Laboratory Cultivation of Caenorhabditis elegans and Other
Nematodes have been cultured continuously in the
laboratory since 1944 when Margaret Briggs
Gochnauer isolated and cultured the free-living hermaphroditic
species Caenorhabditis briggsae
. Work with C. briggsae
and other rhabditid nematodes, C. elegans,
, and R. pellio
, demonstrated the
relative ease with which they could be cultured
(Dougherty, 1960; Vanfleteren, 1980). The culturing
techniques described here were developed for C.
elegans, but are generally suitable (to varying degrees)
for other free-living nematodes. Whereas much of the
early work involved axenic culturing, most of these
techniques are no longer in common use and are not
|FIGURE 1 Caenorhabditis elegans hermaphrodite (A) and
copulating. Several eggs and young larvae are also
In the 1970s, C. elegans
became the predominant
research model due to work by Brenner and coworkers
on the genetics and development of this
species (Brenner, 1974). An adult C. elegans
1.5 mm long and, under optimal laboratory conditions,
has a life cycle of approximately 3 days. There are two
sexes, males and self-fertile hermaphrodites (Fig. 1),
that are readily distinguishable as adults. The animals
are transparent throughout the life cycle, permitting
the observation of cell divisions in living animals using
differential interference microscopy. The complete cell
lineage and neural circuitry have been determined,
and a large collection of behavioral and anatomical
mutants has been isolated (Wood, 1988). C. elegans
has six developmental stages: egg, four larval stages
(L1-L4), and adult. Under starvation conditions or
specific manipulations of the culture conditions, a developmentally arrested dispersal stage, the dauer
larva, can be formed as an alternative third larval stage
(Golden and Riddle, 1984).
Many of the protocols included here and other
experimental protocols have been summarized in
Wood (1988). We also include a previously unpublished
method for long-term chemostat cultures of C. elegans
. General laboratory culture conditions for
nematode parasites of animals have been described
(Hansen and Hansen, 1978), but none of these nematodes
can be cultured in the laboratory through more
than one life cycle. Marine nematodes and some plant
parasites have been cultured xenically or with fungi
(Nicholas, 1975). Laboratory cultivation of several
plant parasites on Arabidopsis thaliana
seedlings in agar
petri plates has also been reported (Sijmons et al.
II. MATERIALS AND
strains, as well as strains of
other free-living nematodes, and bacterial food sources
for them are available from the Caenorhabditis Genetics
Center (250 Biological Sciences Center, University
of Minnesota, 1445 Gourtner Ave., St. Paul, MN 55108).
Most chemicals are obtained from general laboratory
supply companies such as Fisher Scientific and Sigma;
catalog numbers are for Fisher except where noted.
Three sizes of polystyrene petri dishes are used in culturing
nematodes: 35 × 10mm (Cat. No. 8-757-100-A),
60 × 15mm (Cat. No. 8-757-13-A), and 100 × 15mm (Cat. No. 8-757-13). Triple-baffled Fernbach flasks (250
ml, 1 liter, and 2.8 liter, Cat. No. 2554) for liquid culture
are from Bellco. The programmable dispensing pump
(Model DP-200) used when making plates and the
chemostat (Bioflo I) are from New Brunswick Scientific
Company. An IEC clinical centrifuge (Cat. No. 05-101-
5) with rotor 221 and metal shields 303 at setting 4
(RCF approximately 750) for 30s is used for pelleting
Tools for manipulating individual nematodes on
plates are made by anchoring a 1.5-cm piece of 32-
gauge platinum wire (Cat. No. 13-766-10-B) in a 6-in.
inoculating loop holder or in a Pasteur pipette (by
breaking off the pipette at the point where it narrows
and holding in a flame with the wire to seal the end).
For best results the end of the wire used for manipulation
should be flattened and, if necessary, rounded to
remove sharp edges. Observation of nematodes on
plates is performed through a dissecting microscope
with a magnification range of approximately 6 to 50x,
fitted with a variable-angle transmitted light source.
The microscopes in most common use for research are
the Wild M3, M5, and M8 (Leica Inc.).
A. Preparation of Plates
The procedure for preparation of plates is modified
from that of Brenner (1974).
- Cholesterol stock (5 mg/ml): Dissolve 0.5g cholesterol
(Cat. No. C314) in a final volume of 100ml of 95%
- 1M CaCl2: Dissolve 14.7g of CaCl2.2H2O (Cat.
No. C79) in a final volume of 100ml of distilled water
- 1M MgS04: Dissolve 24.65 g of MgSO4.7H2O
(Cat. No. M63) in a final volume of 100ml of distilled
water and autoclave.
- KH2P04 stock (1M): Dissolve 68.04g of KH2PO4 (Cat. No. P285) in approximately 425ml of distilled
water. Add KOH (Cat. No. P250) pellets while monitoring
the pH until pH is 6.0. Bring the volume to
500ml; autoclave in 100-ml aliquots.
- B broth: Add 1.0g of tryptone and 0.5g of NaCl
(Cat. No. S640) to 100 ml of distilled water in a 250-ml
screw-cap flask and autoclave.
- OP50 stock: Inoculate 100 ml of B broth (in screwcap
flask) with Escherichia coli strain OP50, a uracil
auxotroph, and shake overnight at 37°C. Store the
stationary-phase culture at 4°C for up to 60 days.
- NG agar: Add 3 g of NaCl, 17 g of Difco agar (Cat.
No. DF0140-01-0), 2.5g of peptone (Cat. No. DF0118-
15-2), and 975ml of distilled water to a 2-liter
Erlenmeyer flask. Autoclave. Place the flask in a 50°C water bath to prevent solidification while dispensing
medium into plates. Allow the flask to cool to approximately
65°C and add the following, using sterile
technique and swirling the flask after adding each
ingredient: 1 ml cholesterol stock, 1 ml 1 M CaCl2, 1 ml
1 M MgSO4, and 25 ml KH2PO4 stock.
|FIGURE 2 Examples of seeded plates. (A)
Spot plate used for
matings with single males
or with mutant animals that do not mate
(B) Zig-zag plate used for routine strain
crosses. (C) Grid plate
for mutant screens, strain maintenance,
crosses in which progeny are counted.
B. Liquid Culture
- Accurate dispensing of medium is accomplished
most easily with the aid of a programmable dispensing
pump. Fill 60 × 15-mm plates with 13ml of agar,
100 × 15-mm plates with 30ml, and 35 × 10-mm plates
with 4ml. The plates should be bubble free; flame the
surface to remove bubbles.
- Allow the plates to cool overnight; then put
plates at 37°C for 24h. Allow the plates to return to
room temperature; store at 4°C.
- Seed the plates (Fig. 2) with OP50 stock by
spreading approximately 0.05 ml on the surface using
a 1-ml pipette, and incubate overnight at 37°C or for
24-48 h at room temperature. Plates should be at room
temperature before placing worms on them.
The procedure is modified from that of Sulston and
- M9 buffer: Dissolve 3 g of KH2PO4, 6 g of Na2HPO4 (Cat. No. S393), and 5 g of NaCl in distilled water; then
add 1 ml of 1M MgSO4. Bring the volume to 1 liter with
distilled water and autoclave in 100-ml aliquots.
- S basal: Add 5.84 g of NaCl, 50ml KH2PO4 stock,
and 1 ml cholesterol stock to 950ml of distilled water;
autoclave in 100-ml aliquots.
- Potassium citrate stock (1M): Add 105.07 g of citric
acid monohydrate (Cat. No. A104) to 250ml of distilled
water. Add KOH pellets while monitoring the pH until
pH is 6.0. Bring the volume to 500ml and autoclave in
- 100x trace metals: Dissolve 0.69g of FeSO4.H2O
(Cat. No. 1467), 1.86g of Na2EDTA (Cat. No. 02793),
0.197 g of MnCl2.4H2O (Sigma, Cat. No. M-3634), 0.287
g of ZnSO4.7H2O (Cat. No. Z76), and 0.025g of
CuSO4.5H2O (Cat. No. C493) in 1 liter of distilled
water. Autoclave in 100-ml aliquots; store in foilwrapped
- 50% glucose: Add 50g of glucose (Cat. No. D16)
to 50ml of distilled water and autoclave.
- 60% sucrose: Add 120 g of sucrose (Cat. No. S5) to
80ml distilled water and autoclave. Store at 4°C.
- S medium: Add in the order indicated, using
sterile technique, 1 ml potassium citrate stock, 1 ml
100x trace metals, 0.3 ml 1M CaCl2, and 0.3 ml 1M MgSO4 to 100 ml of S basal.
- XI666 medium: Add 20g of Na2HPO4·7H2O (Cat.
No. S373), 4.5g of KH2PO4, 1.2 g of NH4Cl (Cat. No. A661), 16g of tryptone (Cat. No. DF0123-15-5), and 4g
of yeast extract (Cat. No. DF0127-15-1) to 980ml distilled
water in a 2.8-liter baffled flask. Mix, autoclave,
allow to cool, and add 20ml of 50% glucose and 8 ml
of 1M MgSO4.
- XI666 stock: Inoculate X1666 medium with X1666,
a nalidixic acid-resistant, prototrophic, plasmid-free
strain of E. coli. Shake at 37°C overnight. Transfer to
preweighed sterile centrifuge bottles/tubes and centrifuge
at 4000 RCF for 10min. Remove supernatant
and determine weight of bacteria. Resuspend in S
medium to 5% (w/w). Store at 4°C.
C. Chemostat Culture
- With 2 ml of M9 buffer per plate, wash worms off
five 60 × 15-mm plates that have just cleared of bacteria.
Pellet nematodes. Remove supernatant and wash
twice with fresh M9.
- Add the washed worms to 250ml X1666 stock in
a l-liter sterile baffled flask and place on shaker at
20°C. When the medium is cleared of bacteria, centrifuge
at 750 RCF for 5 min.
- Remove supernatant and resuspend in 15 ml of
M9 buffer, divide between two 15-ml tubes, and place
on ice. When cold, add 7.5 ml of cold 60% sucrose to
each tube. Mix by inversion and centrifuge immediately
at 1500 RCF for 5 min.
- Remove nematodes from top of tube immediately
and wash twice with 15 ml of M9 buffer. Place at
20°C on shaker for 30min to allow digestion of bacteria
in nematode intestines.
- Wash twice with M9 buffer and use immediately
or freeze at-70°C.
|FIGURE 3 Chemostat assembly. N, nutrient
reservoir; V, culture
vessel; E, effluent tank; S,
large stir plate; B, large stir bar (in reservoir
keep bacteria evenly suspended); W,
water bath (set at 12°C)
connected to cold finger of chemostat culture
vessel; U, Bioflo I control unit; P, peristaltic
pump; SA, selective
agent; C, compression
fitting (Swagelok Co.) used in the line for
reservoir replacement (see Section III,
C, step 12). Sterility is critical
maintenance of long-term cultures.
: Make in same manner as X1666
stock except use E. coli strain OP50 and resuspend the
pellet in M9 buffer (1/20th volume, or less, of the
Figure 3 is a diagram of the chemostat assembly.
D. Freezing Strains for Long-Term Storage
- Fill nutrient reservoir with 10 liters of 1/5 × S
basal [substitute polyoxyethanyl-cholesteryl sebacate
(Sigma, Cat. No. C-1145) for cholesterol] and add large
- Fill culture vessel with 0.2 vol water. Attach autoclavable
0.2-jiim filters to the vents and air entry
tubing. Autoclave connected nutrient reservoir, culture
vessel, and effluent tank.
- When cool, add the other stock solutions, as to
complete 1/5 × S medium (see Section B), to the nutrient
- Add the concentrate from 12 liters of overnight
OP50 cultures to the nutrient reservoir (final OD600nm =
- Install culture vessel in chemostat control unit
and make connections.
- Start air flow and stir the culture vessel impeller
at 220rpm. Set the heat regulator to 20°C.
- Run the feeding pump until culture vessel is full;
then turn it off.
- Inoculate culture vessel with sterile LI larvae prepared
as described in Section III,E, steps 10 and 11.
- Add OP50 concentrate from a l-liter overnight
culture to culture vessel. Add approximately 1 ml
sterile antifoam A (Sigma, Cat. No. A-5758) as necessary
to minimize foam.
- Monitor the culture every 2 days by removing
a sample and counting replicate aliquots spotted on
plates (see Section III,A). Continue to add sterile
antifoam A to culture vessel as needed. When the
culture reaches ~50 animals per 100/A, turn on the
feeding pump (setting 9).
- Monitor population density and adjust the
speed of the feeding pump as necessary to maintain
a reproducing culture. A dense population (~125
animals/10//I) that is mostly LI and L2 larvae and
roughly one-fourth dauer larvae can be maintained.
- Prepare a replacement nutrient reservoir for use
when the first one is depleted. The effluent tank should
be changed at the same time.
The procedure is modified from that of Brenner
- 1M NaCI: Dissolve 29.22g of NaCl in a final
volume of 500ml distilled water and autoclave.
- S + glycerol: Add 20ml of 1M NaCl, 10ml of
KH2PO4 stock, and 60ml of glycerol (Cat. No. G33) to
110 ml distilled water and autoclave.
E. Isolation of Staged Animals
- Take three contaminant-free 60 × 15-mm plates 1
day after food is depleted and wash worms off plates
with 2ml M9 buffer.
- Add equal volume of S + glycerol and mix by
brief vortexing. Transfer in 0.5-ml aliquots to 2-ml cryovials
(Vangard International, Cat. No. MS4502). Place
vials in a styrofoam freezing box (a styrofoam block
with holes the size of cryovials and a styrofoam lid)
and immediately put at -70°C (cool at approximately
- After 6h the vials can be transferred to liquid
nitrogen or to standard -70°C freezer boxes.
- One vial should be thawed to check for viability,
strain accuracy, and microbial contamination. Thaw
vial by warming between hands until liquid; then pour
contents onto seeded plate. Transfer young healthy
worms to fresh plates the next day.
- Dauer-inducing pheromone stock (modified from
Golden and Riddle, 1984): Take 1 liter of starved liquid
culture. Reduce volume 75% by evaporation under a
stream of air at 100 °C. Centrifuge at 10,000 RCF for
10 min. Dry the supernatant completely at 60 °C. Extract
four to six times with 50ml of 95% ethanol until the
extract is only slightly colored. Combine the extracts
and dry under a stream of air at 60 °C. Back-extract the
resulting oily residue with 10ml distilled water. Filter
through Whatman 3 MM paper and store at 4 °C.
- 0P50 strep: Transfer OP50 stock (see Section III,
A) to preweighed sterile centrifuge bottles/tubes and
centrifuge at 4000 RCF for 10 min. Remove supernatant
and determine weight of bacteria. Resuspend in S
medium to 5% (w/w). Add streptomycin (Sigma, Cat.
No. S-6501) to 50/µg/ml final concentration. Store at
4 °C for a maximum of 2 days.
- Wash the worms off approximately five 60 × 15-
mm plates containing a large number of gravid adults
with approximately 2 ml M9 buffer per plate.
- Combine in a 15-ml Corning polystyrene conical
centrifuge tube and pellet nematodes. Remove all but
8 ml of the liquid.
- Mix together 0.5ml 5 N KOH and 1.2ml 20%
NaOCl (Cat. No. SS290) in a separate tube; combine
with M9 and worms and vortex briefly.
- Remove a small aliquot and monitor under a dissecting
scope while agitating the remaining sample
gently. When 50 to 75% of the adults in the sample
have broken open, pellet nematodes.
- Remove the supernatant, add 8ml of fresh M9
buffer, and pellet. Repeat two more times leaving 0.5-
ml volume after the last wash.
- Resuspend the eggs and pipette onto plates.
This method will generally leave some carcass
This procedure should give greater than 80%
- Make NG agar without peptone (see Section
III,A). Add approximately 25 ∧1/ml dauer-inducing
pheromone stock just before pouring (make only as
much as will be used immediately). Pour 2 ml per 35× 10-mm plate.
- After plates solidify, spot with 10µl OP50 strep
solution and allow to dry.
- Add approximately 100 eggs or allow adults to
lay 100 eggs; then remove the adults and incubate at
25°C for 48-60 h.
- Follow the egg isolation procedure through step
- Bring volume to 10 ml with fresh M9 buffer; incubate
on a rocker for 12h or overnight.
- Feed synchronized L1 larvae from the previous
step. At 20°C mid-L1 larvae can be harvested
after approximately 8h, mid-L2 larvae at 18h,
mid-L3 larvae at 25h, and L4 larvae at 37h (Byerly et al., 1976).
For genetic analysis and maintenance of strains in
active use, the nematodes should be grown on 60 × 15-
mm petri plates. When large numbers of worms are
needed, 100 × 15-mm plates are used. When special
additives are being used that are expensive or in
limited supply, 35 × 10-mm plates are used. Long-term
storage of all strains and those not in active use is best
accomplished by freezing in liquid nitrogen. For biochemical
purposes the nematodes should be grown in
liquid culture. Chemostat culturing enables selection
on a continuously reproducing population whose
density is held constant (Dykhuizen and Haiti, 1983).
Most commonly, selection is for an altered growth rate,
whether due to induced or spontaneous mutations.
Liquid or gaseous selective agents are compatible with
the system described.
More precise synchronization for L4 larvae and
adults can be accomplished by synchronizing through
the dauer stage. If large numbers of dauers are needed,
they can be obtained by slightly modifying the liquid
culturing procedure. The culture should be allowed to
continue for 2-3 days after clearing. After sucrose
flotation, treat with a sterile solution of 1% sodium
dodecyl sulfate (SDS) for 1 hr [resuspend in 22.5 ml of
M9 and add 2.5ml of 10% SDS (dissolve 10g of SDS,
Cat. No. S529, in a final volume of 100ml distilled
water)]. Wash twice with M9 buffer and then repeat
liquid culture protocol starting with step 3.
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