Letters in Applied Microbiology 1990, 10, 171-173 AWCI013

An improved formulation and method of preparation of

crystal violet pectate medium for detection of pectolytic
erwinia
E. J . WOODWARD& K . ROBINSONDepartment of Agricultural Bacteriology, North of
Scotland College of Agriculture, 581 King Street, Aberdeen AB9 I U D , U K

Received I November 1989 and accepted 28 November 1989

W O O D W A R D , E.J. & ROBINSON,K . 1990. An improved formulation and

method of preparation of crystal violet pectate medium for detection of pectolytic
erwinia. Letters in Applied Microbiology 10, 171-173.
An improved procedure for the preparation of CVP medium is described on which
pectolytic erwinia rapidly develop deep, localised pits. The medium can be prepared
quickly and easily and forms a smooth, firm gel suitable for streaking.

Crystal violet pectate medium (RCVP) (Cuppels
& Kelman 1974; Phillips & Kelman 1982) has
been used routinely for isolation and quantifica-
tion of pectolytic erwinia from potato tubers
(DeBoer & Kelman 1975; Perombelon et al.
1987; Robinson & Foster 1987). Traditionally
RCVP contained RalTech sodium polypectate
(RalTech Scientific Services Inc., 3301 Kinsman
Blvd., P.O. Box 7545, Madison, Wisconsin
53707, USA), but when this became unavailable
it was necessary to find an alternative. A substi-
tute medium (BCVP) containing a polypectate
manufactured by Bulmer Ltd (H.P. Bulmer Ltd,
Plough Lane, Hereford HR4 OLE, UK) has
been described by Di & Kelman (1988). Follow-
ing preliminary experiments in this laboratory
BCVP was considered unsatisfactory because
pectolytic activity was slow and poorly defined
compared with that obtained using RCVP.
The purpose of the work described was to
determine modifications in the formulation of
BCVP that would enhance the rate of growth of
pectolytic erwinia and improve the quality of pit
production.
Materials and Methods
MODIFIED FORMULATION A N D
PREPARATION OF CRYSTAL VIOLET
PECTATE MEDIUM (BULMER)
Modified BCVP medium (MBCVP) was made
by adding the following ingredients to 500 ml of
cold distilled water; 1.0 ml 0.075% (w/v)
aqueous crystal violet solution, 3-0 ml 10%
CaCl, .2H,O (freshly prepared), 4.0 g Bacto
agar (Difco), 2.5 g proteose peptone no. 3
(Difco), 1.0 g KNO,, 9.0 g sodium polypectate
(Bulmer, production batch no. 1) and 0.5 ml
10% sodium lauryl sulphate. pH was not adjust-
ed. The medium was then shaken vigorously by
hand and autoclaved for 25 min at 120 psi; the
pressure was released slowly to avoid the forma-
tion of bubbles. Plates were poured and allowed
to dry thoroughly before use. A PTFE-coated
stirring bar (45 x 8 mm) inserted into the media
bottle prior to shaking facilitated eficient break
up of the polypectate and also allowed the
medium to be stirred gently on a magnetic
stirrer after autoclaving.
MEDIUM MODIFICATIONS
Various modifications were made to the formu-
lation of the Di & Kelman (1988) medium.
These included supplementation with proteose
peptone no. 3 (Difco), yeast extract and tryp-
tone (Oxoid); the effect of NaNO, and KNO, ,
the relative concentrations of CaCI, and agar,
and pH in the range 5.5-8.0.
EFFECTS O F MEDIUM COMPOSITION O N
G R O W T H A N D PECTOLYTIC ACTIVITY
Cultures were streaked on to nutrient agar and
incubated at 25°C for 24 h to obtain cells in the
exponential phase of growth. A suspension of
172 E. J . Woodward and K . Robinson
the culture was then made in Ringers solution
and five droplets, each containing in the order
of 4.0 x lo5 cells, were placed separately on to
the surface of well-dried media using a small
inoculating loop. Twenty isolates of pectolytic
erwinia were used comprising ten of Erwinia
carotouora subsp. atroseptica (Eca) and ten of
Erwinia carotouora subsp. carotouora (Ecc).
Results and Discussion
A new method is described for the formulation
and preparation of BCVP (Di & Kelman 1988)
which improves both the rate of growth of Eca
and Ecc and the production of pectolytic pits.
In arriving at the new formulation a variety of
modifications were examined.
In general, colony development on BCVP
requires 48-72 h and consequently pectolytic
activity is limited. Following supplementation
Fig. 1. Streak plate of ECA on MBCVP medium, showing pectolytic activity after 24 h incubation.
with peptone large colonies and distinct, deep
pits were produced in 24 h (Fig. 1). Increasing
peptone concentration from 0.5% to 1% did not
enhance the improvements. The addition of
tryptone or yeast extract had similar effects to
peptone but the medium was less solid and the
pits less distinct.
There appears to be an interrelationship
between the use of NaNO,, K N 0 3 and
peptone. Withdrawal of NaNO, and the addi-
tion of peptone produced deeper, better defined
pits than using NaNO, and no peptone. Substi-
tution of NaNO, with KNO, in the absence of
peptone did not improve pit formation after
24 h incubation, but after 48 h pits were better
defined than with peptone alone and the gel
remained firm. Media containing peptone
without KNO, were liquefied after 48 h.
Retaining firmness of the gel and restricting pit
formation to the area immediately surrounding
the colony are important because they extend
 Improved C V P medium
the time during which plates can be examined
and organisms be isolated from colonies.
The relative proportions of CaCl, and agar
could be altered without significantly affecting
pit development. However, one variation, 3 ml
CaCI,4 g agar, gave a marginal improvement
in pit quality.
When MBCVP was prepared as described the
pH was 6%. Adjustment of pH to values
between 5.5 and 8.0 with and without peptone
had no significant effect on pit formation except
at 5.5 at which both growth and pit formation
were virtually inhibited. No effect of pH on gel
solidity was observed.
The formulation described for MBCVP,
which differs from BCVP in the substitution of
KNO, for NaNO, , the incorporation of 0.5%
peptone and, the CaC1,-agar concentration
produced the best results in respect to rate of
growth, the production of deep well-defined pits
and a firm gel. These modifications when com-
bined with the new ‘cold’ method of pre-
peration, which differs by use of cold rather
than boiling water and mixing by hand rather
than using a blender, produce a smooth, firm
medium suitable for streaking (Fig. 1). The
medium is better than BCVP and is also prefer-
173
able to RCVP which had a rough, granular
texture and was unsuitable for preparing ‘streak’
plates.
References
CUPPELS,D. & KELMAN, A. 1974 Evaluation of selec-
tive media for isolation of soft-rot bacteria from soil
and plant tissue. Phytopatholoyy 64,468475.
DEBOER,S.H. & KELMAN, A. 1975 Evaluation of pro-
cedures for detection of pectolytic Erwinia spp. on
potato tubers. American Potato Journal 52, 117-
123.
Di, Y.B. & KELMAN, A. 1988 Modified Crystal Violet
Pectate (CVP) Medium. In Laboratory Guide ,for
IdentiJication of Plant Pathogenic Bacteria 2nd Ed.
ed. Schaad, N.W. p. 9. Minnesota: American Phyto-
pathological Society.
PEROMBELON, M.C.M., LUMB, V.M. & HYMAN,L.J.
1987 A rapid method to identify and quantify soft
rot erwinias on seed potato tubers. EPPO Bulletin
17,25-35.
PHILLIPS, J.A. & KELMAN, A. 1982, Direct fluorescent
antibody stain procedure applied to insect transmis-
sion of Erwinia carotovora. Phytopatholoyy 72, 898-
901.
ROBINSON,K. & FOSTER, G. 1987 Quantitative assess-
ment of tuber contamination by pectolytic erwinia
and its possible use in the prediction and control of
blackleg. Potato Research 30, 669-674.