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Babesia microti (Franca) Reichenow

PRA-400

Babesia microti strain Naushon is a parasitic protozoan that was isolated in 1986 in Naushon Island, Massachusetts, from a tick. This strain requires in vivo cultivation in a Golden Syrian hamster.
Product category
Protists
Product type
Parasitic protozoan
Classification
Protozoa
Strain designation
Naushon
Genome sequenced strain
Yes
Isolation source
Tick (Ixodes scapularis)
Geographical isolation
United States; Massachusetts; Naushon Island
Applications
Infectious disease research
Vector-borne disease research
Product format
Frozen
Storage conditions
-80°C or colder for 1 week, vapor phase of liquid nitrogen for long-term storage
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Documentation

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Detailed product information

Characteristics

Handling information

Host

in vivo cultivation, Golden Syrian hamster

Handling procedure
Storage and Culture Initiation
Frozen ampules packed in dry ice should either be thawed immediately or stored in liquid nitrogen.  If liquid nitrogen storage facilities are not available, frozen ampoules may be stored at or below -70°C for approximately one week.  Do not under any circumstance store frozen ampules at refrigerator freezer temperatures (generally -20°C).  Storage of frozen material at this temperature will result in the death of the culture.
  1. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes.  Do not agitate the ampule.  Do not leave ampule in water bath after it is thawed.
  2. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected hamster.  Hamster should be primed for faster infection by treatment with cortisone (2 mg/day/hamster) 1-3 days prior to inoculation. Follow the protocol for maintenance of the culture below.  The course of infection may be longer or shorter than usual depending on recovery of the parasite from the frozen state.
Culture maintenance

Yaeger's Anticoagulant
Sodium citrate: 1.33 g
Citric acid: 0.47 g
Dextrose: 3.00 g
Sodium heparin: 0.20 g
Glass distilled H2O to: 100.00 mL
NOTE: Immunosuppression of the host is recommended in order to obtain higher levels of parasitemia.

  1. Immunosuppress hamster(s) by intraperitoneal injection of cortisone (2 mg/day/hamster) 1-3 days prior to inoculation. Cortisone injections are repeated every 2-3 days for the duration of the infection.
  2. Inoculate up to 0.5 mL of infected blood intraperitoneally into a hamster using facility approved methods.   
  3. Monitor the infection daily or at 2-day intervals by examination of blood films stained with 5% Giemsa solution.   
  4. Count the number of infected red blood cells (rbc) versus the total number of red cells under oil immersion and determine the % parasitemia: % parasitemia = infected rbc / rbc X 100. A minimum of 500 red blood cells should be counted. (Note that a red blood cell infected with multiple parasites is counted as a single infected cell.)   
  5. When the level of parasitemia is ≥ 10% the strain should be passaged. Normally this would occur 1-3 weeks post-inoculation, but the rate of infection may vary considerably. (Note that the level of parasitemia before the host will succumb will vary with the strain used. Monitoring parasitemia as described above will alert the experimenter as to when the strain should be passaged.)   
  6. To passage the strain, collect blood from the infected hamster using cardiac puncture or other facility approved method using a syringe and suitable anticoagulant.       
  7. Anesthetize the animal by a facility approved method for anesthesia. Collect blood by cardiac puncture or other facility approved blood collection method.
  8. Add approximately 0.05 - 0.1 mL of anticoagulant solution (Yaeger's or heparin, etc.) to a syringe.      
  9. Transfer blood to a collection tube containing and additional 0.05 – 0.1 mL anticoagulant per mL of anticipated blood collection. Mix the blood and anticoagulant by gentle repeated inversion of the tube to prevent clotting of the blood.
  10. If necessary, blood may be diluted with Alsever’s solution.      
  11. Inject up to 0.5 mL of the infected blood suspension into uninfected hamster(s) to passage or expand the infection as needed. Monitor parasitemia and passage as needed.  
Reagents for cryopreservation
Alsever's Solution
NaCl , 4.2 g
Na3citrate • 2H2O, 8.0 g
Glucose, 20.5 g
Glass distilled H2O to 1.0 L
*Dissolve components in glass distilled H2

O, adjust the pH to 6.1 with 10% (w/v) citric acid and filter sterilize. The solution can be obtained from Sigma-Aldrich (catalog# A3551).

Cryopreservation
  1. Prepare a 30% (v/v) sterile glycerol solution in Alsever's solution.   
  2. Draw approximately 0.05 mL of anticoagulant solution (Yaeger's or heparin, etc.) into a syringe and move it back and forth over the length of the syringe, several times. Remove all air bubbles. Draw blood by cardiac puncture or other facility approved blood collection method from a host animal that has reached desired parasitemia. Transfer blood to a collection tube containing additional anticoagulant (0.05 – 0.1 mL per mL of anticipated blood collection). If clotting occurs during extraction of blood, insufficient heparin was used. Mix the heparinized blood with the 30% glycerol solution in a 2:1 ratio to obtain a final concentration of cryoprotectant of 10% (v/v). Mix slowly by inversion and place the mixture on ice. The freezing process should start 15 to 30 minutes following the addition of the heparinized blood to the cryoprotectant solution.
  3. Dispense 0.5 mL aliquots of blood suspension into 1.0 to 2.0 mL sterile plastic screw-capped cryovials. Place the vials in a controlled rate freezing unit. From room temperature, cool the vials at -1C°/min to -40°C. If the freezing unit can compensate for the heat fusion, maintain rate at -1°C/min through this phase. At -40°C, plunge the vials into liquid nitrogen. Alternatively, place the vial in a Mr. Frosty freezing container. Place the container at -80°C for minimum 4 hours and then plunge vials into liquid nitrogen.
  4. To thaw a frozen ampule, place in a 35°C ± 2°C water bath, until thawed (2-3 min). Immerse the vial just sufficient to cover the frozen material. Do not agitate the ampule.   
  5. Immediately after thawing, aseptically remove the contents of the ampule with a syringe and inoculate an uninfected animal. Follow the protocol for in vivo propagation and maintenance in the Product Information Sheet. The course of infection may be longer or shorter than usual depending on percent recovery of the parasite from the frozen state.

History

Deposited as
Babesia microti
Depositors
C. Ben Mamoun
Chain of custody
ATCC <-- C. Ben Mamoun
Type of isolate
Arthropod
Year of origin
1986

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

Frequently Asked Questions

References

Curated Citations

Piesman J, et al. Concurrent Borrelia burgdorferi and Babesia microti infection in nymphal Ixodes dammini. J. Clin. Microbiol.24(3): 446-447, 1986. PubMed: 3760136

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