The next step is the most important when using a swinging bucket centrifuge: Gradually increase the speed of the rotor to
a maximum of 800 rpm. This gradual increase is only possible if the centrifuge is equipped with a dial, a knob, or a digital
entry button that allows you to increase the speed incrementally. If the rotor speed is increased gradually to the target
speed, the centrifuge bucket will move slowly to a horizontal position and the coverslip will remain in place. If the rotor
speed is increased too rapidly, the coverslip may be dislodged from the sample tube. (Never operate the centrifuge with the
lid open.) Often the lowest rpm setting allows the buckets to swing outward. If the coverslip will not stay in place at the
lowest rpm, use the method suggested for the fixed-angle centrifuge (Figure 9). Also follow the instructions for the fixed-angle centrifuge if the centrifuge does not have variable speeds or if the initial
speed is too fast.
After the required centrifuge time of 10 minutes, turn off the centrifuge, and allow the rotor to come to a complete stop.
Do not touch the rotor or use the centrifuge brake to slow it. Remove the coverslip from the sample tube in one deliberate
upward motion, and place it on a microscope slide. Place one side of the coverslip on the slide first and lower it gradually
at an angle onto the glass slide as described previously to prevent entrapped air bubbles.
We are often asked whether to allow the sample to stand an additional five or 10 minutes after centrifugation. Although data
suggest that an additional 10-minute standing time may increase parasite recovery rates,2 we suggest that if extra time is added to the procedure, the time should be added to the duration of centrifugation.
Centrifugal flotation with a fixed-angle centrifuge requires minor procedure changes from those used with a swinging bucket
centrifuge (see an overview of the fixed-angle technique in Figure 9). Because the sample is placed in the centrifuge at an angle, it is not possible to form a meniscus at the top of the sample
tube or to place a coverslip on the tube. Also, since the tube is not topped with a coverslip, the final speed at which the
sample is centrifuged is not as important, nor is it necessary to start the centrifuge at a slower speed. Centrifuge the sample
at about 1,200 rpm for five minutes. Faster centrifuge rates can be used without adversely affecting the result. We do not
suggest reducing the centrifuge time to less than five minutes.
Allow the centrifuge to stop as described above. Remove the centrifuge tube, place it in a holder, and add flotation solution
to form a meniscus. Follow the same procedure for placing a coverslip as described previously. Allow the sample to stand for
a minimum of 10 minutes before removing the coverslip, placing it on a slide, and examining it for parasite stages. If a sucrose
flotation solution is used, we have found that it may be necessary to allow the tube and coverslip to stand for 15 to 20 minutes
to recover all of the parasites stages. In my clinical experience, it is not necessary to wait longer than 10 minutes when
all other flotation solutions are used.
Examine the coverslip on the slide systematically and thoroughly. A prepared fecal slide is three-dimensional—it has length,
width, and depth. Depth is the most important. It is helpful to focus on the dust on the top of the coverslip and then move
downward. The smaller parasites (e.g. Giardia species, Cryptosporidium species, small coccidia) will be found in the first or top-most layer. The next layer down will contain the large eggs (roundworms,
hookworms, whipworms), oocysts, and larvae (if present).