Sample Incubation Procedure

One of the primary limitations for evaluating the effectiveness of treatment of a cryptosporidium infection is the detection method. Under laboratory conditions, clostridium perfringens is used as an indicator organism for cryptosporidium. The difficulty in using this method in fieldwork is the need for anaerobic incubation at 45°C. In most cases, a commercial lab is required, which is very cost prohibitive, and, in the case of Zinga Island, because of the distance to the mainland and limited transport possibilities, difficult to achieve. A field test was developed to detect clostridium. The field test should be used as a screening tool. Final confirmatory samples should be sent to an accredited lab. One side benefit the solar thermal heat method is that water with a temperature of 40 to 50°C is readily available.

Anaerobic conditions were achieved by using a candle. A mayonnaise jar was emptied and cleaned. Samples were prepared using blood agar (the tryptosesulfate-cycloserine-agar, more specific to clostridium, was unavailable). After preparation, the Petri dishes were affixed to the bottom of the jar with candlewax. A candle was inserted, lit, and the jar closed. The flame used up the oxygen in the jar, creating anaerobic conditions.

A 50-L water barrel was converted into an incubator. The outsides, top and bottom were insulated with four layers of Styrofoam packing material and one layer of silver-backed yellow fiberglass insulation. The barrel was filled with 53°C water and closed.

In order to keep the jar in an upright position, an athletic sock was passed over the jar, knotted at the bottom and tied to rock. The apparatus was inserted in the water bath and sealed.

After 18 hours, the samples were removed. The lake samples, known to have clostridium, showed roughly 200 colonies. Microscopic analysis showed both the rods and the spores typical of clostridium. Although certain species identification was impossible with the limited microscope capabilities, there is a strong probability, given a known presence of clostridia in the sample, that the analysis was correct. The procedure was repeated with the heat-treated sample and no colonies were observed.

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