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AUTOMATION IN MICROBIOLOGY
PRESENTED BY,
Dr.Jithu K Mathew
JR 1
AUTOMATION IN LAB
PARTIAL LAB
AUTOMATION
FULL LAB
AUTOMATION
OUTLINE
SPECIMEN
COLLECION
• Veebot
SPECIMEN
TRANSPORT
• Pneumatic
tube
transport
MICROSCOPY
• automatic
staining
methods
• urine
analyzer
OUTLINE....
PROCESSING
• INNOVA
• InoqulA
• Previ-Isola
• WASP
IDENTIFICATION
• Digital reading
• MALDI-TOF
• VITEK
• VIDAS
• OMNILOG
AST
OUTLINE......
• FULL LAB AUTOMATION
• AUTOMATION IN BLOOD CULTURE
• ADVANCES IN MYCOBACTERIOLOGY
VEEBOT
• Portable robotic device for
automated phlebotomy
• near infra-red visualisation
producing stereoimages and
robotic kinematics introducing
needle and effecting
venepuncture.
• Accuracy,safety,precision
• able to locate small peripheral
vein
PNEUMATIC TUBE TRANSPORT SYSTEM
• CAPSULE PIPELINES
PTTS....
ADVANTAGES
• FAST
• EASY
• LESS LABOUR
DISADVANTAGES
• HEMOLYSIS OF BLOOD
SAMPLES AND CAN
INTERFERE WITH RESULTS
• COST OF INSTALLATION
AND MAINTENANCE
AUTOMATION IN GRAM STAINING
• Smears are prepared and introduced into enclosed system
• 1) specimen fixation
2) stained with crystal violet and thenwashed by deionized water
3) stained with iodine and then washed by deionized water
4) decolorized and counterstained simultaneously by acetone safranin
solution
5) dried by the system.
• Eg :: 1)PREVI color automated gram stainer 2)Aerospray gram
series slide stainer
differences....
• Fixation 1]heat
2] alcohol 95%
• Staining methods
1]Bath stainers-chance of carry over of stain
2]Spray stainers-less wastage
3]cuvette designed staining
• Capacity- 30-120/hour
• Turn over time-3-5 min
AUTOMATED URINE ANALYZER
• UF-100 AND UF-50
• IQ 200 AUTOMATED URINE MICROSCOPY ANALYZER
• IRIS FLOW VIDEOMICROSCOPY
UF-100 ,UF- 50,uf 1000i
• Laser based flow cytometry along with impedance detection ,forward
light scatter and fluorescence to identify cells
• two dyes are used
1] phenathridine - DNA
2]carbocyanine - -ve charged cell membrane
nuclear membrane
mitochondria
• The system aspirates 0.8 mL of urine .
• analyse cells [erythrocyte,leukocytes (WBC) and epithelial cells],
Bacteria and casts .
• use electrical impedance for volume
• forward light scatter for size
• uses a couple of fluorescent dyes for nuclear and cytoplasmic
characteristics
• The formed elements are categorised in a two-dimensional space
(scattergrams) on the basis of their size, shape, volume,and staining
characteristics.
IQ 200 AUTOMATED URINE MICROSCOPY
ANALYZER
• The sample is mixed,aspirated to a flow cell where 500
photomicroscopic images are taken
• Auto particle recognition system analyses the cells in the
photomicrograph based on cell size,shape ,texture and contrast
AUTOMATION IN SPECIMEN PROCESSING
• The currently available speciemen processors include:
1]Innova processor -BD diagnostics
2]InoqulA fullautomation/manual automation [FA/MI] specimen
processing device - BD Diagnostics
3]Previ Isola Automatic plate streaker-Biomerieux
4] Walk Away Specimen Processor[WASP]-Cogan diagnostics
• Process LIQUID -BASED SPECIMENS
INNOVA PROCESSOR
• 5 Drawer * 40 tubes = 200 samples
• 270 agar plates
• Library of streaking patterns
• 1,10,30 microlitre nichrome loop
• specimen can be added as they arrive in lab
• automatic decapper
InoqulA FA/MI
• FOR
 Slide preparation
 autoinoculation of liquid specimen
 manual inoculation of other specimens
• inoculation using magnetic beads
• different atmospheres of incubation
• more isolated colonies than manual
PREVI ISOLAAUTOMATED PLATE STREAKER
• Uncapped bottles
• disposable applicator and pippette for each plate and specimen
• single streak pattern-radial
• 180 plates/hr
• streaked plates are ejected to stacks
• more isolated colonies
WASP
• SCARA Robot to move specimens and plates
• Gram smear preparatory module
• 320-370 plates
• automatic loop changer -1,10,30 microL loops
• full library of streaking pattern
• streaked plates into stacks
• half plates can be streaked and labelled.
DIGITAL PLATE READING
BActerial Rapid Detection using Optical scatter
Technology (BARDOT)
• irradiation of bacterial colonies grown in a Petri dish with a
red laser to generate light scattering patterns. The light
scattering patterns are dependent on the three-
dimensional (3D) morphology of bacterial colonies.
• distinguished Listeria, Staphylococcus, Salmonella,
Vibrio, and Escherichia with classification accuracy of 90–
99% . Five species of Listeria , three species of Vibrio [7],
and seven serogroups of E. coli have been discriminated
with the accuracy of >91%, >96%, and >81%,
respectively.
IDENTIFICATION SYSTEMS
• API 20E/NE identification system
• BBL CRYSTAL
• MicroScan Walkaway
• Vitek system
• Sensititre Gram negative auto identification system
• Phoenix system
• Omnilog ID system
API (ANALYTICAL PROFILE INDEX)
IDENTIFICATION SYSTEM
• Packaged system for identification of
-Enterobacteriaceae -20 E ID
-non fermentative bacilli -20 E
20 NE system
-Gram positive cocci-API Staph Ident
-API Staph
-API ID 32 Staph
API 20 E SYSTEM
• for identification of Enterobacteracea
and non fermenters like
P.aeruginosa,S.maltophila,Acinetobact
er species
• Consists of a plastic strip with 20
cupules containing dehydrated
substrates and a plastic incubation
chamber
• Same day identification-5 hrs or after
24-48 hrs( more accuracy and
precision).
Example :
BBL CRYSTAL ENTERIC /ON FERMENTER ID
SYSTEM //GRAM POSITIVE IDS
• MINIATURISED
IDENTIFICATION
SYSTEM
• consists of fluorogenic and
chromogenic substrates
• reproducibility 96.3%-
100%
• accuracy of 96.9%
VITEK SYSTEM
• VITEK LEGACY SYSTEM AND VITEK 2 SYSTEM
• The VITEK 2 is an automated microbiology system utilizing growth-
based technology.
• 3 formats -VITEK 2 compact-industrial microbiology
-VITEK 2
- VITEK 2 XL
VITEK 2 and VITEK 2 XL
 more focused on the clinical microbiology laboratory
 provide increased levels of automation and capacity for higher volume
laboratories.
 They also provide an option of automatic pipetting and dilution for
antimicrobial susceptibility testing
COMPONENTS
• Integrated modular system
1] filling - sealer unit
2]reader-incubater
3]computer control module
4]data terminal
5]multicopy printer
Reagent Cards
• 64 wells - each contain an individual
test substrate. Substrates measure
various metabolic activities such as
acidification, alkalinization, enzyme
hydrolysis, and growth in the presence
of inhibitory substances
TYPES OF REAGENT CARDS
• 1. GN - Gram-negative fermenting and non-fermenting bacilli
• 2. GP - Gram-positive cocci and non-spore-forming bacilli
• 3. YST - yeasts and yeast-like organisms
• 4. BCL - Gram-positive spore-forming bacilli
• Suspension Preparation
A sterile swab or applicator stick is used to transfer a sufficient number
of colonies of a pure culture and suspend the microorganism in 3.0 mL
of sterile saline in a plastic test tube.
The turbidity is adjusted accordingly and measured using a turbidity
meter called the DensiChekTM
Product McFarland turbidity
range
GN 0.50-0.63
GP 0.50-0.63
YST 1.80-2.20
BCL 1.80-2.20
• A test tube containing the
microorganism suspension is placed
into a special rack (cassette) and the
identification card is placed in the
neighboring slot .
• The cassette can accommodate up to
10 tests (VITEK 2 Compact) or up to
15 tests (VITEK 2 and VITEK 2 XL).
• The filled cassette is placed either
manually (VITEK 2 compact) or
transported automatically (VITEK 2
and VITEK 2 XL) into a vacuum
chamber station. After the vacuum is
applied and air is re-introduced into
the station, the organism suspension
is forced through the transfer tube
into micro-channels that fill all the
test wells
Card Sealing and Incubation
• Inoculated cards are passed by a mechanism, which cuts off the
transfer tube and seals the card prior to loading into the carousel
incubator. The carousel incubator can accommodate up to 30 or up to 60
cards. All card types are incubated on-line at 35.5 + 1.0ºC.
• Each card is removed from the carousel incubator once every 15
minutes, transported to the optical system for reaction readings, and then
returned to the incubator until the next read time.
Optical System
A transmittance optical system allows interpretation of test reactions
using different wavelengths in the visible spectrum. During incubation,
each test reaction is read every 15 minutes to measure either turbidity or
colored products of substrate metabolism. Results are interpreted at 3 hr
of incubation.
MALDI-TOF
• MATRIX ASSISTED LASER DESORPTION/IONISATION -TIME OF
FLIGHT MASS SPECTROMETRY
• Identification Based on PEPTIDE MASS FINGERPRINT
• commercially available systems include
-Vitek MS -[bioMerieux]
-MALDI Biotyper-[Bruker]
• SAMPLES
Culture -bateria,mycobacteria,yeasts,molds
sediment from positive blood culture
sediment from specimens like urine,CSF, stool
• MATRIX SOLUTION USED
alpha cyano 4 hydroxy cinnamic acid
sinapinic acid(3,5-dimethoxy-4hydroxycinnamic acid)
2,5 dihydroxy benzoic acid
• SOLVENT USED
acetonitrile
trifluoroacetic acid
ethanol
highly purified water
MECHANISM
• sample[ A] is mixed with excess
matrix[M] solution.
• laser ionises [M]→MH+
• [MH+] + A→M + AH+
• Ionised particles to mass
spectrometer vaccum chamber
• migration due to potential
difference
• and velocity of migration depends
on the mass to charge ratio
• TOF- determined by arrival of different molecules at the
detector
• Summation of TOF produce a spectrum
DISADVANTAGES
• High capital cost
• Limitations:
Shigella × E.coli
Streptococcus mitis
ADVANTAGES
• Less labor
• less time to identification
• less materials used
• ※good clinical impact
RESULT INTERPRETATION
• Score value
≥2.3 - reliable genus and species
ID
2.0-2.29 - genus reliable species
probable
1.7-1.99 - genus probable
≤1.7 - not reliable
MALDI TOF IN BACTERIOLOGY
• Identification of bacteria from clinical specimens,culture plates,
• bacterial strain typing and taxonomy
• detect antimicrobial resistance-beta lactamase production,carbapenemase
activity.detect the antibiotic mass alteration due to chemical modification
• detection ,identification and inactivation of biological warfare
• food and water safety
APPLICATIONS IN VIROLOGY
• Diagnosis of Influenza,entero virus,HPV,herpes,hepatitis viruses
• genotyping of hepatitis B $ C virus,JC polyoma virus
• for typing ,subtyping,& tracing the lineage of human influenza virus
• detection of mutation in hepatitis B virus
• drug resistance of gancyclovir in CMV
APPLICATION IN MYCOLOGY
• Less advanced - drug resistance and fungal strain typing is not advanced
• Fungi identified are Candida,Cryptococcus,
Dermatophytes,Fusarium,Aspergillus,Pencillium.
SENSITITRE AUTO IDENTIFICATION
SYSTEM
• The sensititre automated reading and incubation
system(ARIS)(TREK diagnostic system) is an automated
system
• uses fluorescent technology to detect bacterial growth
and enzyme activity.
• Consists of 32 biochemical tests and fluorescent tests.
• Each biochemical test
medium along wt appropriate
fluorescent indicator is dried
into individual wells of
sensititre plate.
• Tests are read for the
presence or absence of
fluorescence.
• Results are transmitted to
computer for analysis and
identification.
Results –read after 5 hr
incubation
If identification cannot be
obtained---read after
overnight reincubation.
THE PHOENIX SYSTEM
• A newly developed fully automated identification and
antimicrobial susceptibility test system.
• comprised of disposable panels that combine
identification and AST .
• perform automatic reading at every20 mts,during
incubation.
• Gram negative identification segment uses 45
biochemical substrate
• 16 fluorogenic + 14 fermentation + 8 carbon source + 5
chromogenic + urea and ornithine.
• Identify aerobic GNB in 2-12 hrs.
• Instrument monitor both fluorescent level and visible
spectral changes ,interprets the result.
THE OMNILOG ID SYSTEM
• Fully automated system.
• Uses carbon sourse utilization method.
• Simultaneously incubates ,read and interprets the
microplates.
• Continuously process the sample but allows complete
access at any time during sample run.
• Allows organism to be incubated at their optimal
temperature
• Reads microplate after 4 hr--Pattern is compared to the
identification data base--and an ID is called if enough
positive reactions have developed.
• If no result is obtained after 6 hr,instrument automatically
continues to incubate the plates and read after 16 hr-24
hr.
• database contain 1400 different organisms ,including
501 gram negative species.
VIDAS AND MINIVIDAS
PRINCIPLE
• The assay principle combines an enzyme immunoassay
competition method with a final fluorescent detection
• The Solid Phase Receptacle (SPR®) serves as the solid
phase as well as the pipetting device for the assay.
• Reagents for the assay are ready-to-use and predispensed
in the sealed reagent strips.
• All of the assay steps are performed automatically by the
instrument. The reaction medium is cycled in and out of
the SPR several times.
• The sample is collected and transferred into the well
containing an alkaline phosphatase-labeled antibody (conjugate).
• The antigen present in the sample
and the antigen coated on the interior of the SPR
compete for the available sites on the specific antibody conjugated
to alkaline phosphatase.
During the final detection step, the substrate
(4-Methyl-umbelliferyl phosphate) is cycled in and out of the SPR.
The conjugate enzyme catalyzes the hydrolysis
of this substrate into a fluorescent product
(4-Methyl-umbelliferone), the fluorescence of which is
measured at 450 nm. The intensity of the fluorescence is
inversely proportional to the concentration of antigen
present in the sample
SHERLOCK MICROBIOLOGICAL IDS
• gas chromatography (GC) system dedicated to bacteria
identification by fatty acid methyl ester (FAME) analysis is
the Sherlock Microbial Identification System (MIS)
• The principle of the FAME method rests upon the
assumption that some microorganisms have typical
cellular fatty acid compositions, which can be compared
with the mean fatty acid composition of the strains used to
create the library. After comparison, the identities of
unknown microorganisms are determined.
NOVEL TECHNOLOGIES IN ID
• Surface Plasmon Resonance imaging (SPRi) is an optical
detection technique enabling the real-time and label-free
monitoring of molecular interactions occurring on metallic
layers. Recently, SPRi has been coupled to protein
microarrays to detect food-related bacterial pathogens in
small (about 1 mL) sample volumes. The successful
detection of live bacteria was possible by choosing
antibodies specific to antigens expressed by the bacteria
and located on the cell surface
Live bacteria are
captured on
microarrayed specific
antibodies (spotted in
triplicate onto the
biochip surface) during
the enrichment step.
SPRi data are treated
and plotted as variations
of light reflectivity (ΔR
(%)) over time for each
region-of-interest
(corresponding to
antibody spots arrayed
on the sensor).
AUTOMATED AST
• ID/AST SYSTEMS
• NOVEL TECHNOLOGIES
RAISUS ID/AST SYSTEM
DRAST
ABBOTT AVANTAGE {AV} DS
INK JET PRINTER
MAC CHIP
DIRECT RAPID ANTIMICROBIAL SUSCEPTIBILITY
TESTING[DRAST]
• AST from positive blood culture bottles
• microscopic image analysis
• Growth detection and time lapse for MIC calculation
MAC CHIP
• Micro Fluidic Agarose Channel
chip
• Rapid AST by single cell tracking
Total Lab Automation (TLA)
3 TLA system
1) Kiestra TLA
2) FMLA (Full Microbiology Laboratory Automation)
3) WASP lab
Common elements
Conveyer /track system
Digital camera
Automated incubator
Digital reading station
software
KIESTRA TLA
5 modules-linked by conveyer
Sorter A
Barcod A
Inoql A –processor
Read A –incubator with digital imaging equipment
ergonomics A –work benches
FMLA
• Previ isola automated plate streaker
• Smart incubator system
• Linked via conveyer /track system
WASP LAB
WASP
CO2 & nonCO2 incubator
Linked by conveyer system
Image acquisition station-capture image
Plate with growth ---reloaded on WASP lab for automated
broth inoculation and Kirby bauer disk dispensing
AUTOMATION IN BLOOD CULTURE
CULTURE METHODS
RAPID
IDENTIFICATION
METHODS
OLD NEW
[CONT. MONITORING]
OLDER METHODS
BACTEC BACTEC 9240/9120
NOVEL METHODS
BACT/ALERT
1]Lysis centrifugation intrinsic
fluorescence
2]Integrated Comprehensive
direct droplet
TREK ESP
BACTEC
• Early automated method
• Radiometric method
• non-continuous monitoring
• 3-5m1 sample is incubated at 35.37°C in a sealed
rubber septum vial with a liquid14 C-labelled sterilized
substrate with an activity of: 2 uCi per vial
• If bacteria are present they metabolize carbohydrate or
protein, the components of the substrate as energy
source, releasing14 co2 by catabolizing glucose or by
decarboxylation of aminoacid produced during incubation.
• 14 C02 produced during the incubation period is then aspirated from the
test vial through sterilizing filter into the ionization chamber, the
electrometer present in Bactec unit then measures the current produced
in the ionization chamber.
• Growth index (GI) : The amount of 14 CO2 liberated is proportional to
the amount of the bacterial growth in the nutrient media. A reading of
100 GI corresponds to 0.025 microcurie of14 C.
• A threshold GI may be set which is usually 30 for aerobic vials and 20
for anaerobic vials, a reading above threshold level indicates the
presence of bacteria.
BACTEC 9000 series-
• continuous monitoring system
• CO2 production is detected
• signals when growth detection
• tube with growth can be remov manually and can be
proceeded
CONSTITUENTS IN BACTEC VIAL
• Different media for aerobic culture,anaerobic
culture,fungal culture and paediatric sample
• anticoagulant - 0.025 -0.05% sodium polyanethol
sulfonate-toxic to certain bacteria eg: Neisseria
• Resins-Neutralise antimicrobial activity
except:imipenem,cilastatin
• CO2 & O2 (N2 in Anaerobic medium )
• sensor for detection of fluorescence
The BacT/Alert blood culture system
First continuous monitoring blood
culture system
Each blood culture bottle -10 ml blood
Data unit- A cabinet about the size of a
small refrigerator. Serves as self
contained incubator,shaker and
detection device. Hold 240 or 120
bottles.
Bottle is placed bottom down in to the
receiving well in the data
unit,directed by a bar code on bottle
label.
Bottom of bottle has a colour sensor --separated from blood
broth mixture by a unidirectional CO2 permeable
membrane.
Light sensitive detector is also present .
Micro organism grow in blood broth mixture
CO2 liberated
CO2 + H20 → H2C03↔ [H+] + {HCO3-}
{ACIDIC pH]
Colour of sensor turns from green to yellow
• At 10 mt intervals , light beam from emitting
diode (1 for each well) is projected through an
excitation filter to reflect off CO2 sensitive
sensor.Reflecting light is directed through an
emission filter to a photo sensitive detector
• Sufficient CO2----alter the sensor------
visible/audible alert.
• Positive bottle removed for processing.
• 144 times / bottle / day
• data points are plotted as reflectance unis
versus time and result in a growth curve
The TREK ESP culture system II
Differs from other 2 in the following ways
1) Production of CO2 is measured manometrically.
2) both gas production and consumption are monitored.
3) change in concentration of H2 and O2 addition to CO2
are detected
Lysis centrifugation intrinsic fluorescence
Integrated Comprehensive direct droplet
• based on DNA zyme
detection
• load as low as 1 bacteria
is identified
AUTOMATED SYSTEMS IN
MYCOBACTERIOLOGY
Radiometric test---BACTEC 460
Non radiometric tests---MGIT & MGIT 960
MB/BacT
ESP culture system II
BACTEC MYCO/F LYTIC
BACTEC AFB SYSTEM –BACTEC 460
Principle:
Growth medium for culturing Mycobacteria is
supplemented with a substrate labeled with radioactive
carbon (14C).
When Mycobacteria grow in this medium, they
metabolize the labeled substrate and release 14CO2 into
the atmosphere above the medium inside the sealed
bottle.
The Bactec 460 instrument tests for the presence 14CO2
by removing the gas from the bottle and transferring it to
an electrometer, where the amount of ionization is
converted to a number called a Growth Index (GI).
The rate and amount of 14CO2 (GI) produced is directly
proportional to the rate and amount of bacterial growth in
the medium
PANTA mix to suppress contaminant growth
C14 labeled palmitic acid-growth detector
Vial is inoculated with 0.5 ml processed specimen and
incubate at 35⁰C.
When designated period of incubation is over,3 days
usually,vials are placed on track of BACTEC 460
instrument in preparation for reading
A growth index more than 10 ---positive
Detection time of M.tuberculosis—9-14 days
Rapid growers-less than 7 days
Disadvantage:
High cost
inability to observe colony morphology
Over growth by contaminants
Need for disposal of radioactive material
Can perform rapid drug susceptibility study-adv
Can differentiate M.tuberculosis and bovis from
nontuberculous mycobacteria using blood culture vial
containing NAP(P-nitro-alpha acetylamino-beta hydroxy
propriophenone)
M.tb and bovis can not grow in NAP medium.
MYCOBACTERIA GROWTH INDICATOR TUBE (MGIT) &
MGIT 960
MGIT system consists -16 x 100 mm round bottomed glass
tubes –4 ml modified 7H9 broth base + 0.5 ml OADC
enrichment(oleic acid ,bovine albumin,dextrose and
catalase)+ 0.1 ml PANTA antibiotic mixture.
A fluorescent compound is embedded in silicone on the
bottom of the tube---sensitive to dissolved O2 in broth.
Presence of O2 in uninoculated medium serves to quench
fluorescence.
As bacteria grows ----consumes O2---fluorescence is
unmasked----detected uv light(woods lamp)
Growth may be also detected by non homogenous turbidity
or small grains in culture medium.
0.5 ml of specimen is added to broth—incubate-tubes are
read every other day starting on 2 nd day after
inoculation—read with woods lamp,placing the tube b/w a
positive(Na sulphite solution) and negative control(un
inoculated medium)
Positive tubes—stained for AFB
Negative tubes---reincubated and observed at regular
interval for up to 6 wks.
MGIT 960 I --- a non radiometric ,automated system----uses
BD BBL MGIT media and sensor to detect the
fluorescence that is visually interpreted .
Holds 960 plastic tubes –continuously monitored
DST possible
MB/Bac T Mycobacteria detection system
Similar to BacT/Alert blood culture system.
Bottle—10 ml enhanced middlebrook 7H9broth
CO2,N2,O2 under vaccum.
MAS—0.5 ml+ 0.5 ml specimen
Bottom sensor changes from dark green to yellow in
presence of CO2.
Reflected light is used to monitor CO2 production.
DST can be done
The ESP culture system II
Adaption of ESP blood culture system
Each bottle when placed in a special drawer in incubation
module,is attached to a senor.
Each bottle is continuously monitored for any change in gas
pressure due to metabolic activity of microorganisms in
broth.
Significant changes may be signalled early ,from the
consumption of O2,or later with production of gas by
metabolism
The bottle contain modified middlebrook 7H9
medium,casitone ,glycerol and cellulose sponges.
Sponges—growth platform for mycobacteria like alveoli.
Prior to inoculation add antibiotic mixture –PVNA—
polymyxin B ,vancomycin,nalidixic acid,amphotericin B.
DST possible
The BACTEC MYCO/F LYTIC
Bottle contain a lytic agent to release mycobacteria that are
phagocytosed by WBC.
Incubated and monitored automatically and continuously
like other BACTEC blood culture bottle.
Good culture media for bacteria and fungi in bloodstream.
BacT/Alert 3D
Recovery of a wide range of pathological organisms,
including bacteria, yeasts and mycobacteria.
The BacT/ALERT 3D is the most compact, modular and
flexible blood culture system available, providing a single
platform for the recovery of microorganisms from blood,
sterile body fluids and mycobacterial specimens, both
respiratory and non-respiratory.
AUTOMATION IN MICROBIOLOGY

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AUTOMATION IN MICROBIOLOGY

  • 1. AUTOMATION IN MICROBIOLOGY PRESENTED BY, Dr.Jithu K Mathew JR 1
  • 2. AUTOMATION IN LAB PARTIAL LAB AUTOMATION FULL LAB AUTOMATION
  • 4. OUTLINE.... PROCESSING • INNOVA • InoqulA • Previ-Isola • WASP IDENTIFICATION • Digital reading • MALDI-TOF • VITEK • VIDAS • OMNILOG AST
  • 5. OUTLINE...... • FULL LAB AUTOMATION • AUTOMATION IN BLOOD CULTURE • ADVANCES IN MYCOBACTERIOLOGY
  • 6. VEEBOT • Portable robotic device for automated phlebotomy • near infra-red visualisation producing stereoimages and robotic kinematics introducing needle and effecting venepuncture. • Accuracy,safety,precision • able to locate small peripheral vein
  • 7. PNEUMATIC TUBE TRANSPORT SYSTEM • CAPSULE PIPELINES
  • 8.
  • 9. PTTS.... ADVANTAGES • FAST • EASY • LESS LABOUR DISADVANTAGES • HEMOLYSIS OF BLOOD SAMPLES AND CAN INTERFERE WITH RESULTS • COST OF INSTALLATION AND MAINTENANCE
  • 10. AUTOMATION IN GRAM STAINING • Smears are prepared and introduced into enclosed system • 1) specimen fixation 2) stained with crystal violet and thenwashed by deionized water 3) stained with iodine and then washed by deionized water 4) decolorized and counterstained simultaneously by acetone safranin solution 5) dried by the system. • Eg :: 1)PREVI color automated gram stainer 2)Aerospray gram series slide stainer
  • 11. differences.... • Fixation 1]heat 2] alcohol 95% • Staining methods 1]Bath stainers-chance of carry over of stain 2]Spray stainers-less wastage 3]cuvette designed staining • Capacity- 30-120/hour • Turn over time-3-5 min
  • 12.
  • 13. AUTOMATED URINE ANALYZER • UF-100 AND UF-50 • IQ 200 AUTOMATED URINE MICROSCOPY ANALYZER • IRIS FLOW VIDEOMICROSCOPY
  • 14. UF-100 ,UF- 50,uf 1000i • Laser based flow cytometry along with impedance detection ,forward light scatter and fluorescence to identify cells • two dyes are used 1] phenathridine - DNA 2]carbocyanine - -ve charged cell membrane nuclear membrane mitochondria
  • 15. • The system aspirates 0.8 mL of urine . • analyse cells [erythrocyte,leukocytes (WBC) and epithelial cells], Bacteria and casts . • use electrical impedance for volume • forward light scatter for size • uses a couple of fluorescent dyes for nuclear and cytoplasmic characteristics • The formed elements are categorised in a two-dimensional space (scattergrams) on the basis of their size, shape, volume,and staining characteristics.
  • 16. IQ 200 AUTOMATED URINE MICROSCOPY ANALYZER • The sample is mixed,aspirated to a flow cell where 500 photomicroscopic images are taken • Auto particle recognition system analyses the cells in the photomicrograph based on cell size,shape ,texture and contrast
  • 17.
  • 18.
  • 19. AUTOMATION IN SPECIMEN PROCESSING • The currently available speciemen processors include: 1]Innova processor -BD diagnostics 2]InoqulA fullautomation/manual automation [FA/MI] specimen processing device - BD Diagnostics 3]Previ Isola Automatic plate streaker-Biomerieux 4] Walk Away Specimen Processor[WASP]-Cogan diagnostics • Process LIQUID -BASED SPECIMENS
  • 20. INNOVA PROCESSOR • 5 Drawer * 40 tubes = 200 samples • 270 agar plates • Library of streaking patterns • 1,10,30 microlitre nichrome loop • specimen can be added as they arrive in lab • automatic decapper
  • 21. InoqulA FA/MI • FOR  Slide preparation  autoinoculation of liquid specimen  manual inoculation of other specimens • inoculation using magnetic beads • different atmospheres of incubation • more isolated colonies than manual
  • 22.
  • 23. PREVI ISOLAAUTOMATED PLATE STREAKER • Uncapped bottles • disposable applicator and pippette for each plate and specimen • single streak pattern-radial • 180 plates/hr • streaked plates are ejected to stacks • more isolated colonies
  • 24.
  • 25. WASP • SCARA Robot to move specimens and plates • Gram smear preparatory module • 320-370 plates • automatic loop changer -1,10,30 microL loops • full library of streaking pattern • streaked plates into stacks • half plates can be streaked and labelled.
  • 26.
  • 28.
  • 29. BActerial Rapid Detection using Optical scatter Technology (BARDOT) • irradiation of bacterial colonies grown in a Petri dish with a red laser to generate light scattering patterns. The light scattering patterns are dependent on the three- dimensional (3D) morphology of bacterial colonies. • distinguished Listeria, Staphylococcus, Salmonella, Vibrio, and Escherichia with classification accuracy of 90– 99% . Five species of Listeria , three species of Vibrio [7], and seven serogroups of E. coli have been discriminated with the accuracy of >91%, >96%, and >81%, respectively.
  • 30.
  • 31. IDENTIFICATION SYSTEMS • API 20E/NE identification system • BBL CRYSTAL • MicroScan Walkaway • Vitek system • Sensititre Gram negative auto identification system • Phoenix system • Omnilog ID system
  • 32. API (ANALYTICAL PROFILE INDEX) IDENTIFICATION SYSTEM • Packaged system for identification of -Enterobacteriaceae -20 E ID -non fermentative bacilli -20 E 20 NE system -Gram positive cocci-API Staph Ident -API Staph -API ID 32 Staph
  • 33. API 20 E SYSTEM • for identification of Enterobacteracea and non fermenters like P.aeruginosa,S.maltophila,Acinetobact er species • Consists of a plastic strip with 20 cupules containing dehydrated substrates and a plastic incubation chamber • Same day identification-5 hrs or after 24-48 hrs( more accuracy and precision).
  • 35.
  • 36. BBL CRYSTAL ENTERIC /ON FERMENTER ID SYSTEM //GRAM POSITIVE IDS • MINIATURISED IDENTIFICATION SYSTEM • consists of fluorogenic and chromogenic substrates • reproducibility 96.3%- 100% • accuracy of 96.9%
  • 37. VITEK SYSTEM • VITEK LEGACY SYSTEM AND VITEK 2 SYSTEM • The VITEK 2 is an automated microbiology system utilizing growth- based technology. • 3 formats -VITEK 2 compact-industrial microbiology -VITEK 2 - VITEK 2 XL
  • 38. VITEK 2 and VITEK 2 XL  more focused on the clinical microbiology laboratory  provide increased levels of automation and capacity for higher volume laboratories.  They also provide an option of automatic pipetting and dilution for antimicrobial susceptibility testing
  • 39. COMPONENTS • Integrated modular system 1] filling - sealer unit 2]reader-incubater 3]computer control module 4]data terminal 5]multicopy printer
  • 40. Reagent Cards • 64 wells - each contain an individual test substrate. Substrates measure various metabolic activities such as acidification, alkalinization, enzyme hydrolysis, and growth in the presence of inhibitory substances
  • 41. TYPES OF REAGENT CARDS • 1. GN - Gram-negative fermenting and non-fermenting bacilli • 2. GP - Gram-positive cocci and non-spore-forming bacilli • 3. YST - yeasts and yeast-like organisms • 4. BCL - Gram-positive spore-forming bacilli
  • 42. • Suspension Preparation A sterile swab or applicator stick is used to transfer a sufficient number of colonies of a pure culture and suspend the microorganism in 3.0 mL of sterile saline in a plastic test tube. The turbidity is adjusted accordingly and measured using a turbidity meter called the DensiChekTM
  • 43. Product McFarland turbidity range GN 0.50-0.63 GP 0.50-0.63 YST 1.80-2.20 BCL 1.80-2.20
  • 44. • A test tube containing the microorganism suspension is placed into a special rack (cassette) and the identification card is placed in the neighboring slot . • The cassette can accommodate up to 10 tests (VITEK 2 Compact) or up to 15 tests (VITEK 2 and VITEK 2 XL).
  • 45. • The filled cassette is placed either manually (VITEK 2 compact) or transported automatically (VITEK 2 and VITEK 2 XL) into a vacuum chamber station. After the vacuum is applied and air is re-introduced into the station, the organism suspension is forced through the transfer tube into micro-channels that fill all the test wells
  • 46. Card Sealing and Incubation • Inoculated cards are passed by a mechanism, which cuts off the transfer tube and seals the card prior to loading into the carousel incubator. The carousel incubator can accommodate up to 30 or up to 60 cards. All card types are incubated on-line at 35.5 + 1.0ºC. • Each card is removed from the carousel incubator once every 15 minutes, transported to the optical system for reaction readings, and then returned to the incubator until the next read time.
  • 47. Optical System A transmittance optical system allows interpretation of test reactions using different wavelengths in the visible spectrum. During incubation, each test reaction is read every 15 minutes to measure either turbidity or colored products of substrate metabolism. Results are interpreted at 3 hr of incubation.
  • 48. MALDI-TOF • MATRIX ASSISTED LASER DESORPTION/IONISATION -TIME OF FLIGHT MASS SPECTROMETRY • Identification Based on PEPTIDE MASS FINGERPRINT • commercially available systems include -Vitek MS -[bioMerieux] -MALDI Biotyper-[Bruker]
  • 49. • SAMPLES Culture -bateria,mycobacteria,yeasts,molds sediment from positive blood culture sediment from specimens like urine,CSF, stool • MATRIX SOLUTION USED alpha cyano 4 hydroxy cinnamic acid sinapinic acid(3,5-dimethoxy-4hydroxycinnamic acid) 2,5 dihydroxy benzoic acid • SOLVENT USED acetonitrile trifluoroacetic acid ethanol highly purified water
  • 50. MECHANISM • sample[ A] is mixed with excess matrix[M] solution. • laser ionises [M]→MH+ • [MH+] + A→M + AH+
  • 51. • Ionised particles to mass spectrometer vaccum chamber • migration due to potential difference • and velocity of migration depends on the mass to charge ratio
  • 52. • TOF- determined by arrival of different molecules at the detector • Summation of TOF produce a spectrum
  • 53. DISADVANTAGES • High capital cost • Limitations: Shigella × E.coli Streptococcus mitis ADVANTAGES • Less labor • less time to identification • less materials used • ※good clinical impact
  • 54.
  • 55. RESULT INTERPRETATION • Score value ≥2.3 - reliable genus and species ID 2.0-2.29 - genus reliable species probable 1.7-1.99 - genus probable ≤1.7 - not reliable
  • 56. MALDI TOF IN BACTERIOLOGY • Identification of bacteria from clinical specimens,culture plates, • bacterial strain typing and taxonomy • detect antimicrobial resistance-beta lactamase production,carbapenemase activity.detect the antibiotic mass alteration due to chemical modification • detection ,identification and inactivation of biological warfare • food and water safety
  • 57. APPLICATIONS IN VIROLOGY • Diagnosis of Influenza,entero virus,HPV,herpes,hepatitis viruses • genotyping of hepatitis B $ C virus,JC polyoma virus • for typing ,subtyping,& tracing the lineage of human influenza virus • detection of mutation in hepatitis B virus • drug resistance of gancyclovir in CMV
  • 58. APPLICATION IN MYCOLOGY • Less advanced - drug resistance and fungal strain typing is not advanced • Fungi identified are Candida,Cryptococcus, Dermatophytes,Fusarium,Aspergillus,Pencillium.
  • 59. SENSITITRE AUTO IDENTIFICATION SYSTEM • The sensititre automated reading and incubation system(ARIS)(TREK diagnostic system) is an automated system • uses fluorescent technology to detect bacterial growth and enzyme activity. • Consists of 32 biochemical tests and fluorescent tests.
  • 60. • Each biochemical test medium along wt appropriate fluorescent indicator is dried into individual wells of sensititre plate. • Tests are read for the presence or absence of fluorescence. • Results are transmitted to computer for analysis and identification.
  • 61. Results –read after 5 hr incubation If identification cannot be obtained---read after overnight reincubation.
  • 62. THE PHOENIX SYSTEM • A newly developed fully automated identification and antimicrobial susceptibility test system. • comprised of disposable panels that combine identification and AST . • perform automatic reading at every20 mts,during incubation.
  • 63. • Gram negative identification segment uses 45 biochemical substrate • 16 fluorogenic + 14 fermentation + 8 carbon source + 5 chromogenic + urea and ornithine. • Identify aerobic GNB in 2-12 hrs. • Instrument monitor both fluorescent level and visible spectral changes ,interprets the result.
  • 64. THE OMNILOG ID SYSTEM • Fully automated system. • Uses carbon sourse utilization method. • Simultaneously incubates ,read and interprets the microplates. • Continuously process the sample but allows complete access at any time during sample run. • Allows organism to be incubated at their optimal temperature
  • 65.
  • 66. • Reads microplate after 4 hr--Pattern is compared to the identification data base--and an ID is called if enough positive reactions have developed. • If no result is obtained after 6 hr,instrument automatically continues to incubate the plates and read after 16 hr-24 hr. • database contain 1400 different organisms ,including 501 gram negative species.
  • 67. VIDAS AND MINIVIDAS PRINCIPLE • The assay principle combines an enzyme immunoassay competition method with a final fluorescent detection • The Solid Phase Receptacle (SPR®) serves as the solid phase as well as the pipetting device for the assay. • Reagents for the assay are ready-to-use and predispensed in the sealed reagent strips. • All of the assay steps are performed automatically by the instrument. The reaction medium is cycled in and out of the SPR several times.
  • 68. • The sample is collected and transferred into the well containing an alkaline phosphatase-labeled antibody (conjugate). • The antigen present in the sample and the antigen coated on the interior of the SPR compete for the available sites on the specific antibody conjugated to alkaline phosphatase. During the final detection step, the substrate (4-Methyl-umbelliferyl phosphate) is cycled in and out of the SPR.
  • 69. The conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4-Methyl-umbelliferone), the fluorescence of which is measured at 450 nm. The intensity of the fluorescence is inversely proportional to the concentration of antigen present in the sample
  • 70. SHERLOCK MICROBIOLOGICAL IDS • gas chromatography (GC) system dedicated to bacteria identification by fatty acid methyl ester (FAME) analysis is the Sherlock Microbial Identification System (MIS) • The principle of the FAME method rests upon the assumption that some microorganisms have typical cellular fatty acid compositions, which can be compared with the mean fatty acid composition of the strains used to create the library. After comparison, the identities of unknown microorganisms are determined.
  • 71.
  • 72. NOVEL TECHNOLOGIES IN ID • Surface Plasmon Resonance imaging (SPRi) is an optical detection technique enabling the real-time and label-free monitoring of molecular interactions occurring on metallic layers. Recently, SPRi has been coupled to protein microarrays to detect food-related bacterial pathogens in small (about 1 mL) sample volumes. The successful detection of live bacteria was possible by choosing antibodies specific to antigens expressed by the bacteria and located on the cell surface
  • 73. Live bacteria are captured on microarrayed specific antibodies (spotted in triplicate onto the biochip surface) during the enrichment step. SPRi data are treated and plotted as variations of light reflectivity (ΔR (%)) over time for each region-of-interest (corresponding to antibody spots arrayed on the sensor).
  • 74. AUTOMATED AST • ID/AST SYSTEMS • NOVEL TECHNOLOGIES RAISUS ID/AST SYSTEM DRAST ABBOTT AVANTAGE {AV} DS INK JET PRINTER MAC CHIP
  • 75. DIRECT RAPID ANTIMICROBIAL SUSCEPTIBILITY TESTING[DRAST] • AST from positive blood culture bottles • microscopic image analysis • Growth detection and time lapse for MIC calculation
  • 76.
  • 77. MAC CHIP • Micro Fluidic Agarose Channel chip • Rapid AST by single cell tracking
  • 78. Total Lab Automation (TLA) 3 TLA system 1) Kiestra TLA 2) FMLA (Full Microbiology Laboratory Automation) 3) WASP lab
  • 79. Common elements Conveyer /track system Digital camera Automated incubator Digital reading station software
  • 80. KIESTRA TLA 5 modules-linked by conveyer Sorter A Barcod A Inoql A –processor Read A –incubator with digital imaging equipment ergonomics A –work benches
  • 81.
  • 82. FMLA
  • 83. • Previ isola automated plate streaker • Smart incubator system • Linked via conveyer /track system
  • 85. WASP CO2 & nonCO2 incubator Linked by conveyer system Image acquisition station-capture image Plate with growth ---reloaded on WASP lab for automated broth inoculation and Kirby bauer disk dispensing
  • 86.
  • 87. AUTOMATION IN BLOOD CULTURE CULTURE METHODS RAPID IDENTIFICATION METHODS OLD NEW [CONT. MONITORING] OLDER METHODS BACTEC BACTEC 9240/9120 NOVEL METHODS BACT/ALERT 1]Lysis centrifugation intrinsic fluorescence 2]Integrated Comprehensive direct droplet TREK ESP
  • 88. BACTEC • Early automated method • Radiometric method • non-continuous monitoring • 3-5m1 sample is incubated at 35.37°C in a sealed rubber septum vial with a liquid14 C-labelled sterilized substrate with an activity of: 2 uCi per vial • If bacteria are present they metabolize carbohydrate or protein, the components of the substrate as energy source, releasing14 co2 by catabolizing glucose or by decarboxylation of aminoacid produced during incubation.
  • 89. • 14 C02 produced during the incubation period is then aspirated from the test vial through sterilizing filter into the ionization chamber, the electrometer present in Bactec unit then measures the current produced in the ionization chamber. • Growth index (GI) : The amount of 14 CO2 liberated is proportional to the amount of the bacterial growth in the nutrient media. A reading of 100 GI corresponds to 0.025 microcurie of14 C. • A threshold GI may be set which is usually 30 for aerobic vials and 20 for anaerobic vials, a reading above threshold level indicates the presence of bacteria.
  • 90. BACTEC 9000 series- • continuous monitoring system • CO2 production is detected • signals when growth detection • tube with growth can be remov manually and can be proceeded
  • 91. CONSTITUENTS IN BACTEC VIAL • Different media for aerobic culture,anaerobic culture,fungal culture and paediatric sample • anticoagulant - 0.025 -0.05% sodium polyanethol sulfonate-toxic to certain bacteria eg: Neisseria • Resins-Neutralise antimicrobial activity except:imipenem,cilastatin • CO2 & O2 (N2 in Anaerobic medium ) • sensor for detection of fluorescence
  • 92. The BacT/Alert blood culture system First continuous monitoring blood culture system Each blood culture bottle -10 ml blood Data unit- A cabinet about the size of a small refrigerator. Serves as self contained incubator,shaker and detection device. Hold 240 or 120 bottles. Bottle is placed bottom down in to the receiving well in the data unit,directed by a bar code on bottle label.
  • 93. Bottom of bottle has a colour sensor --separated from blood broth mixture by a unidirectional CO2 permeable membrane. Light sensitive detector is also present .
  • 94. Micro organism grow in blood broth mixture CO2 liberated CO2 + H20 → H2C03↔ [H+] + {HCO3-} {ACIDIC pH] Colour of sensor turns from green to yellow
  • 95. • At 10 mt intervals , light beam from emitting diode (1 for each well) is projected through an excitation filter to reflect off CO2 sensitive sensor.Reflecting light is directed through an emission filter to a photo sensitive detector • Sufficient CO2----alter the sensor------ visible/audible alert. • Positive bottle removed for processing. • 144 times / bottle / day • data points are plotted as reflectance unis versus time and result in a growth curve
  • 96. The TREK ESP culture system II Differs from other 2 in the following ways 1) Production of CO2 is measured manometrically. 2) both gas production and consumption are monitored. 3) change in concentration of H2 and O2 addition to CO2 are detected
  • 98. Integrated Comprehensive direct droplet • based on DNA zyme detection • load as low as 1 bacteria is identified
  • 100. Radiometric test---BACTEC 460 Non radiometric tests---MGIT & MGIT 960 MB/BacT ESP culture system II BACTEC MYCO/F LYTIC
  • 101. BACTEC AFB SYSTEM –BACTEC 460 Principle: Growth medium for culturing Mycobacteria is supplemented with a substrate labeled with radioactive carbon (14C). When Mycobacteria grow in this medium, they metabolize the labeled substrate and release 14CO2 into the atmosphere above the medium inside the sealed bottle.
  • 102. The Bactec 460 instrument tests for the presence 14CO2 by removing the gas from the bottle and transferring it to an electrometer, where the amount of ionization is converted to a number called a Growth Index (GI). The rate and amount of 14CO2 (GI) produced is directly proportional to the rate and amount of bacterial growth in the medium
  • 103. PANTA mix to suppress contaminant growth C14 labeled palmitic acid-growth detector Vial is inoculated with 0.5 ml processed specimen and incubate at 35⁰C. When designated period of incubation is over,3 days usually,vials are placed on track of BACTEC 460 instrument in preparation for reading
  • 104. A growth index more than 10 ---positive Detection time of M.tuberculosis—9-14 days Rapid growers-less than 7 days Disadvantage: High cost inability to observe colony morphology Over growth by contaminants Need for disposal of radioactive material
  • 105. Can perform rapid drug susceptibility study-adv Can differentiate M.tuberculosis and bovis from nontuberculous mycobacteria using blood culture vial containing NAP(P-nitro-alpha acetylamino-beta hydroxy propriophenone) M.tb and bovis can not grow in NAP medium.
  • 106. MYCOBACTERIA GROWTH INDICATOR TUBE (MGIT) & MGIT 960 MGIT system consists -16 x 100 mm round bottomed glass tubes –4 ml modified 7H9 broth base + 0.5 ml OADC enrichment(oleic acid ,bovine albumin,dextrose and catalase)+ 0.1 ml PANTA antibiotic mixture. A fluorescent compound is embedded in silicone on the bottom of the tube---sensitive to dissolved O2 in broth.
  • 107. Presence of O2 in uninoculated medium serves to quench fluorescence. As bacteria grows ----consumes O2---fluorescence is unmasked----detected uv light(woods lamp) Growth may be also detected by non homogenous turbidity or small grains in culture medium.
  • 108. 0.5 ml of specimen is added to broth—incubate-tubes are read every other day starting on 2 nd day after inoculation—read with woods lamp,placing the tube b/w a positive(Na sulphite solution) and negative control(un inoculated medium) Positive tubes—stained for AFB Negative tubes---reincubated and observed at regular interval for up to 6 wks.
  • 109. MGIT 960 I --- a non radiometric ,automated system----uses BD BBL MGIT media and sensor to detect the fluorescence that is visually interpreted . Holds 960 plastic tubes –continuously monitored DST possible
  • 110. MB/Bac T Mycobacteria detection system Similar to BacT/Alert blood culture system. Bottle—10 ml enhanced middlebrook 7H9broth CO2,N2,O2 under vaccum. MAS—0.5 ml+ 0.5 ml specimen Bottom sensor changes from dark green to yellow in presence of CO2. Reflected light is used to monitor CO2 production. DST can be done
  • 111. The ESP culture system II Adaption of ESP blood culture system Each bottle when placed in a special drawer in incubation module,is attached to a senor. Each bottle is continuously monitored for any change in gas pressure due to metabolic activity of microorganisms in broth. Significant changes may be signalled early ,from the consumption of O2,or later with production of gas by metabolism
  • 112. The bottle contain modified middlebrook 7H9 medium,casitone ,glycerol and cellulose sponges. Sponges—growth platform for mycobacteria like alveoli. Prior to inoculation add antibiotic mixture –PVNA— polymyxin B ,vancomycin,nalidixic acid,amphotericin B. DST possible
  • 113. The BACTEC MYCO/F LYTIC Bottle contain a lytic agent to release mycobacteria that are phagocytosed by WBC. Incubated and monitored automatically and continuously like other BACTEC blood culture bottle. Good culture media for bacteria and fungi in bloodstream.
  • 114. BacT/Alert 3D Recovery of a wide range of pathological organisms, including bacteria, yeasts and mycobacteria. The BacT/ALERT 3D is the most compact, modular and flexible blood culture system available, providing a single platform for the recovery of microorganisms from blood, sterile body fluids and mycobacterial specimens, both respiratory and non-respiratory.