**4. Cloning of trypanosomes**

Cloning of trypanosomes is necessary for the production of a homogeneous population of trypanosomes. It is carried out as previously described by Otieno and Darji [23]. Briefly, a sample containing trypanosomes is diluted in PSG pH 8.0, to 1 trypanosome per micro‐ scopic field at 400× magnification. This is followed by addition of 0.5 ml guinea pig se‐ rum. Using a needle, a drop of the trypanosome suspension is placed on a cover slip that is overturned onto a cavity slide moistened with PSG to prevent evaporation of the drop. In the laboratory, the drop is examined under a microscope (400× magnification) by at least three experienced technicians to confirm the presence of a single and viable trypano‐ some, which is aspirated and inoculated into an immunosuppressed Swiss white mouse. Inoculated mice are monitored for parasitemia development and sacrificed at the first peak of parasitemia. Blood is harvested by cardiac puncture for cryopreservation of the clone of trypanosomes.

#### **5. Maintenance of cryopreserved trypanosomes**

#### **5.1. Liquid nitrogen**

Cryopreserved trypanosomes are permanently stored in liquid nitrogen. The samples must always be fully immersed in liquid nitrogen and the levels maintained by frequently refilling the storage Dewars. The refilling period is determined by the frequency at which the Dewars are opened during issue of materials for research. The more frequent they are opened, the more liquid nitrogen vapor is lost, hence the need to refill. Under normal circumstances, refilling is done fortnightly.

#### **5.2. Trypanosomes viability and infectivity tests**

**5.** Pull the piston and mix the contents with the PSG buffer thoroughly

slip, and examine the parasitemia at 400 × magnification

**7.** Infect the experimental (donors) as required by the protocol

due to differences in temperature.

**4. Cloning of trypanosomes**

clone of trypanosomes.

**5.1. Liquid nitrogen**

**5. Maintenance of cryopreserved trypanosomes**

Precaution:

12 Cryopreservation in Eukaryotes

samples.

**6.** Remove the air bubbles and place a drop of the mixture on the microscope slide, cover with a cover

**1.** Use of protective devices while handling cryopreserved samples is mandatory. This is necessary because the samples being handled contain live parasites, some of which are pathogenic to humans. Also, nitrogen at –196°C burns. Industrial gloves are recommended while handling liquid nitrogen. Use of facial masks will protect the user from harmful effect in case of contact with the eyes. All safety precautions should be strictly observed when capillary tubes are withdrawn from the liquid nitrogen; they sometimes burst before they are transferred into the screw capped ampoules to thaw, possibly

**2.** It is recommended that retrieval of the sample should be rapid to avoid the thawing of the remaining

Cloning of trypanosomes is necessary for the production of a homogeneous population of trypanosomes. It is carried out as previously described by Otieno and Darji [23]. Briefly, a sample containing trypanosomes is diluted in PSG pH 8.0, to 1 trypanosome per micro‐ scopic field at 400× magnification. This is followed by addition of 0.5 ml guinea pig se‐ rum. Using a needle, a drop of the trypanosome suspension is placed on a cover slip that is overturned onto a cavity slide moistened with PSG to prevent evaporation of the drop. In the laboratory, the drop is examined under a microscope (400× magnification) by at least three experienced technicians to confirm the presence of a single and viable trypano‐ some, which is aspirated and inoculated into an immunosuppressed Swiss white mouse. Inoculated mice are monitored for parasitemia development and sacrificed at the first peak of parasitemia. Blood is harvested by cardiac puncture for cryopreservation of the

Cryopreserved trypanosomes are permanently stored in liquid nitrogen. The samples must always be fully immersed in liquid nitrogen and the levels maintained by frequently refilling the storage Dewars. The refilling period is determined by the frequency at which the Dewars

It is important to ascertain that the cryopreserved samples remain viable by randomly test‐ ing the infectivity of the parasites in laboratory rodents to ensure that this has been main‐ tained and not lost over long periods of storage. The viability testing is performed by removing a single capillary of each of the cryopreserved trypanosome isolate, thawing at room temperature, cutting the sealed end of the capillary tube using a diamond pencil, de‐ canting the capillary contents on a microscope slide, covering the content with a glass cover‐ slips, and examining for the motility of the trypanosomes under the microscope at 400× magnification.
