question
invasion
answer
attachment to host is first step: use pili
evade host is second step: may produce capsules
evade host is second step: may produce capsules
question
four most critical environmental factors to consider
answer
o2/co2 availability
temperature
pH and moisture content
atmosphere of incubation
temperature
pH and moisture content
atmosphere of incubation
question
capsule
answer
coat that prevents phagocytic cells from engulfing them
question
Aerobic
answer
requires oxygen
question
Two types of toxins
answer
exotoxins and endotoxins
question
Anaerobic
answer
without oxygen
question
Exotoxins
answer
toxic substances, produced by bacteria through metabolizing nutrients, that bacteria secrete into their environment
question
facultative anaerobe
answer
can live with or without oxygen
question
Endotoxins
answer
lipopolysaccharide component of the outer membrane of gram- bacteria that is released after cell death
usually worse than exotoxins
usually worse than exotoxins
question
Aerotolerant
answer
do not utilize oxygen but can survive and grow slowly in its presence
question
what is usually the first step in any micro identification scheme
answer
gram stain
question
Microaerophiles
answer
require oxygen concentration lower than air
question
capnophile
answer
grows best at higher CO2 tensions than normally present in the atmosphere (5-10%)
question
four main categories of gram stain
answer
gram+ cocci
gram- cocci
gram+ bacilli
gram- bacilli
gram- cocci
gram+ bacilli
gram- bacilli
question
most aerobes are actually
answer
facultative anaerobes
only a select few are strict aerobes
only a select few are strict aerobes
question
morphologically indistinct are described as
answer
gram- coccobacilli
gram variable bacilli
pleomorphic
gram variable bacilli
pleomorphic
question
temperature requirements of most medically important bacteria
answer
35-37 (body temp)
question
how do most gram+ react to vancomycin
answer
susceptible
question
how do most gram- react to vancomycin
answer
resistant
question
temperature requirements of nonpathogenic organisms
answer
30 (surface body temp)
yeast likes this temp
yeast likes this temp
question
most common method to determine genus and species of bacteria
answer
determining nutritional and metabolic capabilities
question
temperature requirements for campylobacter
answer
42
question
ph of most clinically relevant bacteria
answer
6.5-7.5
question
enzyme tests
answer
driving force in bacteria metabolism
direct reflection of organisms genes
direct reflection of organisms genes
question
moisture content of bacterial growth
answer
don't dry them out, need humidity
increased solute concentration in media can osmotically shock the bacterial cell and cause lysis
no water = no metabolism
increased solute concentration in media can osmotically shock the bacterial cell and cause lysis
no water = no metabolism
question
catalase test
answer
H2O2 + catalase = h20 + o2
bubble formation is positive for the presence of catalase
used to differentiate between gram+ organisms, mainly staph and strep
staph is +
strep is -
listeria monocytogenes and corynebacterium are +
Enterococci and nonspore forming bacilli are -
bubble formation is positive for the presence of catalase
used to differentiate between gram+ organisms, mainly staph and strep
staph is +
strep is -
listeria monocytogenes and corynebacterium are +
Enterococci and nonspore forming bacilli are -
question
purpose of candle jars
answer
create an atmosphere containing 3-5% co2
used to grow certain fastidious organisms
used to grow certain fastidious organisms
question
oxidase test
answer
identifies bacteria that have cytochrome oxidase which participates in electron transport and in nitrate metabolic pathways which produces indolphenol oxidase
indolphenol oxidase + reagent = dark purple
used to differentiate between gram- organisms
pseudomonas +
enterobacteria -
MUST use BAP or any plate without fermentable sugar
indolphenol oxidase + reagent = dark purple
used to differentiate between gram- organisms
pseudomonas +
enterobacteria -
MUST use BAP or any plate without fermentable sugar
question
most bacteria grow within how many hours upon incubation
answer
24-48
question
indole test
answer
tryptophan broken down into pyruvic acid, ammonia, and indole
used to identify enterobacteria
spot test: blue color
tube test (kovacs reagent): red color
e coli is +
used to identify enterobacteria
spot test: blue color
tube test (kovacs reagent): red color
e coli is +
question
urease test
answer
urease + urea = ammonia + h20 + co2
ammonia increases pH
pH indicator turns from beige to pink
proteus is +
ammonia increases pH
pH indicator turns from beige to pink
proteus is +
question
streaking for isolation
answer
streaked in quadrants so that individual bacterial colonies are obtained and a relative number of organisms can be estimated based on growth past the original area of inoculation
question
why are calibrated loops used to inoculate plates
answer
to quantify colony forming units (cfu)
must spread innoculum evenly over entire plate
technique used for urines
must spread innoculum evenly over entire plate
technique used for urines
question
pyr test
answer
identify gram+ cocci
bright red color
strep pyogenes (group a) and Enterococcus are +
other streps are -
bright red color
strep pyogenes (group a) and Enterococcus are +
other streps are -
question
what are some criteria for establishing the organisms clinical significance
answer
predominance of organism
direct smear results (gram stain)
virulence
body site it was obtained from
direct smear results (gram stain)
virulence
body site it was obtained from
question
Hippurate hydrolysis
answer
production of glycine from hydrolysis
produces deep purple color
used to ID GBS (strep agalactiae)
produces deep purple color
used to ID GBS (strep agalactiae)
question
coagulase test
answer
measure if organism can clot plasma protein fibrinogen
staph aureus is +
staph epidermidis is -
card/slide: agglutination
tube: inoculate, incubate 4 hrs at 37, clumping
staph aureus is +
staph epidermidis is -
card/slide: agglutination
tube: inoculate, incubate 4 hrs at 37, clumping
question
criteria to characterize bacterial growth
answer
colony size
colony pigmentation
colony shape
colony surface appearance
changes in agar
odor
colony pigmentation
colony shape
colony surface appearance
changes in agar
odor
question
subculture
answer
used to obtain a pure culture so that identification can be done
question
purpose of acetamide
answer
deamination process
used to identify nonfermenting gram- rods such as pseudomonas aeruginosa
used to identify nonfermenting gram- rods such as pseudomonas aeruginosa
question
saprophytes
answer
nonpathogenic which benefits from humans as a source of nutrients, warmth, moisture but does not help or harm the host
question
purpose of bile solubility
answer
differentiate strep pneumoniae from alpha hemolytic strep
tube test: clear is + and cloudy suspension is -
plate test: colonies flatten or disintegrate is + and no change is -
tube test: clear is + and cloudy suspension is -
plate test: colonies flatten or disintegrate is + and no change is -
question
purpose of citrate test
answer
identify enterobacteria
positive test goes from green to blue
salmonella and klebsiella are +
positive test goes from green to blue
salmonella and klebsiella are +
question
pathogen
answer
A disease causing agent
question
colonizers
answer
become normal flora
beneficial because they compete with pathogens for space/nutrients, they also keep immune system stimulated
beneficial because they compete with pathogens for space/nutrients, they also keep immune system stimulated
question
purpose of CAMP test
answer
used to identify strep agalactiae (group b)
on BAP, single streak of staph aureus down center of plate
streak sample perpendicular to center line, do not touch but get close
after incubation, an arrowhead hemolysis occurs if +
on BAP, single streak of staph aureus down center of plate
streak sample perpendicular to center line, do not touch but get close
after incubation, an arrowhead hemolysis occurs if +
question
opportunistic
answer
cause disease only if favorable circumstances exist
question
cetrimide purpose
answer
used to identify pseudomonas aeruginosa
slants incubated aerobically at 35-37
examine daily for 4 days, reexamine at day 7 then discard
growth and yellow-green to dark blue-green color is +
no growth is -
slants incubated aerobically at 35-37
examine daily for 4 days, reexamine at day 7 then discard
growth and yellow-green to dark blue-green color is +
no growth is -
question
decarboxylase test purpose
answer
differentiate between enterobacteriaceae
ability of organisms to produce arginine, lysine, or ornithine decarboxylase
purple is +
yellow is -
ability of organisms to produce arginine, lysine, or ornithine decarboxylase
purple is +
yellow is -
question
what do doctors rely on micro to do
answer
isolate, identify, and determine susceptibility of organisms
question
best id methods for micro
answer
rapid
sensitive
accurate
readily available
sensitive
accurate
readily available
question
Esculin Hydrolysis Test purpose
answer
used to identify Enterococcus and strep group D
black slant with loss of fluorescence under wood's lamp is +
tan/brown slant with no loss of fluorescence under wood's lamp is -
black slant with loss of fluorescence under wood's lamp is +
tan/brown slant with no loss of fluorescence under wood's lamp is -
question
fermentation media purpose
answer
determine ability of organisms to ferment a specific carb
peptone media: enterobacteria and coryneforms
pink is + (with gas: e coli, without: shigella)
yellow is -
bhi media: strep and Enterococcus
yellow is +
purple is -
peptone media: enterobacteria and coryneforms
pink is + (with gas: e coli, without: shigella)
yellow is -
bhi media: strep and Enterococcus
yellow is +
purple is -
question
ID methods that use phenotypic criteria
answer
physical/metabolic characteristics that are readily observed
size, shape, color, +/- hemolysis, pigment production, staining characteristics
size, shape, color, +/- hemolysis, pigment production, staining characteristics
question
ID methods that use physiological characteristics
answer
motility, spore formation, capsule production
question
Gelatin Hydrolysis Test purpose
answer
production of proteolytic enzymes that liquefy gelatin
complete/partial liquid is + (proteus vulgaris)
solid is - (enterobacter aerogenes)
complete/partial liquid is + (proteus vulgaris)
solid is - (enterobacter aerogenes)
question
ID methods that use biochemical reactions
answer
carb utilization, amino acid degradation, enzyme production
question
Indole Production Test purpose
answer
ability to split tryptophan to indole
pink/wine color ring is +
yellow/no color change is -
enterobacteriaceae and other gram- bacilli
pink/wine color ring is +
yellow/no color change is -
enterobacteriaceae and other gram- bacilli
question
ID methods that use molecular techniques
answer
analysis of structure and function of various molecular components
question
Litmus Milk Test purpose
answer
fermentation of lactose
casein coagulation
casein hydrolization
litmus reduction
pink: acid
blue: alkaline
purple: no change
white/colorless at bottom of tube: reduction of indicator
coag: acid or alkaline
shrunken pink clot: acid--digestion
shrunken blue clot: alkaline--peptonization
casein coagulation
casein hydrolization
litmus reduction
pink: acid
blue: alkaline
purple: no change
white/colorless at bottom of tube: reduction of indicator
coag: acid or alkaline
shrunken pink clot: acid--digestion
shrunken blue clot: alkaline--peptonization
question
ID methods that use immunologic/serologic techniques
answer
qualitative/quantitative analysis of antibodies/antigens
question
growth at 42C purpose
answer
does organism grow at this temp
growth at both 35 and 42 is +
no growth at 42 but growth at 35 is -
campylobacter
growth at both 35 and 42 is +
no growth at 42 but growth at 35 is -
campylobacter
question
types of micro specimens
answer
swab
tissue
fluid
tissue
fluid
question
MRVP purpose
answer
ability of organisms to produce and maintain stable acid end products from glucose fermentation to overcome buffering capacity
ability of organisms to produce neutral end products from glucose fermentation
red is +
yellow is -
mr: acid fermentation
vp: acetoin production
opposites, will never be + for both
ability of organisms to produce neutral end products from glucose fermentation
red is +
yellow is -
mr: acid fermentation
vp: acetoin production
opposites, will never be + for both
question
Motility Test purpose
answer
determine whether or not an organism is motile (contains flagella)
hanging drop method:
position change relative to each other is +
remain in same relative position to each other is -
semi solid method:
spreading is +
no change is -
hanging drop method:
position change relative to each other is +
remain in same relative position to each other is -
semi solid method:
spreading is +
no change is -
question
large volumes of fluid need to be
answer
concentrated
by centrifugation or filtration
use sediment to inoculate
by centrifugation or filtration
use sediment to inoculate
question
nitrate reduction purpose
answer
differentiates based on ability to reduce nitrate to nitrite or nitrogen gas
read for presence or absence of three metabolic products: gas, no3, and no2
read for presence or absence of three metabolic products: gas, no3, and no2
question
micro specimen preparation
answer
swabs: need two, can use directly or place in broth/saline
tissue: homogenized- ground/cut into small pieces
fluid: concentrated, if filtered, place filter directly on media
tissue: homogenized- ground/cut into small pieces
fluid: concentrated, if filtered, place filter directly on media
question
how should plates be incubated
answer
upside down, lid on bottom
question
ONPG Purpose
answer
ability to produce b-galactosidase
differentiate between late lactose fermentation and true nonlactose fermentation
yellow is +
clear is -
differentiate between late lactose fermentation and true nonlactose fermentation
yellow is +
clear is -
question
mutualism
answer
both organisms benefit
question
optochin test purpose
answer
optochin lyses pneumococci is + (zone of inhibition)
alpha strep is resistant is - (no zone of inhibition)
alpha strep is resistant is - (no zone of inhibition)
question
of medium purpose
answer
oxidative fermentative media
carb usage to produce acid byproducts
six common: glucose, xylose, mannitol, lactose, sucrose, maltose
fermenter: open and sealed tubes yellow
oxidizer: open tube yellow, sealed tube red
nonfermenter/nonoxidizer: open and sealed tubes both red
carb usage to produce acid byproducts
six common: glucose, xylose, mannitol, lactose, sucrose, maltose
fermenter: open and sealed tubes yellow
oxidizer: open tube yellow, sealed tube red
nonfermenter/nonoxidizer: open and sealed tubes both red
question
commensalism
answer
organism benefits and host is unaffected
question
phenylalanine deaminase purpose
answer
ability of organisms to oxidatively deaminate phenylalanine to phenyl pyruvic acid
green is +
clear/tan is -
green is +
clear/tan is -
question
parasitism
answer
organism benefits and host is harmed
question
pathogen
answer
organism that causes disease
question
Salt tolerance test purpose
answer
ability to grow in a high salt concentration
differentiate Enterococcus and no enterococci
turbidity with or without color change from purple to yellow is +
no turbidity and no color change is -
differentiate Enterococcus and no enterococci
turbidity with or without color change from purple to yellow is +
no turbidity and no color change is -
question
TSI purpose
answer
used to determine whether gram- rods utilize glucose and lactose or sucrose fermentatively and forms h2s
yellow: ferment glucose, sucrose, and/or lactose
red/yellow: glucose fermentation only
red/red: glucose, sucrose and lactose nonutilizer
yellow: ferment glucose, sucrose, and/or lactose
red/yellow: glucose fermentation only
red/red: glucose, sucrose and lactose nonutilizer
question
opportunistic pathogen
answer
cause disease only in a compromised host
question
virulence
answer
variety of microbial factors that contribute to an organisms ability and speed in causing a disease
question
Urea Hydrolysis Test purpose
answer
Agar: Differentiate rapid urease-positive bacteria from slower urease-positive and urease-negative bacteria.
change from orange to magenta is +
no color change, light orange, is -
change from orange to magenta is +
no color change, light orange, is -
question
nitrite purpose
answer
ability to reduce nitrites to gaseous nitrogen
no color change is +
red is -
no color change is +
red is -
question
carrier
answer
individual infected with organism that can be transmitted to another person, carriers are usually asymptomatic
question
Coagulase test purpose
answer
differentiate staph aureus from other staphs by the ability to bind to fibrinogen
clumps/clots is +
no clumping/clotting is -
clumps/clots is +
no clumping/clotting is -
question
zoonosis
answer
infection in animals that can be transmitted to humans
question
defensive mechanisms used by humans
answer
skin and mucous membranes
mucous membrane cleansing activity
antimicrobial substances
phagocytosis
mucous membrane cleansing activity
antimicrobial substances
phagocytosis
question
bile esculin agar purpose
answer
used to identify Group-D streptococci and enterococci through presentation of darkened medium
question
lysine iron agar purpose
answer
determines if gram- rod decarboxylayes or deaminates lysine and forms h2s
glucose ferment: butt yellow
lysine decarboxylase: butt purple
oxidative deamination of lysine: burgundy
no reaction: purple
glucose ferment: butt yellow
lysine decarboxylase: butt purple
oxidative deamination of lysine: burgundy
no reaction: purple
question
intact skin and mucous membranes
answer
first line of defense
innate
physical barriers that prevent entry to body
innate
physical barriers that prevent entry to body
question
mucous membrane cleansing activity
answer
mucous removes organisms by trapping them and respiratory tract had cilia to remove foreign material
question
production of antimicrobial substances
answer
lysosomes, found in tears and saliva, disrupts gram+ organisms
IgA found in mucous membranes
IgA found in mucous membranes
question
phagocytosis
answer
pmn's, macros, monos which are WBCs that ingest and kill microbes
except eosinophils
except eosinophils
question
Host susceptibility factors
answer
nutritional status
age
stress
genes
preexisting conditions
age
stress
genes
preexisting conditions
question
microbial factors
answer
invading
toxins
localization
dissemination
toxins
localization
dissemination
question
zone of inhibition
answer
The zone where bacteria can't grow around a given antibiotic
question
susceptibility
answer
lack of resistance
question
common susceptibilities
answer
gram+ : vanc suscept, colistin resist
gram- : vanc resist, colistin suscept
group a strep: suscept to bacitracin
strep pneumoniae: suscept to optochin
gram- : vanc resist, colistin suscept
group a strep: suscept to bacitracin
strep pneumoniae: suscept to optochin
question
vancomycin susceptibility helps to do what when identifying organisms
answer
establish gram status when the organism is an anaerobe and the gram stain is questionable
question
colistin susceptibility helps to do what when identifying organisms
answer
establish gram reactivity especially in anaerobes
question
chromogenic substrate
answer
A colorless subtance that is transformed into colored products by an enzymatic reaction
question
fluorogenic substrate
answer
enzymatic cleavage of these substrates yields a fluorescent compound which can be measured
question
two methods of identifying organisms based on susceptibility
answer
API: manuel
vitek: automated
vitek: automated
question
molecular diagnostics of organisms
answer
involves the use of nucleic acids (genomics) and proteins (proteomics) to diagnose and monitor disease
question
genomics
answer
study of whole genomes
question
proteomics
answer
study of the structure and function of proteins in the human body
question
nucleic acid probes
answer
locate nucleotide sequence to rapidly and definitively identify organisms
question
why are nucleic acid probes beneficial
answer
rapid/definitive
works on fastidious/difficult to grow
safe for dangerous organisms
works on fastidious/difficult to grow
safe for dangerous organisms
question
nucleic acid synthesis
answer
dna/rna chains are copied and reproduced as a complementary sequence
dna -> rna -> protein
dna -> rna -> protein
question
Retroviruses
answer
use reverse transcriptase to copy their RNA genome into DNA
rna -> dna -> rna -> protein
rna -> dna -> rna -> protein
question
Nucleotide
answer
monomer of nucleic acids made up of a 5-carbon sugar, a phosphate group, and a nitrogenous base
question
Ribonucleotides
answer
RNA
question
deoxyribonucleotides
answer
DNA
question
replication
answer
process of copying DNA prior to cell division
question
transcription
answer
synthesis of an RNA molecule from a DNA template
question
how does nucleic acid probes technology work
answer
uses labeled / tagged nucleic acid segments to detect organisms
dna is most popular
dna is most popular
question
what is the first step of DNA probe preparation
answer
isolate a segment of the DNA strand for which the nucleotide base sequence is known
question
prep of nucleic acid probe
answer
DNA cut into small segments using restriction endonucleases
gel electrophoresis is then used to sort fragments
desired fragment is then inserted into carrier (plasmid or virus) which is then cloned by host OR use amplification technique (pcr) to make multiple copies of the desired fragment
plasmids then released from bacterial cell by lysis
DNA fragment then removed from plasmid using the same endonuclease used to insert it
purified by electrophoresis in an agarose gel
gel electrophoresis is then used to sort fragments
desired fragment is then inserted into carrier (plasmid or virus) which is then cloned by host OR use amplification technique (pcr) to make multiple copies of the desired fragment
plasmids then released from bacterial cell by lysis
DNA fragment then removed from plasmid using the same endonuclease used to insert it
purified by electrophoresis in an agarose gel
question
bacteriophage
answer
A virus that infects bacteria
question
plasmid
answer
small, circular piece of DNA located in the cytoplasm of many bacteria
question
annealing process
answer
joining DNA fragment with the plasmid DNA which is done using ligases
question
labeling nucleic acid probes happens after prep of probe and is done by
answer
radioactive isotopes
nonradioactive isotopes (32P and 35S)
biotin
nonradioactive isotopes (32P and 35S)
biotin
question
Radiolabeling
answer
radioactive isotope added to the DNA fragment
adv: sensitive, very tiny quantities needed
dis: special equipment, training, variable activity, decay, disposing
adv: sensitive, very tiny quantities needed
dis: special equipment, training, variable activity, decay, disposing
question
after nucleic acid probe prep and labeling what happens
answer
labeled probe must be separated into separate strands to be used
denaturation/separation of the two strands done with detergents, enzymes, naoh, or heat
denaturation/separation of the two strands done with detergents, enzymes, naoh, or heat
question
what is a successful probe
answer
probe will hybridize/combine with either DNA/RNA of target organism but does not hybridize with nucleic acids from other organisms that may be in sample
question
how are positive reactions or hybridization seen
answer
adding avidin conjugated with an enzyme such as alkaline phosphate
fluorescein attached to antibodies which conjugate to visualize the presence of biotin labeled probe in place of the enzyme labeled avidin
fluorescence can be visualized using uv light, very sensitive
fluorescein attached to antibodies which conjugate to visualize the presence of biotin labeled probe in place of the enzyme labeled avidin
fluorescence can be visualized using uv light, very sensitive
question
How do DNA probes work?
answer
cells are disrupted and DNA strands separated out
DNA denatured
single DNA strand fixed to solid surface
labeled probe added and react with material on solid support
if complementary sequence is there, probe anneals with target DNA
support medium is washed to remove unbound DNA and nonspecific bound DNA
DNA denatured
single DNA strand fixed to solid surface
labeled probe added and react with material on solid support
if complementary sequence is there, probe anneals with target DNA
support medium is washed to remove unbound DNA and nonspecific bound DNA
question
modified gen probe
answer
detects complementary strands of ribosomal rna
question
advantage of modified gen probe
answer
only one copy of complementary dna per cell but thousands of ribosomal rna segments in same cell
rna are already single stranded and ready to hybridize without the denaturing step
has in solution approach, no solid support system
rna are already single stranded and ready to hybridize without the denaturing step
has in solution approach, no solid support system
question
pna probe
answer
peptide nucleic acid probe with fluorescence in situ hybridization (fish) for rapid identification of organisms in blood cultures
no sugar phosphate backbone, making charge more neutral, neutrality allows a more rapid and tight bind
no sugar phosphate backbone, making charge more neutral, neutrality allows a more rapid and tight bind
question
pcr
answer
combination of complementary nucleic acid hybridization and nucleic acid replication to allow a single nucleic acid target to be multiplied 10E7 or great, improving sensitivity
question
PCR process
answer
1. Denaturation: heating sample to 94°
2. Annealing: cool sample to 50-58°
3. Extension: extends the primer-target duplex using DNA polymerase at 72°, adds nucleotides to the 3' end, taq polymerase catalyzes primer extension
2. Annealing: cool sample to 50-58°
3. Extension: extends the primer-target duplex using DNA polymerase at 72°, adds nucleotides to the 3' end, taq polymerase catalyzes primer extension
question
thermal cycler
answer
carries the pcr mixture thru each reaction step at the precise temp and optimal time
question
amplicon
answer
PCR product
question
Real-time PCR
answer
performs amplifcation and detection steps at the same time
question
Restriction Fragment Length Polymorphisms (RFLPs)
answer
Differences in DNA sequence on homologous chromosomes that can result in different patterns of restriction fragment lengths (DNA segments resulting from treatment with restriction enzymes).
less exacting than nucleic acid sequencing
less exacting than nucleic acid sequencing
question
immunologic assays
answer
to observe/measure antigen/antibody reactions with the help of a secondary phenomenon (something must be added in addition to the antigen/antibody binding)
question
titer
answer
reciprocal of the highest dilution that shows visible reaction
question
measurable consequences of immunologic assays
answer
precipitation
agglutination
complement fixation
labeling
agglutination
complement fixation
labeling
question
precipitation immunologic assays
answer
both antigen and antibody are soluble
crosslinking of antigen and antibody results in large lattice formation which eventually becomes insoluble and precipitates out of solution
crosslinking of antigen and antibody results in large lattice formation which eventually becomes insoluble and precipitates out of solution
question
Zone of equivalence
answer
proportion of antigen to antibody is equal or close in concentration and will give a visible precipitate
question
prozone
answer
excess antibody
question
postzone
answer
excess antigen
question
precipitation ring formation
answer
solutions containing antigens and antibodies are carefully layered on top of one another (avoid mixing) in test tube
antigen and antibody diffuse toward each other and form a ring or band of visible precipitate at the zone of equivalence
method does not allow quantitation
antigen and antibody diffuse toward each other and form a ring or band of visible precipitate at the zone of equivalence
method does not allow quantitation
question
Nephelometry
answer
Measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension (10 to 90 degrees, usually 70)
precipitate formed by antigen and antibody produces increased reflection, measured by calculating turbidity
good for high throughput but expensive
inaccurate results from unclean/scratched tubes, hemolysis, lipemia
precipitate formed by antigen and antibody produces increased reflection, measured by calculating turbidity
good for high throughput but expensive
inaccurate results from unclean/scratched tubes, hemolysis, lipemia
question
Ouchterlony Double Diffusion assay
answer
antigen and antibody are put on opposite sides of a gell, they diffuse slowly toward each other on the gell in an inc radius
identity: percipitate lines form a smooth continuous curve connecting the centers of the two wells
partial identify: precipitation lines form a continuous curve with a spur sticking out
no identity: the two precipitation lines criss cross each other
allows semi quantification
identity: percipitate lines form a smooth continuous curve connecting the centers of the two wells
partial identify: precipitation lines form a continuous curve with a spur sticking out
no identity: the two precipitation lines criss cross each other
allows semi quantification
question
radial immunodiffusion rid
answer
Single diffusion of the antigen into a gel that already contains the antibody throughout
quantitative
replaced by elisa due to frequent errors
quantitative
replaced by elisa due to frequent errors
question
Zone electrophoresis
answer
separation of antigens/antibodies using electric current into 5 areas (albumin, alpha 1, alpha 2, beta, and gamma)
strips are stained and read with densitometer
strips are stained and read with densitometer
question
immunoelectrophoresis
answer
combines electrophoresis with immunodiffusion
electrophoresis done first, once separated, antibody prep is added in trough causing antigen/antibody complex to diffuse and form bands of precipitate
used for identification and quantitation
replaced by immunofixation, immunoblotting, and latex agglutination
electrophoresis done first, once separated, antibody prep is added in trough causing antigen/antibody complex to diffuse and form bands of precipitate
used for identification and quantitation
replaced by immunofixation, immunoblotting, and latex agglutination
question
Counterimmunoelectrophoresis
answer
performed in agar where pH allows antigen and antibody to have opposite charges
electric current forces them to migrate toward each other
faster than ouchterlony and increase sensitivity
electric current forces them to migrate toward each other
faster than ouchterlony and increase sensitivity
question
rocket electrophoresis
answer
variation of rid: pH of gel is such so that antibodies remain immobile in gel and antigen has negative charge
height of band precipitate is proportional to antigen concentration
can measure either antigen or antibody
height of band precipitate is proportional to antigen concentration
can measure either antigen or antibody
question
Western blot
answer
electrophoresis followed by stain with antibody attached
used for hard to diagnose or ambiguous diagnoses
widely used to diagnose HIV
used for hard to diagnose or ambiguous diagnoses
widely used to diagnose HIV
question
specific proteins for hiv
answer
gagdna
pol
p17
p55
envgene
p24
kp41
pol
p17
p55
envgene
p24
kp41
question
flocculation test
answer
test for antibody detection based on interaction of soluble antigen with antibody, results in the formation of a precipitate of fine particles
vdrl
reagin produced by syphilis patients
replaced by rpr
vdrl
reagin produced by syphilis patients
replaced by rpr
question
agglutination methods
answer
antibodies cross link with cells causing clumping
more sensitive than precipitation
IgM best agglutinators (10 potential binding sites)
more sensitive than precipitation
IgM best agglutinators (10 potential binding sites)
question
type specific antibody
answer
used to identify or serotype organisms
animals injected with killed organisms causing it to produce polyspecific antibody
animals injected with killed organisms causing it to produce polyspecific antibody
question
agglutination of bacteria/other microbes
answer
bacteria + antibody = small clumps visualized with light microscopy
question
Febrile agglutinin
answer
fever occurs with antigen and antibody come together
question
agglutination using particles coated with antigen or antibody
answer
soluble antigen or antibody is attached to inert particles
bound antigens: antibodies in test cause particles to agglutinate if they are specific for the antigen
bound antigens: antibodies in test cause particles to agglutinate if they are specific for the antigen
question
hemagglutination tests
answer
agglutination of RBCs
active (direct): antigens expressed on RBCs themselves - integral part of cell
indirect: antibodies that bind to RBCs (IgG), a second antibody specific for immunoglobulin is added to induce agglutination, RBCs are carrier particles
passive: soluble antigen adsorbed to rbc then reacted with antibody - modified with tannic acid
micro hemagglutination: soluble antigen adsorbed to RBCs, no longer done
inhibition: antibodies against hemagglutinin molecule on surface preventing agglutination-- no agglutination is positive
active (direct): antigens expressed on RBCs themselves - integral part of cell
indirect: antibodies that bind to RBCs (IgG), a second antibody specific for immunoglobulin is added to induce agglutination, RBCs are carrier particles
passive: soluble antigen adsorbed to rbc then reacted with antibody - modified with tannic acid
micro hemagglutination: soluble antigen adsorbed to RBCs, no longer done
inhibition: antibodies against hemagglutinin molecule on surface preventing agglutination-- no agglutination is positive
question
complement methods
answer
circulate inactive, once active, interact in specific sequence
antigen/antibody complexes in solution/on surface of cells, then active complement with IgG or IgM
if RBCs, lysis will occur
antigen/antibody complexes in solution/on surface of cells, then active complement with IgG or IgM
if RBCs, lysis will occur
question
complement fixation
answer
An immune response in which antigen-antibody complexes activate complement proteins
amount of complement used to proportional to antibody concentration
amount of complement used to proportional to antibody concentration
question
assay for complement
answer
amount of sera needed to lyse 50% sheep RBCs that have been sensitized with IgG -- AKA CH50
measure hemolysis on BAP
measure hemolysis on BAP
question
Immunofluorescence
answer
a method of tagging antibodies with a fluorescent dye to detect or localize antigen-antibody combinations
common dyes: fitc or tritc
common dyes: fitc or tritc
question
Flow cytometry
answer
fluorescence activated cell sorter; laser technology
sorts based on surface antigens, size, granularity
can sort 1000s per second
sorts based on surface antigens, size, granularity
can sort 1000s per second
question
radioimmunoassay (RIA)
answer
used to quantify very small quantities
competition between radiolabeled and unlabeled antigen for the same antibody
unlabeled (cold) antigen increased, more binds to antibody, displacing radiolabeled (hot) antigen
competition between radiolabeled and unlabeled antigen for the same antibody
unlabeled (cold) antigen increased, more binds to antibody, displacing radiolabeled (hot) antigen
question
Enzyme Immunoassay (EIA)
answer
a serological test that uses an enzyme-labeled antibody reactant
sensitive and specific
color change
qualitative and quantitative
qual: color
quant: intensity
sensitive and specific
color change
qualitative and quantitative
qual: color
quant: intensity
question
enzyme-linked immunosorbent assay (ELISA)
answer
can detect unknown antibody or antigen
sensitive, safe, economical, simple, versatile, minimum instrumentation
sensitive, safe, economical, simple, versatile, minimum instrumentation
question
ELISA assay
answer
antigen adsorbed to well/plate
patient sera add (potential 1st antibody)
will bind with antigen if specific for it
unbound washed off
antihuman antibody conjugated to enzyme is added (2nd antibody)
if 1st antibody present, 2nd will bind
unbound washed off
colorless substrate added, if acted on by enzyme, produces color
color is read and concentration is determined
patient sera add (potential 1st antibody)
will bind with antigen if specific for it
unbound washed off
antihuman antibody conjugated to enzyme is added (2nd antibody)
if 1st antibody present, 2nd will bind
unbound washed off
colorless substrate added, if acted on by enzyme, produces color
color is read and concentration is determined
question
Solid phase immunoassays
answer
antibody attached to plate or bead
sample added (possible antigen)
unbound washed off
2nd antibody added which is complexes to enzyme
causes color change
sample added (possible antigen)
unbound washed off
2nd antibody added which is complexes to enzyme
causes color change
question
membrane bound spia
answer
flow thru nitrocellulose, nylon or other membrane used in elisa
antigen or antibody captured in matrix of membrane
causes color change
antigen or antibody captured in matrix of membrane
causes color change
question
colony forming unit (CFU)
answer
a single cell or group of related cells that produce a colony
question
Acetamide utilization
answer
Principle: bacteria cause deamination of acetamide and release ammonia, which increases pH of medium
+ results: royal blue
- results: green
Pseudomonas aeruginosa + Stenotrophomonas maltophilia -
+ results: royal blue
- results: green
Pseudomonas aeruginosa + Stenotrophomonas maltophilia -
question
Cetrimide
answer
toxic substance that inhibits growth of many bacteria
+ growth
- no growth
pseudomonas aeruginosa is +
e coli is -
+ growth
- no growth
pseudomonas aeruginosa is +
e coli is -
question
Citrate Utilization
answer
pos/neg indicated by growth or no growth
pos will sometimes change indicator from green to blue
pos: klebsiella pneumoniae
neg: e coli
pos will sometimes change indicator from green to blue
pos: klebsiella pneumoniae
neg: e coli
question
indole
answer
tryptophan to indole
pos: pink/wine
neg: no color change, clear tan
pos: e coli
neg: klebsiella pneumoniae
pos: pink/wine
neg: no color change, clear tan
pos: e coli
neg: klebsiella pneumoniae
question
lysine iron
answer
K/K: purple/purple : salmonella
K/A: purple/yellow: shigella
Red/yellow: proteus vulgaris
K/A: purple/yellow: shigella
Red/yellow: proteus vulgaris
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