MICRO 3051 Final Lab Exam Study Guide
MICRO 3051 Final Lab Exam Study Guide 81383 - MICR 3050 - 002
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This 11 page Study Guide was uploaded by Stephanie Erickson on Monday February 29, 2016. The Study Guide belongs to 81383 - MICR 3050 - 002 at Clemson University taught by Krista Barrier Rudolph in Fall 2015. Since its upload, it has received 105 views. For similar materials see General Microbiology in Biological Sciences at Clemson University.
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Date Created: 02/29/16
MICR 3051 FINAL LABORATORY EXAM STUDY GUIDE LABS 7– 12 Objectives: 1. Describe how UV light is germicidal (its effect on DNA), and the wavelength at which UV light is the most lethal. a. UV is routinely used to sterilize surfaces b. UV lights is lethal because it causes the formation of pyrimidine dimers – these cause DNA molecule to become deformed so that the DNA polymerase cannot replicate DNA c. The shorter the wavelength the more damaging it is to cells – UV radiation at 260 nm is the most germicidal 2. Describe how we examined the germicidal effects of UV light on S. aureus and B. subtilis. a. We exposed S. aureus (non-endospore forming) and B. subtilis (endospore forming) to UV light for different periods of times and used plastic to block radiation for some trials b. Results should have found: i. Longer exposure time – increased germicidal effect ii. Presence of material that block radiation – decreased germicidal effect iii. Endospore forming bacteria (B. subtilis) was more resistant to UV light than non-endospore forming bacteria (S. aureus) 3. Be able to define the following terms: antiseptic, disinfectant, steriliant, sanitizer, bacteriostatic, and bacteriocidal. a. Antiseptic – inhibit microbial growth or kill microorganisms and are gentle enough to be applied to living tissue (don’t destroy endospores) b. Disinfectant – chemical agents that are applied to inanimate objects (non- heat sensitize) to kill microorganism, can be harmful to human tissue c. Sterilant – destroy all microorganisms, including endospores, sterilize heat-sensitize objects d. Sanitizer – reduce microbial numbers to a safe level but do not completely eliminate all microbes e. Bacteriostatic – agent that only inhibits growth of bacterial cells but does not kill them f. Bacteriocidal – kill bacteria cells 4. Explain the Filter Paper Disk Method of evaluating the relative effectiveness of various disinfectants and/or antiseptics. a. Compares effectiveness of disinfectants b. Plate is streaked evenly with test bacteria c. Filter paper disks are dipped into disinfectant and placed on the inoculated plate d. Agent will diffuse from the disk into the agar, forming a concentration gradient e. If the substance is inhibitory a clear zone of inhibition will surround the disk where no growth has occurred f. Determine the most effective disinfectant by measuring the zone of inhibition and comparing results 5. Distinguish between antibiotics and antimicrobials. a. Antimicrobials – compounds that kill or inhibit microorganisms b. Antibiotics – type of antimicrobial, usually of low molecular weight, produced by microorganisms that inhibits or kills other microorganisms 6. Explain the Kirby-Bauer Method of evaluating the sensitivity of bacteria to certain antibiotics and/or antimicrobials. a. Determines the antibiotic-resistance of a bacterium to an antimicrobial agent b. Uniformly streak a test organism onto a Mueller-Hinton medium c. Deposit paper disks containing different concentrations of various antimicrobial or antibiotic agents d. If agent inhibits growth there will be a zone around the disk where no growth occurs 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. a. Measure the zone of inhibition’s diameter with a metric ruler to the nearest whole millimeter b. R, I, S or is determined based on the measurement of the zone of inhibition – varies for each antimicrobial c. Zone of inhibition: area surrounding disk where no growth occurs d. Resistant i. No plaque around the disk ii. Bacterial growth is normal iii. Antibiotic is not effective – should not be used for treatment e. Intermediate i. Somewhat cloudy plaque around the disk ii. Decreased growth surrounding the disk iii. Antibiotic is somewhat effective: some of the bacteria are immune to antimicrobial while others are still sensitive to its effects, resistant bacteria will survive, while sensitive bacteria will be inhibited – not the best treatment f. Sensitive i. A clear, circular halo founded around the disk ii. No bacterial growth iii. Antibiotic is effective and inhibits or kills bacteria – recommended for treat this bacterial infection 8. Describe the effects of alcohol on bacterial growth. a. Considered an antiseptic: routinely used to disinfect skin, greatly reduces the numbers of microbes present 9. Explain the basis of the Reductase Test, and how it can be used to distinguish between high and low quality milk. a. The basis of the reductase test of milk involves adding dye methylene blue to a milk sample b. Milk is stored at 35 C c. Color of methylene blue changes from blue to clear when the dye is reduced d. Milk that contains a large number of actively growing bacteria will have a lowered oxidation-reduction potential due to the exhaustion of dissolved oxygen by microorganism e. Good quality milk: takes 6 or more hours for color to change from blue to clear f. Poor quality milk: takes 2 hours for color to change from blue to clear 10.Know the meaning and significance of MBRT. a. MBRT: Methylene blue reduction time i. Time it takes for the methylene blue to become colorless/time it takes for milk to be reduced ii. Shorter MRBT = lower quality iii. Longer MRBT = higher quality 11.Understand the role that microbes have in food production and in food spoilage. a. Microbes normally mean that food is spoiled or has potential to cause disease, but could be present because microorganism is used for production b. Foods prepared by microbial fermentation: yogurt, sauerkraut, and summer sausage 12.Understand how to conduct a Standard Plate Count (including determining a dilution scheme) to determine bacterial numbers in a liquid or solid food sample. a. Standard Plate Count (SPC) is the most common method for determining bacterial numbers in a sample b. Sample is diluted in a series of dilution blanks: dilutions of liquid or solid food sample is placed into a liquefied agar medium and poured into petri dish c. Media is incubated for 24-48 hours d. After incubation, colonies are counted e. Number of bacteria in original sample is determined by multiplying the number of colonies by the dilution factor Example: Blender dilution is 1:10. Add 1 mL from blender to 99 mL sterile blank (1:1000) Plate I (1:100) – add 0.1 mL from blender and liquid agar Plate II (1:1000) add 1 mL from sterile blank and liquid agar Plate III (1:10,000) add 0.1 mL from sterile blank liquid agar Incubates the plates then count colonies! Multiply by dilution factor! 13.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. a. CFUs: Colony Forming Units b. Because of the uncertainty in how many actual cells form a colony, SPC are reported in CFUS c. Countable plate: number of colonies falls between 30 and 300 d. If there’s >300, considered TNTC (too numerous to count) 14.Explain how the grape juice fermentation flask was set-up, and know what observations would indicate fermentation had occurred. a. Flask was filled with 100 mL grape juice, 3 mL of yeast culture was added, lead acetate test-strip was taped inside of the flask neck, mouth of flask was sealed with rubber balloon b. Flask was incubated at 15-17 C for 2-5 days c. Evidence of fermentation: gas produced (balloon inflated), pH drops (becomes more acidic), smells like alcohol instead of grape juice 15.Define the process of ammonification. a. Release of ammonia from biological molecules such as proteins, amino acids, nucleic acids, and nitrogenous wastes b. Occurs primarily in the soil as a result of the decomposition of the remains of dead plants and animals and their waste products 16.Describe how to test for the presence of ammonia in soil samples. a. Inoculate one tube of peptone broth with a small amount of soil b. Incubate the tube for 7 days c. Deposit a drop of Nessler’s reagent into two separate depression of a spot plate d. Add loopful of the inoculated peptone broth to one depression and do the same with a control e. Ammonia Results: i. Faint yellow color: small amount of ammonia ii. Deep yellow: more ammonia iii. Brown precipitate: large amount of ammonia f. We also tested for pH i. Blue color: alkaline ii. Yellow color: acidic 17.Describe coliforms and how they are good indicators of fecal contamination in water. a. Coliform definition: gram-negative, facultative anaerobic, non- endospore forming rods that ferment lactose to produce acid and gas b. Criteria to be good indicator of fecal contamination i. Organism occurs primarily in the intestines of humans and some warm-blooded animals and is not found routinely in soil and water ii. Organism can be easily identified by microbiological tests iii. Organism is not as fastidious as the intestinal pathogens, and hence survives a little longer in the 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. a. Presumptive Test: 15 tubes of lactose broth are inoculated with measured amounts of water to see if water contains any lactose-fermenting bacteria. i. Positive test: gas seen in any of the lactose broth tubes – coliforms are presumed to be present in the water sample b. Confirmed test: plates of Levine EMB agar and Endo agar are inoculated from positive (gas-producing) tubes to see if the organism that are gas producing are gram-negative (another coliform characteristic). Both medias inhibit the growth of gram-positive bacteria and cause colonies of coliforms to be distinguishable from noncoliforms. i. Positive test: On EMB agar, coliforms produce small colonies with dark centers (nucleated colonies). On Endo agar, coliforms produce reddish colonies with some green metallic sheen. The presence of coliform-like colonies confirms the presence of a lactose-fermenting, gram-negative bacterium. 19.Know how to determine the MPN of a water sample and understand what it means. a. The presumptive test is used to obtain MPN (most probable number) of coliforms present per 100 mL of water. b. After incubation, record the number of positive (gas-producing) tubes out of the first set of 5 (10 mL), second set of 5 (1 mL), and third set of 5 (0.1 mL) in 1-2-3 fashion. i. If all 5 of the 10 mL tubes produce gas, and then only 4 of the 1 mL tubes, and 0 of the 0.1 mL tubes produce gas record your results as 5-4-0. c. Use the table to obtain your MPN i. In this case the MPN is 170 d. The value of the MPN designates the number of coliforms per 100 mL of water i. In this case there are approximately 170 coliforms per 100 mL e. Also record the confidence interval – this tells you the possible range of coliforms i. MPN is an estimation, the confidence interval tells us we are 95% sure that the number of coliforms is somewhere between 58 and 400 20.Describe the distinguishing characteristics of bacteriophages. a. Bacteriophages are viruses that infect bacterial cells b. They are obligate intracellular parasites – must invade a host cell in order to replicate and reproduce c. Composed of primarily one single kind of nucleic acid molecule encased in a protein coat (capsid) that protects the nucleic acid d. Lack metabolic machinery, such as energy systems, and protein synthesis components necessary for independent replication e. In order to replicate and reproduce, they must use the host cell’s metabolic machinery to synthesize their various component parts f. Exhibit specificity for their host g. Single virus or phage is called a virion h. Structure consists of: i. Nucleocapsid – nucleic acid and capsid ii. Protein sheath – contractile and contains a hollow tube in its center iii. Base plate - where tail fibers and spikes are attached Most of the phage structure is necessary for the delivery of the phage nucleic acid into its host 21.Explain the procedure for determining a bacteriophage titer. a. Label 5 TS broth tubes (9 mL) with dilutions from 1:10 to 1:100,000 b. Transfer 1 mL of phage stock to first dilution blank (1:10) c. Mix well and transfer 1 mL of first dilution to second dilution blank (1:100) d. Repeat up to 1:100,000 dilution e. Transfer 1 mL of phage dilution tube to a liquid agar (9 mL) f. Apply 2 drops of E. coli broth g. Pour contents of onto prospective TSA plates and let harden h. Repeat for all phage dilutions i. Incubate all plates at 37 C for 24 hours j. Count the plaques (clear areas) – remember only to include counts between 25 to 250. k. Multiply the number of plaques by the dilution factor to determine the number of phage particles in the original suspension of phages. 22.Know how to calculate the number of phage particles in a suspension by counting the number of PFUs on plates. a. Count the plaques (clear areas). Multiply the number of plaques by the dilution factor to determine the number of phage particles in the original suspension of phages. Remember only to include counts between 25 to 250. 23.Describe the two mutant isolation procedures we used, and know what type of mutants we were trying to isolate. a. Replica plating is used to demonstrate that mutations occur randomly and spontaneously in a population of cells and are independent events not related to the presence of a drug b. Sterile velveteen cloth is placed over a wood block that is pressed onto a plate that contains no antibiotic and that has the suspected mutant colonies c. Exact colony pattern from the medium lacking streptomycin is replicated by the velveteen onto the medium containing the antibiotic d. Plates are compared to determine which of the colonies on the original plate are spontaneous mutants to streptomycin e. Procedure i. Original organism are spread over nutrient agar with a sterile bent glass rode ii. Plate is incubated to allow colonies to grow iii. After, incubation colonies are picked up with the velveteen colony carrier iv. Nutrient agar is inoculated by lightly pressing the carrier onto it v. Streptomycin is inoculated with same carrier in same manner vi. Plate are compared - colonies grow on streptomycin plate they are considered antibiotic resistant 24.Know the purpose of the gradient plate, and the three plates used in the replica plating procedure. a. Gradient plate – used to isolate and select streptomycin-resistant mutant S. aureus strains; increases frequency of mutations by manipulating the chemical or physical environment b. Mutations occur 1 of 2 ways: i. Spontaneous mutations arise occasionally in all bacteria and develop in the absence of any added agent ii. Induced mutations are the result of the bacterium’s exposure to a mutagen, a physical or chemical agent c. Procedure: i. Tryptic agar is poured into a plate and allowed to harden at an angle ii. Streptomycin agar in then poured into the plate and allowed the harder iii. Creates a gradient from low concentration of streptomycin to high concentration iv. Analyze plate to see the level of antibiotic resistance of the bacteria d. Three plates used in replica plating i. Master plate – the bacteria of interest is streaked on this plate to allow colonies to grow in normal conditions ii. Nutrient agar plate – velveteen colonies are pressed onto this plate after picking up the colonies on the master plate; acts as a control to ensure all colonies are properly transferred iii. Streptomycin – after pressing to the nutrient agar plate, the velveteen pressed is stamped onto this plate, any noted colonial growth demonstrates antibiotic resistance of the bacteria to streptomycin 25.Describe the general characteristics of Staphylococcus aureus as part of the microflora of the human body, and its significance in disease. a. General characteristics: i. Gram-positive, spherical bacteria that form irregular clusters ii. Non-motile, no-spore forming iii. Able to grown in high-salt concentrations iv. Found in nasal membranes, hair follicles, and skin b. Virulence factors: i. Coagulase production: causes serum to form clots ii. Contains DNase, an enzyme that digest DNA iii. Produces hemolysin that causes wide, clear zone of beta- hemolysis on blood agar iv. Produce pigment that has antioxidant properties that prevent host from killing the bacteria (golden color on blood agar) v. Ferments mannitol to produce acid c. Significance: i. Many nosocomial (hospital acquired) infections are caused by S. aureus ii. Mainly causes skin and wound infections iii. S. aureus has developed resistance to many antibiotics (ie. MRSA – methicillin resistant S. aureaus – is becoming a major epidemiological issue 26.Describe how we isolated S. aureus from our noses, and how we can identify it using Blood Agar, MSA, and CHROMagar TM. a. Nose swabbed b. Both Blood Agar and MSA plates were inoculated with nasal bacteria c. The following week, a beta-hemolysis colony from the blood agar plate was used to inoculate the CHROMagar plate d. Results: i. Blood Agar: if S. aureus is present, clear zones will appear around golden-colored colonies indicating beta-hemolysis occurred ii. MSA: MSA contains 7.5% sodium chloride, mannitol, and phenol red indicator. The NaCl inhibits the growth of other bacteria, but S. aureus will still be able to grow. Further, in MSA S. aureus will ferment mannitol, which produces acid causing the phenol red indicator to change color from red to yellow as pH drops. iii. ChromAgar: S. aureus appears as a pinkish-purple color 27.Know the four basic steps to isolate DNA from bacterial cells. a. Bacteria cells are centrifuged to concentrate them b. A detergent is used to break down the cell wall and cell membrane, releasing the DNA c. A protease, along with the heat, is used to denature and digest proteins within the cell d. Cold alcohol is used to precipitate the DNA from the solution, allowing it to be spooled onto a glass or metal rod 28.Define transformation. a. Type of gene recombination, which occurs when a bacterial cell receives small amounts of DNA from the environment 29.Describe the heat shock method we used to artificially transform E. coli with pGLO. a. Transformation is a natural process, but it is rare and occurs infrequently i. Cells that can in take DNA are considered competent b. Treatments can be administered to increase competence c. Ca++ is used to neutralize the negative charges on the DNA and the cell membrane, making it easier for DNA to enter d. Cells are subjected to brief heat shock that helps increase permeability of the membrane to DNA 30.Know the selectable characteristics of pGLO that allowed us to select for transformants. a. Transformation is accomplished in the lab with the use of a plasmid b. The plasmid is defined with certain selectable characteristics (antibiotic resistance, glowing) that it will transfer to the host DNA and can be easily identified in order to determine if transformation was completed successfully c. pGLO: plasmid being used i. Ori: origin of replication, DNA sequence recognized by enzymes that initiate replication of DNA ii. Drug Resistance: bla gene, provides ampicillin resistant iii. Gene of Interest: GFP - a gene that codes for the production of green fluorescent proteins 31.Know the role of araC in pGLO, and why arabinose was added to one of the plates. a. Arabinose promoter controls transcription of GFP gene b. When arabinose is present, it binds to a DNA-binding protein araC, which will then allow protein RNA polymerase to bind to the promoter and transcribe the genes c. Transcribed genes will then be translated to provide ampicillin resistance and the green fluorescence proteins d. If no arabinose is present, the genes will not be transcribed or translated e. Arabinose is added so that araC would bind to it and GFP would be transcribed and glowing phenotype would present **Arabinose is an inducible promoter: it only allows genes to be transcribed when certain conditions exist (in this case, araC presence) 32.Be able to predict the outcomes of a hypothetical transformation. a. +pGLO (LB/amp) i. Will NOT grow on ampicillin plate because araC was not present, and therefore genes were not transcribesd b. +pGLO (LB/amp/ara) i. Colonies WILL grow on ampicillin plate and they will glow because araC was present, which allows for transcription and translation of the bla and GFP genes; MUST HAVE ALL THREE TO GROW AND GLOW c. -pGLO (LB/ amp) i. No plasmid, no change in host cell genotype phenotype, no growth d. -pGLO (LB) i. No plasmid, no change in host cell genotype phenotype, no growth 33.Describe the effects of lysozyme on bacterial cells. a. Lysozyme: enzyme found in tears and saliva the degrades beta 1-4 bonds between the amino sugar molecules in peptidoglycan and thus causes breaks in the lattice and weakening of the cell wall; leads to cell lysis i. Gram positive bacteria: 90% of cell wall is peptidoglycan so they are very susceptible to lysozyme ii. Gram-negative bacteria: not as easily affected by lysozyme because outer membrane prevents lysozyme from reaching the peptidoglycan layer 34.Define communicable, epidemiology, endemic, epidemic, and pandemic. a. Infectious Disease – a disease caused by microorganisms that enter the body and multiply in the tissues at the expense of the host b. Communicable – infectious diseases that are transmittable to other persons i. Can be accomplished by direct or indirect methods 1. Direct: handshaking, kissing, sexual intercourse 2. Indirect: water, food, animals, inanimate objects c. Epidemiology – the study of how, when, where, what, and who are involved in the spread and distribution of diseases in human populations d. Endemic – an infectious disease that exhibits a steady frequency over a long period of time in a particular region (ie. malaria in tropical regions) e. Epidemic – excessive number of newly reported cases in a given time period in a specific area (ie. Chylamidia in the US) f. Pandemic – infectious disease that spreads to occurs on one or more continents (ie. AIDS, H1N1) 35.Define antigen, antiserum, and agglutination. a. Antigen: any substance that causes your immune system to produce antibodies b. Antiserum: a blood serum containing antibodies that work again a specific antibody c. Agglutination: clumping of particles; occurs when antibodies react with soluble antigens and form a precipitate 36.Explain how we used the slide agglutination test (serological typing) to identify S. aureus. a. Remember that coagulation is an important characteristic of S. aureus - 97% of strains of S. aureus have proven to be coagulase positive b. Serological typing uses 3 circle of the agglutination slide – agent is applied and mixed for 20 seconds, then results are read i. Circle #1: One drop of positive control reagent is added – shows clumping (agglutination) ii. Circle #2: One drop of negative control reagent is added – clear background iii. Circle #3: latex reagent and bacteria of interest are added – assess clumping, it is S. aureus then it will agglutinate 37.Distinguish between a positive and negative test for an agglutination reaction. a. Positive test: large clumps of aggregated latex beads is very positive, fine clumps are slightly positive; clear background is very positive, cloudy background is slightly positive b. Negative test: smooth, cloudy suspension, free of agglutination and particles 38.Be able to define and calculate morbidity, mortality, and incidence. Morbidity: illness due to a specific diseases Mortality: death due to a specific disease Incidence: number of new cases of a specific disease Morbidity = no. cases per period x K susceptible population size at midpoint of period Mortality = no. disease-related deaths per period x K no. people with the disease Incidence = no. new cases x K susceptible population size at midpoint of period K is a factor that varies and serves to assist in more clearly reporting the results. Because values are often very small, the number is commonly multiplied by a power of ten so it can be reported as a whole number (2 case per 100,000 people) The final lab exam will consist of the following types of questions: multiple-choice, true/false, fill-in-the-blank (vocabulary without a word bank), matching, short- answer, and practical-type 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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