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Material Safety Data Sheet

Organisms

Organisms this product works with:

Culture Media Supplements

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.

 

 
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