Organisms this product works with:
GARDNERELLA VAGINALIS SELECTIVE MEDIUM
A selective supplement for the isolation of Gardenerella vaginalis.
COLUMBIA BLOOD AGAR BASE
Code: CM0331
Formula |
gm/litre |
Special peptone |
23.0 |
Starch |
1.0 |
Sodium chloride |
5.0 |
Agar |
10.0 |
pH 7.3 ± 0.2 |
Directions
Add 39 g to 1 litre of distilled water. Boil to dissolve and sterilise
by autoclaving at 121°C for 15 minutes.
GARDNERELLA VAGINALIS SELECTIVE SUPPLEMENT
Code: SR0119
Vial contents (each vial is sufficient for 500ml of medium) |
per vial
|
per litre |
Gentamicin sulphate |
2.0 mg |
4.0 mg |
Nalidixic acid |
15.0 mg |
30.0 mg |
Amphotericin B |
1.0 mg |
2.0 mg |
Directions
Reconstitute one vial as directed, aseptically add the contents
to 450 ml of sterile Columbia Blood Agar
Base
cooled to approximately 50°C, and supplement with 50 ml of sterile human,
rabbit
or
horse blood. Mix
well
and pour into sterile Petri dishes. For the double layer technique hold the
medium in a water bath at
50°C.
Description
Gardnerella Vaginalis Selective Supplement, is based on the formulation of
Ison et al.1 and is recommended for the selective isolation of G. vaginalis
from the vaginal discharge of patients with symptoms of Non-specific Vaginitis
(NSV). The symptoms of this mild condition prior to the isolation of the aetiological
agent(s)
are:
1. The absence of recognised pathogens.
2. Foul smelling discharge.
3. pH greater than 4.5.
4. Release of `fish’ odour on the addition of potassium hydroxide
(10%) to the discharge.
5. The presence of `clue’ cells in prepared wet mounts (these
are epithelial cells with a characteristic stippled or granular appearance caused
by Gram
variable bacilli adhering to the cell surface).
Several media and techniques have been described for the isolation of G.
vaginalis. Gardnerella Vaginalis Selective Medium can be used for
the
surface inoculation technique or the double layer technique2.
With added human blood or rabbit blood3, a betahaemolytic reaction is exhibited
by G. vaginalis. This can be used as a preliminary diagnosis feature1.
The addition of `Tween 80’ (0.02% v/v) to the medium containing human blood has
been found to give enhanced beta-haemolytic zones4,5.
G. vaginalis is a Gram variable, small, pleomorphic bacillus which forms
0.25-0.5 mm diameter colonies producing beta-haemolysis on medium containing
human blood.
Technique
Surface Inoculation Method (Isolation)
1. Prepare the selective medium from Columbia Blood Agar Base,
Gardnerella Vaginalis Selective Supplement and defibrinated Horse Blood SR0050,
according to the directions. To demonstrate the characteristic haemolysis
substitute horse blood with human or rabbit blood when preparing
the medium.
2. Using a swab inoculate the vaginal discharge the medium.
3. Incubate, at 35°C for 48 hours in an atmosphere containing
7% carbon dioxide6.
4. Carry out confirmatory tests on all colonies from medium containing horse
blood and on betahaemolytic colonies from medium containing human blood or
rabbit blood.
Double Layer Method (Isolation and Presumptive identification)
1. Prepare two lots of selective medium from Columbia Blood
Agar Base, Gardnerella Vaginalis Selective Supplement and sterile human blood
according to the directions.
2. Use one lot to prepare base medium plates and
place the second lot in a water bath at 50°C.
3. Using the swab inoculate the vaginal discharge on to the surface of the
prepared plates. Allow to dry at room temperature for half an hour.
4. Overlay with 5ml of the selective medium at 50°C.
5. Allow the overlay medium to set.
6. Incubate at 35°C for 48 hours in an atmosphere containing 7% carbon
dioxide.
7. Carry out confirmatory tests on isolates that show a beta-haemolytic
zone. Use an inoculating wire to stab through the agar overlay to reach the
colonies
beneath.
The following tests have been compiled from the literature and personal communication.
Test or Substrate | Test Result |
% Positive |
Oxidase | Negative |
0 |
Catalase | Negative |
0 |
Haemolysis of: | ||
Human blood | Positive |
967 |
Rabbit blood | Positive |
96 |
Horse blood | Negative |
some strains |
Sheep blood | Negative |
07 |
Hippurate hydrolysis | Positive |
92 |
Starch hydrolysis | Positive |
90 |
Metronidazole 50 µg | Susceptible |
90 |
Trimethoprim 5 µg | Susceptible |
100 |
Sulphonamide 1000 µg | Resistant |
0 |
Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on
the label.
Store the prepared plates at 2-8°C.
Appearance
Dehydrated Medium: Straw coloured, free-flowing powder.
Prepared medium: Opaque red-coloured gel.
Quality control
Positive controls: |
Expected results |
Gardenerella vaginalis ATCC® 14018 |
Good growth; grey/white colonies |
Negative control: |
|
Proteus mirabilis ATCC® 29906 |
Inhibited |
* This organism is available as a Culti-Loop®
References
1. Ison C. A., Dawson S. G., Hilton J., Csonka G. W. and Easmon C.
S. F. (1982) J. Clin. Path. 35. 550-554.
2. Spiegel C. A., Eschenbach D., Schoenknech F. and Holmes
K. K.(1980) N. Engl. J. Med. 303. 601-607.
3. King E. A. (1964) `The Identification of Unusual Pathogenic
Gram negative Bacteria’ Center for Disease Control, Atlanta GA (quoted in
Reference 7).
4. Taylor E. and Phillips I. (1983) J. Med. Microbiol. 16.
83-92.
5. Totton P. A., Amsel R., Hale J., Piot P. and Holmes K.
K. (1972) J. Clin. Microbiol. 15. 141-147.
6. Bailey R. K., Voss J. L. and Smith R. F. (1979) J.
Clin. Microbiol. 9. 65-71.
7. Greenwood J. R. and Picket M. J. (1979) J. Clin. Microbiol. 9.
200-204.