lab practical Flashcards
What rapid tests are used in the lab?
Immuno-based assays
What are rapid strep tests for?
Diagnosis of Strep. Pyogenes
what type of antigens do strep tests look for?
group A strepococcal antigens
what binds to an antigen when it has dye attached?
antibodies
what is being detected in an immuno-based assay test?
a protein that is specific to a pathogen or a condition
what is the most common point-of-care testing format?
rapid immunoassays that employ test strips
what are point-of-care tests?
tests that are conducted by healthcare workers or patient without the need for any other lab equiptment
if the specimen being tested contains Group A strep bacteria, the strep antigens extracted from the throat swab will what?
bind to these antibodies
what is the “test” region for the strep test?
retention of the dye-colored antigen/antibody complexes
what indicates that the sample is positive for Group A strep?
colored line at the test line region
what does the test region also contain in the strep A test?
fixed antibodies that will bind directly to dye-labeled antibodies
what does a strep test positive result look like?
2 lines running through the test region and control region
what does a strep negative result look like?
one line in the control region
what does an invalid result look like in a strep test?
one line in the test region
are rapid strep tests foolproof?
no
what is involved in a rapid staph test?
latex bead agglutination tests
how is a rapid staph test done?
coated latex beads are mixed with the test organism and allows to interact
what does a positive rapid staph test look like?
beads will appear and clump up
what does a negative rapid staph test look like?
the beads will reman in a a homogenous suspension
what type of species is the rapid staph test specific to?
staph. aureus
what do all pathogenic strains of S. aureus produce?
a bound coagulase and contain Protein A on their cell surface
what are the latex particles in a rapid staph test coated with?
human fibrinogen and IgG
what are the steps of a rapid strep test?
- add 4 drops of regeant A to the tube
- add 4 drops of reagent B to the tube
- place throat swab in the tube
- squeeze the swab against the tube
- remove group A test strip and put into tube
- read results
what color will the solution be after adding reagent A in a rapid strep test?
light red
what color will the solution be after adding the reagent B in a rapid strep test?
will turn a pale yellow
how many times do you agitate the swab in the tube for the rapid strep test?
10 times
how long is the throat swab left in the tube of the strep test?
1 minute
what is the correct end of the group A test strip?
wavy lines
how long does it take to read the results of a rapid strep test? when do you not read the results?
- 5 min
- 10 min
what are the steps of a rapid staph test?
- make sure the card is flat
- dispense 1 drop of test latex onto both of the circles on the reaction card
- dispense 1 drop of negative control solution onto one circle on the reaction card
- use a sterile loop and smear a colony onto the other test-latex-containing circle
- pick up a hand rock card
- dispose of the card
how long should you hand rock the rapid staph test card?
20 seconds
should the control latex agglutinate?
no
why is the test region placed before the control region on the rapid strep test?
so the antibodies can reach the test region but not reach the control region
what is critical throughout the unknown project?
to use appropriate aseptic technique to prevent introducing environmental organisms into your cultures
what is the initial step of the unknown isolation?
gram staining
what is an essential step for most microbial identification strategies?
obtaining pure cultures
what are the steps of part 1 of the unknown project?
- observation of mixed culture by gram staining
- obtaining isolated colonies
- identify isolated colonies that are pure by observing gram stains
- inoculate stock slants with bacteria from a isolated and pure colony
- confirm the purity of your stock slants
why do we gram stain our unknown project in the beginning?
to confirm mixed culture and assess arrangement
what are the 3 types of agars we use in our isolation streaks?
- nutrient/ TSA
- PEA
- MacConkey
is the TSA gram positive, negative or both?
both
is the PEA gram positive, negative or both?
gram positive
do we use the MacConkey agar to identify pure colonies?
no
what type of test did we do to properly classify gram positive organisms?
catalase test
what is the catalase test used to do?
differentiate staphlyococcal species from strep/enterococcal species
what is the catalase test based on?
how these organisms detoxify hydrogen peroxide
what exactly is catalase?
an enzyme that converts hydrogen peroxide into water and molecular oxygen
what is the catalase test’s first step?
collect a sample of gram positive organism
what is the catalase test’s 2nd step?
place 2 drops of 3% hydrogen peroxide into end of swab
what is the last step in the catalase test?
immediately look for bubbling reaction and record results
what is the presence of bubbles mean in the catalase test?
indicates the presence of catalase and positive test result
is the novobiovin sensitivity and growth characteristics on MH agar s catalase positive or catalase negative test?
catalase positive
what plate is divided in two sections in the Novobiocin sensitivity test?
Muller-hinton plate
what does college gram positive culture create?
a bacterial lawn on top portion
what do you put on the top half of the MH plate?
Novobiocin
is the DNAse agar plate preformed for a catalase positive or negative test?
catalase positive
how is the DNAse agar plate test done?
- collect gram positive and inoculate the DNAse agar plate
2. tape the plate closed and incubate at 37C
what are the steps in the Mannitol salt agar plate?
- label
- inoculate the MSA with a straight line streak
- tape plate closed
what are the steps in the PYR broth?
- label
- inoculate the broth
- incubate
what are catalase positive tests?
- Novobiocin sensivity
- DNAse agar streak
- MSA agar
- PYR broth
what does the PYR broth test for?
aminopeptidase PYR
what does the MSA agar plate test for?
Mannitol Fermentation
what does the DNAse agar test for/
DNAse activity
what does the MH agar plate test for?
Novobiocin sensitivity or resistance
what are the types of catalase negative tests?
- STX sensitivity test
- BEA slant streak
- PYR broth
what type of bactrim STX grow on?
blood agar
what type of media is blood agar?
differential and enriched
what is beta hemolysis?
complete lysis of red blood cells
what is alpha hemolysis?
partial hemolysis of red blood cells
what are the three types of hemolysis in bacterium STX test?
- beta
- alpha
- none
what type of medium is BEA?
selective and differential
what does the BEA test focus on?
positive or negative for esculin hydrolysis
what is considered a positive test in the BEA slant?
50% of slant should be grey/black
is the PYR broth a test in both gram positive cocci tests?
yes
what does a positive result look like in the PYR broth?
a red color will be produced
what does a negative result look like in the PYR test?
light orange color
what does a positive novobiocin sensitivity test?
zone of inhibition > 16mm
what is a negative novobiocin sensitivity test?
less than 16mm it is resistant
what is a positive DNAse agar test results look like?
a clear zone is observed
what is a positive MSA result look like?
zone of inhibitation >20mm
what is a negative blood agar plate result look like?
less than 20mm is resistant
what type of media is MSA?
selective and dfferential
what does the MSA contain?
7.5% NaCl
what is a positive result of MSA test?
phenol red turns yellow
colonies that are >3mm are considered?
large
colonies that are <1mm are considered?
small
what is the universal virulence factor in all gram-negative bacteria?
LPS in outer membrane
what does the universal virulence factor cause?
morbidity and mortality
what is the collection of biochemical tests called that we did on gram negatives?
microgen GNA-ID system
what species of gram negatives did the collection of biochemicals test identify?
enterobacteriaceae species
how many wells were there in the microgen GNA-ID system?
12 wells
what did each well contain?
- dehydration substrates
- indicators in saline
what is the purpose of well 1?
to determine if the organism is able to carry out lysine decarboxylation
why was well 1 overlaid with mineral oil?
to generate an anaerobic environment
what is the purpose of well 2?
to determine if the organism is able to carry out ornthine decarboxylation
why was well 2 overlaid with mineral oil?
well 2 is anaerobic
what is the purpose of well 3?
to see which organisms produce the enzyme thiosulfate reductase
Does Well 3 need an anaerobic or aerobic environment? How is this solved?
- anaerobic
- overlaying mineral oil
what do wells 4,5,6 test for?
the organisms ability to metabolize various carbohydrates
what does well 7 contain?
- ONPG
- nitrate
how many tests does well 7 provide?
2 tests
what does well 7 test for/
first is the apperance of Beta-galactosidase activity
how is the nitrate reduction test preformed in well 7?
- second test after ONPG
- nitrate reagent A added first
- nitrate reagent B added
what happens if no color appears in the nitrate reduction test in well 7?
add a pinch of zinc and it is negative
what is the purpose of well 9?
to detect urease activity
what is the purpose of well 10?
to detect bacteria that ferment glucose but only produce one acid end produce
what test is done in well 10?
VP test
how long does the VP test in well 10 take?
15-30 min
what does well 8 test for?
- presence of tryptophanase
- indole production
what is added to well 8?
kovac’s reagent
what does well 11 test for?
citrate utilization
what does well 12 test for?
trypotphan deaminase activity
define bioinformatics
the use of computational tools to address biological questions
what is our primary collections of bioinformatics tools?
NCBI
what is the NCBI a division of?
the national library of medicine
what does the NCBI serve as?
a gateway for researchers to access informatio
what does BLAST stand for?
basic local alignment search tool
define BLASTn
uses a neucleotide sequence and searches a nucleotide database
define BLASTp
A nucleotide or amino-acid sequence information
what sequence should the Query sequence be entered in?
FASTA format
define electrophoresis
- a technique that separates molecules
- based on size
define PCR
a technique that can exponentially amplify a fragment of DNA in a test tube
what type of PCR reaction will we preform on our unknown?
in silico
how is the amplicon determined in the PCR reaction?
by primers
define sanger sequencing
technique to sequence DNA
what is important when running a PCR test?
dispense the master mix back into PCR tube
what electrodes should be properly connected?
black and red
When the dark blue running dye has travelled ___ of the length of the gel, turn the power supply off and disconnect the _____
- 3/4
- electrodes
what is fermentation?
a collection of bacterial metabolic processes that engage when respiration (aerobic or anaerobic) is not possible
what does fermentation result in?
the production of acidic waste products
What does the production of acidic products such as lactic acid, propionic acid, and acetic acid through fermentation result in?
conditions that alter the characteristics of the raw food product
Fermentation not only increases the concentration of _____ or _______ (SCFAs), but also the diversity of ______ products
- lactate
- short-chain fatty acids
- metabolic
what are the most commonly comsumed fermented products?
diary
what does adding salt do in vegetable ferments?
support the production of a brine
what illness continue to be a significant source of bacterial disease in he US?
foodborne
what are S&S agar plates selective for?
enteric gram negative organisms
Escherichia species are ____ ____ and appear as ____ colonies on S&S Agar
- lactose positive
- red
Both Salmonella and Shigella species are _____ ___ and appear as __________ colonies on S&S Agar
- lactose negative
- clear/tan
Foodborne Pathogens Lab steps
- Take a ________ __ plate and divide the plate in ___.
- Using a ____, roll over the _____ sample to collect organisms. Then swab the ____ portion of the S&S plate.
- Using a sterilize ____ to collect some bacteria from the ____ of the plate and streak them out across the _____ half of the plate to obtain _______ _____
- Using ______, securely close the plate prior to incubating at 37oC for 48 hours
- S&S agar, half
- swab, meat, loop-loop, top, lower, isolated colonies
- parafilm
