biology exam 2

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GE11- What is the max level of coliforms/100 mL for US water standards?
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GE 12- what is the difference between transient and residential species?transient- result of contact with surroundings, highly variable, can be washed off easily resident species- live in or on the skin, utilize skin secretions and wastes as nutrientsGE 12- what does this bacterial population data provide us with an estimation of?density and diversity of resident and transient species on our skinGE 12- What are the two things resident species snack on, and what are the 4 things each has to offer?sweat: urea, AAs, lactic acid, salt sebum: lipids, FAs, glycerol, hydrocarbonsGE 12- what are the 2 types of mediums used, how are they incubated, and what are they selective for?RCMF- incubated anaerobically, selects for Propionibacterium HBA- incubated aerobically, non-selective heated blood agar, allows growth of micrococcaceaeGE 12- What are the forearm dilutions for both mediums?HBA: 10^0, 10^-1, 10^-2 RCMF: 10^-1, 10^-2, 10^-3GE 12-What are the forehead dilutions for both mediums?HBA: 10^-1, 10^-2, 10^-3 RCMF: 10^-2, 10^-3, 10^-4GE 12- what is the equation to go from SA, DF, and colonies to colony forming units per cm^2[(colonies x DF)/(volume plated mL)] x [(volume saline mL)/(sampling area cm^2)=(cfu/cm^2)GE 9- After both colonies have grown, which is the first test?gram stain, isolate one species of gram-positive cocciGE 9- after gram staining, what is the next thing to note? What are the 3 types?hemolysis on fresh culture beta: clear halo, RBC's completely lysed alpha: green halo, incomplete lysis gamma: no halo, no hemolysisGE 9- How often should cultures be re-streaked?once a weekGE 9- What diagnostic test answered which procedure we followed?catalase negative or positiveGE 9- provided isolate results gram: catalase: hemolysis: optochin sensitivity (6 mm disk): Bile esculin: VP and AD:gram: positive catalase: negative Hemolysis: gamma optochin sensitivity: resistant, no growth bile esculin: negative VP and AD: negative and positiveGE 9- what was the provided isolate?streptococcus sanguis groupGE 9- steps of catalase test, results, what it tells yasteps: 1. place one drop of 3% hydrogen peroxide on slide, emulsify loopful of cells results: production of O2 bubbles within few seconds is +, lack of bubbles is - why: aerobic and aerotolerant cells can produce hydrogen peroxide, catalase breaks it down to water and O2 and detoxifies the compoundGE 9/P1- Cytochrome oxidase test steps, results, what it tells ussteps: 1. tube of powdered reagent from the fridge, suspend in 5 mL of water 2. smear visible fresh culture on bibulous paper with toothpick, place drop of reagent on smear results: if paste turns dark blue within 10 sec, + why: cytochrome oxidase catalyzes O2-> H20 reductionGE 9- Coagulase test steps, results, what it tells ussteps: 1. coagulase plasma from freezer, thaw in hands, place loopful of cells in plasma and gently mix 2. incubate at 37* C for 24 hours, check for coagulation results: coagulation + why: produces fibrin barriers around bacterial cells and protects from immune attackGE 9- PYR (L-pyrrolidinyl arylamidase) steps, results, what is tells ussteps: 1. place PYR disk on slide or petri lid 2. add 5-10 uL of water to dish, then heavily inoculate with paste of isolate culture 3. wait 2 min before adding a drop of color developer (p-dimethylaminocinnamaldehyde) to the disk. watch for 1 more min results: pink to red color within 1 min, +, no color change is - why: some streptococci, enterococci, micrococcaceae possess this enzymeGE 9- Antibiotic sensitivity test steps, result, what is tells ussteps: 1. scrape 2-4 medium colonies from plate, suspend cells in 0.5 mL of sterile water in sterile tube 2. dip cotton swab into suspension, roll to remove some liquid, spread evenly over surface of blood agar plate by rolling swab back and forth 3. rotate plate 90* and repeat, then rotate and repeat once more 4. obtain disc of antibiotic using sterilized forceps and place in center of plate. gently tap down with forceps results: measure diameter after 24-48 hrs of incubation, + depends on what type of antibiotic why: sensitivity: no growthGE 9- growth in 5% NaCl steps, results, what it tells ussteps: 1. inoculate tube of BHI broth and tube of BHI broth + 5% NaCl with similar quantity of cells, incubate both at 37* C with shaking 2. vortex BHI + NaCl and compare with BHI broth for turbidity. results: equivalent growth in tubes is + (cells tolerate 5% NaCl), less growth in NaCl tube is - why: test for tolerance to salt, distinguishes staphylococci from other catalase and gram + speciesGE 9/P1- ornithine or AD test steps, result, what it tells ussteps: 1. lightly inoculate tube of OD medium or AG medium, overlay medium with .5 mL sterile mineral oil 3-4 mm deep using P1000 2. incubate at 37* w/o shaking, check every 24 hours for 3 days results: change to purple +, medium that turns yellow or no change is - why: tests for presence of decarboxylase enzymes required for breakdown of AA's to amines and CO2GE 9- bile esculin test steps, result, what it tells ussteps: 1. streak slant of BE agar, incubate at 37*, check every 24 hours for 3 days results: any slants that are less than half-darkened are -, more than half-darkened are + why: some bacteria cleave esculin from glucose and can tolerate bile saltsGE 9/P1- VP test steps, result, what it tells ussteps: 1. inoculate tube of VP broth, incubate at 37* w/o shaking for 6-7 days 2. transfer 2 mL aliquot to empty tube, don't shake or disturb contents 3. add 18 drops Reagent A and vortex for 1 min to oxygenate medium 4. add 6 drops reagent B and vortex briefly 5. place tube in rack and don't move for 20-60 min results: pink or red color change +, negative is yellow to copper after an hour why: tests if the bacteria will ferment glucoseGE 9/P1- acid from mannitol/lactose test steps, results, what it tells ussteps: 1. heavily inoculate BP tube with mannitol broth, incubate w/o shaking at 37* 2. check daily for 6 days for acid production results: color change from purple to yellow, + why: fermentation test in low pHGE 13- what are the 4 characteristics of coliforms?facultatively anaerobic, rod-shaped, gram negative, non-spore-forming bacteria that ferment lactose with gas productionGE 13- what does coliform presence indicate?possible fecal contaminationGE 13- What is the MPN method used for?determine total coliform counts and E. coli counts through use of 3 mediaGE 13- Describe the 3 different mediums and what they're selective forLauryl tryptose broth (LTB): selective for coliforms, presumptive coliform determination brilliant green lactose bile (BGLB): inhibits non-coliforms, used to confirm presence of coliforms E. coli broth (EC): selective for E. coli, detects presence of fecal coliformsGE 13- Day 1 procedure1. obtain water sample 2. arrange sets of 5 LTB tubes (5 replicates per dilution) 3. dilute as needed 4. Add 1 mL of each dilution to each corresponding 5 LTB tubes, do not vortex or shake 5. incubate at 37* for 48 hoursGE 13- Day 3 procedure1. examine LTB tubes for turbidity and gas 2. for all +, transfer sterile loopful of LTB broth to BGLB tube, do not shake/vortex 3. incubate at 37* for 24 hoursGE 13- Day 4,5,6 procedure1. examine BGLB tubes for gas and turbidity 2. for all +, transfer sterile loopful of broth to tube of EC medium 3. incubate at 45.5* for at 24-72 hours Looking for turbidity and gas after 24 hours, but can't say negative until 72 hours has passedGE 13- MPN/100 mL formula[(total number of positive tubes) x (100)]/ sqrt[(combined volume of sample in negative tubes) x (combined volume of sample in all tubes)]GE 13- MPN tablex-x-x is lowest dilution to highest dilution (10-2, 10-3, 10-4) number of positives in the replicatesEN 1- what are denitrifiying bacteria?common aerobic heterotrophic bacteria that, as facultative anaerobes, continue respiration when O2 is depleted by using NO3- as the terminal electron acceptor.EN 1- steps1. add inoculum to tube, refill tube to brim (anoxic), close tightly, incubate at room temp in dark or low light 2. turbidity and N2 gas bubbles evidence growth 3. streak positive enrichments on same medium plus 15 g/L agar, incubate in gas packEN 1- how can you enrich those who can't use nitrate as a nutrient?ask molly idkEN 2- what are the 3 groups cyanobacteria can be divided into1. unicellular forms w/ no specialized cells and little-no motility 2. unibranched filamentous forms with no specialized cells, move by gliding except when surrounded by obvious sheath 3. filamentous forms which produce occasional or numerous heterocysts, sometimes resting spores, gliding motility seen only periodically in special short filamentsEN 2- medium types and pHbasal medium: BG-11 or SWBG-11 SWBG-11 is BG-11 plus 25 g/L NaCl, 3 g/L MgSO4 or 25 g/L of instant ocean ph: 7.5 in unbuffered freshwater medium, 8.0 in marine medium mineral medium that will not support heterotrophs due to absence of organic carbonEN 2- enrichment steps1. inoculate flask of medium with a few drops or loops of source water, incubate in light 2. amphotericin B has been added 5.6 mg/L to inhibit eukaryotic algae which then selects for cyanobacteriaEN 3- What does this enrichment provide for and select against?provides for photoheterotrophic growth of various purple non-sulfur bacteria, selects against other phototrophsEN 3- what is the base medium and what does it select against?medium: DCMU selects against cyanobacteria by inhibiting photosystem IIEN 3- What are the two options for non-fermentable carbon sources to be added to the medium?malate or citrateEN 3- outline the procedure1. add inoculum to tube, fill to brim, replace cap. 2. incubate in front of incandescent lamps 3. check for growth in 6-7 daysP1- how did you receive your species and what was the first step?two species in mixed culture first step is to obtain pure culture on solid medium before doing any testsP1- name the tests available to us (9)acid from lactose acid from mannitol hydrogen sulfide production indole production VP rxn cytochrome oxidase ornithine decarboxylase gelatin hydrolysis catalaseP1- what does each symbol mean in the bergey manual+ = 90-100% strains positive [+] = 76-89% positive d = 26-76% positive [-] = 11-25% positive - = 0-10% positiveP1- hydrogen sulfide production test steps, results, what it meanssteps: 1. use needle to stab tube of SIM agar once, approx 2/3 way deep 2. incubate at 37* for 2 days results: blackening of the medium indicates H2S production why: reduction of sulfur to hydrogen sulfide occurs anaerobically depending on organisms metabolism, when H2S combines with ferrous ions, blackening occurP1- indole production steps, results, what it meanssteps: 1. stab-inoculate SIM tube and incubate at 37* for 2 days 2. add few drops of Kovac's reagent results: pink or red color in reagent layer within several seconds is + why: indole production in growth medium can be detected by addition of dimethylaminobenzaldehydeP1- gelatin hydrolysis steps, results, whysteps: 1. use needle, stub NA tube with heavy inoculum 2. incubate at room temp for up to 1 week, examine daily for liquefaction of the medium, may only be clear at surface results: liquefaction of medium + why: gelatin is hydrolyzed to AA's by organisms that possess extracellular enzyme gelatinaseP2- what is nitrificationammonia (NH3) oxidized to nitrate (NO3-) in 2 processes, both processes closely coupledP2- what is the first step of nitrifcationoxidation of ammonia to nitrite by nirosifying bacteria NH3 + O2 --> NO2- + 3H+ + 2e-P2- what is the second step of nitrificationoxidation of nitrite to nitrate by nitrifying bacteria NO2- + H2O --> NO3- + 2H+ + 2e-P2- what are nitrosomonas and where can they be foundammonia-oxidizers found in soil and fresh waterP2- what are nitrobacters and where can they be foundnitrite-oxidizers found in wastewater treatmentP2- what is the sign of activity for nitrosomonas in pure culturereduction of ammonia or accumulation of nitriteP2- what is the sign of activity for nitrobacters in pure culturedepletion of nitrite or accumulation of nitrateP2- what is the intermediate between nictrobacter and nitrosomona metabolismsnitriteP2- describe an environmental water source's nitrite concentrationboth genera present, no nitrite build up because nitrite oxidizers consume it fast as ammonia oxidizers produce itP2- how did we block the activity in order to determine if ammonia oxidizers were present & howblock activity of nitrite-oxidizers so evidence of ammonia oxidation can be detected by an increase in nitrite concentrations add low concentration of chlorate to inhibit oxidation of nitriteP2- Week 1 4 step inoculation procedure1. pipet 1 mL of sample into each of 2 flasks that each have 15 mL of nitrite medium and each of 2 flasks with 15 mL of ammonia medium 2. clearly label each flask with name, date, type of media. Nitrite A, Nitrite B, Ammonium A, Ammonium B 3. analyze each flask for nitrite concentration using nitrite analysis 4. incubate all flasks in shaker at room temperatureP2- Nitrite analysis procedure1. Get all 4 flasks from the shaker 2. Set up 7 spectrophotometer tubes: 1:100 dilution of Nitrite A, Nitrite B, Ammonium A, Ammonium B, undiluted nitrite B, Ammonium A, Ammonium B 3. using 5 mL glass pipet, add 4.7 mL DI water to each tube 4. add 100 uL of each medium into each glass tube 5. Add 100 uL of DIAZOTIZING reagent, mix with P1000 tip and wait 5 min 6. Add 100 uL of COUPLING reagent, mix with P1000 tip and wait 20 minP2- spectrophotometer steps1. put in blank before every reading, needs to be below 0.10 2. do each standard starting at 1 and moving to 6 3. put each tube in and make sure the reading stops 4. record OD540 readingP2- dilution steps1. place 5 mL of DI water in tube using 5 mL glass pipet 2. set P200 pipettor to 50 uL, remove 50 uL of water from tube, add 50 uL of medium and mixP2- why did we add chlorate to Ammonium B and Nitrite B flasks in week 2/3?inhibit oxidation of nitrite in that flaskP2- what is nitrite analysisNitrite reacts with primary aromatic amines (diazotizing reagents) in acidic solutions to produce diazonium salts salts then converted to colored azo compounds when coupled with aromatic compounds that contain coupling reagents. color intensity of azo compounds is read by spectrophotometerP3- what is the top pathway?1 2 X ---> Z ---> MAPP3- what is the bottom pathway?3 4 W ---> HBC ---> MBCP3- What is the combination of the pathway?MAP ---------5-> prodigiosin (red) MBCP3- Session One: UV mutagenesis procedure1. center plates inside cross linker 2. set energy and press start 3. immediately wrap plates in foil and incubate at 37* for one day, then take of foil and store at end of benchP3- Session 2: Procedure1. examine plates, look for abnormally pigmented colonies 2. patch potential pigment mutants on new plate 3. streak WT and up to 8 mutant colonies on PG plate 4. incubate under incandescent lights overnightP3- Session 3: Procedure1. Examine plates, identify strains 2. choose 2 mutants that maintain pigmentation defect, streak each on separate PG plate to obtain isolated colonies 3. incubate under incandescent lights for one day, then seal with parafilm, refrigerate until assayP3- Session 3: strain typesa. strain looks like WT, pigmentation defect not due to mutation b. strains maintain pigmentation defect but start to darken around the edges where they are close to WT strain or other mutant strains, likely have defects in prodigiosin biosynthetic pathway c. strains maintain pigmentation defect throughout. pigment is not variable by location, mutation could be caused by enzyme late in prodigiosin biosynthetic pathway or from mutation in unrelated genesP3- How is complementation analysis done?cross-feeding technique 2 of our mutants and 5 known mutants that cannot make one of the functional enzymes needed to make prodigP3- Complementation Assay procedure step 11. label bottom of 8 plates with serratia strain being used (A-E, WT, YFM 1 or YFM 2), streak one strain on each plate control plates that will assist in identifying pigments that each of these strains normally producesP3- Complementation Assay procedure step 22. label 21 plates with pairs, streak in a squiggly T pattern make each streak wide at top and narrow at bottom, few mm from each but don't touch the streaks tape plates together, store at room temp right side upP3- Complementation Assay procedure step 33. check streaks for 3 days. compare colors of mutant strains streaked side by side to WT and isolated mutant strains look for darkening of pigment, but not complete conversion to redP2: what type of enrichment is the ammonium medium and what are we measuringammonium medium = nitrosifyer enrichment (NH4+ --> NO2-) measuring gain of weekly nitriteP2: what type of enrichment is the nitrite medium and what are we measuringnitrite medium = nitrifyers enrichment (NO2- --> NO3-) measure loss of weekly nitriteP2: what type of enrichment is the nitrogen-free medium and what are we measuringnitrogen free: nitrogen fixers (N2 --> R-NH2) streak to plates after 1-2 weeksP2: Describe the nitrogen cycle1. Start with N2 2. undergoes nitrogen fixation and N assimilation by reduction, produces ammonia/amines (NH3, R-NH2) 3. interacts with nitrosyfer (nitrosomonas) to produce nitrite (NO2-) 4. interacts with nitrifyer (nitrobacter) to produce nitrate (NO3-) 5. nitrate undergoes denitrification, anaerobic respiration where reduction of oxidized N as e- acceptorP2: mixed culture (nitrosifyers and nitrifyers) in nitrite mediumGraph of nitrite over time, makes a horizontal line halfway up y-axis and then a straight and steep decline horizontal line: lag for nitrifyers, no fuel (NH3) for nitrosifyers decline: growth of nitrifyers (NO2- --> NO3-) addition of chlorate inhibits NO2- oxidation so it can even out again halfway down from the halfway starting pointP2- mixed culture (nitrosifyers and nitrifyers) in ammonium mediumgraph of nitrite over time, starts at very low y-axis level, 45* angle up before flattening it again a little higher on y-axis horizontal: lag for nitrosifyers increase: growth of nitrosifyers NH3 --> NO2- horizontal: growth of nitrifyers (NO2- --> NO3-)P2- mixed culture (nitrosifyers and nitrifyers) in ammonium medium, add chlorategraph of nitrite over time, starts at very low y-axis level, 45* angle up and continues increasing over time horizontal: lag for nitrosifyers or steady state (NH3 --> NO2- --> NO3-) increase: add chlorate to inhibit nitrifyers (NO2- -/-> NO3-), growth of nitrosifyers (NH3 --> NO2-)P2- the bacteria of the nitrogen cycle1. nitrogen fixation: hetero and autotrophic, aerobes and anaerobes (hetero and aerobes circled?) 2. nitrosifyers and nitrifyers use obligate aerobes that use O2 as e- acceptor, most strictly autotrophic (use CO2 for carbon) 3. denitrifyers: obligate or facultative anaerobes, heterotrophic (CO2 for carbon)P2- how does the nitrogen cycle get energy?chemolithotrophy- oxidation of reduced N for energyP3- when does a mutant rescue a pigment?mutant gives color when rescuing bc WT is bright red mutant has been rescued when color shows up rescued: red or color on fringe, goes towards WT, starts to fix pathwayEN 2- what is the hallmark of cyano-photometabolismoxygenicEN 2- what are the 2 purposes of light in cyano-photometabolism?energy generation used primarily in CO2 fixation reductant (e- donor) generation for CO2 fixationEN 3: what environments can purple non-sulfur bacteria grow in?grow in light: photoautotrophs (carbon from CO2), photoheterotrophs (carbon from organics) grow in dark: aerobically via respiration, anaerobically via fermentation or anaerobic respirationEn 3: what is the hallmark of purple non-sulfur photometabolism?anoxygenicGE 13: what is MPN and why is MPN method useful?liquid-based protocol for live cells, work with concentrated or dilute samples, sensitivity/recovery better for low population densities, can be adapted to enumerate wide range of speciesGE 13: MPN procedure1. perform dilution or volume series 2. dilute to extinction 3. test each dilution for presence of targeted organisms by inoculating liquid medium with aliquot from dilutions 4. compare results to probability distribution, yields MPN