LABORATORY DIAGNOSIS OF PROTOZOAN INFECTIONS 



381 



properties, and a mordant must be em- 

 ployed to make it effective. Many differ- 

 ent formulas have been used for hema- 

 toxylin staining solutions. In some, the 

 mordant is mixed with the hematoxylin, 

 while in others it is used separately. 

 Many different compounds are used as 

 mordants, the great majority being salts 

 of heavy metals such as iron, lead, copper, 

 cobalt, tungsten and molybdenum. One of 

 the best hematoxylins is Heidenhain's 

 iron-hematoxylin. A modification of this 

 technic is given below. Starting with the 

 smears in 70% alcohol after passing thru 

 iodine, the staining schedule is: 



50% alcohol 5 minutes 



30% alcohol 5 minutes 



Distilled water S minutes 



2% aqueous iron alum 1 hour 



Distilled water 1 minute 



0.5% aqueous hematoxylin .... 2 hours 



Distilled water Rinse 



Saturated aqueous picric acid . . . Destain until 

 the structures assume the proper intensity of 

 color. This process should be controlled by 

 microscopic examination at intervals. Ten 

 minutes is usually good for intestinal amoebae, 

 but a longer time is necessary for large pro- 

 tozoa such as Balantidhmi . 



Distilled water Rinse --2 changes 



Tap water Until all picric 



acid has come out of the smear. Change the 

 water at intervals. 



30% alcohol 5 minutes 



50% alcohol S minutes 



70% alcohol 5 minutes 



Gradual changes in alcohol concentration 

 are used in all staining and dehydration 

 procedures to avoid distortion of tissues. 

 Hematoxylin-stained smears and sections 

 can be kept in 70% alcohol indefinitely. 



In the classical Heidenhain's hema- 

 toxylin staining procedure, the stained 

 smears are destained with iron alum. In 

 the above procedure, saturated aqueous 

 picric acid is used instead; this requires 

 a minimum of observation (usually none) 

 during the destaining process, and the re- 

 sultant stain is dark blue instead of brown- 

 ish black as with iron alum. 



If desired, longer mordanting and 

 staining times can be used. The smears 

 can be mordanted for 2 hours and stained 

 for 4 hours, or they can be mordanted for 

 4 hours and stained overnight. These give 



a little more precise staining, but not 

 enough to make them worthwhile for rou- 

 tine purposes. 



COUNTERSTAINING 



If desired, the smears can be counter- 

 stained with eosin Y. However, this has a 

 tendency to obscure fine nuclear detail 

 somewhat. To counterstain the smears, 

 transfer them from 70% alcohol to 0. 5% 

 solution of eosin Y in 90% alcohol. The 

 pH of this solution should be brought to 

 5. 4 to 5. 6 by adding 4. ml of 0. 1 N HCl 

 per 100 ml. The acidified solution will 

 not keep more than 10 days to 2 weeks. 

 After that its pH will become too high for 

 satisfactory use. Stain for 45 seconds to 

 3 minutes. Transfer to 95% alcohol to 

 wash out excess dye and then proceed as 

 directed below. 



MOUNTING 



Permanent slides are mounted in a 

 medium which, quite fluid at first, later 

 becomes hard. Most mounting media are 

 immiscible with water, and many with al- 

 cohol. Hence, before mounting, all water 

 and alcohol must be removed from the 

 smears. This cannot be done simply by 

 allowing the smears to dry, for such de- 

 hydration in air would ruin the prepara- 

 tions by distorting the protozoa. Mounting 

 media which have been employed include 

 natural resins such as Canada balsam and 

 damar, and synthetic resins such as 

 euparal, naphrax, permount and clarite. 



Starting with stained coverslips in 

 70% alcohol, pass them thru the following 

 solutions: 



95% alcohol 5 minutes 



100% alcohol 5 minutes 



100% alcohol 5 minutes 



Toluene 5 minutes 



Toluene 5 minutes 



Mount in permount: Place a drop of 

 permount on a clean slide, place the 

 coverslip slantingly, smear side down, 

 alongside the drop, and gently lay it down 

 on the drop, taking care to prevent air 

 bubbles from forming. 



