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

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11. Troubleshooting In this section: �Gram Stain Identification �Culturing Microorganisms �Preparing Inocula �Inoculating MicroPlates �Incubating MicroPlates �MicroStation Reader Troubleshooting Carefully following the instructions in this guide will greatly minimize problems. Occasionally, however, you may get stuck or encounter difficulties. This section addresses the symptoms, causes, and solutions to those occasional problems. If you still can’t figure out the cause of the problem, call Biolog Technical Service. We’re always glad to help. Additional help can be found on Biolog’s website at www.biolog.com. Gram Stain Identification Since Gram stain readings start the chain of steps leading to a MicroStation/ MicroLog identification, it’s essential to perform them according to standard lab protocol and to interpret results correctly. � � � � View the smear with a light microscope, using the oil-immersion objective. Gram-positive bacteria appear blue or violet; gram-negative bacteria appear pink or red. Gram-negative bacteria may appear gram-positive if the smear is too thick and decolorization is incomplete. Gram-positive bacteria may appear gram-negative if the smear is overdecolorized. This can also occur if the culture is not fresh and has reached stationary phase (some Bacillus species are gram positive for only a few divisions after spore germination). In addition, gram-positive bacteria will appear gram-negative if the integrity of their cell walls is damaged. To prevent misidentification, prepare light smears of young, actively growing cultures. Use known gram-positive and gram-negative controls. When you’re determining organism morphology, perform Gram stains from liquid medium. Solid agar can affect the appearance of the organism. MicroStation System/MicroLog User Guide Apr 07 Section 11 � Page 1
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
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MicroStation System MicroLog Versi
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LICENSE AGREEMENT Notice: This is a
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Table of Contents Section Title 1.
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10. Frequently Asked Questions…
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Welcome Section 1 � Page 2 Connec
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2. Installation and Registration In
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Installation and Registration The A
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8. E-mail the User Key File to tech
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MicroStation System/MicroLog Overvi
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Follow directions closely to obtain
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Preparing Samples Preparing an inoc
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Preparing Samples Recognizing fasti
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Preparing Samples Media shorthand:
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Preparing Samples Don’t forget to
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Preparing Samples b. Use a sterile
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Preparing Samples aids in liquefyin
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Preparing Samples Caution! Pipet th
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Preparing Samples Special Procedure
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Preparing Samples Initial culture m
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Reading MicroPlates Set Up tab Sele
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Reading MicroPlates When you’re e
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Reading MicroPlates 2. Enter reacti
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Reading MicroPlates Entering sample
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Reading MicroPlates Select file nam
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Reading MicroPlates Naming Workshee
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Reading MicroPlates This arrow show
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Reading MicroPlates Using Worksheet
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Reading MicroPlates 4. Click Save t
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Reading MicroPlates Wells with high
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Reading MicroPlates Exiting When yo
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Interpreting Results Understanding
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Interpreting Results Mismatch Type
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Interpreting Results What are those
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Appendix 5: Program Printouts 1�
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Appendix 5: Program Printouts Funga
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Appendix 5: Program Printouts Print
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Appendix 6: Dangerous Pathogens (DP
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