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10
Media
AEROBIC CULTURE ANAEROBIC
MEDIA
MYCOBACTERIUM
MEDIA
BactAlert FA
Plus
BactAlert PA
Plus
BactAlert FN
Plus
BactAlert MB Process
SAMPLE
Blood & Sterile
Fluid
Blood Blood & Sterile
Fluid
All Specimens Except
Whole Blood
BOTTLE
CONTAINS
• 30 ml of Broth [BHI and TSB]
• Soduim poly-an-ethol-sulfonate
• Nutrients, Amino acids, Carbohydrate substrates
• APB (Adsorbent Polymeric Beads)
In anaerobic media 40ml
• 10 ml Middle brook
7H9
• Pancreatic Digest of
casein.
• Bovin serum albumin.
• Catalase in purified
water
SAMPLE
VOLUME
5 to 10ml 1 to 4ml 1 to 10ml 0.5 ml after
decontamination](https://image.slidesharecdn.com/automationinbacteriologydr-210902105916/85/Automation-in-bacteriology-dr-sumesh-10-320.jpg)
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Automation in bacteriology (dr.sumesh)
- 1. AUTOMATION IN BACTERIOLOGY PRESENTEDBY: Dr. Sumesh Kumar Dash MODERATOR: Prof (Dr.) Kundan Kumar Sahu
- 2. 2 2 The technique ofmaking an apparatus, a process, or a system operate automatically AUTOMATION Webster's Dictionary
- 3. 3 3 • Clinician needquicker result • Faster identification & AST • Saves time & Manpower • Remove/Decrease manual error • Helpful in processing whenever increased sample load NEED OF AUTOMATION
- 4. 4 4 • Time consuming •Tedious • Run the risk of introducing contaminants during procecing • Incomplete Identification • A limited no. of AST can be detected on one plate. • MIC may not be determined PROBLEM WITH CONVENTIONAL METHOD
- 5. 5 5 • Automated bloodculture • Vitek System • Phoenix System • MicroScan Walkaway system • MALDI-TOF VARIOUS AUTOMATED SYSTEMS
- 6.
- 7. 7 7 AUTOMATED BLOOD CULTURE •Automatically continuous reading of blood through computerized system. • Three such systems • BacT/ALERT 3D (bioMérieux, Durham, NC) • BD BACTEC (BD Diagnostic Systems) • VersaTREK (ThermoScientific, Cleveland, OH) • Each of these systems alerts if the culture is positive in the bottle. • After that we have to do a subculture, Gram stain, Identification & AST
- 8. 8 8 • First nonradiometric continuous monitoring blood culture system. • Contains incubator, shaker & detection device, with a capacity to hold either 120 or 240 bottles. • Sample: Blood, Sterile fluid, Respiratory & Non-respiratory sample • Detect: Aerobic & Anaerobic BacT/ALERT 3D
- 9. 9 9 Principle Detects of CO2 Loweringof the pH of the medium, Colour change in a sensor Detected by photosensitive detector (Every 10mins)
- 10. 10 10 Media AEROBIC CULTURE ANAEROBIC MEDIA MYCOBACTERIUM MEDIA BactAlertFA Plus BactAlert PA Plus BactAlert FN Plus BactAlert MB Process SAMPLE Blood & Sterile Fluid Blood Blood & Sterile Fluid All Specimens Except Whole Blood BOTTLE CONTAINS • 30 ml of Broth [BHI and TSB] • Soduim poly-an-ethol-sulfonate • Nutrients, Amino acids, Carbohydrate substrates • APB (Adsorbent Polymeric Beads) In anaerobic media 40ml • 10 ml Middle brook 7H9 • Pancreatic Digest of casein. • Bovin serum albumin. • Catalase in purified water SAMPLE VOLUME 5 to 10ml 1 to 4ml 1 to 10ml 0.5 ml after decontamination
- 11.
- 12.
- 13.
- 14.
- 15.
- 16. 16 16 consists of aself-contained incubator, agitator and detection device. • BACTEC 9050 • BACTEC 9120 • BACTEC 9240 • BACTEC FX • BACTEC LX BD BACTEC
- 17.
- 18. 18 18 Differs from theother 1. Monitored production of CO2 2. Both gas consumption and production are monitored 3. Changes in the concentrations of H, and O, also detected Units with a capacity of 96 to 240 or up to 528 bottles are currently available. VersaTREK
- 19. 19 19 Pressure of thehead-gas is continuously monitored (Every 12 minutes) Consumption of H and O / Production of CO2 Change in pressure Reading by electrostatic precipitator (ESP) system Lights are illuminated for positive bottle. Principle
- 20. 20 20 • REDOX 1(aerobic) • REDOX 2 (anaerobic) • MYCO Media
- 21. 21 21 • Detects differentgases produce or consume • Detects organism when they enter to log phase. • Detection microorganisms, that may not produce sufficient CO2. Advantages
- 22.
- 23. 23 23 • VITEK 2is the most widely used automated system in India • Can perform both identification and antimicrobial susceptibility testing (AST) of bacteria and yeast VITEK 2
- 24. 24 24 Colorimetric reagent card Eachwell contains an individual test substrate. Substrates in the well measure various metabolic activity Reaction pattern compared with the database Identification within 4–6 hours. Principle For Identification
- 25. 25 25 Microbroth dilution card Opticalreading of growth in presences of AMAs (every 15min) Algorithmic analysis of the reading performed by software MIC Principle For AST AES analyzes MIC give rise to sensitivity pattern, recognized the resistance pattern & also detects phenotypes of most organism in 4 -16hrs
- 26. 26 26 Card AST N280 ASTN281 AST N235 AST P628 Amikacin Amikacin Amikacin Ampicillin Amox/clav Aztreonam Amoxicillin/Clav Ciprofloxacin Ampicillin Cefepime Ampicillin Clindamycin Cefepime Cefoperazone/sulbactam Cefalotin Daptomycin Cefoperazone/sulbactam Ceftazidime Cefixime Erythromycin Cefuroxime Ciprofloxacin Cefoxitin Gentamycin Ceftriaxone Colistin Ceftazidime Gentamycin HL Ciprofloxacin Gentamicin Ceftriaxone Levofloxacin Colistin Doripenem Ciprofloxacin Linezolid Ertapenem Imipenem Ertapenem Nitrofurantoin Gentamicin Levofloxacin Fosfomycin Oxacillin Imipenem Meropenem Gentamycin Rifampicin Meropenem Minocycline Nalidixic Acid Teicoplanin Nalidixic acid Piperacillin/tazobactam Nitrofurantoin Tetracycline Nitrofurantoin Co-trimoxazole Ofloxacin Tigecycline Piperacillin/tazobactam Ticar/Clav Norfloxacin Cotrimoxazole Tigecycline Tigecycline Piperacillin/Tazobactam Vancomycin Co-trimoxazole Ticarcillin Trimethoprim/ Sulfamethoxazole
- 27. 27 27 Procedure 0.5-0.63 MCF 3 ml0.45% saline Results AST • GN - 145µL • GP – 280µL Gram Stain
- 28. 28 28 • Standardized procedure •Fast turn around time • Species level bacterial and fungal identification • AST based on MIC • Detection resistance pattern • Validation of every result every time Advantages
- 29. 29 29 Which drug isbetter ???
- 30.
- 31. 31 31 • Largest capacityautomated system. • Performed both identification & AST with wide range. • Manufactured by Becton Dickenson Diagnostics, Baltimore MD . • Can determine AST upto16-22 antimicrobials BD PHOENIX
- 32. 32 32 • Uses redoxindicator system. • Panels tray contains 126 wells (Identification:AST::51:85) • Instrument reads panels every 20 mins • Turbidity is measured as growth indicator, • Turbidity causes the redox indicator to change from an oxidized (blue) state to reduce (pink). • BCXpert & BD EpiCenter software interpret MIC value according to data base. • Gives result in 6-16hrs Principle
- 33. 33 33 Procedure Label a PhoenixID Broth Mix pure colony Vertex 0.20-0.30M BD PhoenixSpec Nephelometer Pour to ID Panel Label the Phoenix AST Broth Add 1dp Phoenix AST indicator Mix properly Add 25 µL standardized ID broth Pour to AST Panel Place it to instrument
- 34.
- 35. 35 35 • Developed in1980s • Uses broth microdilution method to determine the MIC. • Initially there is 2 major type 1. MicroScan: Reads turbidimetrically, over night incubation 2. Walkaway: Reads fluorometrically, 3.5 – 15hrs • Type of panel o MIC panel o MIC combination panels o Breakpoint combination panels Synergies Plus (2005) • All system uses LabPro software to generate result. MicroScan Walkaway
- 36.
- 37. 37 37 • Matrix-assisted laserdesorption/ ionization time-of-flight • Identify by a, mass spectrometric profile of protein and largely rRNA proteins of organism. • Mass spectrometry is an analytical technique in which chemical compounds are ionized into charged molecules and ratio of their mass-to-charge (m/z) is measured. • 2 systems are commercially available: VITEK MS (bioMérieux) Biotyper system (Bruker) MALDI-TOF
- 38.
- 39. 39 39 • Organic compound:α-cyano-4-hydroxycinnamic acid (or) 2,5-dihydroxy benzoic acid (or) 3,5-dimethoxy-4-hydroxycinnamic acid • Solvents: Ethanol/Methanol or Acetonitrile • Water • The solvents penetrate the cell wall of microorganisms and extract out the intracellular proteins. • When the solvent evaporates, ‘co-crystallization’ of protein molecules and other cellular compounds entrapped within the matrix solution takes place (Horneffer et al., 2001) Matrix
- 40. 40 40 • Numerous gasand solid state lasers have been developed for use . • Most MALDI devices use a pulsed UV laser • N2 source at 337 nm • Neodymium- yttrium aluminium garnet (Nd:YAG) • Emits at 355 nm • IR lasers are also used. • Erbium doped- yttrium aluminium garnet (Er:YAG). Laser
- 41. 41 41 • Sample preparationfor identification depends upon the its cell wall. • Some microbes might be identified directly by MS, called Direct Cell Profiling. E.g. Neisseria spp. (Ilina et al., 2009) Yersinia spp. (Stephan et al., 2011) Vibrio spp. (Eddabra et al., 2012) • Preparatory extraction was necessary for identification of Gram-positive bacteria by MALDI-TOF MS, but not for Gram Negative bacteria (Alatoom et al., 2011; Saffert et al., 2011). • A ‘preparatory extraction’ of microbes with Formic Acid increased the ability of MALDI-TOF MS for identifying Gram-positive species. Sample Preparation
- 42. 42 42 • Mycobacterial coloniescollected in screw-cap tubes containing water and 0.5% Tween 20, were inactivated by heating at 95◦C for 1 h. • Inactivated samples were centrifuged and vortexed with glass beads to disrupt the mycobacterial cell wall. • The resultant pellet was re suspended in formic acid, acetonitrile, and centrifuged again. • Finally, the supernatant was deposited onto the MALDI target plate and overlaid with matrix. • It a procedure which combined inactivation and sample preparation. Mycobacteria EI Khéchine et al. (2011)
- 43. 43 43 Procedure Andrew E. Clarket al. Clin. Microbiol. Rev. 2013;doi:10.1128/CMR.00072-12
- 44. 44 44 ADVANTAGE DISADVANTAGE • Fast• High initial cost of the equipment • Accurate • Identification from pure colony only • Broad mass range • Not useful on specimen • Soft ionisation • AST can’t determined • Less expensive • Trained laboratory personnel not required Adv & Disadv
- 45.
Editor's Notes
- #11 Adsorbent Polymeric Beads Act as an Antibiotic Neutralizing Agent. APB contains numerous pores which increase the surface area. More pores equate to more surface area to bind antimicrobials It Contains: APBs1: Gold Beads (Bind with non-polar antimicrobials to neutralize such as vancomycin and others) APB 2: Brown Beads (bind to positively charged antimicrobials to neutralize such as the aminoglycosides and others) Cysteine (Neutralize Carbapenemase group of antibiotics) .
- #17 Laser technology - directly measures changes in CO2 No intermediary reaction of sensor required. system incorporates stirring bars for agitation
- #20 Oxygen consumption is accelerated at the time replicating organisms enter the log phase of growth. A reading may be possible, therefore, early in the incubation period before a detectable amount of CO, is produced. Testing multiple gases is a theoretical advantage for the electrostatic precipitator (ESP) system, especially for the detection of asaccharolytic microorganisms that may not produce sutficient CO, for detection by the indicator.
- #39 The sample for analysis is prepared by mixing with solution called Matrix. When the matrix crystallizes on drying, the sample entrapped within the matrix also co-crystallizes. The sample within the matrix is ionized in an automated mode with a laser beam. Desorption and ionization with the laser beam generates singly protonated ions from analytes in the sample. The protonated ions are then accelerated at a fixed potential, where these separate from each other on the basis of their mass-to-charge ratio (m/z). The charged analytes are then detected and measured using time of flight (TOF) analyzers. Based on the TOF information, a characteristic spectrum called peptide mass fingerprint (PMF) is generated for analytes in the sample. Identification of microbes done by comparing the PMF of unknown organism with the PMFs contained in the database.
- #41 Ultraviolet 10 nm – 400 nm Visible 400 nm – 700 nm Infrared 700 nm – 1 mm