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Manual blood culture Techniques

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The lecture is a simple one describing the various methods that could be applied in small microbiology laboratories where the automated systems are lacking.

Publicada em: Saúde e medicina
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Manual blood culture Techniques

  1. 1. MANUAL BLOOD CULTURE METHODOLOGY STILL IN USE. Dr Mostafa Mahmoud Ahmed, Ph D Consultant Microbiologist, GDHA, Riyadh, KSA. Associate Prof. of Microbiology & Immunology. Faculty of Medicine – Ain Shams University
  2. 2. BLOOD CULTURE METHODS  Many methods available; the choice of which depends mainly upon resources.  The cheapest one is the manual (conventional).  The expensive and sophisticated automated systems are now in hands.  Note that 90% of blood cultures (BC) are normally negative making comparison between different systems so difficult.  There is no blood system as absolute reference one so, sensitivities and specificities cannot be accurately calculated.
  3. 3. A . Manual System: 1. Conventional broth-based system  Simple  Labor-intensive.  Each bottle contains 50-100 ml of broth supplemented with 0.025-0.05% sodium polyanethol sulfonate (SPS) to prevent clotting.  Two bottles aerobic (vented) and anaerobic ones.  To be inspected daily for signs of growth.
  4. 4.  Signs of growth: turbidity, hemolysis, surface pellicle, or froth (gas production).  Another alternative is blind subculture daily.
  5. 5.  The main disadvantage of the conventional system is the highest rate of contamination during subculture techniques.  Prolonged incubation and subculture is needed to detect Brucella species.
  6. 6. 2-Biphasic systems - Conventional broth that can flood solid agar in a closed system. Different devices available:. i- Hemoline system  Castaneda medium is the principle of it.  Was developed in 1947 in France (Biomerieux).  Contains 40 ml broth and solid agar.  Originally described for aerobic culture, however, anaerobic culture bottles available.  Inoculation of agar is by flooding it by inversion of bottles.  Colonies easily detected on surface of agar by inspection daily for 7 days.
  7. 7. Hemoline system
  8. 8. Hemoline system
  9. 9. ii- Septi-Check system -was developed by Hofhann-La Roche, Switzerland in 1980s. - Now marketed by BD systems in USA. -It was based on the dip slide bacteriologic urine- culture kit. - modified to fit onto 100-ml blood-culture bottles, the paddles being coated with appropriate media (chocolate, MacConkey agar). - The bottles contain 70 ml of broth medium. -The paddle is inoculated by the blood in broth by simple inversion of the bottle.
  10. 10. - The agar surfaces and the broth should be visually inspected for microbial growth twice daily for the first 2 days of incubation, and thereafter once a day up to the seventh day of incubation. - For anaerobic blood cultures, conventional broth bottles that should not be vented are available for this system. - advantages: less contamination on subcultures of positive. - disadvantage: daily inspection. - other biphasic systems available but not popular as the previously described ones.
  11. 11. Septi-check system
  12. 12. 3-Gas capture or broth displacement (Signal) system: - Was developed in 1986 by Oxoid. - Based upon the principle that microbial metabolism produces positive pressure in a sealed bottle, which can be visualized in a secondary plastic cylinder (Signal device) attached to the broth tube via a needle. - Aerobic and anaerobic microorganisms causing sepsis can be isolated from one Signal bottle. - However the use of both aerobic and anaerobic bottles in other systems was superior to single bottle use.
  13. 13. Oxoid signal devices
  14. 14. 4- Lysis-centrifugation system (Isolator) -A single-tube blood-culture system based on lysis, centrifugation and subsequent direct plating on appropriate media was described in 1978. - it is now distributed by Oxoid Isolator System. - The Isolator 10 tubes contain the following reagents in 0.7 mL of aqueous solution: saponin (cell-lysing agent), propylene glycol (blocks the foaming tendency of saponin), and SPS (anticoagulant).
  15. 15. - At least 6 ml of blood should be inoculated into these tubes, which must be processed as soon as possible, or at most 8 h after the inoculation of blood. - Tubes are centrifuged in the laboratory, preferably at 3000 g for 30 min. The supernatant is discarded, and the pellet is inoculated on solid media. - The Isolator 1.5 used for pediatric patients inoculated with a maximum of 1.5 ml of blood, and is processed without centrifugation. - Easy manipulation and detect more pathogens.
  16. 16. Isolator Blood Culture System
  17. 17. Some characters of commercial BC systems:
  18. 18. Thank you Alsharjah UAE 2014