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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 thioglycollate broth
determines if thioglycollates are active; green=O2, pink=no O2 resazaurin
used to determine how many bacteria are present quantitative plate count
____ counts are counts that include only "live" bacteria viable
____ counts are counts that include living and dead bacteria total
a sequence of dilutions using previously diluted solutions to make current solutions; a series of sequential dilutions serial dilution
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 cell growth
putting any microbe in or on a media inoculation
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" microaerophiles
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 30-300
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 GasPak Jar
cold loving bacteria; 5℃ - 20℃ psychrophile
moderate/warm loving bacteria; 20℃ - 45℃; 2 common optimal temps: 25℃/Room temperature, 37℃/Body temperature mesophile
hot loving bacteria; 45℃ - 55℃ thermophile
a type of mesophile that can grow at 0C; range from about 0C-30C psychrotroph
What is the genus of the thermophile we grew in class? Bacillus (stereothermophilus)
What bacteria Genus makes a red pigment at 25C? Serratia (marcescens)
temp: minimum, optimum, and maximum temp requirements are also known as? cardinal temperatures
solid growth on plate lawn
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 thermocycler
PCR step/cycle: heat to "unzip"/melt target DNA; disrupts the hydrogen bonds b/w the two complementary DNA strands and cause their seperation denaturation
PCR step/cycle: reaction mixture cooled which allows the primers to base pair with the target DNA sequence annealing (sticking)
PCR step/cycle: temp raised which causes polymerase to add nucleotides to synthesize the new complementary DNA strands extension
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 control ladder
PCR test: what is the white ball with TAq Polymerase made of? magnesium chloride
stopping/slowing growth ___static
killing the microbe ___cidal
controlling microbes on a non-living surface (fomite) disinfectant
controlling microbial growth on a living surface antiseptic
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 phenols (Lysol)
chemical that works by oxidizing agents that work by denaturing proteins or by forming oxidizing agents halogens (iodine)
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
qualitative vs quantitative? goes beyond yes or no result, i.e. effectiveness of ATB against bacteria, measuring the zone of inhibition quantitative
against life; many are produced by living organisms antibiotic
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 resistance
microbe somewhat sensitive to the antibiotic intermediate
microbe is killed by antibiotic sensitive
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 thymine dimer
white light (room light) causes ____?; reverses mutation (separates/unlocks the locked dimer) photoreactivation
time for UV to kill Serratia marcescens 1-minute
UV light testing: colonies within UV-exposed area begin to develop when exposed to white light (room light); light-activated DNA repair enzyme photoreactivation photolyase
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 ______ Thioglycollate
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 MacConkey
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 thio
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 selective media
media that contains an indicator that indicates difference b/w microbes growing on the media differential media
crystallized form of something; high-osmotic pressure (ability to draw fluids) a salt
aka fast test; looking for antibody immunity ELISSA
What does ELISSA stand for? Enzyme Linked Immuno Absorbance Assay
ELISSA test: looking for antigen in pt's blood direct
ELISSA test: looking for lab-created antigen indirect
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 chromogen
Created by: nurse savage
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