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Micro - The Appropriate Use of the Microbiology Laboratory (ii) Lab…
Micro - The Appropriate Use of the Microbiology Laboratory (ii) Lab techniques cont...
Culture
5 steps
inoculation
incubation
for 10-15 days
environment NB
optimum temp is usually 37 (30 for fungi, 42 for campylobacter)
gas pack jars
most are aerobic
N gonorrhoeae needs CO2
isolation
inspection
identification
growth media
solid
plates for ID + emuneration (ordered listing)
slopes for longterm culture (e.g. Lowenstein-Jensen for TB)
agar can be...
enriched to support growth of fastidious bacteria (e.g. chocolate agar for H influenzae)
selective to enable growth of 1 organism while supressing others (e.g. deoxycholate citrate agar for salmonella/shigella)
differential (distinguishes on the basis of metabolic characteristics, e.g. MacConkey for E Coli / pseudomonas)
chromogenic (newest, distinguishes organisms based on colour, e.g. for UTIs)
Sabuoraud agar
for fungi + filamentous bacteria
e.g. Aspergillus fumigatus (child ear infection)
liquid
broth
can be enriched for max sensitivity
ID
morphology
growth requirements
biochemistry
antigen testing (e.g. pneumococcal)
MALDI-TOF mass spectrometry
matrix-assisted laser desorption + ionisation time of flight
all bacteria has 16S rRNA released by matrix
ID of big vs small particles based on TOF (bigger = slower)
rapid: 15-30s per sample
! false +ves for pneumococcus
if +ve for "mixed enterobacteraciae" - no infection
Non-culture diagnostic methods
ELISA
antigen detection
used to ID C diff + H pylori
quick (2-3 hrs)
no culture needed
Molecular techniques
based on nucleic acid (universal to all organisms)
definitive
fast
no culture needed
quantifies load
can detect antibiotic-resistance genes (e.g. oxa 48)
PCR
amplifies gene
requires known target sequence
works for non-viable organisms
sensitive (minute amounts detectable)
expensive
rapid
reliable
RT-PCR for RNA viruses
DNA needs to be extracted (use Roche MagNA pure - magnet)
targets can be
specific genes (+ve or -ve result)
universal gene (16S rRNA, always +ve so ID based on sequence variations)
easily contaminated
PCR inhibitors in biological fluid (e.g. blood)
doesn't distinguish between alive + dead organism (unless detecting RNA)
real time PCR - detection via fluorescent reporter
in future: NGS (whole genome)
good for fastidious organisms
TB
HIV
hep C
Neisseria