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Automated methods in the Microbiology Lab-Issues and Troubleshooting!

Automated methods in the Microbiology Lab-Issues and Troubleshooting!. Dr. P. L. Kashyap Under guidance of: Dr. Rohini Kelkar DEPT. OF MICROBIOLOGY TATA MEMORIAL HOSPITAL, MUMBAI. Overview. Background/history Plate streakers and Gram stainers Blood cultures

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Automated methods in the Microbiology Lab-Issues and Troubleshooting!

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  1. Automated methods in the Microbiology Lab-Issues and Troubleshooting! Dr. P. L. Kashyap Under guidance of: Dr. RohiniKelkar DEPT. OF MICROBIOLOGY TATA MEMORIAL HOSPITAL, MUMBAI

  2. Overview • Background/history • Plate streakers and Gram stainers • Blood cultures • Automated Susceptibility Testing • Automated ID testing • Automated Urine Analysers • Molecular assays • MALDI-TOF

  3. Background • Manual streaking of plates has not really changed since solid agar plates were first used • Microscopy is mostly unchanged • Incubators while probably more reliable are essentially the same • Plate reading has not really changed over the years

  4. Obstacles to automation in Microbiology • Microbiology is too complex to automate: blood, sterile body fluids, urine, catheter tips, tissues, prosthetic devices, lower respiratory tract specimens • Variations in the processing of specimens: tissue digestion, urine colony count, impression smears preparation • Machines are programmable but humans are flexible • Costs of automation • Microbiology labs are small for automation

  5. Pre analytical • Specimen separators • Media pouring • Issues with each are unique

  6. Media preparation-Aseptic precautions

  7. Automated media preparation

  8. Issues and solutions • Sterility at every point or risk of contamination • Tubings need to be autoclaved • U-V light needs to be regularly inspected and maintained

  9. Conventional blood culture 7 days

  10. Blood Cultures • Standard in most diagnostic labs, varying sizes. • Better detection times • More advanced media • Reduced total incubation before calling a bottle a final negative • Suppliers: BacT/Alert (BioMerieux), BACTEC FX (BD), Versa TREK

  11. Issues with blood culture systems • False positives due to high TLC count • Continuous power supply needed • Cost

  12. Conventional ID and susceptibility testing 24-48 hours

  13. Automated ID and Sens • Automated ID testing available since 1977 • Biochemical substrates miniaturised and read by colorimetric or fluorometric means • Available in many labs • Multiple Antibiotics in different dilutions available on Cards or panels to ascertain MIC • Suppliers: Microscan Walkaway (Dade Behring) Vitek2 (BioMerieux) BD Phoenix (BD)

  14. Issues with identification systems • Pure culture • Simple tests are not covered like oxidase, catalase, motility, pigment production • Variability of identification in rarer organisms • Only organisms covered in database • Inoculum size can affect MIC interpretations • MICs of some antibiotics are not actual MICs but extrapolations from related antibiotics

  15. Automated Urine Analysers • Automated Dip-strip inoculation and reading • Cell counts performed automatically – either by flow cytometry or (more recently) high resolution optics taking pictures of cells • High Negative predictive value for urine cultures

  16. Issues with urine analysers • Back to basics • Appropriate specimen collection • Early transport

  17. Molecular methods • GeneXpert- not just for TB, • MRSA, C. difficile, etc.

  18. Issues with molecular methods • Cannot differentiate live from dead • Negative results do not rule out infection

  19. MALDI-TOF(Matrix assisted LASER Desorption Ionisation-Time of Flight Mass spectrometry) • Protein based spectral identification of bacteria • Identifications available in literally minutes – not hours • Tiny amount of bacterial growth needed – not affected by media or incubation conditions • Minimal cost per test (in cents not even dollars), virtually no consumables • Suppliers : Bruker, BioMerieux

  20. MISSION IMPOSSIBLE! Is it really?

  21. Issues with MALDI-TOF • Pure culture • In mixed cultures, will pick up the dominant organism • Antimicrobial susceptibility not possible • Viruses not identified • Identifies only organisms in database

  22. Plate Streakers • Select appropriate media • Loads the samples • Spreading the inoculum to obtain isolated single colonies following incubation • Suppliers: WASP (Copan) Previ-Isola (BioMerieux) Innova (BD) and Inoqula (KIESTRA) • Not all systems include Gram stain preparation

  23. Way ahead……………….. • Total lab automation???????

  24. Obstacles to automation in Microbiology • Microbiology is too complex to automate: blood, sterile body fluids, urine, catheter tips, tissues, prosthetic devices, lower respiratory tract specimens • Variations in the processing of specimens: tissue digestion, urine colony count, impression smears preparation…….……….BUT THE SIMPLER METHODS CAN BE AUTOMATED • Machines are programmable but humans are flexible • Costs of automation • Microbiology labs are small for automation..........Not all!!!

  25. CONCLUSION-TWO QUOTES “The first rule of any technology used in a business is that automation applied to an efficient operation will magnify the efficiency. The second is that automation applied to an inefficient operation will magnify the inefficiency.” -Bill Gates

  26. CONCLUSION-TWO QUOTES

  27. THANK YOU

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