MICRO 3050 Final Lab Study Guide
MICRO 3050 Final Lab Study Guide 81382 - MICR 3050 - 001
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81382 - MICR 3050 - 001
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This 9 page Study Guide was uploaded by Katie Kessler on Thursday November 26, 2015. The Study Guide belongs to 81382 - MICR 3050 - 001 at Clemson University taught by Krista Barrier Rudolph in Fall 2015. Since its upload, it has received 228 views. For similar materials see General Microbiology in Biological Sciences at Clemson University.
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Objectives MICR 3051 FINAL LABORATORY EXAM STUDY GUIDE LABS 7 12 1 Describe how UV light is germicidal its effect on DNA and the wavelength at which UV light is the most lethal Lab 7 exercise 28 page 135 gt 2 and B gt gt gt 3 In this lab we examined the germicidal effects of UV light on S aureus and B subtilis This is when she assigned us a bacteria number and an amount of time ie bacteria 15 with an exposure time of 60 minutes The shorter the wavelength the more damaging it is to the cells The most lethal wavelength is 260 nm When DNA absorbs UV light it causes a formation of the pyrimidine dimers This causes the DNA to become deformed so that DNA polymerase cannot replicate DNA strands past the dimer nor can they be transcribed Describe how we examined the germicidal effects of UV light on S aureus subtilis The germicidal effects were measured with different exposure times some people were given plastic to block the radiation B subtilis is an endospore former and S auerus isn t vegetative cell You should have seen more of a resistance in B subtilis and more germicidal effects in S aureus Be able to define the following terms antiseptic disinfectant steriliant sanitizer bacteriostatic and bacteriocidal gt VVV V gt Antiseptic Substances such as alcohol or betadine that inhibit microbial growth or kill microoganisms These are gentle enough to be applied to living tissue Disinfectant These are chemical agents that are applied to inanimate objects such as oors walls and table tops to kill microorganisms Sterilant destroys all microbial life Ex ethylene oxide Sanitizer Agent that reduces microbial growth to a safe level Bacteriostatic if an agent only inhibits growth of bacterial cells but doesn t kill them Bacteriocidal Agents that kill bacterial cells 4 Explain the Filter Paper Disk Method of evaluating the relative effectiveness of various disinfectants andor antiseptics Lab 7 exercise 32 page 139 gt gt This method is used to determine if an antiseptic or disinfectant inhabits growth of a test organism and compares efficiency In this procedure a plate of suitable medium is streaked with a test organism Filter paper discs are impregnated with the agent by dipping the disk in a germicide or disinfectant and placing the disk on an inoculated plate Then the agent will diffuse from the disk into the agar forming a concentration gradient If the substance is inhibitory a clear zone of inhibition will occur around the disk and it is assumed that no growth has occurred 5 Distinguish between antibiotics and antimicrobials Antibiotics this is an antimicrobial that kills of inhibits microoganisms These are produced by microoganisms Antimicrobials kill or inhibit microoganisms 6 Explain the KirbyBauer Method of evaluating the sensitivity of bacteria to certain antibiotics andor antimicrobials Lab 7 Exercise 31 page 145 gt It is preformed by uniformly streaking a standardized inoculum of the test organism on MuellerHinton medium and then paper disks contain specific concentrations of the antimicrobial or the antibiotic on the agar surface The antimicrobial diffuses out of the disk into the agar forming a concentration gradient If the agent inhibits or kill the test organism there will be a zone around the disk where no growth occurs and that is called the zone of inhibition 7 Understand how you would measure a zone of inhibition what a zone of inhibition is and how you would determine if a bacterium is R I or S to an antimicrobial agent gt Measure a zone of inhibition Measure it with a metric ruler to the nearest millimeter gt What is a zone of inhibition one around the disk were no growth occurs gt Determining if the bacterium is R I or S to an antimicrobial agent 0 Resistant No plaque around the disk which means the bacteria is growing normally Thus the antibiotic will be ineffective 0 Intermediate A somewhat cloudy plaque indicates that not all the bacteria in the area around the disk have been killed This means that there are some members of the bacterial population that are sensitive to this particular antibiotic but others that are genetically immune to its effects If an antibiotic to which the bacteria show quotintermediatequot sensitivity is used it is likely that the sensitive members of the bacterial population will be killed and the resistant ones will survive resulting in the selection of a population resistant to that particular antibiotic o Sensitive A clear circular quothaloquot technically known as a quotplaquequot or zone of inhibition will appear around the antibiotic disk indicating an absence of bacteria The antibiotic has inhibited their growth andor killed them meaning that this particular antibiotic should be effective against the infection 8 Describe the effects of alcohol on bacterial growth Lab 7 exercise 30 page 155 gt Chemicals sufficiently nontoxic to use on liVing tissue gt Alcohol is an antiseptic it greatly kills and reduces the numbers of microbes and is safe for use on liVing tissue 9 10 11 12 13 Explain the basis of the Reductase Test and how it can be used to distinguish between high and low quality milk Lab 8 Exercise 47 page 159 gt Reductase Test estimates the microbiological quality of raw milk This is based on temperatures lower than 35 degrees Celsius gt The test is based on the change in color of the methylene blue from blue to clear when dye is reduced gt Good quality milk methylene blue is nor colorless in 6 hours gt Poor quality milk methylene blue is reduced within 2 hours Know the meaning and significance of MBRT Methrylene Blue Reductase Test the time it takes to become colorless The shorter the time the lower the quality of milk Understand the role that microbes have in food production and in food spoilage gt Bacteria can cause food spoilage by breaking down the food producing acids or other waste products during this process gt Signs of food spoilage may include an appearance different from the food in its fresh form such as a change in color a change in texture an unpleasant odor or an undesirable taste Understand how to conduct a Standard Plate Count including determining a dilution scheme to determine bacterial numbers in a liquid or solid food sample Lab 8 exercise 44 page 175 gt Conduct a standard plate count The standard plate count is a reliable method for enumerating bacteria and fungi A set of serial dilutions is made a sample of each is placed into a liquefied agar medium and the medium poured into a petri dish The agar solidifies with the bacterial cells locked inside of the agar Colonies grow within the agar as well as on top of the agar and below the agar between the agar and the lower dish The procedure described above produces a set of pour plates from many dilutions Most common method for determining bacterial numbers in a sample Blender 110 Add 1 ml from blender into the 99ml sterile blank 1 1000 Plate I 1 100 add 01 ml from blender and add agar Plate 11 1 1000 add 1 ml from sterile blank and then add agar Plate 111 110000 add 01 ml form sterile blank and add agar Incubate all three plates and then count colonies I recommend watching this example It helped me a lot with the dilution schemes httpswwwvoutubecomwatchvsAJERdePQ VVVVVVV Be able to calculate the number of bacteria in a sample based on the number of CFUs on various plates Know what constitutes a countable plate For example say you have 33 colonies in 10A2 All you do is change it to decimal form and make the 2 a positive 2 Therefore your answer will look like 330 10quot2 CFUmL 14 Explain how the grape juice fermentation ask was setup and know what observations would indicate fermentation had occurred Lab 8 Exercise 50 page 179 gt The grape juice was in a ask sealed with a rubber balloon before incubation Lead and acetate test strip is taped to the inside of the neck of the ask 3 ml of yeast was added into the 100 ml it is incubated for 25 days at 1517 degrees Celsius The balloon should swell up and when it it removed odor of gas should be available Fermentation occurs if there is a drop in the pH and if there is gas C02 in the balloon and there is a smell of alcohol instead of grape juice 15 Define the process of ammonification Lab 8 exercise 48 page 183 gt Ammonification It is the release of ammonia from nucleic acids proteins and nitrogenous wastes This process occurs in the soil as a result of decomposition of the dead plants and animals and their waste products 16 Describe how to test for the presence of ammonia in soil samples gt page 185 to test for this drop Nessler s reagent into a spot plate Then add the inoculated peptone broth If there is faint yellow color small amount of ammonia If there is a deep yellow more ammonia If there is a brown precipitate large amount of ammonia 0 To check the pH of the tube blue color alkaline Yellow color acidic 17 Describe coliforms and how they are good indicators of fecal contamination in water Lab 9 exercise 45 page 189 gt Coliforms gram negative faculatative anaerobe nonendospore forming rods that ferment lactose to produce acid and gas gt How are they a good indicator of fecal contamination in water 1 it occurs primarily in the intestines of humans and some warm blood animals It is not normally found in soil or water 2 the organism can be easily identified by microbiological tests and 3 it is not as fastidious as intestinal pathogens and it survives longer in water samples 18 Explain how the presumptive and confirmed tests are conducted and how they are used to determine the presence of coliforms in a water sample Know the media used for each and what a positive test looks like gt gt Presumptive 15 tubes of lactose are inoculated with water to see if they produce gas If there is gas present that is an indicator of coliforms Confirmed EMB agar and Endo agar are inoculated from the positive tubes from the presumptive test to see if the organisms are gram negative Both medias inhibit gram positive EMB agar demonstrates small colonies with dark centers Endo agar coliforms are reddish with some green metallic presence 19 Know how to determine the MPN of a water sample and understand what it means gt Most probable number is determined by the presumptive test gt This tells you the MPN index 100 ml and low and high confidence limits For example 300 has a 78 100 ml MPN index and 2122 low and high gt confidence limits By definition it is a method that is wellestablished and fully documented method of estimating the number of Viable microorganisms in product in which the microorganisms are randomly distributed 20 Describe the distinguishing characteristics of bacteriophages Lab 9 exercise 21 page 203 VVVVVV VV Bacteriophage a Virus that infects bacterial cells Obligate intracellular parasites they must invade a host to replicate and reproduce Encased in a capsid that protects the nucleic acids Hollow core Spike Tail fiber Bacterial eaters Site specific 21 Explain the procedure for determining a bacteriophage titer VV VV Vims stock Have 5 dilutions 110 1100 11000 110000 1100000 Do a serial dilution from a phage stock into each tube Put 1 ml of dilution into agar and mix together add 2 drops of Ecoli Olml 09ml 0 IOquot IO d 22 Know how to calculate the number of phage particles in a suspension by counting the number of PFUs on plates Count the number of plaques and multiply by the dilution factor to determine the number of phage plaquesml in the original sample Remember to only count the plates with colonies between 25250 23 Describe the two mutant isolation procedures we used and know what type of mutants we were trying to isolate Lab 9 exercise 65 page 209 gt We used Replica Plating and gradient plating to find streptomycin resistant mutants Velveteen surface sterilized pressed on master plate Velveteen with cells from original colonies is pressed to minimal medium plate I Colony growth w l Original master plate Replica plate complete medium minimal medium Present on Missing on complete medium replica plate I I Auxotrophic mutant Copyright 2006 Pearson Benjamin Cummings All rights reserved 24 Know the purpose of the gradient plate and the three plates used in the replica plating procedure gt Replica plating uses nutrient agar streptomycin agar E Coli agar gt Gradient Plate is used to determine the presence of spontaneous mutations such as the resistance to streptomycin because they Will grow in high concentrations of the antibiotic 25 Describe the general characteristics of Staphylococcus aureus as part of the micro ora of the human body and its signi cance in disease V VVV VVVVVVV Gram positive Present in the noseskinmucous membrnes Nonendospore Facultative anaerobe Grows in high salt concentrations Hemolytic on blood agar Causes food poisoning or toxic shock syndrome skin infections scalded skin syndrome Ferments mannitol and lactic acid Pathogen It plays a significant role in virulence bc it lyses red blood cells and damages leukocytes heart and renal muscle Oxygen cant be produced in the immune system bc it kills bacteria 26 Describe how we isolated S aureus from our noses and how we can identify it using Blood Agar MSA and CHROMagarTM gt gt gt gt gt Isolation swab the nose inoculate plate on MSA and staphylococcus medium 110 We isolated it from noses using a sample from our noses at fomite inanimate object egg and a control positive result from CHROMagar is mauvecolored ugly pink S aureus on blood agar golden color S aureus on MSA produces acid turns red to yellow S aureus on CHROMagar pinksmall mauve colonies 27 Know the four basic steps to isolate DNA from bacterial cells gt Overnight culture of E coli has been incubated centrifuge to pellet cells Decant supernant and resuspend the pellet and add SDS and proteinase K solution mix DNA Extraction m gtrgt 7 39 3 J I Cells are lysed using a detergent that disrupts the plasma membrane Cell contents Cell debris is The DNA is are treated with pelleted in a precipitated protease to centrifuge The with ethanol It forms viscous strands that can be spooled on a glass rod destroy protein and RNAase to destroy RNA supernatant liquid containing the DNA is transferred to a clean tube 28 De ne transformation gt bacterial mechanism for the transfer of genetic material in which free DNA of one genotype is taken in through the cell surface of bacteria of another genotype and is incorporated into the recipient cell chromosome gt Easier definition foreign DNA placed into a circular plasmid 29 Describe the heat shock method we used to arti cially transform E coli with pGLO gt Heat shock method Mark plates with LB LBamp LBampara LBamp has one positive and one negative LBampara is pGLO and LB is pGLO Two microcentrifuge tubes are needed one pGLO and one pGLO 250 uL of transformation solution is added to each tube and placed on ice E coli is placed in the pGLO tube and pGLO is put in its tubes and then both placed back on ice pGLO DNA is put in the pGLO tubes These are incubated on ice for 10 mins then put in a water bath at 42 C for exactly 50 seconds They are then put back on ice for another 2 min 250 uL of LB brother are added to the pGLO tubes All are incubated at room temp for 10 min pGLO is inoculated onto LB plate 200 uL pGLO is inoculated onto LBamp plate 200 uL pGLO 200 uL onto LBamp and LBampara Plates are then incubated at 37 C for 24 hours after spread out on all plates V VVVV Vvvvv Why is this significant A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell 30 Know the selectable characteristics of pGLO that allowed us to select for transformants gt pGLO contains ori the bla gene which provides resistance to ampicillin and the gene that codes for GFP pGLO is used to introduce GFP into E coli 31 Know the role of araC in pGLO and why arabinose was added to one of the plates gt araC is a DNA binding protein that arabinose which controls the transcription of the GFP binds to gt In the presence of arabinose araC allows protein RNA polymerase to bind to the promoter and transcribe the genes which will in turn be translated to provide ampicillin resistance and the GFP if there is no arabinose is present the genes will not be transcribed or translated gt Arabinose was added so that araC would bind and GFP would be present 32 Be able to predict the outcomes of a hypothetical transformation gt Like autolysins degrades the B14 bonds between the amino acid molecules in peptidoglycan causing breaks in the lattice and weakening the cell wall As a result the solute pressure of cytoplasm can cause the cell membrane to rupture 33 34 35 36 37 38 Describe the effects of lysozyme on bacterial cells gt Gram positive Break down the peptidoglycan and cause cell to lyse gt Gram negative May not be able to damage gram negative because it has an outer membrane The peptidoglycan is not directly exposed De ne communicable epidemiology endemic epidemic and pandemic gt Communicable Disease that is transmissible from person to person gt Epidemiology the study of how when where what and who are involved in the spread of disease and distribution of disease in the population Endemic an infectious disease that exhibits a steady spread over a particular region of the world for a long period of time Epidemic Spread of a disease in a particular place Pandemic when a disease spreads between one or more continents Define antigen antiserum and agglutination gt Antigen any substance that causes your immune system to produce antibodies against it Antiserum a blood serum containing antibodies against specific antigens injected to treat or protect against specific diseases gt Serological typing through aggulation S aureus is positive when blue gt Agglutination tells you if there is Saureus turns blue clumps Explain how we used the slide agglutination test serological typing to identify S aureus gt Agglutination formation of a visible precipitate from the antibodyantigen complex gt We added a drop of positive control to circle 1 one drop of negative control and one drop of latex reagent to 3 circles colonies emulsify Distinguish between a positive and negative test for an agglutination reaction gt Positive Any form of clumping bigger clumps is more positive gt Negative No agglutination means that the cell does not produce virulence factors Be able to define and calculate morbidity mortality and incidence gt Morbidity illness due to a specific disease gt Mortality Death due to a specific disease gt Incidence number of new cases of a specific disease VVV gt The final lab exam will consist of the following types of questions multiple choice truefalse fillintheblank vocabulary without a word bank matching shortanswer and practicaltype questions in which you will have to look at something from the lab procedures such as a plate tube positive result etc and answer a question about it Good Luck
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