeline 1600s – Leeuwenhoek invented microscopes 1860s • Scientists asking: where do bacteria come from? – by this point they understood that it is not by spontaneous generation (idea that life can form spontaneously) Louis Pasteur – disproved spontaneous generation (including bacteria) • Experiment: took 2 sterile flasks containing broth o Exposed one flask to air – microorganisms grew o Did not expose the other flask to air – no microorganisms grew • Pasteurization – process of mild heating to kill microorganisms that make things susceptible to spoilage o Heated at 70C/160F 1870s • Scientists also asking: can bacteria make people sick? Robert Koch – creator of Koch’s Postulates o Still a cornerstone of microbiology o Demonstrated that a specific disease can be caused by a specific microorganism o Established set of criteria that must be met before specific disease is said to be caused by an infectious agent • Koch’s Postulates 1. Suspected pathogenic organism should be present in ALL CASES of the disease and absent from healthy animals. 2. Suspected organism should be grown in a pure culture. 3. Cells from the pure culture of the suspected organism should cause disease in a healthy animal. 4. The organism should be re-‐isolated and shown to be the same as the original organism. Late 1800s • Had been established that microorganisms can cause disease o This begs the question: can we reduce human illness by killing microorganisms? Joseph Lister – applied the concept of disease-‐causing bacteria to surgery/post-‐surgical infections • Soaked surgical instruments in phenol, which greatly reduced the number of post-‐ surgical infections Next question – can microorganisms already in the body be killed, thus killing the disease? Paul Erlich – found that arsenic will kill syphillus bacteria but also makes people ill o Thus the advent of the chemotherapeutic agent – a chemical with therapeutic value Late 1930s • “Antibiotics” not a term • Sulfa drugs were used, but were not referred to as antibiotics o Sulfa drugs inhibit growth of folic acid, which bacteria need to grow • World War II – “antibiotics” still not a term o Penicillin packets poured on wounds at D-‐Day Alexander Fleming – discovered Penicillin on accident • Was growing bacteria on agar plate, which unexpectedly grew a green fuzzy material after several weeks • Where the fuzz existed, there was no bacteria • Learned that the fuzz was a common food mold called Penicillium • Active chemical was termed Penicillin 1950s • Antibiotics were thought of as miracle drugs • At the end of WWII, they became available to civilians General principles of antibiotics 2 states of bacteria • Vegetative – when eating, multiplying, or metabolizing • Spore/dormant (only some bacteria) – when exposed to unfavorable conditions o Metabolically inactive o Come out of dormancy once conditions are favorable again – dormancy can last for thousands of years Antibiotic – a metabolite produced by one microorganism that inhibits a specific metabolic pathway in another microorganism • This is a microorganism’s way of killing a competing microorganism for self-‐preservation Types of antibiotics • Inhibition of bacteria’s cell wall synthesis • Inhibition of bacteria’s DNA synthesis • Inhibition of bacteria’s protein synthesis • Inhibition of bacteria’s metabolism Most biomass on earth is microbial • Most prokaryotes reside underground in the oceanic and terrestrial subsurfaces – many of these are unexplored o This may be the answer to formulating new antibiotics when diseases become resistant to current antibiotics Microorganisms can be both beneficial and harmful to humans • We tend to emphasize harmful microorganisms (i.e. pathogens), but the vast majority of microorganisms in nature are beneficial The prokaryotic cell in detail Distinguishing features of eukaryotic cells (everything but bacteria) • Nucleus – set of parallel membranes that contain chromosomes (23 pairs in humans) o DNA is linear o DNA is wrapped around histones (proteins) o 50% of DNA from each parent § Maternally inherited DNA shows a clear family history that has only been altered by chance mutations • Mitochondria – site of ATP (energy) production o Has its own DNA o All mitochondria come from the mother o Multiplies in cells depending on how much energy a cell needs o Cells vary in number of mitochondria they possess • Membrane-‐bound organelles Distinguishing features of prokaryotic cells (bacteria) • DNA is not enclosed in membrane-‐bound nucleus • DNA circular, not linear • DNA is not wrapped around histones • Almost always have cell walls composed of peptidoglycan o This is of great medical importance o Human cells do not contain peptidoglycan, so substances that inhibit peptidoglycan synthesis harm bacteria but not human cells § E.g. Penicillin • Has plasmids – circular DNA outside of chromosomes o Antibiotic resistance is often transferred by plasmids External to cell wall of bacteria § Capsule/slime layer/glycocalyx § Not all bacteria § In order for bacteria to cause disease, it must be able to stick to your tissue • Ex: cough, diarrhea, UTI – body attempts to expel bacteria § A very thick capsule makes an infection more difficult to treat • For antibiotics to inhibit cell wall formation, protein synthesis, etc., must be able to get into bacteria § The capsule is what interacts with your immune system • Vaccine production is geared at injecting capsule of the bacteria into your body so that your immune system creates antibodies to it • Capsule vaccine candidates – create vaccines with purpose of inhibiting capsule’s ability to adhere to mucus o Flagella § Not all bacteria § Long hair-‐like projection § May be just one or multiple § Enable movement § Flagella vaccine candidates – create vaccines with purpose of inhibiting flagella’s ability to adhere to mucus o Fimbriae § Not all bacteria § Fine hairlike structures that aid in attachment § Ex: Streptococcus mutans • Adheres to tooth enamel with its fimbriae • When the bacteria metabolize sugar, create acid that creates hole in enamel à cavity § Ex: Neisseria gonorrhoeae • Uses fimbriae to attach to urinary tract o Pili § Not all bacteria have them § Hairlike structures, longer than fimbriae/shorter than flagella § Promote exchange of plasmids § Possible prevention of major diseases is to inhibit the production of capsule, flagella, fimbriae, pili o Lipopolysaccharide (LPS) § Molecule extremely important to human health § Found only in gram-‐negative bacteria § Toxic to animals – especially humans § Pyrogenic compound – causes fever, septic shock, cell constriction • Ex: syringe packaging says “non-‐pyrogenic” – means it has been tested for LPS § Also referred to as an endotoxin • Actively releases toxins into environment (e.g. tetanus, botulism) § LPS can be shed by bacteria – released into environment – especially when bacteria die § Once LPS is in circulatory system, there’s no way to get rid of it • Can’t be destroyed – can only treat symptoms § Anything put into the body must be tested for LPS • Use Limulus amoebocyte lystate assay (LAL) to test for presence of LPS – causes gel to form around bacteria with LPS § Sometimes treatment of bacteria with LPS does not involve killing the bacteria because when the cell dies all LPS will be released into the body Prokaryotic cell wall § Composed of peptidoglycan, which is a polymer – anything composed of repeating subunits § Composed of 2 sugar derivatives and 4 different amino acids o Sugars: N-‐Acetylglucosamine M (NAM) and N-‐Acetylmuramic acid (NAG) Gram-‐positive bacteria § Have a thick layer of peptidoglycan in cell wall o Consists of alternating NAM & NAG o Peptidoglycan retains violet stain from Gram test § More receptive to antibiotics than gram-‐negative because they have no outer membrane § Also have teichoic acids § So from outside to inside: teichoic acids à thick pep. layer à thin plasma membrane Gram-‐negative bacteria § Have a thin layer of peptidoglycan in cell wall § Have a thick plasma membrane § From outside to inside: thick plasma membrane containing LPS à thin pep. layer à thick inner plasma membrane § Turns pink after Gram test Gram stain – traditionally has 4 steps 1) Crystal violet, 2) Gram’s iodine, 3) ethanol, 4) saffron Damage to bacterial cell walls is an extremely important concept in the treatment of bacterial infections § Peptidoglycan inhibitors do not damage eukaryotic cells, as they do not contain peptidoglycan Some medically important bacteria lack a cell wall § Mycoplasm pneumonia – common in young children § Chylamidia pneumonia – common in young adults § Penicillin would have no effect on these Cell membrane § 2-‐layered structure composed primarily of lipids and proteins § Surrounds cell – if broken, cell may die § Basically same structure in prokaryotic and eukaryotic cells, but eukaryotic cell membranes have sterols, which make it more rigid o Eukaryotic cells are larger and have membranes more prone to bursting, like a full water balloon § Phospholipid bilayer -‐ each layer is composed of repeating structures with polar head (made of phosphate and glycerol) and 2 nonpolar fatty acid tails o The heads are polar and thus are hydrophilic (seek out water) o 2 fatty acid tails attached to head – are nonpolar and thus hydrophobic (avoid water) § The hydrophilic heads orient themselves to the outside of the cells, where water is § Interspersed throughout the membrane are integral and structural proteins In order for a drug to work, it must get through the plasma membrane § Group translocation – substance is chemically altered in passage across the membrane o A phosphate is usually added to it § Active transport – compound enters cell unchanged § If a drug is chemically changed when passing through the membrane, it may be ineffective once inside the cell o However, sometimes a chemical change is beneficial Ideal cancer drug – nontoxic but adheres to cancer cells (nonexistent so far) Bacterial DNA is a singular circular chromosome § 500x longer than cell but has no histone proteins § DNA is supercoiled Ribosomes § Site of protein synthesis § Differ in size from eukaryotic cells § Some antibiotics function in attaching to the end of a ribosome, thus inhibiting protein synthesis Plasmids – circular, extrachromosomal DNA Endospore -‐ highly resistant differentiated bacterial cell produced by certain gram-‐positive bacteria Important spore-‐forming bacteria Clostridium tetani § Causes tetanus – releases toxin that keeps muscles from relaxing (AKA tetany) – lockjaw § C. tetani spores can only multiply in absence of oxygen – i.e. they are anaerobic § Rusty nail has spores on its surface, but they can’t germinate until they enter a deep puncture wound o Other bacteria in the wound are aerobic and use up the present oxygen, making a favorable environment for the anaerobic C. tetani spores Clostridium botulinum § Produces toxins that prevent muscles from contracting (flaccid paralysis) § Can cause food poisoning o Improperly canned foods o When aerobic bacteria use up the present oxygen, the anaerobic spores can now germinate § Less likely in acidic foods § Honey causes botulism in infants o Adults have trillions of flora in intestines that overpower spores – prevent germination o Infants do not have that flora, so C. botulinum causes flaccid paralysis -‐ suffocation o SIDS – unknown cause o In a few cases have been able to culture C. botulinum in fecal sample § Botox – causes facial muscles to relax o Too much can cause facial drooping Bacillus anthracis § Causes anthrax § Aerobic bacteria § When airborne, is breathed in à spores germinate in lungs § Biological weapon fears Cell chemistry Microorganisms are 70%-‐90% water 55% protein, 20% RNA Rest is made of lipids, polysaccharides, lipopolysaccharides, DNA 4 classes of molecules in bacteria Simple sugars § Monomeric (simple) constituents of polysaccharides § Open chain or ring structure § Backbone of nucleic acids (RNA and DNA) Fatty Acids § Monomeric constituents of lipids § Hydrophilic and hydrophobic ends § Basic structure: CH -‐CH3-‐CH 2H =COO2 2 Nucleotides § Monomeric constituents of nucleic acids § DNA: 2-‐deoxyribonucleic acid § Lacks oxygen at 2’ carbon (H at 2’ carbon) § RNA: ribonucleic acid § Has oxygen at 2’ carbon (OH at 2’ carbon) § Each nucleotide composed of 3 separate units § 5-‐carbon sugar (ribose or deoxyribose) § Nitrogenous base § Either one-‐ring structure (pyrimidine base) or two-‐ring structure (purine base) § Molecule of phosphate (PO ) 2 § RNA vs. DNA § RNA is single-‐stranded § RNA: 5-‐carbon sugar is called ribose sugar (has oxygen) § DNA: 5-‐carbon sugar is called deoxyribose sugar (lacks oxygen) § Nitrogenous bases in RNA include uracil and in DNA include thymine § 3 types of RNA § mRNA (messenger) § tRNA (transfer) § rRNA (ribosomal) § Single stranded nucleic acid – 3 things to remember § At 5’ end, the phosphate group is exposed § Phosphate group is attached to 5 carbon § At 3’ end, the OH or H is exprded § OH or H is attached to 3 carbon § Location of nitrogenous bases § Is either a one-‐ring structure (pyrimidine) or two-‐ring structure (purine) Amino Acids § Monomeric constituents of protein § All amino acids have a COOH (carboxylic acid) and NH (amin2 group § Amino acids differ only in the nature of the side group – represented by “R” o 20 different amino acids § Centrally located carbon § H molecule § Many amino acids can be linked by a peptide bond – this forms a polypeptide o This linkage forms a protein o When amino acids link, amino group of one attaches to carboxylic acid group of the other o One H from amino group and OH from carboxylic acid group are cast off à H O 2 o Since water is cast off, this process is called dehydration synthesis Proteins occur in 4 levels of organization o Primary – straight line – not functional o Secondary – twisted – not functional o Tertiary – twisted to the point that molecule becomes globular – functional o Quaternary – even more twister – functional Denaturation o When heat is added to a protein, bonds are broken and the protein regresses to a linear (primary) appearance o Once cooled, will probably not return to original form Enzymes • Type of protein that lowers the energy required for a reaction • Biological catalysts – do not change themselves when active • Crucial in function of drugs to cure diseases • Many drugs are enzyme inhibitors o Most antibiotics function through enzyme inhibitors • Indicated by a name ending in “-‐ase” o e.g. DNA polymerase • Many require a cofactor in order to function o Cofactor-‐ non-‐protein component § e.g. iron, zinc, vitamin B12 • Enzyme function o Enzyme has active site into which a substrate (any material onto which an enzyme acts) can fit o Enzyme and substrate fit together, forming an enzyme-‐substrate complex o The substrate separates from the enzyme as a changed substance – i.e. the product o Enzyme is not changed and is ready to begin the process again • Remember: enzymes are protein, and the active site is a very small part of the protein Inhibition of enzyme activity • Competitive inhibition (active site) o Inhibitor can fit into the active site rather than the substrate, so different product is produced o Ex: sulfa drugs • Non-‐competitive (allosteric) inhibition o Inhibitor doesn’t fit into the active site, but blocks it so that no product is produced o Ex: cyanide These are effective ways to control bacterial and viral growth • Look for enzymes to selectively inhibit • Cancer growth is problematic because it lacks unique enzymes • Viral growth is difficult to control because they redirect the inhibitor and use it to create more of the virus Biochemical pathways Definition: a sequence of enzymatically catalyzed chemical reactions Cell (microbial) metabolism • Catabolic reactions – produce energy in form of ATP o Breakdown of materials • Anabolic reactions – use energy to synthesize products Catabolism – primarily sugars o Occurs in both eukaryotes and prokaryotes o 3 general stages 1. Glycolysis – process of breakdown of sugars to pyruvate • “lysis” = breaking down 2. Krebs cycle – main products are NADH and FADH 2 3. Electron transport chain o Production of NADH and FADH results in 2 nsport of oxygen to electron transport chain o Therefore, Krebs cycle can’t occur in absence of oxygen Energy production • Transfer of energy from nutrients is by removal of electrons • This usually involves the removal of hydrogen • This is called dehydrogenation • The major carriers of H are: • Nicotinamide adenine dinucleotide (NAD) • Flavin adenine dinucleotide (FAD) ***need to know full names for test*** • The energy carried by these molecules is used to make ATP from ADP in the electron transport chain o ETC is located in the membranes of the mitochondria § Since bacteria have no mitochondria, ETC is located in plasma membrane • Damage to the plasma membrane inhibits ATP production § Most ATP is produced in the ETC • There are many different proteins, polysaccharides, and lipids o All are degraded through activity of a few common metabolic pathways o Most microorganisms remove electrons from carbs (polysaccharides) as primary source of energy § Glucose – most common carb source § Adding sugar to a wound gives bacteria a preferred energy source over body tissue o Proteins and lipids will suffice in absence of polysaccharides o Lipid catabolism: Beta oxidation § Body uses oxidation when there are no carbs o Proteins broken into amino acids in order to pass through cell membrane § Body digests its own tissue when there are no carbs or lipids General principles of respiration Aerobic and anaerobic respiration are similar in principle Fermentation is fundamentally different from those processes Aerobic respiration • Definition: uses oxygen as the terminal electron acceptor in a membrane-‐bound pathway for ATP generation • Most prokaryotes and all eukaryotes • Requires external terminal electron acceptor o Oxygen enters from outside the body • Catabolism – breaking down • Basic steps: Glycolysis à Krebs cycle à electron transport chain o Product: ATP • Electrons are in the form of hydrogen • At the end of electron transport chain, H must bond to something o Oxygen is waiting at the end of ETC to accept H o Hydrogen and oxygen bind to produce H2O Anaerobic respiration • Uses compounds other than oxygen as the terminal electron acceptor in a membrane-‐ bound pathway for ATP generation o Ex: nitrates, sulfates • Yields less ATP than aerobic because anaerobic respiration does not include Krebs cycle o Anaerobic bacteria don’t grow as quickly • Anaerobic bacteria are problematic for deep wound infections • Periodontal disease o Gingival pockets between teeth start to erode (receding gums) – especially in the elderly o Bacteria accumulate with poor dental hygiene o Inflamed gingiva – gingivitis o Solution: cut back gums Fermentation • Catabolism of substrates by mechanism other than respiration – not all cells can do it • Bacteria and yeast yield ethyl alcohol (EtOH) o Many other important substances are produced, especially acids • Typical fermentation reaction o Only small amount of ATP produced – is all produced during glycolysis o Polysaccharides à Monosaccharides à Pyruvate § These three steps comprise glycolysis § Pyruvate breaks down in one of two ways: • Pyruvate à lactic acid and other acids o e.g. yogurt, sauerkraut, pickles o Inhibit growth of pathogenic bacteria (i.e. spoilage) o Yogurt contains acidophilus • Pyruvate à acetaldehyde à ethanol o Commercially important, e.g. citric acid, acetone • Doesn’t require oxygen so all fermentation pathways are anaerobic o Oxygen may be present but not used • Remember: in fermentation, ATP produced only in glycolysis • Fermentation begins with glycolysis and ends with formation of end products o EtOH is only one possible product -‐ other possibilities include lactic acid, acetic acid o Contain stored energy – not completely broken down o Does not require external electron receptor o Organic substrate acts as electron donor and product of that substrate acts as electron acceptor Enzymes affect respiration and fermentation • Things that affect enzyme activity o Temperature § 98.6F (37C) – optimal temperature for enzyme function in human body § Most pathogenic bacteria also do best with 98.6F § Fever: body trying to fight sickness – might inhibit pathogenic enzyme activity o Substrate concentration o pH Microbial growth and control • Microbial growth means increase in number, not size • Most antibiotics are directed at inhibiting DNA, RNA, and protein synthesis • Binary fission is a type of asexual reproduction • E. coli divide every 20-‐30 minutes under optimum conditions o Enormous growth potential – in 10 hr, 1 cell à 1 million cells Bacterial growth • Bacteria have enormous growth potential • Introduce bacteria to new environment à go through growth curve o Lag à exponential growth (log)à stationary à decline (death) • Lag – new environment, DNA and RNA synthesis, but little or no cell division • Log or exponential – maximum cell multiplication o Each increment on Y axis represents 10x multiplication of previous value o Most sensitive to antibiotics at this time – inhibits DNA/RNA/cell wall/protein synthesis § Esp. Penicillin o Maintained for relatively short time – use up space, resources – self-‐limiting • Stationary à death o Toxin production is greatest, nutrients at lowest level • Death – more cells dying than multiplying Effect of environmental factors on growth – 4 main factors § Temperature o 37C (normal body temp) – pathogens grow best at this temp o High fever – not optimal growth o Freezing inhibits growth but doesn’t kill bacteria o Some bacteria grow at boiling temps o Listeria monocytogenes: important human pathogen that grows at refrigerator temps § Psychrophiles – bacteria that grows best below 15C • Mesophiles – most pathogens – up to 39C • Thermophiles – at about 60C o Water heaters, hot springs, etc. • Hyperthermophiles – 100C o Survive due to amino acid substitutions in enzymes o Proteins not denatured by heat Eukaryotic cells: highest survival temp is some amoebae at 60C • pH o Most bacteria: 6.5 -‐ 7.5 o Most environments: 5 – 9 o Fungi are acid tolerant, ex: fruit (fungi are eukaryotes) o Few bacteria grow below pH of 4 § Ex: gastric fluids, vinegar § Exception: Helicobacter pylori – causes ulcers in stomach at pH of 1-‐2 • Relatively new discovery – ulcers now treated with antibiotics instead of antacids • Carried by cats – zoonotic bacteria? • Found in wells o Water availability • Hypertonic vs. hypotonic environment • Refers to amount of dissolved salt in environment vs. in bacteria • Water always wants to reach equilibrium • Hypertonic: higher concentration of salt outside of bacteria § If any bacteria are present, water will flow out of it – bacteria dies • Surface of skin: high concentration of salt, especially with sweat • Staphylococcus aureus – grows in hypertonic environments § Causes MRSA (skin infection) • Oxygen requirements o Obligate aerobes – need oxygen to multiply o Obligate anaerobes – only multiply without oxygen o Facultative anaerobes – can multiply without oxygen, but will use it if present § Ex: E. coli Sending electrical current through bacteria may destroy capsule/slime • Continuing research • This will affect sterilization of I.V. catheters and heart valves Culture media Chemically defined media • Exact chemical composition is known • Very difficult to make – not typically used in undergrad lab courses Complex media • Varies from batch to batch and contains complex additives • e.g. serum, blood, milk, meat byproducts o skim milk agar • Agar plates o Made from seaweed • TSA – trypticase soy agar – commonly used agar • Blood agar – most commonly used medium in hospital o Sheep blood • Chocolate agar – not really chocolate o Blood turns brown when cooked
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