Cultivation of Caenorhabditis elegans on new cheap monoxenic media without peptone

							Source of cholesterol^a
	Plant cooking oil						Chicken egg						Pig fat						Mushroom						Soil
Component (Amount/ L)	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM
Plant cooking oil (100 µL)	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ
Chicken egg (0.56 g)							Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ
Pig fat (0.4 g)													Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ
Autoclaved mushroom solution (20 mL)		Ѵ		Ѵ		Ѵ		Ѵ		Ѵ		Ѵ		Ѵ		Ѵ		Ѵ	Ѵ	Ѵ	Ѵ
Non-autoclaved mushroom solution (20 mL)																						Ѵ	Ѵ	Ѵ
Pure mineral mixture (1 mL of 1 M CaCl₂, 1 mL of 1 M MgSO₄, 25 mL of 1 M KPO₄)	Ѵ	Ѵ					Ѵ	Ѵ					Ѵ	Ѵ					Ѵ			Ѵ
Soil solution ^b(10 mL)					Ѵ	Ѵ					Ѵ	Ѵ					Ѵ	Ѵ			Ѵ			Ѵ	Ѵ
Agar (17 g)	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ
NaCl (4 mL of 0.75 g/ mL)	Ѵ	Ѵ			Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ	Ѵ
Media type	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM	NcM
	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25

1 Notes: ^aPure cholesterol was replaced with chicken egg, pig fat and/or mushroom extract. Mushroom may have nutrients in addition to cholesterol. ^bThe soil solution was intended as a replacement for the pure minerals (salt mixture) in NGM, however it may have included additional nutrients.

Schematic of media preference assay. OP50-seeded plugs of the eight indicated NcMs (grey circles) are placed onto a 9 cm-diameter plain agar plate 1 h before the preference assay. At the start of the assay, 30 well-fed young adult F2 worms are placed within the center circle (CT) and then allowed to migrate. After 2 h, the locations of the worms were recorded.

Table S1.

Brood sizes of C. elegans on 25 test media and the NGM control.

Media	Number of tested hermaphrodites	Total brood size (Mean ± SE)	Dunnett’s post-hoc P-value
NGM	39	254.7 ± 12.8	Control
NcM1	57	144.9 ± 8.8	<0.001
NcM2	50	123.8 ± 8.7	<0.001
NcM3	55	157.5 ± 10.6	<0.001
NcM4	55	123.8 ± 14.4	<0.001
NcM5	25	142.9 ± 14.8	<0.001
NcM6	42	240.5 ± 18.0	0.0228
NcM7	17	171.1 ± 18.3	<0.001
NcM8	26	265.8 ± 8.7	0.5468
NcM9	32	194.5 ± 9.1	<0.001
NcM10	29	152.3 ± 17.3	<0.001
NcM11	24	163.9 ± 12.5	<0.001
NcM12	21	234.0 ± 8.8	0.1817
NcM13	50	128.9 ± 14.2	<0.001
NcM14	47	183.6 ± 7.5	<0.001
NcM15	43	203.6 ± 7.9	<0.001
NcM16	40	198.6 ± 9.3	<0.001
NcM17	38	177.2 ± 17.5	<0.001
NcM18	44	251.7 ± 16.2	1.0
NcM19	38	163.2 ± 12.9	<0.001
NcM20	35	48.7 ± 12.9	<0.001
NcM21	26	178.9 ± 12.4	<0.001
NcM22	35	183.4 ± 11.6	<0.001
NcM23	16	138.4 ± 17.3	<0.001
NcM24	39	178.7 ± 9.5	<0.001
NcM25	45	167.0 ± 8.2	<0.001

1 Note: Gray shading highlights the three media on which worms had brood sizes not different from NGM. Pink shading highlights NcM6 which was only slightly inferior to NGM. Worms on all the remaining NcM media yielded fewer worms than NGM control (yellow). The standard errors (SE) and error bars for each data set are the SE among P0 batches and not the SE across individual P0s.

Brood sizes of *C. elegans* and *C. tropicalis* on test media and the NGM control. (A) The total self-brood sizes of *C. elegans* are presented as means ± 1 SE. Brood sizes differed among the media (P < 2e−16, F = 97.0, one-way ANOVA test). The Dunnett’s post-hoc test was used for brood-size comparisons of the test media against the NGM control (^*** P < 0.001; n.s, not significant, i.e., P > 0.05), see Table S1 for exact P-values. Chl, cholesterol. (B) The total self-brood sizes of *C. tropicalis* differed among the 3 NcMs and NGM (P = 6.3e−10, F = 17.9, one-way ANOVA test). The Dunnett’s post-hoc test is used for brood-size comparisons of the media against the NGM control (^*** P < 0.001; *P < 0.5), see Table S2 for exact P-values. Note: The standard errors (SE) and error bars for each data set are the SE among P0 batches and not the SE across individual P0s.

Table S2.

Brood sizes of C. tropicalis on different media and the NGM control.

Media	Number of tested hermaphrodites	Total brood size (Mean ± SE)	Dunnett’s post hoc P-values
NGM	37	23.3 ± 5.0	Control
NcM6	42	54.8 ± 14.6	<0.001
NcM12	37	48.2 ± 8.4	<0.001
NcM18	32	67.2 ± 15.7	<0.001

1 Note: Worms had bigger brood sizes on all three NcMs than NGM control (yellow). Note, the standard errors (SE) and error bars for each data set are the SE among P0 batches and not the SE across individual P0s.

Table S3.

Lifespan assays of C. elegans on 8 different media.

Media	Number of tested hermaphrodites	Lifespan (days) Mean ± SE	Log-rank P-value
NGM	73	15.2 ± 0.4	Control
NcM6	70	14.8 ± 0.3	1.0
NcM8	72	14.5 ± 0.4	1.0
NcM9	34	21.9 ± 0.9	4e−09
NcM12	66	15.6 ± 0.4	1.0
NcM18	50	15.1 ± 0.5	1.0
NcM20	63	16.9 ± 0.5	0.39
NcM25	57	18.8 ± 0.6	2.4e−05

1 Note: Gray shading highlights the media on which worms had longer lifespans than NGM control (red).

Table S4.

Number of surviving C. elegans worms on 8 different media at each day of the lifespan assays.

	Number of surviving worms
Day	NGM	NcM6	NcM8	NcM9	NcM12	NcM18	NcM20	NcM25
1	73	70	72	34	66	50	63	57
2	73	70	72	34	66	50	63	57
3	73	70	72	34	66	50	63	57
4	73	70	72	34	66	50	63	57
5	72	69	72	34	66	49	63	57
6	72	69	72	34	65	49	63	57
7	70	69	70	34	64	48	62	55
8	70	69	70	34	64	48	61	54
9	70	68	70	34	62	46	59	52
10	67	68	63	32	61	46	59	52
11	63	68	55	31	60	45	58	51
12	58	62	48	30	57	42	56	51
13	52	55	41	30	54	39	52	51
14	46	42	36	30	49	33	51	50
15	33	23	32	30	37	17	47	49
16	21	12	18	30	26	13	36	39
17	12	5	14	28	15	9	28	38
18	11	4	8	28	11	6	21	33
19	10	3	7	28	7	5	14	30
20	6	2	3	27	3	4	10	28
21	5	1	3	21	0	2	7	17
22	2	0	1	16		1	2	9
23	2		1	14		1	1	8
24	2		1	11		1	0	5
25	1		1	7		1		0
26	0		0	4		1
27				3		1
28				2		1
29				2		0
30				1
31				0
Censored	7	10	8	6	14	5	17	23

Lifespan assays of *C. elegans* and *C. tropicalis* on different media and the NGM control. (A) *C. elegans* worms grown on NcM9, NcM20, and NcM25 survived longer than on the NGM control (all P ≤ 0.01, log-rank test), while worms grown on NcM6, NcM8, NcM12, and NcM18 had similar lifespans as the control (P ≥ 0.1, log-rank test). Detailed lifespan data are in Tables S3 and S4. (B) *C. tropicalis* worms grown on NcM6 and NcM12 have similar lifespans as the NGM control (both P > 0.05, log-rank test), while worms grown on NcM18 have shorter lifespans than the control (P < 2e−5, log-rank test), see Tables S5 and S6.

Table S5.

Lifespan of C. tropicalis on 4 different media.

Media	Number of tested hermaphrodites	Lifespan (days) Mean ± SE	Log-rank P-value
NGM	107	14.96 ± 0.42	Control
NcM6	71	16.35 ± 0.51	0.4
NcM12	76	15.49 ± 0.53	1.0
NcM18	133	12.3 ± 0.35	1.3e−05

1 Note: Gray shading highlights the media on which worms had lifespans not different from NGM control (red).

Table S6.

Number of surviving C. tropicalis worms on 4 different media at each day of the lifespan assays.

	Number of surviving worms
Day	NGM	NcM6	NcM12	NcM18
1	107	71	76	133
2	107	71	76	133
3	107	71	76	133
4	107	71	76	133
5	107	71	76	133
6	107	71	76	133
7	105	71	76	133
8	103	70	73	112
9	97	68	67	91
10	85	63	62	77
11	81	60	61	64
12	75	57	57	53
13	66	53	52	44
14	56	47	45	30
15	46	42	36	24
16	36	33	30	21
17	27	24	21	20
17	22	20	14	13
19	17	15	12	7
20	13	13	10	5
21	7	10	10	4
22	4	10	4	2
23	3	5	4	2
24	2	2	4	2
25	2	1	3	1
26	2	0	1	1
27	1		1	0
28	1		1
29	1		1
30	0		0

Table S7.

P-values of generalized linear model of each destination group and preference percentage.

Source	All	NcM6	NcM8	NcM9	NcM12	NcM18	NcM20	NcM25
P _{(percentage~ destination)}	0.342	0.879	0.67	0.0205	5.84e−05	0.571	0.2198	0.1003

Table S8.

P-values of the Dunnett’s test for media preference of C. elegans.

Source	NcM6			NcM8			NcM9			NcM12			NcM18			NcM20			NcM25
Comparison Destination	mean ± SE	NcM6^a	NGM^b	mean ± SE	NcM8^a	NGM^b	mean ± SE	NcM9^a	NGM^b	mean ± SE	NcM12^a	NGM^b	mean ± SE	NcM18^a	NGM^b	mean ± SE	NcM20^a	NGM^b	mean ± SE	NcM25^a	NGM^b
NcM6	5.8 ± 0.7	Ref	<0.001	10.8 ± 0.7	1.0	0.6302	15.8 ± 1.4	0.02654	1.0	15.8 ± 1.8	0.84529	0.41764	14.1 ± 4.2	0.723	0.93	12.5 ± 3.0	0.9786	<1e−05	6.7 ± 1.7	1.0	0.3859
NcM8	10.8 ± 3.4	0.956	0.00641	11.7 ± 5.3	Ref	0.7559	10.0 ± 2.6	0.29024	0.9642	5.8 ± 1.4	0.03458	1.0	9.9 ± 2.1	1.0	1.0	9.2 ± 1.8	1.0	<1e−05	22.5 ± 5.6	0.0711	0.9923
NcM9	0.8 ± 0.7	0.0956	<0.001	3.3 ± 1.2	0.39	0.324	0.0 ± 0.0	Ref	0.0571	6.7 ± 3.3	0.05005	1.0	13.3 ± 5.0	0.845	0.98	2.5 ± 1.4	0.174	<1e−05	6.7 ± 2.4	1.0	0.3861
NcM12	9.2 ± 3.0	0.997	0.00139	17.5 ± 3.2	0.756	1.0	22.5 ± 4.5	<0.001	0.4818	21.7 ± 3.6	Ref	0.05008	10.7 ± 1.7	0.997	1.0	11.7 ± 1.9	0.9986	<1e−05	15.0 ± 4.8	0.5534	0.9984
NcM18	9.2 ± 4.5	0.997	0.00304	8.3 ± 3.4	0.984	0.2928	20.8 ± 4.8	0.00216	0.7125	29.2 ± 7.9	0.63337	0.00142	8.2 ± 1.8	Ref	1.0	4.2 ± 0.7	0.4101	<1e−05	1.7 ± 0.8	0.9984	0.0938
NcM20	7.5 ± 3.0	1.0	0.00300	8.3 ± 3.8	0.984	0.2929	8.3 ± 0.8	0.48166	0.821	2.5 ± 1.4	0.00731	0.97096	14.1 ± 2.7	0.731	0.935	10.0 ± 3.1	Ref	<1e−05	13.3 ± 4.6	0.7350	0.9756
NcM25	1.7 ± 0.8	0.985	<0.001	4.2 ± 1.4	0.505	0.0491	2.5 ± 1.4	0.99881	0.1598	5.0 ± 0.8	0.0239	0.99996	3.3 ± 1.1	0.855	0.61	2.5 ± 1.4	0.1741	<1e−05	5.0 ± 2.8	Ref	0.2531
NGM	32.5 ± 8.0	<0.001	Ref	17.5 ± 2.5	0.756	Ref	14.2 ± 6.4	0.05703	Ref	6.7 ± 2.0	0.0499	Ref	9.9 ± 1.1	1.0	Ref	33.3 ± 3.1	<0.001	Ref	18.3 ± 8.0	0.2531	Ref
Center	5.8 ± 3.0	1.0	<0.001	1.7 ± 1.4	0.214	0.0137	0.8 ± 0.7	1.0	0.0818	1.7 ± 0.8	0.00482	0.92278	6.6 ± 3.5	1.0	0.982	0.8 ± 0.7	0.0619	<1e−05	0.0 ± 0.0	0.9756	0.0536

1 Notes: ^aThe preference percentage of the media where destination equaled source was used as a reference for comparing the means of each test media. ^bThe mean preference percentage of the NGM destination was used as a reference for comparing the means of each test media. Ref indicates reference media for comparison.

Media preference assays. Plots are the percentage of the worms located on the media destinations 2 hours after placing worms in the center of the test assay plates. GLM was used to analyze the correlation between the destinations and percentages of worms migrating to the destinations for all the seven-media sources (P = 1.0) and 10 destination-groups (P = 0.342, see more in Table S7). Dunnett’s post-hoc test was used to analyze P-values for each media destination group (in this figure) or each media destination in comparison with the source and NGM (Table S8). Names of media sources are labeled at the top of each subplot. X-axis, names of media destinations; CT, center; in every assay, a small number of tested worms are not observed (“Lost”).

Table S9.

Brood size of C. elegans on Acinetobacter sp. CFBb9.

Media	Number of tested hermaphrodites	Total brood size (Mean ± SE)	Dunnett’s post hoc P-values
NGM	29	248.0 ± 9.7	Control
NcM6	38	200.2 ± 2.9	<1e−10
NcM12	39	185.6 ± 7.7	<1e−10
NcM18	38	227.7 ± 7.8	<1e−10

1 Note: Yellow shading highlights the NGM control. Brood sizes were lower on NcM plates. The standard errors (SE) and error bars for each data set are the SE among P0 batches and not the SE across individual P0s.

Table S10.

Lifespan of C. elegans on Acinetobacter sp. CFBb9.

Media	Number of tested hermaphrodites	Lifespan (days) Mean ± SE	Log-rank P-value
NGM	90	13.41 ± 0.26	Control
NcM6	96	18.97 ± 0.35	<2e−16
NcM12	83	18.81 ± 0.63	9.4e−16
NcM18	92	17.93 ± 0.41	<2e−16

1 Note: Yellow shading highlights the NGM control. Lifespans were longer on NcM plates.

Table S11.

Number of surviving C. elegans worms on Acinetobacter sp. CFBb9 at each day of the lifespan assays.

	Number of surviving worms
Day	NGM	NcM6	NcM12	NcM18
1	90	96	83	92
2	90	96	83	92
3	90	96	83	92
4	90	96	83	92
5	86	96	83	92
6	86	96	83	92
7	86	95	83	92
8	85	95	77	87
9	85	93	73	86
10	85	93	69	86
11	81	93	68	85
12	64	91	60	83
13	46	91	60	83
14	29	91	60	74
15	15	81	59	69
16	6	71	58	62
17	3	64	58	60
18	0	59	56	51
19		51	51	36
20		41	50	25
21		28	47	17
22		8	36	9
23		5	15	1
24		0	1	0
25			0

Growth assays of *C. elegans* on a natural bacterial strain, *Acinetobacter* sp. CFBb9. (A) The total brood sizes of worms differed among the four different types of media (P < 2e−16, F = 175.8, one-way ANOVA test), with that of worms growing on NcM6, NcM12, or NcM18 being lower than on NGM (***P < 0.001, Dunnett’s post-hoc test), see Table S9 for exact P-values. The standard errors (SE) and error bars for each data set are the SE among P0 batches and not the SE across individual P0s. (B) Worms grown on NcM6, NcM12, and NcM18 have longer lifespans than on NGM (all P < 0.001, log-rank test), see Table S10 for exact P-values.

Table 2.

Approximate cost of reagents for making 1 L of media or approximately one hundred 6-cm plates.

	Cost^a (VND)
Components (amount/L)	NcM6	NcM8	NcM12	NcM18	NGM
Cholesterol (1 mL of 5 mg/mL)					200
Plant cooking oil (100 µL)	2
Chicken egg (0.56 g)		56	56
Pig fat (0.4 g)				28
Autoclaved mushroom (20 mL)	115	115	115	115
Peptone (2.5 g)					3000
Pure mineral mixture (1 mL of 1 M CaCl₂, 1 mL of 1 M MgSO₄, 25 mL of 1 M KPO₄)		3788			3788
Soil solution (10 mL)	0		0	0
Agar (17 g)	34,000	34,000	34,000	34,000	34,000
NaCl (4 mL of 0.75 g/ mL)	3,000	3,000	3,000	3,000	3,000
Distilled water with reverse osmosis (1 L)	2,000	2,000	2,000	2,000	2,000
Total cost in VND	39,117	42,959	39,171	39,143	45,988
Total cost in US$^b	1.70	1.87	1.70	1.70	2.00

1 Notes: ^aCost in Vietnam Dong (VND) based on current prices on May 31, 2020 in Hanoi, Vietnam. ^bBased on the exchange rate (23,000 VND to $1 US on May 31, 2020.

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DOI: https://doi.org/10.21307/jofnem-2021-036 | Journal eISSN: 2640-396X | Journal ISSN: 0022-300X

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Language: English

Page range: 1 - 23

Published on: Jan 1, 2021

Published by: Society of Nematologists, Inc.

In partnership with: Paradigm Publishing Services

Publication frequency: 1 issue per year

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© 2021 Tho Son Le, T. T. Hang Nguyen, Bui Thi Mai Huong, H. Gam Nguyen, B. Hong Ha, Van Sang Nguyen, Minh Hung Nguyen, Huy-Hoang Nguyen, John Wang, published by Society of Nematologists, Inc.
This work is licensed under the Creative Commons Attribution 4.0 License.

Volume 53 (2021): Issue 1 (January 2021)

Cultivation of Caenorhabditis elegans on new cheap monoxenic media without peptone

Figures & Tables

Table 1.

Figure 1:

Table S1.

Figure 2:

Table S2.

Table S3.

Table S4.

Figure 3:

Table S5.

Table S6.

Table S7.

Table S8.

Figure 4:

Table S9.

Table S10.

Table S11.

Figure 5:

Table 2.

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