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
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.
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).
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
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
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).
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
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
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
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.