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Product Name:
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IP:1182:2 (xenic)
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Ownership statement:
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This reagent is the property of the U.S. Government.
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Manufacturer:
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BEI Resources
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Taxonomy:
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Protozoa Classification: Entamoebidae, Entamoeba
Species: Entamoeba histolytica
Strain: IP:1182:2 (xenic)
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Additional Information:
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E. histolytica is a pathogenic protozoan parasite and causative agent of amebiasis, an intestinal infection that predominantly infects humans and other primates in developing countries, with symptoms ranging from asymptomatic colonization to extraintestinal, disseminated disease.4,5,6 The E. histolytica life cycle consists of a highly resistant environmental cyst with a protective, chitin-rich cell wall and a dividing trophozoite, which establishes infection through excystation in the colon.5,7 Infection occurs through shedding of cysts in feces and the ingestion of cysts via contaminated water and vegetables.7 E. histolytica has been shown to cause host tissue damage through amoebic trogocytosis in a mouse model.8
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Material Provided:
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Each vial of NR-20088 contains approximately 0.5 mL of culture in cryopreservative. Please see Appendix I for cryopreservation instructions.
Note: NR-20088 is a xenic culture.
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Packing/Storage:
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NR-20088 was packaged aseptically in cryovials and is provided frozen on dry ice. The product should be stored at -130°C or colder, preferably in the vapor phase of a liquid nitrogen freezer. If liquid nitrogen storage facilities are not available, frozen cryovials may be stored at -70°C or colder for approximately one week.
Note: Do not under any circumstances store vials at temperatures warmer than -70°C. Storage under these conditions will result in the death of the culture.
To ensure the highest level of viability, the culture should be initiated immediately upon receipt. Any warming of the product during shipping and transfer must be avoided, as this will adversely affect the viability of the product. For transfer between freezers and for shipping, the product may be placed on dry ice for brief periods, although use of a portable liquid nitrogen carrier is preferred. Please read the following recommendations prior to using this material.
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Growth Conditions:
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Growth Media:
Trypticase – Yeast Extract – Serum – Gastric Mucin Medium (TYSGM-9)9 supplemented with 5% heat-inactivated adult bovine serum (HIBS) (Appendix II) or Trypticase – Yeast Extract – Gastric Mucin Medium (TYGM-9) supplemented with 5% heat-inactivated adult bovine serum (HIBS), 1.8% rice starch solution and 10% Tween 80 ethanolic solution (v/v) (Appendix III)
Note: 1,000 I.U./mL penicillin and 1,000 µg/mL streptomycin may be added to the growth medium to control bacterial density.
Incubation:
Temperature: 35°C to 37°C
Atmosphere: Xenic and microaerophilic
Propagation:
Note: Increase the concentration of HIBS in the media to 20% during the initial recovery of the culture from cryopreservation.
1. To establish a culture from the frozen state, aseptically add 0.5 mL of growth medium containing 20% HIBS without antibiotics to the frozen vial of NR-20088. Place the vial in a 35°C to 37°C water bath for 2 to 3 minutes, until thawed. Immerse the vial just enough to cover the frozen material. Do not agitate the vial.
Note: Manipulations with the frozen vial should be done quickly to avoid warming of the culture at a suboptimal rate.
2. Transfer the vial contents to a glass one-dram (3.5 mL) screw-capped vial and add 2.5 mL of additional TYSGM-9 containing 20% HIBS. Tighten the cap and incubate in an upright position for 2 to 4 hours at 35°C to 37°C.
3. Ice the vial for 10 minutes and gently invert 10 times. Centrifuge at 200 to 350 × g for 5 minutes.
4. Carefully aspirate the supernatant without disturbing the cell pellet and transfer to a second dram vial. Resuspend the pelleted material containing the Entamoeba and starch with 3 mL of growth medium containing 5% HIBS. Gently invert 10 times.
5. Incubate the two dram vials at a 15° horizontal slant at 35°C to 37°C with the caps screwed on tightly. Observe the culture harboring Entamoeba daily until trophozoites are observed (1 to 3 days). If trophozoites are not apparent after 3 days, re-feed the culture with the bacteria-enriched culture prepared in step 4 supplemented with 5% HIBS. Supplement with penicillin/streptomycin solution if the bacterial density becomes too high.
6. Subculture by transferring 2 mL of culture to a new dram vial. Add 1.5 mL of bacteria-enriched culture prepared in step 4 supplemented with 5% HIBS. Gently invert 10 times. Supplement with penicillin/streptomycin solution if the bacterial density becomes too high.
7. Incubate the second dram vial at a 15° horizontal slant at 35°C to 37°C with the cap screwed on tightly. Observe the culture daily until trophozoites are observed (1 to 3 days).
Please see Appendix I below for cryopreservation instructions.
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Disclaimers:
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You are authorized to use this product for research use only. It is not intended for human use. Use of this product is subject to the terms and conditions of the BEI Resources Material Transfer Agreement (MTA). The MTA is available on our Web site at www.beiresources.org. While BEI Resources uses reasonable efforts to include accurate and up-to-date information on this product sheet, neither ATCC® nor the U.S. Government makes any warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. Neither ATCC® nor the U.S. Government warrants that such information has been confirmed to be accurate. This product is sent with the condition that you are responsible for its safe storage, handling, use and disposal. ATCC® and the U.S. Government are not liable for any damages or injuries arising from receipt and/ or use of this product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, the U.S. Government, ATCC®, their suppliers and contributors to BEI Resources are not liable for damages arising from the misidentification or misrepresentation of products. |
References:
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1. Chadee, K. and E. Meerovitch. “Entamoeba histolytica: Lymphoreticular Changes in Gerbils (Meriones unguiculatus) with Experimentally Induced Cecal Amebiasis.” J. Parasitol. 71 (1985): 566-575. PubMed: 2865345.
2. Chadee, K. and E. Meerovitch. “Entamoeba histolytica: Early Progressive Pathology in the Cecum of the Gerbil (Meriones unguiculatus).” Am. J. Trop. Med. Hyg. 34 (1985): 283-291. PubMed: 2858986.
3. Clark, C. G., Personal communication.
4. Pritt, B. S. and C. G. Clark. “Amebiasis.” Mayo Clin. Proc. 83 (2008): 1154-1159. PubMed: 18828976.
5. Parija, S. C., J. Mandal and D. K. Ponnambath. “Laboratory Methods of Identification of Entamoeba histolytica and Its Differentiation from Look-Alike Entamoeba spp.” Trop. Parasitol. 4 (2014): 90-95. PubMed: 25250228.
6. Ngui, R., et al. “Differentiating Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii Using Nested Polymerase Chain Reaction (PCR) in Rural Communities in Malaysia.” Parasit. Vectors 5 (2012): 187. PubMed: 22947430.
7. Marie, C. and W. A. Petri Jr. “Regulation of Virulence of E. histolytica.” Annu. Rev. Microbiol. 68 (2014): 493-520. PubMed: 25002094.
8. Ralston, K. S. ”Taking a Bite: Amoebic Trogocytosis in Entamoeba histolytica and Beyond.” Current Opin. Microbiol. 28 (2015): 26-35. PubMed: 2627708.
9. Diamond, S. L. “A New Liquid Medium for Xenic Cultivation of Entamoeba histolytica and other Lumen-Dwelling Protozoa.” J. Parasitol. 68 (1982): 958-959. PubMed: 6290631.
10. Stanley, S. L., Jr. “The Entamoeba histolytica Genome: Something Old, Something New, Something Borrowed and Sex Too?” Trends Parasitol. 21 (2005): 451-453. PubMed: 16098811.
11. Loftus, B. J. and N. Hall. “Entamoeba: Still More to be Learned from the Genome.” Trends Parasitol. 21 (2005): 453. PubMed: 16099723.
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Citation:
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Acknowledgment for publications should read "The following reagent was obtained through BEI Resources, NIAID, NIH: Entamoeba histolytica, Strain IP:1182:2 (xenic), NR-20088."
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Biosafety Level:
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2
Appropriate safety procedures should always be used with this material. Laboratory safety is discussed in the following publication: U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories (BMBL). Current Edition. Washington, DC: U.S. Government Printing Office.
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