Micro automation Flashcards
Traditional microbiology
Susceptiblitity studies of isolation
primary isolation
Prior to 1980, the standard was
traditional microbiology:
Agar plates and enrichment
broths for primary isolation
Test tubes for biochemical
testing to identify isolates
Kirby-Bauer plates and broth
dilution tubes for susceptibility
studies of isolates
Current Instrumentation and
Automation In Microbiology
Blood culturing detecting systems
Blood culture detection systems
Bacterial identification and susceptibility systems
Total Lab Automation
Immunoassay systems
Molecular methods
Helpful when organisms cannot be successfully cultured
Rapid information for physician
Blood culture system advantages
Early detection of bacterial growth
Detection of slow-growing bacteria (e.g.
Mycobacteria)
Decreased tech time
Blood culture system disadvantages
High initial cost of instrument
Cannot detect some fastidious organisms
Blood culture system
Bactec fX
Uses fluorescence detection technology
Each bottle has a chemical sensor for
CO2
Growth of organism = increased CO2 =
increased fluorescence
Increase in fluorescence is proportional to
amount of CO2
Instrument monitors for increases in
fluorescence every 10 minutes
Bactec FX media
Media- resin media
(neutralization of
antimicrobials)
Aerobic
Anaerobic
Pedi
Myco/F Lytic
And other
specialty media
BacT Alert
(bioMerieux)
Noninvasive, continuous
monitoring; barcodes
pH sensitive membranes in
bottom of bottles
pH change resulting from CO2
production indicated by color
change
Instrument measures CO2
production colorimetrically
Tests each bottle 144 times/day,
plots growth curve from plot of
reflectance vs. time.
BacT Alert
Each bottle contains an internal
colorimetric sensor on
the bottom that detects carbon dioxide
as a measure of bacterial growth. If
bacteria are in the patient’s blood, they
will produce CO2 and the sensor will
change from blue-green to yellow.
Bact/ Alert virtuo
Next generation
from bioMerieux
Bottles loaded and
unloaded
automatically-
robotics
Media for Bactec
TSB, supplemented
Pedi, Aerobic and anaerobic bottles
With antimicrobial-agent adsorbing
polymeric beads
Aerobic and anaerobic also available
without antimicrobial beads
(historically charcoal was used—no longer
available)
Also Middlebrook 7H9 for Mycobacteri
VersaTrek ( Trek diagnostic systems
Continuous monitoring (aerobic every 12
minutes, anaerobic every 24 minutes)
Gases detected in headspace pressure
Internal computer monitors changes and
determines when to flag a bottle as
positive
Blood culture systems
Once blood culture is positive then
Once blood culture is positive,
identification proceeds in the normal way
Micro steps of diagnosis
1st step- gram stain and subculture
2nd step Notify
3rd step PNA FISH PCR
PNA FISH
Peptide Nucleic Fluorescence In Situ
Hybridization (PNA FISH)
Mutiplex PCR panels
One panel instrument
Biomeriuex filmarray
●One panel with 27 targets – 8 Gram +, 10 Gram -, 5 yeast, 3 antimicrobial resistance markers
Luminex Verigene
Two panels – GN – 9 organism calls and 6
antimicrobial resistance markers and GP – 13
organism calls and 3 antimicrobial resistance markers
Genmark
Three panels – GN has 21 organism calls and 6
antimicrobial resistance markers, GP has 20 organism
calls and 4 antimicrobial resistance markers, Fungal-
14 yeast and 1 fungus
Automated Rapid systems for Microbial Identification
Use what reactions
Types of panels
Most use turbidity, colorimetry, and
fluorescent assay principles
Panels of freeze-dried or lyophilized
reagents provided in microtiter trays or
sealed cards
Panels are incubated, read, and
interpreted by the system software
Automated Raped systems for microbial identification
TAT
Interfaces with
Reported as statistical prediction of correct
identification, based on database
Rapid turnaround time (usually < 24 hours)
Interface with laboratory or hospital
information systems
Microscan W/A
Robot arms move trays to reader at the correct
time.
Oil overlay and reagent are added to the proper
wells
Read, interpret and print panel results
Does require CLS interpretation (exceptions)
Rapid fluorescence panels and conventional
panels available
Automated Rapid systems for Microbial Identification
Vitek 2 system
a “Space-age product”–created in 1960 by
McDonnell Douglas
64-microwell cards contain substrates for
identification of bacteria and AST
Suspension and card placed into a rack
(cassette), card is filled and placed into an
incubator-reader module
Optically scanned and read every 15 minutes
Automated Rapid Systems for
Microbial Identification
The computer soft ware collects
Interfaces with what
Turn around time is what
The computer software collates the
information and matches to a database;
reports in percentages of isolate IDs
Interfaces with susceptibility cards
Turnaround time usually < 8 hours
Automated Rapid ID systems
Microscan system
Substrates conjugated with fluorophores
and substrates with a fluorescent pH
indicator
Wells inoculated with suspension of
organism
Incubated 2 hrs
what also provides rapid ID
Vitek also provides Rapid ID
VITEK cards
Microscan panels combine
separate cards for AST
Microscan panels combine ID and AST
ID/AST and Blood Culture
Instrument Maintenance
Preventive maintenance program
Temperatures (daily) –some blood
culture instruments
Instruments have daily, weekly, and
monthly maintenance defined by
manufacturer
Quality control
QC organism (weekly for AST
cards/panels) if IQCP (Individual
Quality Control Plan) in place
Tests panels/cards
Reagents
Instrument function
Streamlined QC also possible with
VITEK2 ID cards
New lot/new shipment of panels/cards
MALDI TOF
Matrix Assisted Laser Desorption
Time Of Flight
MALDI TOF components
Desorption–> Desolvation and ionization–>( proton transfer) to mass analyzer
Molecular Technology –
Sample-to-Answer Systems
Single target PCR assays
Multiplex panels (Syndromic Panels)
Fully automated sample to answer
system
Point-of-care
Single target PCR systems
Cepheid GeneXpert
Examples for screening include:
Methicillin-resistant S. aureus (MRSA)
Vancomycin-resistant enterococci (VRE)
CarbaR – carbapenem resistance
Cepheid GeneXpert
Examples for diagnosis include
Clostridioides difficile (C.diff)
Influenza
Inflenza/RSV
Norovirus
COVID-19 (Emergency Use-EU approval)
Cepheid GeneXpert
Test menu includes
19 Clinical Microbiology tests (screening and diagnostic)
Syndromic panels ( Multiplex PCR)
Based on a particular site/general diagnosis
(multiple instruments available):
Diarrheal disease (parasites, bacteria, and
viruses)
Respiratory disease (viruses and bacteria)
Meningitis (bacteria, viruses, yeast)
Septicemia (already discussed in blood cultures)
Molecular Assays
Advantages
Molecular assays are more sensitive
and specific
Molecular assays are faster
Molecular assay interpretation is more
objective
Molecular Assays
Disadvantages
Expense for reagents and
instrumentation
False positives possible if proper
contamination control is not used
Types of multiplex pcr panels
●BCID panels – direct from positive blood culture
bioMerieux FilmArray
Luminex Verigene
GenMark
Automated Immunoassays
Bio-Rad Evolis (One example)
●Hepatitis testing
●MMR immunity (IgG and IgM)
●EBV serology
●HIV serology
Self-contained microplate processor—
Automated EIA testing
Fully Automated System - Roche cobas 6800
HIV-1, HBV, HCV, CMV, HIV 1 and 2 Qual
COVID-19 (Emergency Use-EU approval)
