Identification Procedures

1
Identification Procedures

• Germ tube Procedure
• Place inoculum in 0.5 mL of germ tube medium such
  as serum or plasma (human, rabbit, sheep, other)
• Incubate tube for 2-3 hours at 35-37oC
• A drop of yeast-medium mixture is placed on a
  microscope slide, overlaid with a coverslip and
  examined microscopically for the presence of germ
  tubes

2
Identification Procedures

• Germ tube Procedure
• Germ tubes have the following characteristics
• Thin (about 1/2 - 1/3 the diameter of the parent
  cell) structures that are at least 2-4 times
  longer than parent cells
• May be straight but usually slightly curved
• No constriction between parent and germ tube
• Pseudohyphae and elongated blastoconidia that can
  be confused with germ tubes

3
Identification Procedures

• Germ tube Procedure
• Pseudohyphae characteristics
• Width is 1/2 or more of the parent cell
• Length is less than twice the diameter of the
  parent cell
• Pseudohyphae tend to be straight
• Progress to tree-like appearance
• A constriction exists where the pseudohyphae
  joins the parent cell

4
Identification Procedures

• Germ Tube Procedure
• Blastoconidia characteristics
• Buds are wider than germ tubes
• Buds are oval not elongated
• Buds are usually not curved from axis of cell
• There is a definite constriction between the
  parent cell and the bud blown out

5
Germ Tubes
Bud
Pseudohyphae (Arrow)
6
Blastoconidia (lower left) vs germ tubes
7
Identification Procedures

• Germ tube Procedure
• Precautions regarding performance
• Too heavy an inoculum can lead to false negative
  results (the amount of yeast in the medium should
  render it barely perceptibly cloudy)
• Incubation should not be extended beyond three
  hours because some true hyphae producing fungi
  may cause false positive results

8
Identification Procedures

• Germ tube Procedure
• Comments regarding performance
• If the test is positive, no further testing is
  needed for presumptive ID of C. albicans
• If test is negative further tests are needed
  because up to 5 of C. albicans isolates are germ
  tube negative
• Germ tube negative yeasts should next be
  inoculated onto a corn meal tween 80 medium

9
Identification Procedures

• Corn Meal Tween 80 (CMT 80) test
• When inoculated on CMT 80 by the Dalmau
  technique, yeasts will produce various structures
  that normally are not produced on many other
  media
• These structures include pseudohyphae, true
  hyphae with or without arthroconidia and, in the
  case of Candida albicans, chlamydoconidia
• Simple budding yeasts will need to identified
  biochemically

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