File:32. Детекција на бактериофаги.ogg

English: Detection of bacteriophages

Procedures:

1. Decimal dilutions of a sample of wastewater or water containing coliforms are prepared in Tris by transferring 1 ml from the sample in a test tube containing 9 ml sterile Tris buffer.

2. 4 test tubes filled with 3 ml aliquots of agar are to be liquefied in an autoclave and allowed 15 min to reach 45 degrees centigrade with a water bath (45-48 degrees centigrade).

3. The first test tube is to receive a 1 ml sample of an E.coli culture in its log phase and 1 ml of the undiluted water sample. Gentle movement between the palms for a few seconds should achieve homogenization, after which the content is to be poured into a sterile Petri dish with a previously solidified layer of nutrient agar. Gentle circular movements should allow the liquid agar to cover the surface of the plate.

4. Once solidified, the plates may be incubated inversely at 37 ^оС for 48 hours.

RESULTS: Lytic phages present in the water sample are demonstrated by adding E.coli to the water sample and soft agar. These phages attack the bacterial cells and cause cell lysis as they finish their reproduction cycle. As bacteria produce colonies, these phages may disrupt normal colony formation by creating a blank sphere within a colony called a “plaque”. Each plaque is theoretically caused by the activity of a single progenitor phage. Soft agar is used in order to foster this visualisation of the viral distribution across bacterial cells.

Planned and performed by Sofija Kostandinovska, Institute of Biology, FNSM, Ss. Cyril and Methodius University, Skopje, Macedonia.