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Dehydrated Culture Media

DIAGNOSTIC SENSITIVITY TEST AGAR (DST AGAR)

Code: CM0261

A susceptibility test agar for antimicrobial testing.

Typical Formula*

gm/litre

Proteose peptone

10.0

Veal infusion solids

10.0

Glucose

2.0

Sodium chloride

3.0

Disodium phosphate

2.0

Sodium acetate

1.0

Adenine sulphate

0.01

Guanine hydrochloride

0.01

Uracil

0.01

Xanthine

0.01

Aneurine

0.00002

Agar

12.0

pH 7.4 + 0.2 @ 25°C

 
* Adjusted as required to meet performance standards

Directions
Add 40 g to 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes.
For blood agar, cool the base to 50°C and add 7% of Defibrinated Horse Blood SR0050. Mix with gentle rotation and pour into Petri dishes (12 ml for a 9 cm dish) or other containers.
RECONSTITUTION AND MIXING SHOULD BE PERFORMED IN A FLASK AT LEAST 2.5 TIMES THE VOLUME OF MEDIUM TO ENSURE ADEQUATE AERATION OF THE BLOOD.

Description
Diagnostic Sensitivity Test Agar was developed in Oxoid as a dual purpose medium which would satisfy both diagnostic and susceptibility requirements.

The diagnostic role was supported by the nutritional amino-acid base with glucose to encourage early growth. The inclusion of the buffers (disodium phosphate and sodium acetate) helped prevent excessive movements of pH values which could result from utilisation of glucose or amino-acids. Such pH movements would interfere with haemolytic reactions1 and the MIC values of pH-susceptible antimicrobials2.
Long before the mechanisms of folate antagonism had been discovered, the addition of the bases adenine, guanine, uracil and xanthine were shown to improve the performance of the medium as an antimicrobial test medium.

Aneurine, added as a general purpose vitamin, improved the growth of several organisms especially staphylococci.

The agar used in the formulation has been specially processed to allow unimpeded diffusion of antimicrobials from discs3.
DSTA is now primarily used for susceptibility tests and its role in diagnostic microbiology i.e. the primary isolation of organisms from clinical samples, has diminished.

An essential requirement for satisfactory antimicrobial susceptibility media is that the reactive levels of thymidine and thymine must be sufficiently reduced to avoid antagonism of trimethoprim and sulphonamides4.

DSTA meets this requirement and in the presence of lysed horse blood (or defibrinated horse blood if the plates are stored long enough to allow some lysis of the erythrocytes) the level of thymidine will be further reduced. This is caused by the action of the enzyme thymidine phosphorylase which is released from lysed horse erythrocytes5. Thymidine is an essential growth factor for thymidine-dependent organisms and they will not grow in its absence or they will grow poorly in media containing reduced levels6. It is important that users of DSTA are aware of this limitation of thymidine which now exists in the medium and the effect it will have on a small proportion of organisms.

Details of the function of the medium and the methodology used for antimicrobial susceptibility tests are discussed in the Section `Susceptibility Testing’.

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 agar plates at 2-8°C.

Appearance
Dehydrated medium: Light straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

Positive controls:

Expected results

With blood 
Streptococcus pneumoniae ATCC® 6303 *

Good growth; grey/green mucoid colonies with alpha-haemolysis

Staphylococcus aureus ATCC® 25923 *

Good growth; white/grey coloured colonies

Without blood 
Enterococcus feacalis ATCC® 29212 *Good growth; straw colonies
Pseudomonas aeruginosa ATCC® 27853 *Good growth; straw colonies with green pigmentation

Negative control:

 

Uninoculated medium

No change

* This organism is available as a Culti-Loop®

Precautions
Diagnostic Sensitivity Test Agar has reduced thymidine activity and this will affect its performance as a primary isolation medium.

References
1. Expert Committee on Antibiotics (1961) World Health Organization Technical Report Series No.210. WHO, Geneva.
2. Bechtle R. M. and Scherr G. H. (1958) Antibiotics and Chemotherapy 8(12). 599-606.
3. Marshall J. H. and Kelsey J. C. (1960) J. Hyg., Camb. 58. 367-372.
4. Ferone R., Bushby S. R. M., Burchall J.J., Moore W.D. and Smith D. (1975) Antimicrob. Agents Chemotherap. 7. 91-98.
5. Ferguson R. W. and Weissfeld A. S. (1984) J. Clin. Microbiol. 19. 85-86.
6. Stokes E. J. and Ridgway G. L. (1980) `Clinical Bacteriology’ 5th Edn. Arnold. London. p.54.

 
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