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Cultivation of Caenorhabditis elegans on new cheap monoxenic media without peptone

Tho Son Le
Tho Son Le
, 
T. T. Hang Nguyen
T. T. Hang Nguyen
, 
Bui Thi Mai Huong
Bui Thi Mai Huong
, 
H. Gam Nguyen
H. Gam Nguyen
, 
B. Hong Ha
B. Hong Ha
, 
Van Sang Nguyen
Van Sang Nguyen
, 
Minh Hung Nguyen
Minh Hung Nguyen
, 
Huy-Hoang Nguyen
Huy-Hoang Nguyen
 oraz 
John Wang
John Wang
   | 01 sty 2021
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Data publikacji: 01 sty 2021
Zakres stron: 1 - 23
DOI: https://doi.org/10.21307/jofnem-2021-036
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© 2021 Authors, published by Sciendo.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1:

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.
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.

Figure 2:

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.
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.

Figure 3:

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.
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.

Figure 4:

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”).
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”).

Figure 5:

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.
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.

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

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

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

Media components of NcMs and NGM.

Source of cholesterola
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

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

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

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

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

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

Costa (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

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

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

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

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

Source NcM6 NcM8 NcM9 NcM12 NcM18 NcM20 NcM25
ComparisonDestination mean ± SE NcM6a NGMb mean ± SE NcM8a NGMb mean ± SE NcM9a NGMb mean ± SE NcM12a NGMb mean ± SE NcM18a NGMb mean ± SE NcM20a NGMb mean ± SE NcM25a NGMb
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
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