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Helicobacter pylori (Marshall et al.) Goodwin et al.
51932™

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Product category
Bacteria
Strain designation
Tx30a
Type strain
No
Genome sequenced strain
Yes
Isolation source
Clinical isolate
Geographical isolation
United States; Texas
Applications
Enteric disease research
Infectious disease research
Product format
Frozen
General
Specific applications
Enteric Research
Preceptrol
No
Characteristics
Comments
Does not produce cytotoxin
Handling information
Medium
ATCC Medium 260: Trypticase soy agar/broth with defibrinated sheep blood
Temperature
37°C
Atmosphere
Microaerophilic
Handling procedure
1. This organism is shipped frozen in dry ice.  Just prior to use, thaw vial in water at approximately 37oC.  When thawed, a drop of the suspension may be used to do an immediate wet mount to observe the unique morphology of this organism and verify its viability by checking for motility.
2. Aseptically transfer the thawed suspension into a fresh #18 broth (3-5 ml).  Mix well.  This suspension can now be used to inoculate agar slant(s), plate(s), or the preferred biphasic culture.  Two #260 plates should be inoculated, one for microaerophilic growth and the second for aerobic growth.  No growth should occur on the plate incubated aerobically.

3. To obtain a biphasic culture, add 0.6 ml of the suspension to a # 260 slant.  The resulting pool at the bottom of the slant is where the best, most rapid growth will occur.

4. Incubate at 37oC under microaerophilic conditions using an anaerobe jar with an active catalyst and a microaerophilic gas generator pack, or other acceptable method, to obtain microaerophilic conditions.  Incubate tubes with cap loose.

5.       Within 3 days, good growth should be obtained in the broth pool at the bottom of the slant.  Additional incubation may be required for colonies to appear on agar plate.  Further subcultures can be made using the broth pool as the inoculum source.  Subcultures to biphasic cultures will require only 24 to 48 hours of incubation for good growth.

Handling notes
This is a slowing organism that requires moist conditions for best growth.  Growth at the broth/agar interface of the biphasic slant should occur within 3 days, but only light turbidity will be seen.  To observe growth, examine a wet mount of the broth under phase microscopy.  This organism is a medium size, regular to slightly curved to weak spiral, motile, Gram negative rod.
          Growth on agar takes longer than the biphasic culture.  Colonies are small smooth, transparent, butyrous, circular and low convex with an entire edge.  It is essential to use fresh, moist plates.  The cells do not Gram stain well using traditional procedures.  For best results, use a basic fuchsin counterstain in place of the safranin.

          Once good growth in obtained, transfer or freeze the culture.  Adding an equal amount of 20% sterile glycerol to pooled broth from several biphasic slants, followed by freezing in liquid nitrogen or ?ultra-low temperature? freezer is recommended.

          Additional information on this culture is available on the ATCC web site at www.atcc.org.

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