ACANTHAMOEBA BROTH

Cat. no. K225 Acanthamoeba Broth, 15x103mm Tube, 5ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Acanthamoeba Broth, also known as Peptone-Yeast Extract-Glucose (PYG) Medium, is recommended for the maintenance of Acanthamoeba stock cultures.

SUMMARY

Free-living amebae are found naturally in moist soil, marine water, and fresh water, and feed on the bacteria and nutrients found in these environments. They can infect humans through the skin, olfactory epithelium, sinuses, corneal abrasions, or respiratory tract and are responsible for a variety of diseases, including cerebral edema, focal necrosis, granulomatous reactions in skin, multiple necrotic foci, corneal ulceration, and hemorrhagic necrosis. In addition, Acanthamoeba spp. infection can cause amebic keratitis, granulomatous amebic encephalitis (GAE), sinusitis, and cutaneous lesions. Increased infection has been noted in patients undergoing immunosuppressive therapy or chemotherapy, as well as individuals infected with human immunodeficiency virus (HIV). Treatment for amebic infections vary based on the site(s) of infection and apparent disease, but early detection is key for successful patient outcomes.

Acanthamoeba spp. exist in both cyst and trophic stages, and do not have a flagellate stage. Acanthamoeba cysts are approximately 10-30µm in diameter and are resistant to low temperatures (0-2ºC.), chlorination, biocides, and antibiotics. Trophozoites are approximately 25-40µm in diameter and contain common organelles, including smooth and rough endoplasmic reticula, mitochondria, a Golgi complex, microtubules, digestive vacuoles (including a contractile vacuole), and free ribosomes.(1,2)

Acanthamoeba Broth contains peptones and yeast extract (which supply nitrogenous and other nutrients necessary for amebae growth) and buffers to allow the growth of Acanthamoeba spp.

FORMULA

Ingredients per liter of deionized water:*

Acanthamoeba Broth:
Peptone 20.0gm
Yeast Extract 2.0gm
Sodium Citrate 1.0gm
Magnesium Sulfate 0.98gm
Sodium Phosphate 0.355gm
Potassium Phosphate 0.34gm
Ferrous Ammonium Sulfate 0.02gm

Final pH 6.5 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is temperature sensitive; protect from excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Acanthamoeba isolated on Free-Living Amebae Media (Cat. no. G225 and R225):

1. If a lab wishes to preserve a culture of Acanthamoeba spp. for future reference or as a positive control, mark an area on the plate where many amebae are present.

2. Using a loop, scrape the agar in this area and transfer the inoculum to a tube of Acanthamoeba Broth.

3. Tighten cap and incubate at room temperature.

4. Every 2-4 weeks, mix suspension and pipet 0.25ml into a new tube of Acanthamoeba Broth. Tighten cap and incubate at room temperature.

Frozen Acanthamoeba isolate from ATCC®:

1. Thaw ampule by placing in a 35ºC. waterbath.

2. Immediately after thawing, transfer contents of ampule into a tube of Acanthamoeba Broth.

3. Tighten cap and incubate at room temperature.

4. Every 2-4 weeks, mix suspension and pipet 0.25ml into a new tube of Acanthamoeba Broth. Tighten cap and incubate at room temperature.

INTERPRETATION OF RESULTS

To check for propagation of Acanthamoeba spp. a wet-mount can be made and viewed under oil-immersion (100x) or the high dry objective (40x) using phase microscopy (see photographs below). Observe for trophozoites with acanthapodia (spine-like pseudopods) on the surface (characteristic of Acanthamoeba spp.).

Acanthamoeba trophozoite

A. castellanii (ATCC® 30010) trophozoite. Organelles and acanthapodia are visible. 1000x phase-contrast.

Acanthamoeba trophozoite

A. castellanii (ATCC® 30010) trophozoite. Organelles and acanthapodia are visible. 1000x phase-contrast.

LIMITATIONS

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as other culture media, slides, cover slips, pipets, microscope, incinerators, and incubators, etc. are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Phase-Contrast Observations
Time Temperature Atmosphere
Acanthamoeba castellanii
ATCC® 30010**
* 48hr 15-30°C Aerobic Presence of acanthapodia

USER QUALITY CONTROL

Physical Appearance

Acanthamoeba Broth should appear clear, and amber in color.

REFERENCES

1. Garcia, L.S., 2007. Diagnostic Medical Parasitology, 5th ed. American Society for Microbiology, Washington, D.C.

2. Health Protection Agency, 2007. Isolation and identification of Acanthamoeba species. National Standard Method W 17 Issue 2. www.hpa-standardmethods.org.uk/pdf_sops.asp.

3. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

4. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

5. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

6. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.


ATCC is a registered trademark of the American Type Culture Collection.

012617gr