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

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STOP! Is your suspension density accurate? Did you calibrate using Biolog’s Turbidity Standards? Preparing Inocula Preparing Samples Once your microbe is isolated and cultured, prepare a liquid inoculum. Refer to instructions enclosed with Biolog MicroPlates for detailed directions. In general: • The inoculum MUST be within the range specified by the turbidity standards accompanying your database (+ 2% T). • The inoculum MUST be uniformly suspended. If a bacterial organism forms clumps, you will need to use special techniques to achieve a homogenous suspension. (see “Dry Tube Method” page 4.11) Table 4-2 will help you decide which suspension medium to use: Organism Type Inoculating Fluid Turbidity Standards Inoculum Density Gram Negative Non-Enteric GN/GP-IF GN-NENT 52% T Gram Negative Enteric GN/GP-IF + T GN-ENT 61% T Gram Negative Fastidious GN/GP-IF + T GP-COC & GP-ROD & GN-FAS Gram Positive Cocci Gram Positive Rods (non-spore forming) Gram Positive Rods (spore forming bacillus) GN/GP-IF + T GP-COC & GP-ROD & GN-FAS 20% T 20% T GN/GP-IF GP-ROD SB 28% T Anaerobes AN-IF AN 65% T Yeasts Sterile water YT 47% T Filamentous Fungi (and select yeast species) FF-IF FF 75% T TABLE 4-2: SELECTING THE CORRECT INOCULATING FLUID, TURBIDITY STANDARDS, AND CELL DENSITY Adding thioglycolate to inoculating fluid Some microbes (see Table 4-2) require the addition of thioglycolate (T) to the inoculum. Thioglycolate acts as an anticapsule agent; it decreases production of bacterial capsules so that strains give more consistent patterns. Biolog provides you with droppers of thioglycolate. To add thioglycolate: MicroStation System/MicroLog User Guide Apr 07 Section 4 � Page 7
Preparing Samples Don’t forget to add thioglycolate when working with: GN Enteric GN Fastidious GP Cocci & Rods Remember, your microbes are alive. Treat them with care. Use fresh cultures. Caution! Use special care! Do not excessively vortex FF-IF suspensions. The FF-IF is viscous. It will trap air bubbles that will interfere with absorbance readings. 1. Hold reagent dropper upright and point tip away from you. Using a dissecting hemostat fully crush ampule close to it’s center one time only. Tap the bottom on benchtop a few times. Caution: Do not manipulate dropper any further, as the plastic may puncture, causing injury. 2. Invert the dropper for convenient drop-by-drop dispensing of reagent. 3. Dispense precisely 3 drops of concentrated thioglycolate into your inoculating fluid. Do not use more than 3 drops per 18-20 ml. This gives you a 5 mM final concentration. 4. Each dropper contains about 15 drops. You can continue to use an open dropper for the rest of the day, and then discard the remainder. Adding sodium salicylate to inoculating fluid If gram-positive bacteria are false positive even after you’ve added thioglycolate to the inoculating fluid, try adding sodium salicylate: 1. Add thioglycolate as described above. 2. Add 1 ml of sterile 100 mM sodium salicylate to 18-20 ml of GN/GP- IF. 3. Proceed with sample preparation. Preparing inocula Once you’ve added thioglycolate (if necessary), prepare the inocula: 1. Establish the appropriate turbidity range on your turbidimeter by adding and subtracting 2% T to the percent transmittance measured with the appropriate turbidity standard. 2. Dip a sterile swab into the inoculating fluid to moisten it. 3. Lift cells from the agar by rolling the swab over the colonies, rather than sliding across them. Be sure not to pick up any agar. 4. Twirl the swab against the inside surface of the tube (above the fluid line) to gently dissolve colonies. Section 4 � Page 8 MicroStation System/MicroLog User Guide Apr 07
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Page 3 and 4: LICENSE AGREEMENT Notice: This is a
Page 5 and 6: Table of Contents Section Title 1.
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Page 47 and 48: Reading MicroPlates Set Up tab Sele
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Combining Data Files Data Managemen
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OK, Out, Hold, Atypical Sorting lab
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Remember! When building your own da
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Data Management and Compiling Datab
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DATABASE WINDOW Data Management and
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Arrow indicates selected entry Data
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Export Help Mark this choice if you
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8. Administration and Security In t
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Important! • If you test Password
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Log-In Privileges Administration an
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Remember that the password must be
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9. Technical Notes In this section:
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Making Biolog Universal Growth Agar
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� � � � Technical Notes Gen
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10. Frequently Asked Questions In t
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Q A Q A Q A Frequently Asked Questi
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Troubleshooting Symptom Cause Solut
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Troubleshooting Preparing Inocula S
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Troubleshooting MicroStation Reader
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Glossary Dendrogram Cluster diagram
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Glossary Spore, fungal Sterile, fun
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Culture Media Atmosphere Temperatur
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Appendices: Notes Notes: Section 13
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Appendix 4: Database Species Lists
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Appendix 5: Program Printouts Appen
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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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