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| Species | Procedure | Confirmation of axenic state | Larvae use | Reference |
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| S. venezuelensis | (i) L3 larvae were collected from Bearman culture
(ii) Larvae were washed in 0.25% sodium hypochlorite solution for 10 min
(iii) Larvae were exposed to benzylpenicillin (180 mg/L) and ceftazidime (1 mg/mL) (30 or 60 min) | Larvae were incubated at 25°C and 37°C for 7 days in tubes containing thioglycolate broth with brain and heart infusion | NIH germ-free mice infection | [15] |
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| S. venezuelensis | (i) L3 larvae were collected from Bearman culture
(ii) Larvae were washed 6 times for 20 minutes each in distilled water containing penicillin (100 IU/mL), streptomycin (0.1 mg/mL), and fluconazole (0.8 mg/mL) | Maintained in blood agar culture for 24 h at 28°C | 96-well plate assay containing water | [16] |
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| S. ratti | (i) L3 larvae were washed 3 times in PBS buffer | — | 6-well plate assay containing 4 mL PBS buffer (pH 7.3) | [17] |
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| S. stercoralis | (i) A concentrated suspension of L3 larvae was placed in a water bath at 37°C.
(ii) 1 volume of low melting point agarose (3% low melting point agarose in BU saline; 50 mM Na2HPO4; 22 mM KH2PO4; 70 mM NaCl) at 37°C was mixed with 2 volumes of concentrated parasite suspension (this operation was performed with tubes in the water bath at 37°C to avoid solidification).
(iii) A 100 mm Petri dish(s) was placed on ice, and 5 mL of parasite suspension in 1% agarose pipetted into the center of the plate avoiding contact with the sides of the plate
(iv) After solidification, 10–20 mL of BU saline at 37°C was added to the plate and incubated at 37°C for 30 min Viable worms migrate from the agar matrix into the surrounding medium during this period
(v) The liquid medium was then collected and transferred to a 15 mL conical centrifuge tube Worms can sediment by either gravity for 15 min or by centrifugation at 500 rpm for 5 min | — | In vitro assays | [18] |
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| S. stercoralis | (i) L3 larvae were collected from agar culture
(ii) Larvae were washed several times in saline solution (PBS) with penicillin (100 U/mL) and streptomycin (0.1 mg/mL) | — | Gerbil (Merionesunguiculatus) infection | [19] |
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| S. venezuelensis and S. ratti | (i) L3 larvae were incubated at room temperature in water with Amphotericin B (0.25 μg/mL) and ceftriaxone (20 μg/mL) | — | 96-well plate assay with melted agar at 1% | [20] |
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| S. stercoralis | (i) L3 larvae were washed two times in M9 buffer
(ii) Supernatant was removed and worm pellet was resuspended in the residual volume
(iii) The worms were added to a conical centrifuge tube containing Percoll solution at 40% supplemented with 25 mM Heppes and 10,000 U penicillin/10 mg streptomycin
(iv) The suspension was centrifuged at 1000 rpm for 2 min and washed twice with M9 buffer | — | In vitro assays | [18] |
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| S. stercoralis | (i) L3 larvae were collected from charcoal cultures and washed by centrifugation and resuspension in sterile RPMI with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.1 mg/mL gentamicin
(ii) Afterwards, they were placed for 20 min in a 2% solution of low-melt agarose, and PBS (supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.1 mg/mL gentamicin) was added after agarose solidification
(iii) Larvae that migrated into the PBS solution were then harvested | — | L-3 antigen solubilization | [21] |
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