Diagnostics Labs

Download Antibiotic Resistance: Methods and Protocols by Stephen H. Gillespie PDF

By Stephen H. Gillespie

Medical and diagnostic microbiologists aspect molecular and actual equipment for learning the turning out to be challenge of antibiotic resistance in micro organism, and facilitate new antibiotic learn courses to aid redress the matter. The innovations variety from those who supply quick analysis via DNA amplifications and phage demonstrate, to these for plotting the transmission of resistant organisms and investigating their epidemiology. in addition to illuminating the fundamental biology of antimicrobial resistance, in addition they strengthen and enforce now diagnostic and epidemiological instruments.

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7 mL microcentrifuge tubes. 8. Sterile plastic PCR tubes. 9. 5, 1 mM EDTA), stored in small aliquots at –20°C. 10. 10 mg/mL Proteinase K (Boehringer Mannheim) in TE, stored in small aliquots at –20°C. 11. 5, 50 mM EDTA, 2% (w/v) N-lauryl sarcosine, 1% (w/v) sodium dodecyl sulfate, 30 mg/L dithiothreitol, stored at room temperature. 12. 1% N-lauryl sarcosine. Gentle heating may be required to dissolve the guanidine thiocyanate. This solution is stable at room temperature, however precipitates may form in cool rooms.

A culture of 104 organisms/mL RIF resistant Mtb containing 4 µg/mL of RIF. Immediately, remove 1 mL aliquot of each control culture for DNA extraction. 5. Continue to cultivate the remaining control cultures at 37°C. On day 4 and day 7 of incubation, remove 1 mL aliquot for DNA extraction. 2. Sputum Processing 1. Collect and handle sputum specimens according to Centers for Disease Control and Prevention/National Institutes of Health (CDC/NIH) guidelines or equivalent for any potentially biohazardous contamination.

Gel fixing: place the plate in a shallow plastic tray, cover with fix/stop solution and agitate for 20 min at room temperature (see Note 14). The fix/stop solution is saved to terminate the developing reaction. 3. Wash the gel three times for 2 min each in ultrapure water with agitation. The gel plate is carefully drained after each wash (see Note 15). 4. Transfer the gel to staining solution and agitate for 30 min at room temperature. 5. Rinse the gel by dipping briefly (>5 s) into ultrapure water, draining and then place immediately into a tray of prechilled developing solution (see Note 16).

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