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Micro-Lab Pr. 2
|what kind of broth is needed to check temperature?
|just a nutrient broth; no special broth needed
|differential media; used to determine O2 requirements of bacteria; a semi-solid gel with an O2 indicator in the media
|determines if thioglycollates are active; green=O2, pink=no O2
|used to determine how many bacteria are present
|quantitative plate count
|____ counts are counts that include only "live" bacteria
|____ counts are counts that include living and dead bacteria
|a sequence of dilutions using previously diluted solutions to make current solutions; a series of sequential dilutions
|equipment for quantitative plate counts
|stock (bacteria), seven 9-mL sterile water test tubes, eight sterile pipettes, four sterile petri dishes
|quantitative plant count procedure
|transfer 1-mL of stock to tube 1 w/pipette, transfer 1-mL from tube 1 to tube 2 (and so on thru tube 7), transfer 1-mL from tube 4 (w/the original pipette for tube 4) to plate 4 (and so on thru tube 7 to plate 7), add agar to dish 4-7 and swirl gently
|an increase in the number of cells; cell division
|putting any microbe in or on a media
|bacteria that require total 02; what does it look like in test tube?
|strict aerobes; bacteria all the way at top; with a green ring
|bacteria that require a total absence of O2; what does it look like in test tube?
|strict anaerobes; clear at top of specimen, all bacteria below that
|bacteria that can grow in the presence or absence of O2 and do NOT use O2 for growth; what does it look like in test tube?
|aerotolerant anaerobes; cloudy throughout
|bacteria that grow better in the presence of O2 than no O2; what does it look like in test tube?
|facultative anaerobes; looks like a lightning bolt in test tube
|bacteria that require small amounts of O2 for growth and are killed by "regular" amounts of O2; "mud bacteria"
|Why is O2 toxic to some bacteria?
|anaerobes lack certain enzymes that are essential for bacteria to survive in presence of oxygen
|How do aerobic cells deal with toxic O2 compounds (byproducts of aerobic respiration)?
|they're eliminated by enzymes (which are commonly found in aerobic bacteria)
|this procedure is done to ensure that only a limited number of colonies develop in the plate; if too many colonies present on a plate, some cells are overcrowded and do not develop
|serial dilutions; quantitative plate count
|number range of bacteria on a plate appropriate for determining cell count/mL
|a sealed jar system which uses a foil gas generator, a palladium catalyst, and a methylene blue indicator strip; if microbe grows in this---it is anaerobic
|cold loving bacteria; 5℃ - 20℃
|moderate/warm loving bacteria; 20℃ - 45℃; 2 common optimal temps: 25℃/Room temperature, 37℃/Body temperature
|hot loving bacteria; 45℃ - 55℃
|a type of mesophile that can grow at 0C; range from about 0C-30C
|What is the genus of the thermophile we grew in class?
|What bacteria Genus makes a red pigment at 25C?
|temp: minimum, optimum, and maximum temp requirements are also known as?
|solid growth on plate
|Quantitative plate count: formula for determining bacterial count
|# colonies x dilution factor divided by volume plated = colonies or cells/mL
|What does PCR stand for?
|polymerase chain reaction
|What is PCR used for?
|DNA replication in a test tube; replicate ocean bacteria, trace tainted produce, trace outbreaks in healthcare settings
|any replication done with heat; a specialized machine used to rapidly heat and cool samples in PCR
|PCR step/cycle: heat to "unzip"/melt target DNA; disrupts the hydrogen bonds b/w the two complementary DNA strands and cause their seperation
|PCR step/cycle: reaction mixture cooled which allows the primers to base pair with the target DNA sequence
|PCR step/cycle: temp raised which causes polymerase to add nucleotides to synthesize the new complementary DNA strands
|What is agarose gel electrophoresis used for?
|visualize and measure DNA
|used to visualize DNA properties under the UV light
|ethidium bromide card
|process of using electricity to move something; to visualize and measure DNA
|agarose gel electrophoresis
|name of the piece of equipment that the ethidium bromide is put into
|gel electrophoresis apparatus (or gel box)
|first lane on ethidium bromide card
|PCR test: what is the white ball with TAq Polymerase made of?
|killing the microbe
|controlling microbes on a non-living surface (fomite)
|controlling microbial growth on a living surface
|antimicrobial susceptibility test; used for measuring effectiveness of antimicrobials against pathogenic microbes
|Kirby-Bauer method (aka Disc Diffusion method)
|type of chemical that works by denaturing the proteins and breaking the cell membranes--> cell death
|chemical that works by oxidizing agents that work by denaturing proteins or by forming oxidizing agents
|chemical that works by dissolving lipids in cell membrane and by denaturing proteins; kill gram -
|alcohol (isopropol alcohol)
|work by breaking down the bacterial cell membrane (since it combines with a phospholipid) and denatures the proteins
|quaternary ammonium compounds (Cepacol)
|completely clear zone around disc in Kirby-Bauer method; the "kill zone"
|zone of inhibition
|qualitative vs quantitative? yes or no result
|qualitative vs quantitative? goes beyond yes or no result, i.e. effectiveness of ATB against bacteria, measuring the zone of inhibition
|against life; many are produced by living organisms
|unit of measurement for Kirby-Bauer technique; measuring what?
|mm; diameter of zone of inhibition
|the ability of a microbe to withstand the effects of an antibiotic
|microbe somewhat sensitive to the antibiotic
|microbe is killed by antibiotic
|Kirby-Bauer method result: chemical testing--> qualitative or quantitative?
|qualitative (did it work--yes or no?)
|Kirby-Bauer method result: ATB sensitivity testing--> qualitative or quantitative?
|quantitative (effectiveness? numerical data based on measuring zone of inhibition)
|Can you 'eyeball' ATB sensitivity testing result?
|no, need to measure diameter of zone of inhibition then use table/chart to determine result (for correct ATB)
|Which microbe digests blood? has it's own special plate---name of plate? Looking for what?
|S. pyogenes; BAP; look for hemolysis
|taking two Ts and binding/gluing them together; exposure to UV causes _____ to lock together therefore not allowing the proper base pairing to occur--->effects bacterial replication and can cause mutation in cells
|white light (room light) causes ____?; reverses mutation (separates/unlocks the locked dimer)
|time for UV to kill Serratia marcescens
|UV light testing: colonies within UV-exposed area begin to develop when exposed to white light (room light); light-activated DNA repair enzyme
|a method of separating out bacteria to be able to pick out individual colonies, therefore each separate colony is a small pure culture
|streak plate method
|Streak Plate method steps
|flame inoculating loop and cool for all 3 steps (1st time--gently shake, 2nd/3rd cool loop in side of plate), tight lines in 1st section, looser lines each time in 2nd and 3rd sections, initial swipe in 2 and 3 is taken from previous section
|basic growth media
|TSA (Tryptic Soy Agar)
|differentiates hemolytic types, indicator-blood, alpha/beta/gamma
|TSA (Tryptic Soy Agar) + 5% Sheep's blood (BAP)
|media type: differentiates fermentation types; indicator is ____ ____
|PR Sugar, Phenol Red
|differentiates O2 requirements category, indicator ______
|selects for halophiles (staph species), inhibitor-high salt concentration, differentiates-mannose fermentors (b/w staph species), indicator-phenol red
|MSA (Mannitol Salt Agar)
|MSA media turns yellow
|S. aureus (mannose fermenter)
|MSA media with no color change
|S. epidermititis (non-fermenter)
|selects for G+, inhibitor- ____, differentiates-hemolytic types, indicator-blood
|PEA-B (Phenyl Ethyl Alcohol)
|selects for G-, inhibitor-bile salts, differentiates-lactose fermentors (E. coli), indicator-neutral red; lactose fermentors (E. coli) turn magenta, non-lactose fermentors--no color change
|selects for G-, inhibitor-dye, differentiates-lactose fermentors, indicator-dye; lactose fermentors (E. coli) turn metallic green, non-fermentors--no color change
|EMB (Eosin Methylene Blue)
|means sulfur group somewhere
|Kirby-Bauer method steps
|rub microbe all over TSA plate, place infused discs (chemical or ATB discs) on plate, check for zone of inhibition
|Which media(s) are differential only?
|TSA + 5% sheeps blood, Thioglycollate
|Which media(s) are selective and differential?
|MSA, PEA-B, MacConkey, EMB
|type of media that contains an inhibitor that inhibits the growth of everything but what you're looking for
|media that contains an indicator that indicates difference b/w microbes growing on the media
|crystallized form of something; high-osmotic pressure (ability to draw fluids)
|aka fast test; looking for antibody immunity
|What does ELISSA stand for?
|Enzyme Linked Immuno Absorbance Assay
|ELISSA test: looking for antigen in pt's blood
|ELISSA test: looking for lab-created antigen
|results of ELISSA test
|positive, weak positive, or negative
|Do you need exact timing for ELISSA test?
|yes--can get false results d/t color sticking more and more in sample
|quality control of ELISSA test
|at least 3 replicates for each specimen
|Why would ELISSA results be weak positive instead of full positive?
|time of exposure, how big of dose, previous exposure (titers), how sick host was
|dye used in ELISSA test