1921
Volume 38, Issue 3
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645
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Abstract

Abstract

A cryopreservation protocol for muscle stage larvae is described. Larvae are pretreated in 10% bile at 37°C for 1 hr to induce an increase in surface permeability, then incubated in 20% v/v ethanediol at 37°C for 10 min, transferred to 0°C for a second incubation step of 15 min in a v/v mixture of 33% ethanediol: 33% methanol: 34% saline at 0°C followed by rapid cooling (≈5,100°C min) of aliquots distributed onto glass coverslips. The larvae are thawed by dropping the coverslips into 2 ml saline at room temperature (20°C) and immediately agitating, which simultaneously dilutes (1:100) the cryoprotectants. Groups of 6 mice were infected with muscle stage larvae by gastric intubation. Five days post-infection 14.5 ± 3.2% of control untreated unfrozen MSL and 16.5 ± 4.1% of unfrozen bile and cryoprotectant treated controls were recovered as adult worms from the small intestine. Of the cryopreserved larvae, 1.1 ± 0.4% were recovered as adults, which represents 7.6% compared to the untreated unfrozen controls. When the bile pretreatment step was omitted fewer adult worms (0.09 ± 0.04%) were recovered and no second generation muscle stage larvae were produced. Modifying this technique by omitting the first incubation step in 20% ethanediol and extending the second incubation step to 25 min yielded 72.3% recovery of adult worms 5 days post-infection compared to unfrozen controls. The reproductive capacity index of bile treated cryopreserved was 2.5 ± 0.6 compared to 51.8 ± 18.8 for normal muscle stage controls.

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/content/journals/10.4269/ajtmh.1988.38.558
1988-05-01
2017-09-24
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http://instance.metastore.ingenta.com/content/journals/10.4269/ajtmh.1988.38.558
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  • Accepted : 16 Oct 1987

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