Industrial Micro & Biotechnolo
Industrial Micro & Biotechnolo MICR 4354
Weber State University
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I MICRO 4354 Chap 2 p 1 Chapter 2 Microbial Growth and Nutrition Microbial nutrition A Nutritional classification is based on sources of energy electrons hydrogen and carbon 4 Energy a chemotrophs use chemical energy b phototrophs use light energy Electrons a organotrophs use organic molecules b lithotrophs use inorganic molecules Carbon a autotrophs use CO2 b heterotrophs use organic molecules There is no such thing as a universal medium B Macronutrients H N LN 4 U 0 Nutrients needed in high concentrations for four major cellular polymers a Proteins b Lipids c Nucleic acids d Polysaccharides carbohydrates Nutrients needed are carbon hydrogen oxygen nitrogen phosphorus and sulfur CHONPS Heterotrophs are more common than autotrophs in industrial microbiology a substrate 2 1020 gL b Sugars are very common substrates Hydrogen and oxygen usually obtained from water a 02 gas needed for aerobic respiration b 02 gas needed for sterol synthesis Nitrogen can make up 15 or more of cell dry weight a inorganic ammonium or N2 b organic urea amino acids Phosphorus and sulfur from inorganic salts a Phosphorus usually g 100 mgL b Sulfur 2030 mgL C Main elements 1 2 Required at levels of a few milligrams per liter 1020 mgL Calcium iron potassium magnesium sodium D Trace elements 1 2 3 Cobalt copper manganese molybdenum nickel selenium zinc Some organisms may have additional requirments eg Wo Required at very low levels pgL E Nutrient uptake 1 membranebound transport mechanisms required for uptake of nutrients by microbial cell a mechanism must be specific no use to transport unusable compound b membrane is selectively permeable c transport of dilute solutes against a gradient 2 LN 4 MICRO 4354 Chap 2 p 2 passive diffusion molecules move from a region of higher concentration to one of lower concentration as a result of random thermal agitation a rate is dependent on size of concentration gradient b requires relatively high external concentration c inefficient not extensively employed d gases and small uncharged molecules facilitated diffusion diffusion process aided by a carrier a permeases embedded in membrane mction as carrier proteins b more efficient than passive diffusion 1 more rapid at lower concentrations 2 saturatable levels off compared to linear increase with passive dif ision 3 no energy requirement still gradient driven still dif ision c permeases may mction by changing conformation allowing buildup of internal concentration 1 allows lipidinsoluble molecules to enter cell 2 can work in reverse if internal concentration is high enough d does not appear to be very important in procaryotes used with glycerol larger role in eucaryotes for sugar and amino acids active transport energylinked transport of solute molecules a works against a concentration gradient 1 works well even with low nutrient concentrations in the environment 2 can accumulate nutrients 1001000X greater concentration than external environment b employs carrier proteins 1 high specificity 2 saturatable c can use ATP other high energy phosphate compounds or proton motive force d many compounds have multiple transport systems may have selective advantage in different environments e typically used with sugars and amino acids f ATPbinding cassette transporters ABC transporters protein carriers that span the membrane and require energy usually ATP for activity 1 specific for molecule to be transported 2 located in the periplasm of gram negative cells or on the outside of the cell membrane of gram positive cells 3 probably involved in chemotaxis g active transport can also be powered by a proton or sodium gradient 1 symport linked transport of two substances in the same direction H linked uptake of amino and organic acids 2 antiport linked transport of two substances in opposite directions NaH 3 occurs in G and G group translocation molecule is transported and chemically altered at the same time a phophenolpyruvatezsugar phosphotransferase system PTS best studied 1 transports sugars into procaryotes 2 phosphorylates sugar using phosphoenol pyruvate PEP as phosphate donor systems are fairly complex with cytoplasmic and membrane enzymes involved present in facultative anaerobes and anaerobes rare in aerobes except for some Bacillus species PPquot MICRO 4354 Chap 2 p 3 6 Polymers high molecular weight compounds are hydrolyzed by extracellular enzymes a b Extracelluar enzymes include proteases amylases cellulases lipases peroxidases etc Many industrial applications 11 Microbial growth kinetics A General growth 1 growth increase in cell number or cell mass usually refers to number in case of bacteria 2 most bacteria reproduce by binary ssion a b c cell numbers increase by powers of 2 generation time amount of time required for population to double reproduce doubling time generation time 3 growth of lamentous bacteria or those that grow in aggregates are dif cult to model B Batch growth 1 closed system nite amount of nutrients and accumulation of wastes a b c simple familiar growth format provides data for growth curves growth is limited by physiology and medium 2 bacterial growth curve plot of cell growth over time usually in a batch culture or closed system a 0 lag phase period of little or no cell division 1 quotgearing upquot phase 2 intense metabolic activity particularly DNA and enzyme synthesis log or exponential phase period of most rapid growth where cells are in continual state of cell division 1 generation time reaches a constant minimum based on genetic potential and medium limitations 2 cells most active metabolically 3 microorganisms particularly sensitive to adverse conditions stationary phase period of equilibrium between growth and death of cells 1 depletion of nutrients and accumulation of waste products 2 changes in pH death phase period when more cells are dying than dividing 1 logarithmic decline phase constant proportion dies with time 2 population can diminish or become extinct 3 Growth is usually virtually always modeled during the exponential phase a b c by biomass X by number N Growth rate u is dependent on biomass cell concentration generation time is not 4 cells are selfduplicating units so growth is a rstorder reaction a rate of increase is proportional to number of cells 1 1st order reaction dependent on the concentration of one reactant substrate 2 zero order reaction independent of reactant concentration dependent on catalyst concentration 3 mixed order dXdt uX 1 X cell concentration 2 t time 3 u speci c growth rate a constant of proportionality units reaction occurs during transition om one phase to another reciprocal time hr l MICRO 4354 Chap 2 p 4 5 Integration and log conversion linearize the growth equation y ax bFig 22 b and c a mooX1 mt t1gt or X X 39 e b IOgIO X2 39 IOgIO X1 9 39 t12303 c u slope 2303log10 X2 log10 X1t2 t1 6 growth can also be considered in terms of multiplication a NzN1 2n 1 or In NzN1 n ln2 2 n number of generations to produce N2 N1 b n ln2 ut2 t1 so u n ln2t2 t1 1 when n 1 t2 t1 is the generation time g or td 2 u ln2g 0301g 7 Growth rate is related to nutrient concentration in hyperbolic manner analogous to Michaelis Menton kinetics developed by Monod a u u 39 sgtltK s 1 u specific growth rate at limiting nutrient concentration S 2 pm growth rate at saturating nutrient concentrations 3 KS concentration of nutrient at a growth rate of 12 umx b when S gtgt K p um c can be applied to batch or continuous culture 8 diauxy can occur if two subtrates are present and one is used preferentially C Application and optimization of batch fermentations 1 nutrient concentrations decrease while products increase so balanced growth never occurs 2 batch systems are easier to run than continuous systems and are much more common 3 downtime is a significant factor in economics with batch processes a harvesting biomass and spent broth b cleaning the vessel c introducing fresh medium d sterilization e inoculation f lag before product accumulation the yield coefficent Y is a measurement of biomass or product that results per unit of substrate consumed g or mole a X YXSSS where X biomass usually gL YwS yield coefficient g biomass g substrate consumed S initial substrate concentration Sr residual substrate remaining in the fermentation vessel b YpS yield coefficient for products g or mole product g or mole substrate consumed c yield coefficients are important because they indicate the efficiency of substrate conversion 5 determination of the maximum specific growth rate umx is important because primary products are correlated strongly with exponential phase a the Monod equation above or 215 can be linearized b doublereciprocal plots think LineweaverBurk can be used to determine the kinetic constants 1 1H KsHleSH 1m 2 other linearizations eg EadieHofstee work as well D Continuous growth kinetics 4 MICRO 4354 Chap 2 p 5 1 open systems more like natural systems than batch systems are a nutrients are added spent media including cells are removed b initial stage is similar to batch culture as cells grow to maximum density c eventually steadystate conditions are reached where the cell density and growth rate are constant bacteria maintained in exponential phase growth rate dependent on dilution rate rate of introduction of fresh medium cell density dependent on concentration of limiting nutrient if dilution rate is greater than the growth rate cells are washed out uring steadystate growth growth rate is equivalent to wash out production of cell mass dNdt MN rate of cell loss through over ow dNdt fv DN where f ow rate V culture volume fV dilution rate D c MN DN or u D when the culture is in steady state growth washout d the critical dilution rate is when D is greater than pm 3 the Monod equation can be applied to continuous culture a D umax SrKS 8 eqn 224 where KS Monod constant substrate concentration at 12 pm Sr residual substrate concentration in bioreactor at steady state b so Sr KS DLmax D eqn 225 1 states the fundamental relationship between substrate concentration 8 and dilution rate D 2 since Sr can be controlled by dilution rate growth is controlled by a ratelimiting nutrient 3 chemostat continuous cultivation system where growth is controlled by the rate of a limited nutrient entering the system so cell density is maintained by the concentration of the limiting substrate being consumed 4 turbidostat continuous cultivation system where growth is controlled by monitoring the turbidity of the culture and adjusting dilution rate to keep cell density constant c as D increases u increases and Sr increases Fig 25 4 the concentration of biomass or product during continuous cultivation can be related to the yield coef cient a Y YxsSR Sr YxsSR DKSWJLmx D where gt lt biomass or product concentration SR initial substrate concentration concentration in input b again during steady state growth biomass is controlled by substrate feed concentration and dilution rate 1 in the absence of inhibition cell density is controlled by substrate concentration in the feed a D constant b Sr constant 2 as D increase Sr increases and biomass decreases III Measuring growth A Measurements can be direct or indirect viable or total 1 direct counts cells FPQWWF P U3 391 U m MICRO 4354 Chap 2 p 6 2 indirect measures a property or characteristic of cells direct total counts 1 direct microscopic counts with a counting chamber 2 Coulter counter a automatic counter that counts particles within a size range b uses changes in resistance as particles in an electrolyte pass between electrodes c problems with cell clumping and debris direct viable counts 1 plate counting techniques a pour plates and spread plates 1 assumes that inoculum is homogeneous and cells are not clumped 2 culture is usually serially diluted to provide inoculum with 30 300 cells b can take some time for results 2 modifications of standard methods indirect viable counts 1 Most Probable Number MPN a statistical estimation based on number of cultures showing growth in serially diluted series b useful with cells that won39t grow on solid media c general form of equation 2 Ixipi 1113 1 2 2lo ini where x MPN ocI volume tested TI number of total tests pI number showing growth d tables are readily available for 3 5 and 10tube MPN setup 2 ATP bioluminometry a ATP is extracted from cells and reacted with the enzyme luciferase to produce light b the light output is measured with a bioluminometer c the method is rapid and sensitive can detect as little as 1 yeast cell or 10 bacterial cells indirect total count methods 1 turbidity spectrophotometry a according to the BeerMmbert Law log T A b absorbance is proportional to cell density doubling density decreases transmittance 10 fold but absorbance is linear c requires cell densities above 106 cellsml d can be standardized with viable counts or total counts 2 metabolic activity a accumulation of product b depletion of substrate c useful for ecological studies 3 dry weight a can be nearly direct in the case of unicellular organisms standardized with direct method b especially useful for filamentous mgi IV MICRO 4354 Chap 2 p 7 c can be extended to cellular components 1 protein 2 DNA 3 membrane lipids cell yields can be determined for a limiting nutrient Y mass of cellsmass of substrate consumed 1 basis of microbial assays for vitamins and other growth factors 2 can be extended to YATP if pathways known Effects of environmental conditions on microbial growth P A temperature 1 every organism has a minimum temperature a maximum temperature and an optimum temperature psychrophile opt g 15 C a oceans avg temp 5 C b organisms growing under ice in polar regions snow alga c membranes rich in unsaturated fatty acids 3 mesophile opt about 35 C a best studied group b corresponds to pathogens thermophile opt about 60 C hot springs water heaters compost heaps and digestors membranes rich in saturated fatty acids changes in enzyme structures 5 extreme thermophile optz 85 C archeans thermal vents steam vents increased GC high intracellular potassium concentration with the counterion 23 diphosphoglycerate 6 eukaryotes rarely grow above 55 C P 4 09pr 0900 pH 1 each organism has a pH range Where growth is possible along with an optimum pH 2 most life occurs around neutrality but ranges from 2 10 3 acidophiles live at low pH 4 alkalinophiles live at high pH 5 neutraphiles live around pH 7 6 internal pH tends to be around neutrality C water activity 1 describes the availability of water to the organism 2 aW ratio of vapor pressure of air over a substance or solution divided by vapor pressure of water at the same temperature 3 high water activity dilute environment 4 low water activity inhibits bacterial growth a high solute concentration b method of preservation MICRO 4354 Chap 2 p 8 5 halotolerant able to survive with salt but less than optimal growth 6 halophiles require salt for growth a often archeans b modi ed cell walls and lipids 7 osmophiles live in environments with low water activity could be due to nonsalt solutes 8 xerophiles live in dry environments 9 osmohiles halophiles and xerophiles usually contain a compensating solute to balance the osmotic strength of the external solute a polyols 1 arabitol in fungi 2 glycerol in alga b potassium c amino acids 1 glutamic acid 2 proline d other organisms may use different internal solutes oxygen concentration 1 bacteria vary in their need for and tolerance 2 obligate anaerobes are killed by oxygen 3 aerotolerant anaerobes can grow in the presence of oxygen but cannot use it as an electron acceptor 4 facultative anaerobes can use oxygen but can also grow without it 5 microaerophiles require oxygen but at lower levels than in air 2 10 6 aerobes require oxygen for growth 7 toxic forms of oxygen a superoxide anion 02 1 occurs during reduction of O2 to H20 2 highly reactive 3 can cause oxidative destruction of lipids and other biochemical components b peroxide 022 1 formed during respiratory processes 2 often used as a disinfectant c hydroxyl free radical OH 1 formed by ionizing radiation 2 formed by reaction of superoxide anion with peroxide 8 several enzymes to protect against oxygen toxicity a catalase 1 H202 H202 2H20 02 2 present in aerobes and facultative anaerobes b peroxidase 1 H202 NADH H 9 2H20 NAD 2 no oxygen produced c superoxide dismutase 1 02 02 9 H202 02 2 present in aerobes facultative anaerobes and aerotolerant anaerobes 9 MICRO 4354 Chap 2 p 9 enzymes especially those with reduced metal iron groups may be sensitive to oxygen V Control of growth A Terminology 7 9915 9 sterilization destruction of living cells viable spores viruses viroids disinfection killing inhibition or removal of organisms sanitization reduction of microorganisms to safe health levels antiseptic chemical agents applied to tissues to kill or inhibit pathogens cide kills organisms lytic lyses organisms static inhibits growth of organisms B Effectiveness of antimicrobials 1 population death is generally exponential or logarithmic 2 efficiency of antimicrobials in uenced by at least 6 factors a population size larger population requires more time to die b population composition different degrees of resistance between different organisms or structures 1 spores more resistant than vegetative cells 2 Mycobacterium tuberculosis acidfast more resistant than most bacteria 3 young cells more readily destroyed than older cells c concentration of antimicrobial 1 usually greater concentration greater effectiveness 2 70 ethanol more effective than 95 ethanol d exposure duration 1 longer exposure greater death 2 sterilization reduction of survival probability to g 10 e temperature increase usually increases effectiveness of chemical f local environment 1 heat kills better at acid pH 2 efficiency higher with lower organic matter C Heat 1 moist heat a boiling kills viruses bacteria fungi b 10 minutes boiling kills vegetative cells not endospores c thermal death time TDT shortest period of time to kill all organisms at a specific temperature under de ned conditions decimal reduction time D or D value 1 important to food industry 2 usually assumed population of 1012 cells reduced to 100 3 ifD for C botulinum spores is 0204 minutes at 121 C 12D 25 minutes 4 z value increase in temperature required to reduce D to 1 10 its value a for C botulinum z 10 C b at 111 C D 204 minutes 12D 245 minutes e pressurized steam autoclave 1 autoclave fancy pressure cooker 2 combines wet heat with pressure allows temperatures above 100 C 3 temperatures above 100 C required to destroy endospores 4 chamber lled with saturated steam for 121 C 15 psi P time to kill 90 2 3 4 MICRO 4354 Chap 2 p10 5 materials exposed for 2 15 minutes f pasteurization 1 reduces microorganism numbers but retains avor of foods especially dairy beer and other beverages 2 brief heating followed by rapid cooling 3 63 66 C for 30 minutes batch or older method 4 ash pasteurization 72 C for 15 seconds g tyndallization discontinuous boiling or fractional steam sterilization 1 heat material to 90 100 C for 30 minutes on 3 consecutive days incubated at 37 C 2 1st heating destroys cells but leaves endospores 3 2nd and 3rd heating destroys germinating endospores Dry heat sterilization a 160 170 C for 23 hours b cell constituents oxidize c less effective than moist heat Filtration a physical removal of microorganisms 1 excellent for heat sensitive materials 2 can be used with gases b depth filters thick layers of fibrous or granular material 1 twisting channels of small diameter 2 microbes removed by physical entrapment and adsorption to filter material c membrane filters thin 01 mm membranes 1 made of cellulose acetate cellulose nitrate polycarbonate polyvinylidene chloride or other synthetic materials 2 vegetative cells removed with 02um pore size Radiation a alters DNA causing lethal mutations b UV ultraviolet light 260 nm 1 doesn39t penetrate glass dirt films water many plastics 2 often used to sterilize cabinets or entire rooms c ionizing eg gamma 1 penetrates objects 2 also called cold sterilization 3 widely used with food D Chemical methods 1 phenolics N a early use by Lister b Lysol contains a mixture of phenolics c denature proteins and disrupt cell membranes d excellent for surfaces but can cause skin irritation alcohols a bactericidal and mgicidal but not sporicidal b denature proteins and dissolve membrane lipids halogens a iodine most common followed by chlorine b tincture of iodine 22 iodine in waterethanol solution of potassium iodide 4 U 0 l a b a 5 99 e ald a b MICRO 4354 Chap 2 p 1 1 effective antiseptic 2 stains and may damage skin 3 iodophor complex of iodine organic carrier a water soluble stable nonstaining b slow release to prevent skin burns and irritation chlorine disinfectant of choice for municipal water supplies and swimming pools 1 added in many forms forms hypochlorous acid 2 oxidizes several materials destroying cells but not endospores 3 Halzone tablets used for personal drinking water 4 excellent household disinfectant a 1100 dilution of household bleach 13 ozgal 07 nonionic detergent 1 02 gal b cleans and kills bacteria heavy metals Hg Ag As Zn Cu used to be common germicides 1 most heavy metals are bacteriostatic not bactericidal 2 currently using less toxic more effective germicides 3 1 silver nitrate added to eyes of infants to prevent ophthalmic gonorrhea being replaced by erythromycin which is also effective against Chlamydia and Neisseria 4 silver sulfadiazine used on burns 5 copper sulfate used as algicide in lakes and swimming pools combine with proteins sul iydryl groups inactivating them quaternary ammonium compounds detergents organic molecules nonsoaps that serve as wetting agents and emulsifiers 1 amphipathic molecules 2 effective cleansing agents cationic detergents more antimicrobial than anionic detergents 1 quaternary ammonium compounds most popular 2 positively charged quaternary nitrogen with long hydrophobic aliphatic chain disrupt membranes and may denature proteins kill most cells but not endospores or M tuberculosis often used as disinfectants for food utensils small instruments and skin antiseptics ehydes formaldehyde and gluteraldyde most common b combine with and deactivate proteins c 2 glutaraldehyde commonly used to disinfect hospital equipment gases a ethylene oxide is both microbicidal and sporicidal 1 combines with cell proteins 2 penetrates packing materials even plastic wraps explosive usually done in special sterilizer E Evaluation of agents EPA regulates disinfectants FDA controls antiseptics phenol coefficient 1 a potency compared with phenol 1 dilutions inoculated with Salmonella typhi and Staphylococcus aureus and incubated 2 tubes subcultured at 5 min intervals 3 phenol coefficient obtained from highest dilution that kills bacteria after 10 min but VI 3 2 3 MICRO 4354 Chap 2 p 12 not after 5 min 4 reciprocal of concentration divided by that for phenol for phenol coefficient value b can be misleading because conditions of test are not realistic 1 clean medium 2 pure cultures use dilution test more realistic than phenol coefficient a stainless steel cylinders are contaminated with bacterial mixtures dried brie y immersed in test disinfectants for 10 min transferred to culture media and incubated 2 days b disinfectant concentration that kills all organisms with 95 confidence level is determined disinfectants can also be tested under quotin usequot conditions Antimicrobial chemotherapy General mechanisms of activity 1 pathogen damage can occur through several mechanisms P LN 4 U 0 a most selective antibiotics interfere with cell wall synthesis b high therapeutic index since cell walls not sound in eucaryotes Cell wall synthesis inhibition a penicillin ampicillin carbenicillin methicillin cephalosporins b inhibit enzymes for peptidoglycan crosslinking activate cell wall lytic enzymes c bacitracin inhibits CW synthesis by interfering with lipid carrier that transports precursors across the plasma membrane protein synthesis inhibition a streptomycin gentamicin bind to 30 ribosome subunit and causes misreading of mRNA b chloramphenicol binds to SOS ribosomal subunit inhibits peptidyl transferase blocking peptide fond formation c tetracyclines bind to 30 interfere with aminoacyltRNA binding d erythromycin binds to SOS inhibits peptide chain elongation e high therapeutic index because drugs differentiate between procaryotic and eucaryotic ribosomes nucleic acid synthesis inhibition a rifampicin b inhibits DNAdependent RNA polymerase blocking RNA synthesis c often toxic to eucaryotic systems also Cell membrane disruption a polymyxin B b binds to cell membrane disrupts structure and permeability metabolic antagonism antimetabolites a sulfa drugs compete with PABA inhibits folic acid synthesis b trimethoprim inhibits dihydrofolate reductase blocking tetrahydrofolate synthesis c dapsone interferes with folic acid synthesis d isoniazid may disrupt pyridoxal or NAD metabolism and mctioning inhibits synthesis of mycolic acid quotcord factorquot Several factors determine effectiveness of antimicrobial drugs a drug must reach site of infection so delivery system important 1 penicillin G unstable in stomach acid 2 gentamicin aminoglycosides not well absorbed through gut and must be injected intramuscularly 3 parenteral routes nonoral administration MICRO 4354 Chap 2 p 13 b concentration must exceed MIC 1 dependent on amount administered 2 speed of uptake 3 rate of elimination from body 4 best if drug is absorbed slowly over a long period and excreted slowly c infecting organism 1 dormant bugs less susceptible 2 pathogen must have proper target site d many agents less effective due to resistance mechanisms spread quickly via plasmids B Classes of antibiotics 0 9 sulfa drugs a structural analog similar to metabolic intermediate b similar to PABA necessary for synthesis of folic acid quinolones a synthetic drug broad spectrum bactericidal b inhibits DNA replication and repair transcription c nalidixic acid uoroquinolones cipro oxacin norfoxacin o oxacin penicillins a Blactam ring is common feature side chains vary b penicillinase destroys ring c block peptidoglycan crosslinking leading to lysis d many people are allergic cephalosporins a originally isolated from Cephalosporium mgus b Blactam ring like penicillins c useful for people allergic to penicillin d broad spectrum tetracyclines a naturally produced by Streptomyces or semisynthetic b bind to 30 ribosomal subunit inhibiting protein synthesis c bacteriostatic d broad spectrum aminoglycoside antibiotics Streptomyces make streptomycin kanamycin tobramycin Micromonospora purpurea synthesizes gentamicin bind to small ribosomal subunit inhibiting protein synthesis bactericidal most effective against gram negatives quite toxic to humans Widespread resistance erythromycin a macrolide synthesized by Streptomyces erythraeus b broad spectrum bacteriostatic most effective against G c bind to 23S rRNA of SOS ribosomal subunit inhibiting protein elongation d macrolides have 12 to 22carbon lactone rings chloramphenicol a synthetic but originally from Streptomyces venezuelae b acts like erythromycin Hares dupe MICRO 4354 Chap 2 p 14 c broad spectrum bacteriostatic d quite toxic to humans C Mechanisms of drug resistance P LN 4 U drug cannot enter cell a G unaffected by penicillin G because it can39t penetrate the outer membrane b changes in binding proteins render cells resistant chemical modification a penicillinase hydrolyzes the Blactam ring b groups can be added which inactivate drugs modification of target a changes in 23S rRNA protects against chloramphenicol or erythromycin b change binding site for sulfanilamide genes for drug resistance can be chromosomal or on plasmids a spontaneous mutations in chromosome are rare b chromosomal changes usually result in changes in drug receptors preventing binding c R plasmids resistance plasmids often code for enzymes that destroy or modify drugs 1 implicated in resistance to aminoglycosides penicillins cephalosporans erythromycin tetracyclines sulfonamides chloramphenicol and others 2 plasmids transferred rapidly through populations 3 single plasmid can carry resistance to many drugs Overuse of antibiotics has led to many resistant strains a increase drug concentrations to destroy susceptible and spontaneous mutants b use two drugs together c limit use especially broadspectrum antibiotics D Anti mgal drugs 1 2 3 eukaryotic so drugs often toxic to humans most fungi have ef cient detoxi cation mechanisms often target membrane sterols or cell walls E Determining activity levels 1 2 dilution susceptibility tests a a series of broth tubes containing a range of antibiotic concentrations inoculated with test organism b minimal inhibitory concentration MIC growth after 1620 hr c minimal lethal concentration MLC growth in subculture d cidal drugs usually kills at 24x MIC static drugs kill at much higher concentrations if at lowest concentration that prevents growth no lowest concentration that kills the organism no a disk diffusion tests a antibiotic impregnated disks are placed on agar previously inoculated with the test bacterium 1 antibiotic diffuses forming a gradient 2 resistant organisms grow up to the disk 3 susceptible organisms grow some distance from the disk displaying a clear zone around the disk a wider the clear zone more susceptible b zone width is a mction of initial concentration solubility diffusion rate MICRO 4354 Chap 2 p 15 susceptibility of organism c zone Width cannot be used to compare 2 antibiotics b KirbyBauer most used disk diffusion test 1 MuellerHinton agar inoculated with lawn of bacteria 2 disks placed on surface 3 incubation at 35 C for 1620 hr 4 diameters of zones measured and compared to tabulated values to determine degree of microbial resistance a plot MIC vs zone diameters for different strains b determine from plot if treatment dosage would result in MIC