Entamoeba Medium

Intended Use:

Recommended for cultivation of Entamoeba histolytica.

Composition**

Final pH (at 25°C): 7.0±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Liver, infusion from

Directions

Suspend 33.0 grams in 1000ml purified/distilled water. Heat to boiling to dissolve the medium completely. Distribute in tubes and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Allow tubes to solidify in a slanted position. Cover about half of the slant with fresh sterile horse serum-saline mixture (1:6) and add a 5 mm loopful of rice powder, which has been sterilized in an oven at 160°C for one hour. SCORCHING OF THE MEDIUM SHOULD BE PREVENTED.

Principle And Interpretation

Amoebiasis occurs throughout all areas of the world, the causative agent of which is Entamoeba histolytica, the only amoeba pathogenic for humans (1,2,3). In nature, the organism exists in the cyst form. It enters the body through contaminated food or water. The organism passes through the stomach as cyst and the trophozoite amoebas emerge in the intestine. E. histolytica destroys the tissue of the large intestine, causing lesions, deep ulcers and diarrhea (1). Entamoeba Medium formulated by Cleveland and Sanders (4) and Cleveland and Collier (5), is used for the cultivation of E.histolytica.

This medium is highly specific for E.histolytica, which grows luxuriantly on this medium. Other intestinal amoebae do not grow readily on this medium. The technique of overlaying the medium with fresh sterile horse serum-saline mixture, as reported by Cleveland and Collier, was reported to be the best method of isolation of E.histolytica (6). Proteose peptone and HL infusion from provide amino acids and other nitrogenous substances that support growth of E.histolytica. Sodium chloride maintains the osmotic balance of the medium and sodium alpha-glycerophosphate acts as a phosphorous source.

Type of specimen

Clinical samples - Stool, urine samples, etc.

Specimen Collection and Handling:

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (7,8).

After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions :

In Vitro diagnostic Use only. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations :

1. Indiviual microorganism may differ in nutritional requirement and show variable growth patterns.
2. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user's unique requirement.
3. Further serological and biochemical testing is required for complete identification.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance Light yellow to brownish yellow homogeneous free flowing powder

Gelling Firm, comparable with 1.1% Agar gel.

Colour and Clarity of prepared medium Dark amber coloured, slightly opalescent gel forms in tubes as slants

Reaction Reaction of 3.3% w/v aqueous solutions at 25°C. pH : 7.0±0.2

pH 6.80-7.20

Cultural Response Cultural characteristics observed after an incubation at 25-30°C for 68-72 hours.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (7,8).

Reference

1. Alcamo I. E., 2001, Fundamentals of Microbiology, 6th Ed., Jones and Bartlett Publishers.
2. Bruckner D. A., 1992, Amebiosis. Clin. Microbiol., Rev. 5: 356-369.
3. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
4. Cleveland and Sanders, 1930, Arch. Protiskenkunde, 70:223.
5. Cleveland and Collier, 1930, Am. J. Hyg., 12:606.
6. Sixth Annual Year Book 1935-36, P. 130, Suppl., Am. J. Pub. Health,1936, 26, No. 3.
7. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
8. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.