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

Recommendations

Info

Culturing Microorganisms Symptom Cause Solution Poor overall growth Using nonrecommended media Use Biolog-recommended media. Isolate takes several days to form a colony Bacterium will not grow on BUG + B or forms pinpoint colonies Slow growth and/or weak pattern formation Mixed growth on agar plates Some environmental organisms take several days to become visible on growth plate, at which point culture may be too old for successful ID Fastidious gram-negative bacteria need special culture conditions Some environmental bacteria may be oligotrophic or temporarily in an oligotrophic state (i.e., they will only grow on low nutrient media such as R2A) Sub-optimal growth temperature and/or humidity and/or atmosphere Sticky bacterial surfaces Filamentous fungi mixed with bacteria Troubleshooting Subculture one or two passages in broth medium. Set up two or three agar plates to obtain sufficient growth. Use Chocolate agar and incubate with elevated CO2 at 35-37° C. Try to subculture the bacterium from R2A and see if they will gradually adapt to growing on BUG + B. If not, it is a species that is not included in our MicroStation/MicroLog database. Agricultural bacteria may be grown on BUG without blood. Use specified incubating temperatures (see Section 4 and Appendices). If your unknown organism came from a cold or warm environment, grow it first at its environmental temperature, then try to grow it at 35-37° C, 30° C, or 26° C. Add a pan of water to provide humidity in your incubator. Incubate the MicroPlate at the same temperature as the growth plate. Transfer a colony into a few ml of GN/GP-If or sterile water, vortex for several seconds, and streak out from the cell suspension onto a medium that aids the detection of subtle differences in colony morphology. Take a single colony and streak for isolation on a separate plate. Repeat as necessary until you have a pure culture. Cultures can be streaked on media containing antibacterial antibiotics. If this fails, use 1 drop of saline suspension of the mixed culture to inoculate each of four tubes of Sabouraud broth (1.0 ml). Add in series either 1,2,3 or 4 drops of concentrated HCl. Streak out from each tube. MicroStation System/MicroLog User Guide Apr 07 Section 11 � Page 3
Troubleshooting Preparing Inocula Symptom Cause Solution Incorrect turbidity Inaccurate %T measurements Trouble making a homogenous suspension Too few positive reactions in MicroPlates Turbidimeter out of calibration Tubes not properly blanked Significant scratches or smudges on tube Nonuniform suspension of cells Mucoid or clumpy cells Detergent traces in tube Calibrate accurately. Use correct standards. Make sure standards have not expired. Once you blank a tube, do not rotate it while making suspension. Blank each suspension tube (tubes are not optically precise and can vary tube-to-tube and with rotation). Use suspension tubes only once, then discard. Visually inspect tubes before using. Wipe outside of tubes with a tissue before placing in turbidimeter. Use 7” swab to mix cells all the way to the bottom of the tube. The light path that looks through the tube is only viewing the bottom of the tube. For Spore Forming Gram positive Rods, Follow the recommendations on pages 4-10 to 4-12. For Non-Spore formers: 1. Roll swab over young colonies in 3 rd and 4 th quadrants. 2. Mash colonies against side of a sterile tube (above meniscus). 3. Add several ml of inoculating fluid and wash the sides of the tube with a cotton swab. 4. Mix well, then examine for clumps. If clumps are present, allow them to settle. 5. Transfer this concentrated inoculum into a fresh Inoculating Fluid tube until the recommended turbidity is reached. * Be careful not to transfer any colony clumps into the Inoculating fluid. 1. For difficult organisms such as Gornia or Tsukamurella, suspend colonies as a dense suspension in a small amount of inoculating fluid as above. 2. Incubate as needed at 35° C (do not exceed 10 minutes). Vortex (if clumps still present, pull off supernatant and repeat). 3. Bring supernatant volume up to 18 ml and mix until homogenous. Use suspension tubes only once, then discard. Use of “bad” water Use high quality purified water without preservatives. Section 11 � Page 4 MicroStation System/MicroLog User Guide Apr 07
Page 1 and 2:
MicroStation System MicroLog Versi
Page 3 and 4:
LICENSE AGREEMENT Notice: This is a
Page 5 and 6:
Table of Contents Section Title 1.
Page 7 and 8:
10. Frequently Asked Questions…
Page 9 and 10:
Welcome Section 1 � Page 2 Connec
Page 11 and 12:
2. Installation and Registration In
Page 13 and 14:
Installation and Registration The A
Page 15 and 16:
8. E-mail the User Key File to tech
Page 17 and 18:
MicroStation System/MicroLog Overvi
Page 19 and 20:
Follow directions closely to obtain
Page 21 and 22:
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Preparing Samples Preparing an inoc
Page 31 and 32:
Preparing Samples Recognizing fasti
Page 33 and 34:
Preparing Samples Media shorthand:
Page 35 and 36:
Preparing Samples Don’t forget to
Page 37 and 38:
Preparing Samples b. Use a sterile
Page 39 and 40:
Preparing Samples aids in liquefyin
Page 41 and 42:
Preparing Samples Caution! Pipet th
Page 43 and 44:
Preparing Samples Special Procedure
Page 45 and 46:
Preparing Samples Initial culture m
Page 47 and 48:
Reading MicroPlates Set Up tab Sele
Page 49 and 50:
Reading MicroPlates When you’re e
Page 51 and 52:
Reading MicroPlates 2. Enter reacti
Page 53 and 54:
Reading MicroPlates Entering sample
Page 55 and 56:
Reading MicroPlates Select file nam
Page 57 and 58:
Reading MicroPlates Naming Workshee
Page 59 and 60:
Reading MicroPlates This arrow show
Page 61 and 62:
Reading MicroPlates Using Worksheet
Page 63 and 64:
Reading MicroPlates 4. Click Save t
Page 65 and 66:
Reading MicroPlates Wells with high
Page 67 and 68:
Reading MicroPlates Exiting When yo
Page 69 and 70:
Interpreting Results Understanding
Page 71 and 72:
Interpreting Results Mismatch Type
Page 73 and 74:
Interpreting Results What are those
Page 75 and 76:
Interpreting Results NOTE: A1 value
Page 77 and 78: Interpreting Results Slide the scro
Page 79 and 80: Interpreting Results VS. CURRENT MI
Page 81 and 82: Interpreting Results “Other” fi
Page 83 and 84: Data Management and Compiling Datab
Page 85 and 86: Combining Data Files Data Managemen
Page 87 and 88: OK, Out, Hold, Atypical Sorting lab
Page 89 and 90: Remember! When building your own da
Page 93 and 94: DATABASE WINDOW Data Management and
Page 97 and 98: Arrow indicates selected entry Data
Page 103 and 104: Export Help Mark this choice if you
Page 109 and 110: 8. Administration and Security In t
Page 111 and 112: Important! • If you test Password
Page 113 and 114: Log-In Privileges Administration an
Page 115 and 116: Remember that the password must be
Page 117 and 118: 9. Technical Notes In this section:
Page 119 and 120: Making Biolog Universal Growth Agar
Page 121 and 122: � � � � Technical Notes Gen
Page 123 and 124: 10. Frequently Asked Questions In t
Page 125 and 126: Q A Q A Q A Frequently Asked Questi
Page 127: Troubleshooting Symptom Cause Solut
Page 131 and 132: Troubleshooting MicroStation Reader
Page 133 and 134: Glossary Dendrogram Cluster diagram
Page 135 and 136: Glossary Spore, fungal Sterile, fun
Page 137 and 138: Culture Media Atmosphere Temperatur
Page 139 and 140: Appendices: Notes Notes: Section 13
Page 141 and 142: Appendix 4: Database Species Lists
Page 175 and 176: Appendix 5: Program Printouts Appen
Page 177 and 178: Appendix 5: Program Printouts 1�
Page 179 and 180:
Appendix 5: Program Printouts Funga
Page 181 and 182:
Appendix 5: Program Printouts Print
Page 183 and 184:
Page 185 and 186:
Page 187 and 188:
Appendix 6: Dangerous Pathogens (DP
show all

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

Create successful ePaper yourself

Delete template?

Save as template?