MicroStation System, MicroLog Version 4.2 - DTU Systems Biology ...

Troubleshooting
Symptom Cause Solution
Gram variability Smear is too thick Make a single-cell-layer smear.
Run positive and negative controls.
All smears appear Overwarming of slides Air dry slides completely before warming.
gram negative
Don’t use a flame to fix slides.
Using old cultures Prepare slides from fresh log-phase growth cultures.
Over-decolorizing Repeat Gram stain, using specified timing.
Run positive and negative controls.
Morphology unclear Growth on agar media
affects morphological
appearance
Do a Gram stain from broth culture.
Too-intense color Under-decolorizing Use decolorizer made by same company that makes
your stains.
Prepare decolorizer or acetone/alcohol at several
concentrations. Test solutions at 5, 10, and 15 seconds
with known positive, negative, and weakly positive
cultures.
Run positive and negative controls.
Additional differentiation techniques
Make sure you’re using proper Gram stain procedure. If you still get
ambiguous results, use the following table as a guide:
Test Reactions
Vancomycin sensitivity (30 µg disks) Gram-negative bacteria � resistant
Gram-positive bacteria � most are sensitive
Growth on MacConkey and CNA plates Gram-negative bacteria � MacConkey +, CNA –
Gram-positive bacteria � MacConkey –, CNA +
KOH test (3%)* Gram-negative bacteria � suspension becomes thick
and stringy
L-alanine aminopeptidase activity (use
Gram-negative bacteria � +
commercially available test strips impregnated
with a colorimetric substrate for this enzyme)*
Gram-positive bacteria � –
*For test procedures, see Bailey & Scott’s Diagnostic Microbiology, Baron E.J. and Finegold, S.M., C.V. Mosby
Co., 1990, pages 102-103.
Section 11 � Page 2 MicroStation System/MicroLog User Guide Apr 07