Plant and Soil 78, 23-28 (1984). Ms.

Fr 4
© 1984 Martinus Nijhoff/Dr W. Junk Publishers, The Hague.

A modified sucrose fractionation procedure for the isolation of

frankiae from actinorhizal root nodules and soil samples
DWIGHT BAKER
Charles F. Kettering Research Laboratory, 150 E. South College St., Yellow Springs, OH
45387, USA

and DAVID O'KEEFE

Plant Science Division, University of Wyoming, Laramie, WY 82071, USA

Key words Actinorhizal plants Bacterial isolation techniques Frankia Nitrogen fixation
Root nodules Soil Sucrose density fractionation

Summary The isolation and pure culture of the symbiotic nitrogen-fixing frankiae has always
been difficult. In the past the isolation of these actinomycetes directly from soil samples has
proven impossible and isolations from root nodules of many genera has been only poorly
successful. We report here a modified sucrose fractionation procedure which increased the
success of isolations from root nodules and which permitted the isolation of Frankia directly
from soil samples. Crushed nodule suspensions or soil suspensions were incubated briefly in
0.7% phenol (carbolic acid) just before application to a sucrose density gradient. This phenol
incubation decreased the number of contaminating eubacteria and fungi but more importantly
increased the number of Frankia developing on the isolation plates. If the phenol incubation
was used solely without sucrose fractionation no Frankia were isolated, suggesting the death of
the organisms due to phenol toxicity. The use of selective nitrogen-deficient media proved
important for the isolation of frankiae from soils.

Introduction
The isolation and cultivation of in vitro of actinorhizal micro-
symbionts has always been problematic l . Reasons for difficulties
in isolating the symbiotic frankiae are I) the very slow growth of these
bacteria in relation to other actinomycetes and eubacteria, 2) the
preponderance of rapidly growing contaminating eubacteria and actino-
mycetes associated with a soil-borne structure, 3) the unknown media
preferences of frankiae making the transition from a symbiotic to
a heterotrophic or saprophytic metabolism, and 4) the inhibiting nature
of phenolic compounds released from host plant cells during isolation
procedures. Currently, several isolation procedures are employed which
overcome one or more of these difficulties with variable success. The
sucrose fractionation technique 2 , the serial dilution or modified dilu-
tion techniques S,7, the selective incubation or microdissection tech-
niques 4 ,l2, the osmium tetroxide technique 8 and the filter exclusion
technique 3 are most commonly used to isolate frankiae from actino-
rhizal root nodules. These techniques have proven particularly useful
with the host genus Alnus and to a lesser degree with Myrica,
23
24 BAKER AND O'KEEFE

Comptonia and Elaeagnus. No procedure has proven successful in

isolating frankiae directly from soils to date.
Isolations of other soil-borne actinomycetes have been improved if
phenol (carbolic acid) is incubated with the soil suspension 9 ,ll . In this
study a modified sucrose fractionation technique was devised utilizing
phenol which not only increased the relative success of isolations from
root nodule material but also permitted the isolation of frankiae
directly from soils. Recommendations are presented for culture media
preferences of several Frankia types.

Methods

Collection procedures
Root nodules and root zone soil samples were collected from actinorhizal plants from
natural populations in Table 1. Nodules and soil samples were chiIIed with ice at the time of
collection and then frozen in the laboratory until isolations were undertaken.

Isolations from nodules

Small portions of root nodule tissue were surface sterilized in a 2% solution of glutaral-
dehyde containing Tween 20 as a surfactant. The nodule pieces were then rinsed twice with
sterile distilled water (SDW) and crushed in a sterile mortar and pestle to form a crude nodule
suspension. To this point no variation from the original sucrose fractionation technique! was
made. The crude nodule suspension was then made to contain 0.7% phenol (v/v) using liquified
phenol and incubated for 10 min. Control suspensions contained no phenol. After incubation
a smalI sample of the nodule suspension was applied to a sterile discontinuous sucrose density
gradient composed of three layers: bottom layer, 60% (2.5 M); middle layer, 45% (1.6 M), top
layer 30% (1 M). Concentrations of sucrose solutions were determined by refractometer. The
use of a refractometer was important in this step. Other workers l2 have not used a refracto"
meter and failed to achieve proper separations. The gradients were centrifuged to equilibrium
using either a low speed or an ultracentrifuge equipped with a swinging bucket rotor. At com-
pletion the gradient tubes were pierced and the lower interface (45/60) collected. This fraction
was used to inoculate selective culture media (see below) by a pour plate procedure at a dilution
of 1:100. The solidified plates were sealed with Parafilm (American Can Corp., Greenwich,
CT USA) and incubated at 28°C. Observations were made at weekly intervals after inoculation
and results recorded at 8 weeks as number of Frankia colonies per isolation plate. Morphological
criteria were used to identify the organisms as Frankia 2.

Isolations from soils

Five grams of soil were suspended in 50 ml SDW and incubated for 30 min at room tempera-
ture. The suspension was then made to contain 0.7% phenol as described above and incubated
for an additional 10 min. A small sample of the soil suspension was applied to a discontinuous
sucrose gradient and the procedure continued as for the isolation from nodules.

Culture media
A defined propionate minimal medium (DPM) containing (g/l) KH 2PO 4' 1.0; MgSO 4 •
7H 20, 0.1; CaCl 2 • 2H2 0, 0.01; sodium propionate, 1.2; and Hoagland's microelement stock,
1 ml/l and FeS0 4 -EDTA stock 1.8 ml/l" adjusted to pH 6.8, was used for all soil and nodule
samples. In addition a second complex medium was used for each soil and nodule sample as
follows: Frankia agar (FA)l for samples from Alnus, Myrica, Cercocarpus and Purshia; Czapeks
medium supplemented with 0.2% yeast extract (YCZ)lO for samples from Casuarina; or "S"
agarlO for samples from Ceanothus. Cycloheximide was added to media before sterilization at a
concentration of 100 J.lg/ml, whenever necessary to inhibit growth of fungi.