Apr26 M3-Diagnostic Microbiology Laboratory Flashcards
diff bacteria dx methods
- microscopy
- culture
- bacterial ID (biochem tests, ID systems)
- susceptibility testing
- serology
- nucleic acid-based detection (PCR)
steps of gram stain
- heat-firxed smear with specimen stained with crystal violet (purple)
- add iodine fixor (KI)
- alcohol to decolorize
- counterstain with safranin
gram positive cocci in clusters is what organism
staph aureus
gram negative rods: think of what organism
e.coli
gram negative diplococci think of what organism
neisseria meningitidis
acid-fast stain is used for what
mycobacteria spp (includes TB)
acid-fast stain is based on what (what will you stain)
will stain the many LIPIDS on the mycobacteria CELL WALL
acid-fast stain steps
- stain with carbolfuschin (red) + heat (stain mycobacteria red if have any)
- acid alcohol to decolorize
- counterstain with methylene blue (stain background blue for contrast)
partially acid fast organisms
- Nocardia spp (gram+ rod)
- Actinomyces spp (gram+ rod)
- *gram + rods**
how do you do your acid fast stain with partially acid fast organisms
use lower concentration of acid alcohol to decolorize
appearance of mycobacteria spp on acid fast stain (Ziehl-Neelsen)
red rods
5 types of culture media
- general purpose
- enriched
- selective
- differential
- specialized
goal of general purpose culture + agar used
- detect most aerobic and facultatively anaerobic organisms
- sheep’s blood agar
enriched culture: goal + agar used
- grow organisms like Haemophilus influenza that don’t grow on sheep blood
- added nutrients for fastidious organisms
- chocolate agar
selective culture: goal + agar used
- bring out specific bacteria (and suppress others)
- MacConkey agar if want to bring out gram+ and suppress gram-
differential culture: goal + agar used
- group microbes based on characteristics seen on medium (diff based on hemolysis and who ferments lactose)
- sheep blood agar, MacConkey agar
specialized culture: goal + agar used
-additives (Abs to suppress specific pathogens and growth factors to select others) to isolate specific pathogen
alpha hemolysis charact + organisms
- partial hemolysis of RBCs, greenish hue
- strep pneumoniae and strep viridans
beta hemolysis charact + organisms
- hemolysin produced breaks down RBCs
- beta hemolytic strep (Group A,B,C,G strep) and staph aureus
gamma hemolysis charact and organisms
- no hemolysis
- enterococcus
purple vs no colour meaning on MacConkey plate
- purple = lactose fermenters (pH stain and reaction with purple crystal violet in the medium
- no color = not lactose fermenter
typical lactose fermenter
E.coli
next step after plating specimens on selected agars
must incubate them
4 environments of incubation for culture and what organisms prefer these
- aerobic
- microaerophilic (less O2) = campylobacter
- CO2 rich = streptococcus
- anaerobic environment (C.diff, dies in presence of O2)
what do you get when ask for blood culture
two bottles
- one for aerobic org test
- one for anaerobic
what does a blood culture bottle contain
- liquid nutritionally enriched media
- device or resin for antibiotic removal
- anticoagulant
what is done with blood cultures after collection
incubated at 35C for 5 days in continuous monitoring system
2 ways the continuous monitoring system senses culture growth in a bottle
- pH sensitive membrane in bottom of bottle, senses change bc of CO2 released from metabolism
- can detect color change
(3. can also use sensors)
biochem tests for rapid bacterial identification
- catalase
- oxidase
- coagulase
- motility medium
- triple sugar iron agar
- indole
- PYR
catalase test and typical positive bacteria
- check if bacteria has the catalase enzyme to make H2O2 into H2O and O2
- staph aureus is +. (strep is neg)
oxidase test and typical positive
- check if bacteria produce enzyme oxidase. if it does, will catalyse the rx and make BLUE color
- pseudomonas is +
coagulase test and typical positive
- rabbit plasma + add bacteria to see if it has coagulase by checking if plasma coagulates (coagulase makes fibrinogen into fibrin)
- staph aureus is +
motility medium test principle
- start with initially yellow medium and add column of bacteria in bottom in middle
- column doesn’t move = not motile
- column disperses but still visible = partially motile
- all dispersed = motile
triple sugar iron agar test
- inoculate bacteria in a glucose vial, a sucrose vial and a lactose vial
- check they ferment what
specific thing other than fermentation that triple sugar iron agar test can help detect and it indicates what bacteria
- if a bacteria produces H2S, will see black color
- typical of salmonella
2 identification systems to identify bacteria
- API (different reactions occur and computer identifies bacteria based on pattern of rxs. positives and negatives)
- automated system (put in machine, machine does tests, gives answer)
specimen for nose and sinus
- nasopharyngeal aspirate or swab
- sinus lavage
- biopsy
specimen for throat and pharynx
pharyngeal swab or tonsil swab (abscess)
specimen for lungs and bronchi
- sputum
- blood culture
- BAL (bronchoalveolar lavage), trans-tracheal aspiration, lung biopsy
middle ear specimen
trans-tympanic aspiration
stomach and duodenum specimen
biopsy
intestines specimen
- feces
- rectal swab
- blood culture (typhoid fever)
urinary tract specimen
- mid-stream urine or catheter
- supra-pubic aspiration
CNS specimen
- CSF
- blood culture
eye specimen
purulent discharge
blood specimen
blood culture and any other suspected primary focus
wound specimen
- drainage aspiration
- biopsy
bone and joint specimen
- joint tap
- synovial biopsy
- marrow or bone biopsy
grey moist glistening colonies in CSF indicates what
Neisseria meningitidis
what’s done in lab on day 1
- blood culture bottles incubation (when you took blood sample)
- gram stain on other specimen, inoculation of agars + incubation
- so basically culture in bottle + agar depending on specimens*
what’s done in lab on day 2
- read agar cultures
- if got pure agar cultures, start Abx susceptibility testing to guide right Abx choice AND for public health matters
2 susceptibility testing methods
- E-test (strip with vanco on it that you put on the culture carpet)
- disk diffusion or Kirby-Bauer (add Abx discs on the culture carpet)
- further the bacteria = higher the susceptibility (bigger diameter around disc of Abx)*
3 ways to measure the MIC of an organism (which is the ultimate goal of susceptibility testing)
- measure directly with E-test (strip has values listed indicated vanco conc on that section of the strip) and automated systems
- infer MIC by disk diffusion
3 classifications given to an organism based on its MIC to an Abx
- sensitive (usual achievable conc is enough to treat)
- resistant (not inhibited by usually achieved conc OR organism is resistant at this not clinically efficient concentration)
- intermediate: MIC approaches attainable blood levels but not the efficacy that is usually seen against sensitive organisms
for an intermediate organism, when will the Abx be the most useful
- in body sites where the drug is concentrated (in urine, like fluoroquinolones)
- when a higher dosage than usual can be used (beta-lactams)
blood culture day 1, 2 and 3 (1 being growth detection)
- gram stain + subculture blood in agar
- identification tests + subculture for susceptibility testing
- final ID available (if not already) and susceptibility available
(so 1. gram 2 and 3 are ID and susceptibility)
serology def and when used more
- measure pt’s Ab response to an infection or Ag (or detect Ag in patient sample)
- used more when hard to culture or long incubation
3 methods of serology
- ELISA (enzyme linked immunosorbent assay) (Indirect or Sandwich)
- immunofluorescence assays
- latex agglutination
indirect ELISA method
- Ag coated beaker
- add serum pt
- wash to remove not bound stuff
- add Ab to Ab #1
- add enzyme to make color if 2 Abs bound
Sandwich (direct) ELISA method
- Ab coated beaker
- add serum infected with Ag (direct)
- wash (remove Ag not bound)
- add Ab to the Ag
- add enzyme to make color if sandwich Ab-Ag-Ab made
immunofluorescence assay methods
- indirect fluorescent antibody test (IFA)
- direct fluorescent antibody test (DFA)
indirect fluorescent antibody test (IFA)
- indirect, detects Ab in patient sample
1. Ag precoated slide
2. add pt serum
3. add fluo labelled Ab to the Abs in patient serum
organisms that are tested for using immunofluorescence assays (IFA)
- treponema pallidum (syphilis)
- toxoplasma gondii, a paraiste (toxoplasmosis)
direct fluorescent antibody test (DFA) method
- direct, tests for Ag in the blood or wtv
1. Ab precoated slide
2. add pt serum
3. add fluo labelled Ab to the Ag
organisms usually detected with direct fluorescent antibody test (DFA)
-HSV
-VZV
(influenza, RSV, pneumocystis jeroveci, T. pallidum)
latex agglutination goal
detect a streptococcal infection in the serum and identify the beta hemolytic strep group
latex agglutination method
- have diff beads coated with Abs to strep group A,B,C,G
- add pt serum
- Abs on beads agglutinate with patient serum Ags of strep (or Ags on beads agglutinate with Abs in pt serum)
nucleic-acid base identification (PCR): what’s the goal
- dx hard to culture infections (TB, respiratory viruses and their types: influenza and others)
- ID colonies (MRSA)
- investigate outbreaks
- genotyping (MRSA, C.diff)
idea of PCR
- RT-PCR is used to detect mRNA expression
- a cDNA library is obtained
- the cDNA library is amplified
steps of PCR once you have DNA (or cDNA)
- heat to denature the dsDNA (separate strands)
- cool for the primes to base pair with their region of interest (mekA gene on plasmin, in MRSA)
- raise temp for polymerisation
- repeat cycle
what is the primer for when you try to do PCR on MRSA
mekA gene is on a plasmin in MRSA. the primer of PCR anneals to that
