Asparagine utilization

Purpose

This test detects the inability of Xanthomonas to utilize asparagine as a sole source of carbon and nitrogen. This is a diagnostic feature for separating Xanthomonas from the closely related pseudomonads, which can use asparagine as a sole source of carbon and nitrogen.

Ingredients

Solution #1

Ingredient	1 L	500 ml
Distilled water	100 ml	50 ml
K2HPO4	8 g	4 g
KH2PO4	2 g	1 g

Solution #2

Ingredient	1 L	500 ml
Distilled water	100 ml	50 ml
MgSO4 • 7H2O	2 g	1 g
FeSO4 • 7H2O	0.5 g	0.25 g
NaC1	1 g	0.5 g
MnSO4	0.02 g	0.01 g
N2-free H2SO4	1 drop	0.5 drop (10μl)

Solution #3

Ingredient	1 L	500 ml
Distilled water	100 ml	50 ml
Na2MoO4 • 2H2o	0.02 g	0.01 g

Solution #4

Ingredient	1 L
Distilled water	100 ml
CaSO4	0.16 g

L-Asparagine solution

Ingredient	1 L	500 ml
Autoclaved Distilled water	950 ml	470 ml
L-Asparagine dissolved in a 10ml water blank	2 g	1 g

Instructions

1. All glassware must be acid-soaked, then thoroughly rinsed in 4 changes of distilled water, and dried before use.
2. Prepare the 4 solutions.
3. Filter-sterilize the L asparagine solution into the autoclaved water.
4. Then filter-sterilize, in the following order: solutions #3,#4,#2,#1, at a concentration of 10 ml/liter (5 ml/500 ml) into the sterile asparagine solution.
5. Dispense in 5 ml quantities in test tubes.
6. Inoculate from weak suspensions. Include a positive growth control (a non-Xanthomonas species).
7. Examine tubes for growth after 4, 7, and 10 days of shaker incubation. Weak growth is checked by transferring a 2 mm loopful of the 10 day old culture to fresh tubes of the asparagine medium and to YS broth. Repeat if weak growth occurs.

References

• Dye, D. W. 1962. The inadequacy of the usual determinative tests for the identification of Xanthomonas spp. New Zealand Journal of Science 5: 393-416.

Contributed by

From the Culture Media for Plant Pathogenic Fungi and Bacteria, University of Massachusetts: Contributed by Robert L. Wick