PLATE CULTURE METHOD
Purpose
Isolates and quantitates micro-organisms (colony forming units) which may be identified by using routine microbiology laboratory methods.
Materials Required
Ø Brush with Biofilm Sample in test tube or suitable container
Ø Sterile Phosphate Buffered Saline (PBS)
Ø 2 Blood Agar Plates
Ø 1 Sabouraud Plate
Ø Sterile Plastic Spreaders or equivalent
Ø 10 and 100 ul micropipette and tips
Ø Waterbath Sonicator and / or Vortex Mixer
Ø Incubator
Preliminary Instructions
1) Retrieve Biofilm sample by following the FAS Endoluminal Brush Instructions for Use.
2) Place the Brush / Biofilm sample into a test tube or an equivalent container.
Procedure
1) Add 1ml of Sterile PBS to the container holding the Brush / Biofilm sample (“Sample”).
2) Sonicate the Sample in a sonicating waterbath for 1 minute (room temperature).
Note: If a sonicating waterbath is not available, proceed directly to Step 3.
3) Vortex for 15 seconds.
Note: If a sonicating waterbath is not available, vortex for 1 minute.
4) Transfer 10 ul of the PBS / Biofilm solution on to Blood Agar Plate and another 10 ul on to a Sabouraud Plate. Spread over the surface of the plate using a sterile plastic spreader.
5) Repeat Step 4 using 100 ul of PBS / Biofilm solution on to the second Blood Agar Plate.
6) Incubate all plates overnight at 37◦ C.
7) Examine plates for bacterial / yeast growth, record and report growth to clinicians according to the hospital’s standard procedure.
8) Re-incubate and re-examine plates at 48 hours. Record and report results as above.
CAUTION: If a Biofilm sample has been taken from a catheter which is impregnated internally with antimicrobial compounds, then special procedures are necessary so that micro-organisms are not inhibited during the culture procedure – leading to erroneous results. It is recommended that plates be made with culture media containing neutralising agents that prevent inhibition. A suitable media has been defined by Schmitt et al (1996) J Clin Microbiol 34: 508-511.
Interpretation of Quantitative Culture: Data collected and evaluated has provided the following guidelines for the interpretation of culture results (Reference: Kite: J Clin Path 1997 50: 278-281).
< 100 CFU: Not clinically significant
> 100 CFU: Clinically significant
> 100,000 CFU: Diagnostic of CRS
