Lab Flashcards
what antiseptics/disinfectants disrupt the lipid bilayer
alcohols, soaps, detergents
what antiseptics/disinfectants are oxidizing agent
hydrogen peroxide, halogens, metals, aldehydes, ethylene oxide, benzalkonium chloride
what antiseptics/disinfectants break H bonds
acids and bases
what is the function of pasteurization
heat to reduce number of microbes but not to zero
besides time, what is the main difference between vat, high temperature, and ultra pasteurization
temperature
what are the temperature differences between vat, high temperature, and ultra pasteurization
vat= 145F (63C)
high temp= 161F (72C)
ultra= 280F (138C)
how does an autoclave work
it holds water at 121C and 2x atmospheric pressure for 15 minutes to destroy all microbes including spores
what type of setting is UV radiation used in for sterilization
hospital
how does ionizing/gamma radiation work
creates free radicals to damage DNA
what is gamma radiation used for
to sterilize items that can’t be put through an autoclave (ex: plastics)
what is involved in liquid filtration
sterilized liquids by passing them through a filter with 2 micrometers
when is liquid filtration used
when heating would kill something you wanted to preserve
how does a hemocytometer/Petroff-Hausser Counter with microscopy work to count bacteria
it uses a microscope slide with grids
the volume of each square is known so you can count the number of cells in each square and multiple to determine the number of bacteria in the liquid sample
how does spectrophotometry work
a line is shined though the liquid sample and the amount of light blocked is used to determine the number of bacteria in the sample by comparing it to McFarlands 0.5 standard
what are 2 limitations with spectrophotometry
production of biofilm increases bacteria size so you may be over-counting
can’t be used with samples that are too dark or too opaque
what is the concentration and number of bacteria in McFarlands standard
0.5
1x10^8 bacteria/mL
how many bacterial colonies must be present on a plate to count
30-300
what are 2 limitations to counting bacteria on a plate
biofilm causes bacteria to stick together so each colony may represent more than 1 bacteria
having more than 300 colonies is likely that some colonies represent more than 1 bacteria leading to undercounting
what is the dilution standard for antibody/antigen samples
1:2
in titers, the __ the second number, the more antibody present (higher or lower)
ex: is 1:16 or 1:64 a greater titer
higher
1:64
what causes zeta potential in RBC
sialic acid on the RBC surface creates a net - charge that IgG is too small to overcome, therefore IgM must be used for crosslinking
what antibody does blood typing use
anti-RBC IgM
what is the question asked for the indirect Coomb’s test
is there IgG that binds to the RBC
*used maternal serum
what is the question asked for the direct Coomb’s test
does the RBC have IgG stuck to it
*uses fetal RBC
how does radial immunodiffusion differ from double immunodiffusion
radial determines the amount of antigen in the sample
double immunodiffusion tells us if the antigen is present or not
do immunoassays use monoclonal or polyclonal antibodies
mono
what is ELISA used for
to detect protein/antigen in serum using antibodies
*can be used to determine titer
what does ELISPOT TSpot test for
how many cells dump out the protein of interest
*more spots on plate= more cells that released the protein/cytokine
ex: if T cells make IFN gamma
“is IFN gamma released in response to tuberculosis antigen?”
what is the function of immunohistochemistry
used to access tissue location a protein localizes in
what results does fluorescence activated cell sorting (FACS) give is
percent of cells that express a specific marker and level of expression
(flow cytometry)
what does neutrophil functional assay tell us
if oxidative burst is functioning and NADPH oxidase is made (shift in flow cytometry= working)
what is the function of leukocyte function assay
determine if B and T cells have normal activity by using something you know will activate all B or all T cells
what 2 things can be used to stimulate T cells in leukocyte function assay
PHA
antibodies against CD3 and CD28
what is used to stimulate B cells in leukocyte function assay
antibody against antibody
(ex: antibody against IgG)
how do you measure T cell proliferation
used fluorescence based assay, dye the cytoplasm, stimulate T cells, notice degree of decreasing fluorescence
how would you perform a functional assay for apoptosis
isolate monocytes/T/B cells
treat with PHA to stimulate T cells
culture with IL-2 for T cell proliferation
add anti-FAS antibody to trigger apoptosis
use TUNEL assay to detect fragmented DNA
*undergoing apoptosis= cell will fluoresce
how can you test for T cell maturation
if cells are actively undergoing VDJ recombination in the thymus, the T cell receptor excision circles will be detected in the blood
if they are absent, there is no VDJ recombination= no T cells= can be used to detect SCIDs
what is the function of CH50 test (complement hemolysis 50%)
test if complement is working or if it has been used up
less complement= CH50 closed to 1:2
what is the Cr51 release assay used to test for
CD8+ T cell function
when should modified acid-fast staining be performed
for actinomyces, fungi, and some parasites
if everything turns pink, what happened with the gram stain
too much decolorizer
if everything is purple, what happened with the gram stain
not enough decolorizer
what bacteria does MacConkey agar select against
gram + and bacteria that can’t grow in the presence of bile
what does it mean if MacConkey agar turned pink
bacteria ferments lactose
what does eosin methylene blue agar select against
gram +
what does if mean if the eosin methylene blue agar turned dark green/black
the bacteria ferments lactose
what does mannitol salt agar select against
bacteria that need high water activity to grow
what does it mean if mannitol salt agar turns yellow
the bacteria ferments mannitol
what bacteria show on ESBL agar
e.coli O157.H7 is pink
MRSA is blue
all other bacteria are white
TSI slants are used as a differential for what 3 characteristics
acid production (fermentation)
gas production
precipitation of metal
what 2 media allow for precipitation of a metal
Hektoen enteric agar
tellurite containing blood/chocolate agar
what is charcoal buffered yeast agar used for
charcoal absorbs toxic metabolites generated by growing bacteria
what is chocolate agar used for
to grow bacteria that need components or RBC to grow but don’t have the enzymes to break down RBC on their own
what is the function of PCR
look for presence or absence of 1 specific gene
(ex: antibiotic resistance or virulence factor gene)
what is the function of rt-PCR
determine viral load of a virus with an RNA genome
what is the function of western blot
what infection is it often used for
looks for the amount of one protein made
HIV
what is pulse field gel electrophoresis (PFGE) used for
determine whether the organism is of the same strain
(used to identify foodborne illness outbreaks)
what is multi-locus sequence typing used for
to measure evolutionary relatedness by identifying point mutations
*more detailed than PFGE
what is ribosomal RNA sequencing used for
identify organisms at the species level
what are the steps to determine if molecular Kochs postulates have been met
mutate isolate to knockout the gene
perform artificial transformation to reintroduce a plasmid containing the gene
if reintroduction of the gene into the bacteria leads to the bacteria causing symptoms again, the pathogenic gene has been identified and postulates have been satisfied
how much blood should be collected for culture in adults vs children vs neonates
adults: at least 20mL
children: at least 10mL
neonates: 2mL
how can culturing be used to determine if meningitis is caused by bacterial or fungal infection
compare the glucose concentration in the blood vs in CSF
Viral= normal glucose
Bacterial or fungal= low glucose
once blood is collected, how is it distributed in vials
half in a viral for anerobic organisms
half in a vial for aerobic organisms
when is a broncho-alveolar lavage used
if a sample needs to be taken but no sputum is being produced for collection
how does ViTEK identify organisms
based on culture characteristis
how does MALDI-ToF identify organisms
based on molecular signatures of different bacteria/fungi/parasites
how does BioFire identify an organism
by testing for the presence or absence of multiple genes simultaneously
why is MALDI-ToF not often used in hospital
it is expensive for the machine and subscription to databases
what is the main advantages of MALDI-ToF to identify an organism
results within 2 hrs= faster diagnosis
what are the 2 main advantages of BioFire to identify an organism
organisms don’t have to be grown (uses the direct sample)
primers cover the most common pathogens with the symptoms present
what agar is used for the Kirby Bauer test
Mueller Hinton
how does minimum inhibitory concentration differ from minimum bacterial concentration
MIC- lowest concentration to prevent bacterial growth
MBC- lowest concentration to kill bacteria
what is the difference between primary cell culture vs diploid cell strain vs continuous cell line
primary- cells taken from a living animal, keep alive as long as possible
diploid- cells of single cell type can be cultured approx. 50 times
continuous- malignant cells can be cultured an infinite number of times
what is the cytopathic effect with viruses
even if we can’t see visible virus, we know there was infections based on changes in the cell that the virus made
if a virus is a DNA viral, inclusion bodies will be found in __. If RNA, they will be found in __
DNA- nucleus
RNA- cytoplasm
what is used to determine the number of viruses in a sample
viral plaque assay
more virus= more cells killed= more plaques
for viruses, samples need to be collected within __ days of symptoms
3-7
viral samples for nucleic acid detection must be kept at __C or frozen at __
4C
-70C
for detection of various viral strains, what are primers used to detect
hemagglutinin and neuramidase (each are strain specific)
for use of ELISA to detect viral proteins, how is it performed
add specific antibodies to blood
antigen will bind to antibody
add a secondary antibody to visualized bound antigens
what question is hemagglutination answering
how much virus is there
how is hemagglutination interpreted
the more virus, the more hemagglutinin, the more clumping (seen fuzzy in well plate)
what question is hemagglutinin inhibition answering
is there antibody that binds to the virus
how is hemagglutination inhibition results interpreted
higher hemagglutinin inhibition= more antibody= less clumping (seen as a button on well plate)
what method of identification would not be used to identify a virus
electron microscopy
how do cassette based tests work
viral proteins are bound to a nitrocellulose membrane
antibodies bind to these proteins
the control line is loaded with IgG so it will always show since the sample will always have IgG
the test line has the peptide of interest so the patient’s antibodies will stick to the antigen
protein A colloid gold conjugated binds to the Fc portion of the antibody allowing for a visible colored time for test results
ELISA and flow cytometry (FACS) both measure protein expression. how do they differ in their measurement
ELISA- protein in serum/culture
flow cytometry (FACS)- protein expression on cells
is fluoride always toxic
no, it depends on the concentration
what would we use to filter bacteria from solution containing it’s toxin
liquid filtration
what size of filtration will not allow bacteria to pass
0.2micrometers
what would we use to check a bacteria sample in saline that is MRSA after inoculation on a plate
McFarland
what agar would be used to isolate intestinal bacteria that are gram -
MacConkey
what does the color change on eosin methylene blue agar indicate about the bacteria
lactose fermenting
what culture method should be used to see if something from a pond can grow on skin
Mannitol salt agar
what culture method would you use to test if an organism can live on human skin
mannitol salt agar
if a bacteria that is beta hemolytic when near bacteria that lyse RBC is grown on its own, what type of agar is needed
chocolate
what test would you use to compare 2 types of pathogens based on 1 gene
PCR
if an adult is suspected of having bacteremia, how would you diagnose it
draw 20mL blood and divide half and half into an anaerobic tube and an aerobic tube
why would a urine sample contain erythrocytes and gram positive and negative bacteria, but no leukocytes
contamination by normal vaginal flora and menstrual blood
why would we use a monoclonal antibody
it has high specificity
what benefits does immunohistochemistry provide
it allows us to visualize specific locations of cells and tissues that were targeted
what would be the secondary reagent in an indirect fluorescent antibody test
fluorochrome bound to an anti-human Ig
