By EDWARD J. BUECHER 2 AND EDER L. HANSEN
Biochemical and physiological studies of
nematodes often require sufficient material
for analysis (5, 6). Two important limitations
of axenic culture methods are the need
for proteinaceous components (1, 8) and
proper gas exchange (9). Proteinaceous
supplements are tedious to prepare. Suitable
gas exchange is obtained by using thin layers
of medium (9), necessitating glassware of
considerable size if large populations are desired.
The plant-parasitic nematode Aphelenchus
avenae can withstand vigorous aeration in
buffer (3). When air was continuously
bubbled through a culture of A. avenae in 20
ml of a defined medium supplemented with
fresh chick embryo extract and serum (7),
the population increased from 400 to 11,000
per ml in 4 weeks. This aeration system was
therefore applied to mass culture of other
nematodes.
The free-living nematodes Caenorhabditis
elegans, Turbatrix aceti, and Panagrellus
redivivus, and the insect-parasitic nematodes
Neoaplectana glaseri and N. carpocapsae
(DD136 strain) were tested for growth under
continuous aeration. Sufficient air flow was
bubbled through the medium to continuously
mix the nematodes. Air was sterilized by
passage through a Millipore DA gas filter.
Gas washing bottles (Kontes Glass Co.,
Vineland, New Jersey) of 125, 250, or 500ml with open-end dispersion tubes were
used as the culture vessels. The basal medium
consisted of 3% soy peptone, 3% yeast
extract (4), and 0.7% dextrose; it was
autoclaved for 20 min at 121 C. MEM
vitamins® 100 × solution (Grand Island
Biological Co., Grand Island, New York),
5 ml per 100 ml of medium, and autoclaved
antifoam emulsion Y-30® (Dow
Coming Corp., Midland, Michigan), 0.2 ml
per 200 ml of medium, were added aseptically.
Filtered preparations of heated liver
extract (10) or yeast extract ( 1 ) were added
as supplements of 5 to 10 mg per ml; medium
containing yeast extract was adjusted to pH
3.8 with glacial acetic acid. When either
sodium caseinate (Nutritional Biochemicals
Corp., Cleveland, Ohio) (W. Hieb, personal
communication) or glycogen (Fisher Scientific
Co., New York, New York) was used as
the supplement, it was added at 10 mg per
ml and autoclaved with the basal medium.
Filtered hemin chloride (2) was then added
at l0 ~g per ml.
The inoculum size varied from 30 per ml
for N. carpocapsae to 2,000 per ml for T.
aceti; cultures were incubated at room temperature
(20 to 25 C). Growth was determined
from nematode counts; the final count
was made after 3 weeks when no further increase
in population was observed. The resuiting
populations are summarized in Table
1. Each species went through one or two
generations. Counts were up to 10 times
greater than in control test tube cultures, and
increased up to 600-fold over the inoculum.
Weights of lyophilized nematodes from the
170-ml liver-supplemented cultures were 500
to 720 mg. The 500-ml culture of C. elegans yielded a wet weight of 5 g and a lyophilized
weight of 1 g of nematode tissue.
Stoll obtained high populations of N.
glaseri with shaking of 3,5 mm-deep cultures
(11). In a 100-ml culture with constant
aeration we obtained a twofold increase in
population (Table 1). The protein used, 5
mg per ml, was lower than optimum for this
species.
Constant aeration appears to remove ammonia
and thus retard a rise in pH and subsequent
death noted in test tube cultures. In
mass cultures less than 1% of the nematodes
were dead at harvest compared to 50% in
test tube controls. Exceptions were cultures
of C. elegans in sodium caseinate medium,
in which 10% were dead, and T. aceti in
yeast medium, in which 4% were dead.
The tedious task of preparing protein extracts
can be eliminated by using glycogen or
sodium caseinate. Sodium caseinate is particularly
useful in that it is an autoclavable,
inexpensive, commercially available supplement
that supports high nematode populations.
It also supported maturation of C.
briggsae, C. elegans, P. redivivus, T. aceti
and N. carpocapsae in test tube cultures.
Retreived from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2619865/pdf/199.pdf
