Blastocystis: Isolation, Xenic Cultivation, and Cryopreservation

C. Graham Clark1, C. Rune Stensvold2

1 Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, 2 Department of Microbiology and Infection, Statens Serum Institut, Copenhagen
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 20A.1
DOI:  10.1002/cpmc.18
Online Posting Date:  November, 2016
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Abstract

Blastocystis is an intestinal parasite that is very easily isolated in culture from fresh stool samples. In fact, the parasite grows so readily in culture that short‐term in vitro culture is sometimes used as a diagnostic tool in the absence of DNA‐based methods. While axenizing Blastocystis cultures remains a significant challenge, the parasite can be propagated for several months in the presence of metabolically active bacteria (xenic culture). Hence, culture can be used for maintaining live Blastocystis strain libraries. This enables the production of a stable resource of reference material, which for instance can be used for DNA‐based assays and research. Blastocystis isolates can also be cryopreserved with a view to reestablishing them in culture. Here, we provide protocols for xenic in vitro culture and cryopreservation of Blastocystis. © 2016 by John Wiley & Sons, Inc.

Keywords: cell culture; diagnosis; molecular epidemiology; parasite; preservation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: General Principles and Jones's Medium
  • Alternate Protocol 1: Use of Lysgm Medium
  • Alternate Protocol 2: Use of Robinson's Medium
  • Basic Protocol 2: Cryopreservation of Blastocystis
  • Support Protocol 1: Thawing Cryopreserved Blastocystis Cells
  • Commentary
     
 
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Materials

Basic Protocol 1: General Principles and Jones's Medium

  Materials
  • Na 2HPO 4·12H 2O
  • KH 2PO 4
  • NaCl
  • Yeast extract (e.g., Sigma, cat. no. Y1625)
  • Horse (e.g., Sigma, cat. no. H6762) or adult bovine (e.g., Sigma, cat. no. B9433) serum
  • Stool sample of interest
  • 100‐ml and 1000‐ml bottles
  • Cell culture tubes with nonvented caps, sterile (borosilicate glass [e.g., Corning, cat. no. 99449 or Fisher, cat. no. 14‐959‐35A] or polystyrene [e.g., Corning, cat no. 430157 or Greiner, cat. no. 186161])
  • Wooden dowel rods (∼4 mm diameter) or similar
  • Tube rack
  • 37°C incubator
  • Microscope capable of 400× magnification, standard or inverted
  • Glass slides and coverslips

Alternate Protocol 1: Use of Lysgm Medium

  Materials
  • K 2HPO 4
  • KH 2PO 4
  • NaCl
  • Yeast extract (e.g., Sigma, cat. no. Y1625)
  • Liver extract (e.g., Oxoid, cat. no. LP0027)
  • Gastric mucin type IV (e.g., Sigma, cat. no. M2378)
  • Adult bovine serum (e.g., Sigma, cat. no. B9433)
  • 100‐ml and 1000‐ml bottles

Alternate Protocol 2: Use of Robinson's Medium

  Materials
  • Erythromycin powder (e.g., Acros Organics, cat. no. 227330050)
  • Bacto Peptone (e.g., BD Biosciences, cat. no. 211677)
  • Potassium hydrogen phthalate
  • 40% (w/v) NaOH
  • NaCl
  • Citric acid
  • KH 2PO 4
  • (NH 4) 2SO4
  • MgSO 4·7H 2O
  • 85% (w/v) lactic acid (e.g., Sigma, cat. no. 252476)
  • Standard Escherichia coli strain (e.g., NCTC 10418)
  • Adult bovine serum (e.g., Sigma, cat. no. B9433) or horse serum (e.g., Sigma, cat. no. H6762)
  • 0.2‐µm syringe filters (e.g., Sigma, cat. no. F8773)
  • 100‐ml and 1000‐ml bottles
  • 37°C incubator
  • Cell culture tubes

Basic Protocol 2: Cryopreservation of Blastocystis

  Materials
  • Growth medium
  • Isolate of Blastocystis to be frozen
  • Culture medium base (i.e., medium without serum; see protocol 1 and Alternate Protocols protocol 21 and protocol 32)
  • Heat‐inactivated serum
  • Dimethylsulfoxide (DMSO; e.g., Sigma, cat. no. D2650)
  • Disinfectant
  • Isopropanol
  • Cell culture tubes
  • 50‐ml capped plastic tubes
  • Centrifuge with swinging bucket rotor
  • Cryovials (e.g., NUNC, cat. no. 363401) and rack
  • 1000‐ml flask
  • Appropriate temperature incubator
  • Cell freezer (e.g., Nalgene Mr. Frosty; Sigma, cat. no. C1562)
  • −80°C freezer
  • Liquid nitrogen storage

Support Protocol 1: Thawing Cryopreserved Blastocystis Cells

  Materials
  • Culture medium
  • Cryopreserved inoculum (see protocol 4)
  • 37°C water bath
  • Floating water bath rack of appropriate size for cryovials
  • Cell culture tubes
  • Appropriate temperature incubator
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Literature Cited

Literature Cited
  Clark, C.G. and Diamond, L.S. 2002. Methods for cultivation of luminal parasitic protists of clinical importance. Clin. Microbiol. Rev. 15:329‐341. doi: 10.1128/CMR.15.3.329‐341.2002.
  Diamond, L.S. 1982. A new liquid medium for xenic cultivation of Entamoeba histolytica and other lumen dwelling protozoa. J. Parasitol. 68:958‐959. doi: 10.2307/3281016.
  Jones, W.R. 1946. The experimental infection of rats with Entamoeba histolytica. Ann. Trop. Med. Parasitol. 40:130‐140. doi: 10.1080/00034983.1946.11685270.
  Robinson, G.L. 1968. The laboratory diagnosis of human parasitic amoebae. Trans. R. Soc. Trop. Med. Hyg. 62:285‐294. doi: 10.1016/0035‐9203(68)90170‐3.
  Samarawickrema, N.A., Upcroft, J.A., Thammapalerd, N., and Upcroft, P. 2001. A rapid‐cooling method for cryopreserving Entamoeba histolytica. Ann. Trop. Med. Parasitol. 95:853‐855. doi: 10.1080/00034980120111181.
Key Reference
  Stensvold, C.R., Arendrup, M.C., Jespersgaard, C., Mølbak, K., and Nielsen, H.V. 2007. Detecting Blastocystis using parasitologic and DNA‐based methods: A comparative study. Diagn. Microbiol. Infect. Dis. 59:303‐307. doi: 10.1016/j.diagmicrobio.2007.06.003.
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