Protocol for survival assay of Caenorhabditis elegans to Pseudomonas aeruginosa PA14 infection

Summary

The nematode Caenorhabditis elegans is a powerful model organism for studying the molecular and cellular mechanisms of innate immunity governed by the intestine. Here, we present a protocol to perform C. elegans survival assays to infection by the bacterial pathogen Pseudomonas aeruginosa PA14. Specifically, we describe steps for preparing C. elegans strains and PA14 bacteria for survival assays. This protocol will assist researchers to study genes involved in intestinal innate immunity and gut defense against pathogen infection.

For complete details on the use and execution of this protocol, please refer to Liu et al.¹ and Zheng et al.²

Graphical abstract

Highlights

• •Preparation of C. elegans strains and P. aeruginosa PA14 bacteria for survival assays
• •Steps for examining C. elegans survival of different genotypes upon PA14 infection
• •Instructions for data collection and analyses of C. elegans survival assay

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The nematode Caenorhabditis elegans is a powerful model organism for studying the molecular and cellular mechanisms of innate immunity governed by the intestine. Here, we present a protocol to perform C. elegans survival assays to infection by the bacterial pathogen Pseudomonas aeruginosa PA14. Specifically, we describe steps for preparing C. elegans strains and P. aeruginosa PA14 bacteria for survival assays. This protocol will assist researchers to study genes involved in intestinal innate immunity and gut defense against pathogen infection.

Before you begin

This protocol describes the reagents, media, and experimental steps required for C. elegans survival assays to study the immune response and host-pathogen interactions. The pathogen P. aeruginosa PA14 is classified as a bio-safety level 2 (BSL-2) pathogen and all experiments related to P. aeruginosa PA14 should be carefully performed in the BSL-2 laboratory. Well-fed worms are critical for reproducible results. In addition, this protocol can also be used to screen and identify the genes involved in intestinal innate immunity and gut defense against pathogens by using RNA interference (RNAi).

Institutional permissions

The animals, the nematode of C. elegans, were used in this study and do not require institutional permission.

Preparation of Luria Broth (LB) media and plates

Timing: 3 days

In the following part, the media, buffers, and chemicals required for worm maintenance and survival assays are prepared. Experimental C. elegans strains were maintained at 20°C; referring to the procedures in Wormbook (www.wormbook.org).^3,4

• 1.
  Prepare stocks of sterile Luria Broth (LB) plates (100 mm) and liquid media before starting the protocol.

  • a.
    Wrap LB plates in parafilm and store at 4°C for up to one month before use (antibiotic free).
  • b.
    Autoclave 75 mL of liquid media sealed in 250 mL conical flasks with a sterile membrane at 121°C for 30 min according to “materials and equipment”.

Note: Dry LB plates at 22°C–25°C for 12–16 hours and ensure the bacterial lawn growth.

Note: Streak Escherichia coli OP50 or P. aeruginosa PA14 on LB plates with or without ampicillin antibiotic in accordance with bacterial resistance.

Note:E. coli OP50 and P. aeruginosa PA14 should be streaked in independent laminar hoods, respectively, to avoid cross-contamination.

• 2.Streak E. coli OP50 and P. aeruginosa PA14 on LB plates. Incubate the plates at 37°C for 12–16 h.

Note:P. aeruginosa PA14 streaking plates should be prepared freshly, we should start to prepare them in advance 5 days prior to transferring L4 stage C. elegans to P. aeruginosa PA14 lawn.

Note:E. coli OP50 and P. aeruginosa PA14 must be incubated in independent incubators, respectively, to avoid cross-contamination.

Note: OP50 plates can be stored at 4°C for up to two weeks before use.

Note: Freshly prepared OP50 bacterial culture is critical for obtaining reproducible results.

Note:P. aeruginosa PA14 streaked on LB plates can be stored at 4°C for up to 7 days before use.

Preparation of NGM plates and survival assay plates

Timing: 3 days

• 3.Prepare two bottles of 500 mL of Nematode Growth Media (NGM) and autoclave at 121°C for 30 min (see ‘‘materials and equipment’’ section).
• 4.From the first 500 mL NGM media bottle, pour 11 mL of NGM media into 45 petri dishes (60 mm).
• 5.Pour the other 500 mL NGM media with an addition of 50 μg/mL 5-fluoro-2′-deoxyuridine (FUdR) at final concentration into 110 petri dishes (35 mm).

Note: Approximate 4.2 mL NGM media containing FUdR for each 35 mm plate.

Note: For NGM media preparation, please see step in the section of materials and equipment.

Note: Dry the NGM plates in a laminar hood at 22°C–25°C for 12–16 hours, then store at 4°C in a plastic box with a lid for up to one month before use.

• 6.Inoculate 75 mL LB media in a 250 mL conical flask with a single colony of E. coli OP50 from the freshly streaked OP50 LB plate. Incubate at 37°C for 16 h with shaking at 200 rpm.
• 7.
  Seed 250 μL of fresh prepared E. coli OP50 bacteria in the center of 60 mm NGM plate and spread by swirling the plate.

  • a.
    Dry the OP50 lawn in a laminar hood at 22°C–25°C for 6–8 h.
  • b.
    Grow the OP50 lawn on the NGM plate upside-down in a plastic box with a lid at 37°C for 24–36 h.

CRITICAL: The OP50 or P. aeruginosa PA14 lawns of NGM plates are separately grown in different incubators.

Note: Do not let the OP50 bacteria cover the full surface of NGM plate. Reserve some uncovered surface for subsequent operation, for instance bleaching, egg laying, or transferring worms.

• 8.Inoculate 5 mL of LB media in a sterile test tube with a single colony of P. aeruginosa PA14. Incubate at 37°C for 12–16 h with shaking at 200 rpm.

Note: Approximately 60 h prior to the initiation of survival assay, we start to prepare the P. aeruginosa PA14 lawn of NGM plates from a single P. aeruginosa PA14 colony.

Note: The test tube should ensure aeration for P. aeruginosa PA14 bacteria growth.

Note: Do not allow the P. aeruginosa PA14 grow in LB media for longer than 16 h at 37°C.

Note: Static bacterial cultures should be avoided.

• 9.
  Seed 150 μL of fresh P. aeruginosa PA14 bacteria on the 35 mm NGM + FUdR plates and swirl to ensure that the P. aeruginosa PA14 culture covers the full surface of plate.

  • a.
    Dry the P. aeruginosa PA14 full-lawn plates for about 20 min in the laminar hood at 22°C–25°C before transferring them upside-down into a plastic box.
  • b.
    Incubate these plates at 37°C for 24 h and subsequently 25°C for 8–12 h.
  • c.
    Place the plates on a clean bench at 22°C–25°C for 1 h before survival assay.

CRITICAL:P. aeruginosa is a human opportunistic pathogen and Biosafety Level 2 (BSL2) pathogen. Follow the recommended institutional guidelines and procedures for working with P. aeruginosa PA14. The P. aeruginosa PA14 survival assay plates can be discarded only after sterile treatment by autoclaving at 121°C for 30 min.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
Non-pathogenic Escherichia coli strain	CGC	OP50
P. aeruginosa strain	D. Wang Lab	PA14
Chemicals, peptides, and recombinant proteins
5-Fluoro-2'-deoxyuridine (FUdR)	Sigma	Cat#F0503
Cholesterol	Sigma	Cat#C8667
Ampicillin	Sangon Biotech	Cat#A610028-0025
NaOH	Sinopharm	Cat#H1Q0101
Agar powder	Solarbio	Cat#A8190
Peptone	Solarbio	Cat#P8450
NaCl	Sigma	Cat#S7653
Tryptone	Oxoid	Cat#LP0042
Yeast extract	Oxoid	Cat#LP0021
K2HPO4	Sigma-Aldrich	Cat#60353
KH2PO4	Sigma-Aldrich	Cat#60218
MgSO4	Sigma-Aldrich	Cat#M2643
CaCl2	Sigma-Aldrich	Cat#C5670
Experimental models: Organisms/strains
Caenorhabditis elegans: N2 (wild type)	CGC	N/A
unc-25(e156)	CGC	CB156
age-1(hx546)	CGC	TJ1052
pmk-1(km25)	CGC	KU25
Software and algorithms
Prism 8.0 software (v8.4.3)	GraphPad	https://www.graphpad.com/
Other
20°C incubator	Longyue (Shanghai)	LBI-400-L
25°C incubator	Zhujiang (Taihong)	LRH-300
37°C incubator	Maikenuo (Changzhou)	SPX-250
Cell culture tube	BKMAMLAB	110408005
35 mm Petri dish	Bontai	35 mm
60 mm Petri dish	Bontai	60 mm
100 mm Petri dish	Bontai	100 mm
Shaking incubator	Zhicheng Inc.	ZWYR-D2403
Laminar hood	Zhicheng Inc.	ZHJH-C1109C
ZEISS stereomicroscope	ZEISS	Stemi 580
Millipore 0.22 μm PES filter	Millipore	SLGPR33RS
OASIS-lifespan assay	Yang et al.	https://doi.org/10.1371/journal.pone.0023525

Materials and equipment

Equipment: This protocol requires two laminar hoods to manipulate E. coli OP50 and pathogen P. aeruginosa PA14 strains independently and two 37°C incubators to culture them separately. These precautions will avoid cross-contamination. A laminar hood and a 20°C incubator are used to handle and maintain the worms. The other laminar hood and a 25°C incubator are used to handle P. aeruginosa PA14 and perform survival assays upon P. aeruginosa PA14 infection. Standard stereomicroscopes are used to maintain the worms and count worms following the survival assay procedure.

Data collection and analysis: All data is recorded in the pre-designed notebook and Microsoft Excel and analyzed by online software OASIS, then graphed using GraphPad Prism.

1 L of Nematode Growth Media (containing FUdR)

Reagent	Final concentration	Amount
NaCl	0.03%	3 g
Agar	0.16%	16 g
Peptone	N/A	3.5 g
ddH2O	N/A	975 mL
MgSO4 (1 M)	1 mM	1 mL
CaCl2 (1 M)	1 mM	1 mL
KPO4 (1 M, pH 6.0)	25 mM	25 mL
Cholesterol (5 mg/mL)	5 μg/mL	1 mL
5-Fluoro-2′-deoxyuridine (FUdR)	50 μg/mL	400 μL

Autoclave the 1 L of NGM media at 121°C for 30 min and then cool to 55°C. Add 1 mL MgSO₄, 1 mL CaCl₂, 25 mL KPO₄, 1 mL Cholesterol, and 400 μL FUDR before pouring the media to the petri dishes. Dry NGM plates in a laminar hood at 22°C–25°C for 12–16 h, then store at 4°C in a plastic box with lid for up to one month before use.

CRITICAL: Do not add the MgSO₄, CaCl₂, KPO₄, cholesterol, or FUdR into the media until the temperature cools to 55°C. These chemicals should then be fully mixed with media.

1 L of Luria broth (LB)

Reagent	Final concentration	Amount
NaCl	1.0%	10 g
Tryptone	1.0%	10 g
Yeast extract	0.5%	5 g
ddH2O	N/A	Up to 1 L

Autoclave 75 mL liquid media sealed in 250 mL conical flask at 121°C for 30 min, then store at 22°C–25°C.

1 L of LB agar plates

Reagent	Final concentration	Amount
NaCl	1.0%	10 g
Tryptone	1.0%	10 g
Yeast extract	0.5%	5 g
Agar	1.5%	15 g
ddH2O	N/A	Up to 1 L

Autoclave the 1 L of LB agar media at 121°C for 30 min and cool to 55°C, add ampicillin (if needed) and mix well, then pour into 10 cm petri dishes (21 mL per dish). Leave LB plates at 22°C–25°C to cool down and dry for 12–16 h. Move LB plates into a plastic box with lid and store them at 4°C for up to one month before use.

Bleach solution

Reagent	Final concentration	Amount
NaOCl solution	N/A	20 mL
5 M NaOH	1 M	20 mL
ddH2O	N/A	20 mL

Bleach solution can be stored at 4°C for up to 1 month or at 22°C–25°C for one week in the dark.

Other solutions

Name	Regents
1 M MgSO4	MW: 120.37. 1 M in ddH2O. Autoclave and store at 22°C–25°C up to one month before use.
1 M CaCl2	MW: 110.98. 1 M in ddH2O. Autoclave and store at 22°C–25°C up to one month before use.
1 M KPO4	108.3 g KH2PO4, 35.6 g K2HPO4, add ddH2O to 1 L. Autoclave and store at 22°C–25°C up to one month before use.
5 mg/mL Cholesterol	Dissolve in ethanol. Filter sterilized and store at 4°C.
5-Fluoro-2′-deoxyuridine (FUdR)	125 mg/mL in ddH2O. Filter sterilized and store at −20°C. Final concentration in NGM is 50 μg/mL.

Step-by-step method details

Inoculate OP50 in LB media and prepare NGM plates for worm maintenance

Timing: 30 min

In the section, E. coli OP50 culture will be prepared for worm maintenance. The details for this step please refer to “before you begin”.

Synchronize worms and prepare PA14 lawn plates for survival assay

Timing: 2 days

• 1.
  Prepare worms for subsequent synchronization: Bleach worms on OP50 plates.

  • a.
    Add one drop of bleach solution (see ‘‘materials and equipment’’ section) on the edge of the NGM plate near to OP50 lawn.
  • b.
    Quickly pick 35–40 gravid adults and put them in the bleach solution to break the worms.

    Note: The adult worms will be broken, bleach solution will be getting dry, and eggs will get out of the worm bodies.

    Note: To avoid contamination, worms should be bleached on the outside region of, but near to OP50 lawn in NGM plates.

  • c.
    The larval worms after hatched will move out of the region that beach solution dropped and to OP50 lawn.
  • d.
    Keep the bleached eggs at 20°C for 3 days. These eggs will develop into gravid adults in approximately 72 h.
• 2.
  Synchronize worms:

  • a.
    Transfer 40 gravid adults to a new OP50 plate and leave them to lay eggs for 1 h at 20°C.
  • b.
    Remove the adult worms and incubate the plates containing eggs at 20°C for approximate 60–65 h until the eggs develop to the late L4 larval stage.

Note: For temperature sensitive strains, worms should be incubated at the appropriate temperature.

• 3.Inoculate 5 mL of LB media in a sterile 14 mL test tube with a single colony of P. aeruginosa PA14 according “before you begin” and incubate for 16 h at 37°C with shaking at 200 rpm (Figure 1).

CRITICAL: The higher density of P. aeruginosa PA14 culture, the faster worms will be killed.

• 4.
  Prepare survival assay plates: Troubleshooting 1.

  • a.
    Seed 150 μL of fresh PA14 bacteria culture on 35 mm NGM + FUdR plates as described in “before you begin”.
  • b.
    Incubate P. aeruginosa PA14 lawn plates at 37°C for 24 h and then at 25°C for 8–12 h.
  • c.
    The plates are kept at 22°C–25°C for 1 h before being used for survival assay.
  • d.
    The plates with different incubation times at 37°C will show different colors (Figure 2) and exhibit distinct toxicity to worms (Figure 3; please also see Table 1).

Note: For one biological replication, one P. aeruginosa PA14 NGM + FUdR plate is required for each strain.

Note: To separate the mechanisms between pathogen sensing/avoidance and pathogen clearance/intestine defense, the survival assay of C. elegans should be performed on the full lawn of pathogen P. aeruginosa PA14.

CRITICAL: All strains should be examined for at least three biological replicates.

Figure 1.

P. aeruginosa PA14 bacteria liquid cultures

LB media control (left) or inoculated with a single P. aeruginosa PA14 colony (right) for 12–16 h. P. aeruginosa PA14 cultures turn to dark green with aeration at 37°C for 12–16 h.

Figure 2.

An example of survival assay plates incubated at 37°C for different durations

Empty plate (left) and seeded P. aeruginosa PA14 culture onto NGM plates containing FUdR and incubate at 37°C for 12 h (middle) or 24 h (right), respectively.

Figure 3.

Percent survival of wild-type worms on differently cultured P. aeruginosa PA14 plates

Percent survival of wild-type worms on P. aeruginosa PA14 survival assay plates incubated at 37°C for 12 h (A) and 24 h (B). All experiments were repeated at least three times. Statistical significance was determined by log-rank test for survival assays. ∗∗p < 0.01.

Table 1.

The raw data of wild-type worms exposed to the indicated P. aeruginosa PA14 plates^b

Hours	PA14 @ 37°C_12 h				PA14 @ 37°C_24 h
	Dead	Alivea	Censored	Percent survival	Dead	Alivea	Censored	Percent survival
0	0	50		100	0	53		100
12	0	50		100	0	53		100
24	0	50		100	0	53		100
36	0	50		100	0	53		100
48	0	49	1	100	4	48	1	100
60	3	45	1	98	6	42		87.5
72	6	39		88	11	31		68.75
84	8	31		72	10	21		39.58
96	11	20		52	8	13		20.83
108	8	12		32	7	6		4.17
120	5	7		18	6	0		0
132	4	3		8
144	3	0		0

^aThe worms that died or lost because of vulva burst or crawling off the plates were censored, which resulted in a total counted number of examined worms was different from the number of transferred worms, more or less (approximately 50). (Also see below).

^bThis table includes the raw data shown in Figure 3.

Survival assay upon P. aeruginosa PA14 infection

Timing: 7 days

In this section, the synchronized late L4 worms are transferred to survival assay plates to perform the survival assay of worms upon pathogen P. aeruginosa PA14 infection.

• 5.Transfer approximately 50 late L4 worms to each survival assay plate of different genotypes, and cultivate the plates containing the worms at 25°C to start survival assay.
• 6.
  Worms are scored for survival every 12 h. Troubleshooting 3.

  • a.
    In the beginning of survival assay before worms begin to die, the number of alive worms should be counted and recorded every 12 h.

Note: Worms should be censored if they exhibit a burst vulva or leave the plate.

Note: Worms are scored as dead if they fail to respond when the nose and the tail of worms are touched by an eyebrow worm picker for three times. Carefully watch for worm movement. If the worm does not respond, such as normal locomotion, curling or contraction the tail, or foraging and pumping behavior in the head, pick it out of the survival assay plate and score as a dead worm.

• 7.Analyze data and compare the difference between two groups as described in the ‘‘quantification and statistical analysis” section.

Expected outcomes

In this study, we tested the survival of wide-type worms on different culture plates of P. aeruginosa PA14, such as survival plate with incubation at 37°C for 12 h or 24 h. The data show that the survival median of wild-type worms incubated for 24 h was 85.15 ± 2.9 2 h. And the survival median of wild-type worms incubated for 12 h was 99.25 ± 3.24 h (Figure 3; Table 4). This indicated that the virulence of P. aeruginosa PA14 cultured at 37°C for 24 h was higher than that for 12 h. The stable virulence of P. aeruginosa PA14 is crucial to getting reproducible data.

Table 4.

The example of essential statistics after statistical analysis

Strain (genotype)	TD Mean ± SEM (hours)	# Worms scored	# Censored worms	p Value (log-rank test)	Figure
PA14 @ 37°C_12 h	99.25 ± 3.24	48	2		Figure 3
PA14 @ 37°C_24 h	85.15 ± 2.92	52	1	p = 0.0026
Wild-type	88.50 ± 2.54	48	2		Figure 4A
unc-25(e156)	72.27 ± 2.40	45	6	p = 0.0001 vs. wild-type
age-1(hx567)	119.75 ± 2.79	48	2	p = 9.9 × 10−9 vs. wild-typea
Wild-type	92.00 ± 2.74	54	1		Figure 4B
unc-25(e156)	70.64 ± 2.35	44	8	p = 2.7 × 10−7 vs. wild-type
pmk-1(km25)	56.47 ± 1.80	51	2	p = 9.9 × 10−9 vs. wild-typea

^ap value 9.9 × 10⁻⁹ is provided when OASIS calculated p value is 0.0e+00.

Meanwhile, we tested mutants with deficiency in γ-aminobutyric acid (GABA) biosynthesis of unc-25(e156). GABA is synthesized in GABAergic neurons through decarboxylation of glutamate by glutamic acid decarboxylase (GAD) encoded by unc-25 in C. elegans.⁵ In the experiment, we used age-1 mutants as a control that exhibit robust enhanced resistance to P. aeruginosa PA14 infection. Loss-of-function of unc-25 mutants showed enhanced sensitivity to P. aeruginosa PA14 infection as compared to that of wild-type worms. On the contrary, age-1 mutants showed enhanced resistance to P. aeruginosa PA14 infection (Figure 4A; please also see Table 2). Alternatively, pmk-1 mutants can be used as a control strain that exhibits hypersensitivity to PA14 infection as compared to that of wild-type worms upon P. aeruginosa PA14 infection (Figure 4B; please also see Table 3).⁶

Figure 4.

Percent survival of indicated genotypic worms exposed to P. aeruginosa PA14

Percent survival of wild-type, unc-25, and age-1 (A) or wild-type, unc-25, and pmk-1 (B) mutant worms on P. aeruginosa PA14 survival assay plates incubated at 37°C incubation for 24 h. All experiments were repeated at least three times. Statistical significance was determined by log-rank test for survival assays. ∗∗∗p < 0.001.

Table 2.

The raw data of the indicated genotypic worms exposed to P. aeruginosa PA14^b

Hours	Wild-type				unc-25(e156)				age-1(hx546)
	Dead	Alivea	Censored	Percent survival	Dead	Alivea	Censored	Percent survival	Dead	Alivea	Censored	Percent survival
0	0	50		100	0	51		100	0	50		100
12	0	50		100	0	51		100	0	50		100
24	0	50		100	0	51		100	0	50		100
36	0	50		100	0	50	1	100	0	50		100
48	0	50		100	6	42	2	86.67	0	50		100
60	5	44	1	89.58	11	30	1	62.22	0	49	1	100
72	10	33	1	68.75	13	15	2	33.33	0	49		100
84	12	21		43.75	8	7		15.56	2	47		95.83
96	8	13		27.08	5	2		4.44	8	38	1	79.17
108	9	4		8.33	2	0		0	11	27		56.25
120	4	0		0					8	19		39.58
132									9	10		20.83
144									7	3		6.25
156									3	0		0

^aPlease see the note above.

^bThis table includes the raw data shown in Figure 4A.

Table 3.

The raw data of the indicated genotypic worms exposed to P. aeruginosa PA14^b

Hours	Wild-type				unc-25(e156)				pmk-1(km25)
	Dead	Alivea	Censored	Percent survival	Dead	Alivea	Censored	Percent survival	Dead	Alivea	Censored	Percent survival
0	0	55		100	0	52		100	0	53		100
12	0	55		100	0	52		100	0	53		100
24	0	55		100	0	52		100	0	53		100
36	0	55		100	0	52		100	5	47	1	90.20
48	0	55		100	8	42	2	81.81	20	27		50.98
60	6	49		88.89	10	30	2	59.09	15	11	1	21.57
72	8	40	1	74.07	11	16	3	34.09	7	4		7.84
84	13	27		50	9	6	1	13.63	4	0		0
96	10	17		31.48	6	0		0
108	8	9		16.67
120	6	3		5.56
132	3	0		0

^aPlease see the note above.

^bThis table includes the raw data shown in Figure 4B.

Quantification and statistical analysis

Data collection and statistical analysis.

• 1.Data are recorded and collected using the below table templated sheet.
• 2.Open a new excel file and enter the data into the excel table for subsequent analyses.
• 3.Upload data excel file and perform statistical analyses by an online website tool OASIS (https://sbi.postech.ac.kr/oasis/?tdsourcetag=s_pcqq_aiomsg).
• 4.Collect essential information of the statistics, for instance the information exhibited in Table 4.

Limitations

This protocol describes how to prepare and perform survival assays to pathogenic bacteria in the laboratory. However, the outcomes of survival assays can be affected by several factors, including the plate moisture content, temperature, humidity and airflow of the experimental room, toxicity of P. aeruginosa PA14, feeding status and stage of worms, and other related steps.⁷ The results of C. elegans survival assays exposed to P. aeruginosa PA14 infection also can be influenced by any type of bacterial contaminant before or during experiments. The critical factor that affects the result of survival assay upon PA14 infection is toxicity of P. aeruginosa PA14.⁸ The toxicity of P. aeruginosa PA14 will vary due to culture duration of the pathogen (Figure 3). Generally, the toxicity of P. aeruginosa PA14 is positively correlated to its culture duration.

Troubleshooting

Problem 1

How to prepare P. aeruginosa PA14 lawn NGM + FUdR plates with stable toxicity for reproducible survival assays. (Related to step 3 and step 4).

Potential solution

• •P. aeruginosa PA14 should be freshly thawed and streaked on fresh LB plates from a frozen stock in −80°C freezer or liquid nitrogen. A newly streaked P. aeruginosa PA14 colony plate should be stored at 4°C for up to 7 days before use.
• •NGM plates containing FUdR should be dried at 22°C–25°C for 12–16 h, then stored at 4°C in a plastic box with lids for up to one month before use.
• •According to “before you begin” step 7, culture P. aeruginosa PA14 in LB media at 37°C for no more than 16 h.
• •After seeding newly prepared P. aeruginosa PA14 culture onto NGM plates containing FUdR, leave these plates without lids in a laminar hood until the P. aeruginosa PA14 culture to be absorbed completely.
• •Move the plates into a 37°C incubator and continuously culture for 24 h. Then move the plates into 25°C incubator to balance temperature for 8–12 h.

Problem 2

The reproducibility of the same experiment is low. (Related to step 2, 3, and 4).

Potential solution

• •The well-fed, synchronized late L4 worms should be used for survival assay.
• •The worms should be maintained on newly prepared OP50 NGM plates.
• •The worms should be gently and carefully transferred to survival assay plates with a worm pick.
• •Freshly thaw and streak P. aeruginosa PA14 stock on a fresh LB plate to maintain stable toxicity after the previously streaked P. aeruginosa PA14 plate has been used for more than one week.

Problem 3

The status of the worms can be scored as dead. (Related to step 6).

Potential solution

• •The assay requires scoring worms as dead or alive every 12 h by using an eyebrow picker to gently touch the nose and the tail of worms. If the worm does not respond to the gentle touch, such as pharyngeal pumping behavior, head foraging movement, or tail swing, the worms are scored as dead and are removed from the survival assay plate.
• •In the experiment, some worms died and their whole body became clear and transparent following P. aeruginosa PA14 infection. This phenotype is easily observed by turning down the light of stereomicroscope.
• •The worms that died because of vulva burst or crawling off the plates were censored.

Problem 4

The number of dead worms exceeded the total number of worms before starting the assay. (Related to before you begin step 5).

Potential solution

• •In the survival assay, the synchronized late L4 larval worms are used to examine the susceptibility to pathogen P. aeruginosa PA14 infection. If FUdR loses efficacy and does not prevent progeny from hatching, the total number of dead worms will exceed the number of transferred worms in the beginning of the assay.

Problem 5

The worms burrow into agar of the survival assay plates. (Related to materials and equipment).

Potential solution

• •Discard the survival assay plate.
• •The concentration of agar should be 1.6%–2% (v/v), and do not puncture the agar when transferring the worms to the P. aeruginosa PA14 plates.

Problem 6

The worms crawled off the wall of survival assay plates. (Related to materials and equipment and step 4).

Potential solution

• •The survival assay plates may be too wet.
• •The NGM plates containing FUdR should be dried in 37°C for 12–16 h in a plastic box with a lid. They can be stored at 4°C for subsequent experiments.
• •Use absorbent paper to remove water droplets on the lid before seeding the P. aeruginosa PA14 onto the survival assay plates.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Haijun Tu (haijuntu@hnu.edu.cn).

Technical contact

Technical questions about this protocol should be directed to the technical contacts, Junqiang Liu (jiasuduliu@126.com) and Haijun Tu (haijuntu@hnu.edu.cn).

Materials availability

This study did not generate new reagents.

Data and code availability

This study did not generate data or code.