Demonstration of the Life Cycle  of Hymenolepis diminuta: The Crowding Effect

        Tapeworms in general, reveal a decreased number of proglottids in individual worms relative to an increased worm burden.  Hymenolepis diminuta, a tapeworm normally infecting rats and occasionally humans (usually children), represents a good organism to demonstrate this effect.  The object of the exercise is to infect a number of rats with a known number of mature cysticercoids and to determine what effect increased populations or "crowding" has on the proglottid number of each worm.

PROCEDURE

1.  Obtain the beetles of  Tenebrio molitor which are infected with         Hymenolepis diminuta.  These can be obtained from Carolina Biological Supply or another biological supply house.  Alternatively, uninfected beetles may be obtained and infected by feeding them feces of an infected rat. The feces will contain egg capsules.  Already- infected rat feces can also be obtained from the above sources. 
2.  Deprive the rats of water for 24 hours but allow them dry food. 
3.  Dissect a number of beetles in a depression slide or Syracuse dish and obtain as many mature cysticercoids as are required.  Mature cysticercoids have elongated tails and are most likely to lead to an infection.
4.  The cysticercoids should be washed by adding fresh distilled water to the dish in which they have been isolated, followed by removal of the distilled water and accompanying debris.  This step should be repeated a number of times until the depression slide contains only cysticercoids.  At this time, you can evaluate the maturity of the worms and select a specific number with which to infect the rat.
5.  The rats, which have been thirsted for 24 hours, should be tempted with a clean-water-filled eyedropper.  With patience, the rats will eventually take a drop of water from the end of the pipette, after which you should withdraw the untainted water and substitute it with a pipette filled with water containing the desired number of cysticercoids.
6.   Infect the  individual rats with one, five, 10, 15, and 20 cysticercoids each .
7.  The pre-patent period is 18 days.  The pre-patent period is that interval between infection and full maturation of the worms.  By the end of 18 days, the worms should be delivering egg capsules as evidenced by their presence in the feces.  A fresh fecal pellet should be checked beginning at about the 18th day post-infection to insure the rat has been successfully infected. 
8.  Sacrifice the rats with overdoses of a euthanizing solution, dissect and remove the entire length of the small intestine.
9. Slice the small intestine lengthwise, being careful not to cut through the worms.
10.  Untwist the worms from each other and transfer them to separate Syracuse dishes.
11.  Kill the worms by submersing them in hot water (about 70C).  This relaxes them and makes counting proglottids easier.  An alternative approach would be to count the proglottids of one worm, weigh that worm, and then determine the mass of all the worms in that individual rat.  Then, apply the following formula.

        number of proglottids                             X          
        in one tapeworm    
        _________________        =        ____________

        mass of  that worm                       mass of all the
                                                            worms in that rat

12.  After you have completed the above, you may want to make permanent slides of specific segments of a few worms as follows.

    Slide Preparation for Hymenolepis diminuta

l.  Drop the whole worm (one at a time) into a 70C water bath and swirl with an applicator stick.  (You have already done this in the above procedure).  This is to insure the worms dies relaxed and extended.  Grasp the larger end of the worm (gravid proglottids) with an applicator stick you break in half or bacteriological loop and lay it on a  foot long glass plate being careful to straighten the worm and not twist it.  Dangling the worm from the applicator stick/bacteriological loop and bringing it slowly to the glass plate seems to work best.  Lay parallel rows of worms and cover the worms with glass slides.  The slides will flatten out the worms.  Squirt AFA (absolute ethanol-formalin-acetic acid) fixative along the edge of the glass slides until the worms are fully immersed.  After about 30 minutes, remove the glass slides and cut the worms into convenient lengths, suitable for placing on a slide.  Be careful of the scolices--there is only one to a worm.  The desired end result is to have a scolex, and sections of immature, mature, and gravid proglottids on a single slide.  Transfer the worm sections to small containers with fresh fixative and continue fixation for 90 minutes longer.
2.  Transfer the worms to 70% ethanol for 60 minutes.  This solution is a holding solution and the worms may be stored for extended periods of time.  THIS IS THE ONLY SOLUTION IN WHICH YOU MAY PAUSE.
3.  Transfer the worms to a 30% ethanol solution for 60 minutes.
4.  Stain the worms with Harris hematoxylin until they appear darker than you would expect necessary.  Dilute the stock stain (1 part stain/9 parts distilled water) and stain from 2-6 hours. The worms should not be touching each other during the stain. The worms should occasionally be turned over or gently agitated in the stain to prevent uneven staining.  By this time, the worms become brittle, so undue handling should be avoided.
5.  Wash the worms 2-3 times in distilled water.  To do this, pour off the previous solution and add distilled water.  Then, pour off the distilled water and add fresh distilled.  Repeat this 2-3 times.
6.  Dehydrate the worms in ascending ethanols of 30% and 50% for 30 minutes each.
7.  Destain the worms in 70% acid ethanol until a light, reddish purple color appears, changing the alcohol  if it becomes heavily colored.
8.  Neutralize the worms by placing them in 70% alkaline alcohol.  The worms will become decidedly bluish.
9.  Dehydrate the worms by passing them through an ascending series of 80%, 95% and 100% ethanols for 60 minutes each.  The 100% ethanol dehydration should involve three changes of solution for 20 minutes each.
9. Clear the worms by placing them in xylene or toluene until the unreacted stain is removed.  Both xylene and toluene have been shown to have carcinogenic properties in lab animals and should only be handled under a fume hood. 
10.  Mount the worm segments on clean, glass slides using permount as a mounting medium.  If the permount is too viscous, thin it with a small amount of xylene or toluene. 
11.  Allow the slides to air-dry over the next few days and label appropriately.
12.  Construct graphs which depict number of cysticercoids swallowed vs. proglottid number.