Lab Final Exam Questions with correct Answers

Lab Final Exam Questions with correct Answers define biofilm Is location in biofilms important? If so, why? -------------- define quorum sensing Heterogeneous - a lot of microbes living together Location in biofilm is important - most growth outside (more oxygen and food) *inside need different paths of fermentation Outside - fast growers Inside - slow growers Some dead Can have horizontal gene transfer Competent - capable of taking up DNA through transformation Bioluminescent - bacteria doing it ------------- The ability of bacteria to sense the presence of other bacteria via secreted chemical signals. Steps of Biofilm formation Biofilms - bacteria / plaque on teeth Molecules in the environment that eventually stick First attachment isn't too strong - then quartum sensing (call for people to come here) attracted to come to the biofilm structure Desorption: the release of an adsorbed substance from a surface. Produce an exopolymer (proteins, nucleic acids, msotly like a glycoprotein sugary stuff) More and More and then as they start to build up and making this biofilm strucutre (water structure - channels) big enough you might have some detachment / erosion / sloughing Dispersion - so stressed that biofilms can leave and go cause problems generalized steps of biofilm formation attachement colonization development active dispersal purpose of biofilm water channels there can be an issue with nutrient availability on the inner portion of a biofilm. So holes in the film form so substrate can pass through biofilm water channels there can be an issue with nutrient availability on the inner portion of a biofilm. So holes in the film form so substrate can pass through ammonification Conversion of organic Nitrogen in dead organisms back to ammonium. the formation of ammonia compounds in the soil by the action of bacteria on decaying matter How we tested for ammonification How does Nessler's reagent work? 1. innoculated tube of peptone broth with soil 2. incubate 7 days room temp 3. add to Nessler's reagent spot plate. Nessler's reagent - This pale solution becomes deeper yellow in the presence of ammonia. At higher concentrations, a brown precipitate may form. Define germicidal a substance or other agent that destroys harmful microorganisms; an antiseptic (i.e. UV) What UV wavelengths are best for killing microbes? Why? 260 nm - 270 nm range because absorbed by nucleic acid (of 100mn - 400nm) Procedure for determining germicidal effect of UV on S. aureus and B. subtilis define mutagen What do they interact with that results in mutation? A chemical or physical agent that interacts with DNA and causes a mutation. ( ie. UV light) Define Anitmicrobial agent Define disinfectant Define antiseptic Define antibiotic An antimicrobial is an agent that kills microorganisms or stops their growth Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms Antiseptic are antimicrobial agents that are applied to living tissue to destroy microorganisms An antibiotic is a substance that inhibits the growth of or kills microorganisms in the body. --- An ANTIBIOTIC is a low molecular substance produced by a microorganism that at a low concentration inhibits or kills other microorganisms. Procedure for Filter Paper Disk Method of measuring relative effectiveness of disinfectants/antiseptics Procedure for Kirby-bauer test note the Mueller-Hinton agar Müller-Hinton agar is a microbiological growth medium that is commonly used for antibiotic susceptibility testing. Define zone of inhibition and explain how its measurement can be used to determine if a bacterium is Resistant, Intermediate, or Sensitive to an antimicrobial agent. (Kirby-bauer test) Note the pattern for resistant, intermediate, sensitive ,_ , Procedure for determining the effectiveness of soap, alcohol and sanitizer Percent reduction equation Left hand is control right hand is experimental took percent reduction for left and right thumb prints Methylene Blue Reductase Test what is it used to test? what is a positive and negative result? What is MBRT? Scientific explaination milk quality blue color reduces after incubation = positive (is contaminated with bacteria) blue color is not reduced after incubation = negative (not contaminated with bacteria) (Methylene Blue Reduction Time) MBRT is a method for interpreting the results of a methylene blue reductase test. The longer it took the milk+dye solution to decolorize, the GREATER the quality of milk ----- loss of oxygen in the milk due to reduction by microbes causes color in the methylene blue die to dissipate Understand how to conduct a Standard Plate Count for a liquid and solid food sample (including how to set up a 1:10 and a 1:100 serial dilution). (lab 8) Be able to calculate the number of bacteria in a liquid or solid sample based on the number of CFUs on various plates. For example, if a plate containing a 1/1,000,000 dilution of the original ml of sample shows 150 colonies, then 150 represents 1/1,000,000 the number of CFUs present in the original ml. Therefore the number of CFUs per ml in the original sample is found by multiplying 150 x 1,000,000 as shown in the formula below 150 x 1,000,000 = 150,000,000 Alcohol fermentation equation starting from pyruvate where are acetaldehyde, pyruvate decarboxylase, ethanol , and alcohol dehydrogenase in the process? where is NADH + H+ -- NAD+ Colony # range for a countable plate Between 30 and 300 colonies What microbe does fermentation Grape fermentation flask setup Confirmative indications after incubations yeast smell, gas in balloon, test strip if present. pH Coliform characteristics shape? G-/G+? What are they able to ferment? what are the fermentation products? Rod-shaped Gram-negative non-spore forming and motile or non-motile bacteria which can ferment lactose with the production of acid and gas when incubated at 35–37°C. 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. LACTOSE BROTH - 10% gas production in Durham tube EMB AGAR - Green metallic sheen ENDO AGAR - pink/red growth with a metallic sheen Know how to determine the MPN of a water sample and understand what it means For example: a water sample tested shows a result of 3–2–1 (3 × 10 ml positive, 2 × 1 ml positive, 1 × 0.1 ml positive) gives an MPN value of 17, i.e. the water sample contains an estimated 17 coliforms per 100 ml bacteriophage viruses that infect bacteria plaque Plaque, in microbiology, a clear area on an otherwise opaque field of bacteria that indicates the inhibition or dissolution of the bacterial cells by some agent, PFU definition PFU equation Plaque Forming Unit *Concentrations of viral suspensions measured by the number of plaques Lysozyme effect on bacterial cells where are they usually found An enzyme, present in mucus, tears, and saliva, that destroys bacteria by dissolving their cell walls. Staphylococcus aureus is a major bacterial human pathogen that causes a wide variety of clinical manifestations.[1] Infections are common both in community-acquired as well as hospital-acquired settings and treatment remains challenging to manage due to the emergence of multi-drug resistant strains such as MRSA (Methicillin-Resistant Staphylococcus aureus).[2][3] S. aureus is found in the environment and is also found in normal human flora, located on the skin and mucous membranes (most often the nasal area) of most healthy individuals.[1] S. aureus does not normally cause infection on healthy skin; however, if it is allowed to enter the bloodstream or internal tissues, these bacteria may cause a variety of potentially serious infections.[1] Transmission is typically from direct contact. However, some infections involve other transmission methods.[4] Staphylococcus aureus is Gram-positive bacteria (stain purple by Gram stain) that are cocci-shaped and tend to be arranged in clusters that are described as "grape-like." On media, these organisms can grow in up to 10% salt, and colonies are often golden or yellow (aureus means golden or yellow). These organisms can grow aerobically or anaerobically (facultative) and at temperatures between 18 C and 40 C. Typical biochemical identification tests include catalase positive (all pathogenic Staphylococcus species), coagulase positive (to distinguish Staphylococcus aureus from other Staphylococcus species), novobiocin sensitive (to distinguish from Staphylococcus saprophyticus), and mannitol fermentation positive (to distinguish from Staphylococcus epidermidis) Staphylococcus aureus (including drug-resistant strains such as MRSA) are found on the skin and mucous membranes, and humans are the major reservoir for these organisms.[3][5] It is estimated that up to half of all adu procedure for e how we isolated S. aureus from our noses, and how we can identify it using Blood Agar, MSA, and CHROMagar Blood Agar = Beta-hemolytic MSA = Yellow CHROMagar = Staphylococcus aureus → pink to mauve Other bacteria → Colourless, blue or inhibited positives v. negatives The mutatant of which bacteria did we isolate with the replica plating and gradient plating procedures? Streptomycin resistant bacteria replica plating method for isolating mutants purpose of the three plates Master plate: from the previous lab, stock nonselective replica plate: orientation standardize reference selective replica plate: orienation locked and only grows the mutant or non mutant. Compare with the nonselective plate to see what colonies are not growing. streptomycin resistant bacteria gradient plating procedure for isolating mutants purpose of the plate which experiment was it used in The antibiotic in the top layer, diffuse into the bottom layer producing a gradient of antibiotic concentration from low to high. A gradient plate is made by using Streptomycin in the medium. E. coli, which is normally sensitive to Streptomycin, will be spread over the surface of the plate and incubated for 24 to 72 hours. After incubation colonies will appear on both the gradients. The colonies develop in the high concentration are resistant to the action of Streptomycin, and are considered as Streptomycin resistant mutants. DNA extraction steps 1. Break cells open to release DNA -vortex/centrofuge to separate cells -salt soln protects DNA backbone -detergent added and breaksdown lipids in cell membrane/nuclei 2. Separate DNA from proteins/other cellular debris -filtering or proeases to degrade DNA associated proteins 3. Precipitating DNA with alcohol -presence of salt+alcohol makes DNA insoluble. Ethanol or isopropanol 4. Clean the DNA 5. Confirming presence and quality of the DNA -purity and concentration: spectrometer (optical density). Gel electrohporesis can indicate quality and presence. heat shock definition heat shock method we used overview what temp and time was used? (42C for 50sec) the effect of subjecting a cell to a higher temperature than that of the ideal body temperature of the organism from which the cell line was derived. improves permeability of membrane/wall(?) Know the role of araC in pGLO, and why arabinose was added to one of the plates araC - activate GFP arabinose - activates araC protein which activates GPF gene promoter transformation process in which one strain of bacteria is changed by a gene or genes from another strain of bacteria chemical transformation - Ca+ is used to increase membrane permiability to uptake plasmids with DNA in it. Heatshock often used to further increase chances electroporation - electric field added to the competent cells to make holes (pores) in the cell walls for plasmids to go thru. Outgrowth repairs the holes In both cases the bacteria is then put on selective media to select transformants via antibiotic resistance from the plasmids Be able to predict the outcomes of a hypothetical transformation infectious disease A disease that is caused by a pathogen and that can be spread from one individual to another. pGLO - the plasmid with 3 genes - araC -GFP -Amp/bla araC = switch that turns on GFP gene (start protein production) in the presence of the sugar arabinose GFP = green fluorescent protein (UV) amp/bla= gene that codes for beta lactamase, Ampicilin resistance --- LB plate - just LB broth LB/amp - LB broth and ampicilin present in plate LB/amp/ara - LB broth, ampicilin, and arabinose present in medium