NBL/BAL/Sputums Processing

Mucoid/mucopurulent/purulent/bloodstained Sputums only:

• Add equal volume of a 0.1% solution of dithiothreitol or N-acetyl L-cysteine (NALC) to sputum (dilution (a) )
• Agitate and incubate for 10-20 mins
• Dilute 10 µL of homogenised sputum (dilution (a)) in 5mL of sterile distilled water (to produce dilution (b) )
• Inoculate 1µL loopful of this dilution (b) onto each type of routine (CBA and choc) plates
• For cystic fibrosis, ITU and immunocompromised also inoculate 1 µL of the sputasol/sputum dilution (a) to additional plates (MSA/PYO/BCEP)
• For patients with cystic fibrosis who have no previous B. cepacia colonisation, inoculate 100 µL of dilution (a) onto B. cepacia medium and spread inoculum over the entire surface of the agar plate.
• For Fungal culture, inoculate 100 µL of dilution (a) onto fungal culture media and spread inoculum as above

BAL/NBLs - that require centrifugation:

• Centrifuge at 1200 xg for 10 mins
• Tip off all but 0.5 mL of supernatant and re-suspend centrifuged deposit in remaining fluid
• Using a 10µl sterile loop inoculate each agar plate (CBA/BACI/CLED/CAND/SAB & LEG) with the specimen

If specimen is mucoid - add supotasol (1:1 dilution) after first centrifugation, incubate and then inoculate plates -> this changes colonial count! Only >10 cols significant; if diluted >5 cols significant

Routine: CBA/Bacitracin
Bronchiectasis/NBL/BAL gets an extra CLED/CAND/SAB
Cystic Fibrosis patients gets extra plates - MSA/PYO/BCEP

Lung abscess get an extra: NEO+Met

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