Ova and Parasite (O&P) Examinations

Introduction | Macroscopic | Microscopic |

Microscopic Examination - Ova and Parasite Examination

Introduction | Direct Wet Smear | Concentration | Permanent Stained Smear

Permanent Stained Smear

Introduction | Trichrome Stain | Iron Hematoxylin Stain | Modified Iron Hematoxylin Stain | Procedure Review

Trichrome Stain

Procedure Notes

The single most important step in the preparation of a well-stained fecal smear is good fixation. If this has not been done, the protozoa may be distorted or shrunk, may not be stained, or may exhibit an overall pink or red color with poor internal morphology.
Slides should always be drained between solutions. Touch the end of the slide to a paper towel for 2 s, to remove excess fluid, before proceeding to the next step.
Incomplete removal of mercuric chloride (Schaudinn's fixative and PVA) may cause the smear to contain highly refractive crystals or granules which may prevent finding or identifying any organisms present. Since the 70% ethanol-iodine solution removes the mercury complex, it should be changed at least weekly to maintain the strong tea color. A few minutes is usually sufficient to keep the slides in the iodine-alcohol; too long a time in this solution may also adversely affect the staining of the organisms.
When using non-mercury-based fixatives, the iodine-alcohol step (used for the removal of mercury) and the subsequent alcohol rinse can be eliminated from the procedure. The smears for staining can be prerinsed with 70% alcohol and then placed in the trichrome stain or they can be placed directly into the trichrome stain as the first step in the staining protocol.
Smears that are predominantly green may be due to the inadequate removal of iodine by the 70% ethanol (steps 4 and 5). Lengthening the time of these steps or more frequent changing of the 70% ethanol will help.
To restore weakened trichrome stain, remove cap and allow the ethanol to evaporate (carried over on the staining rack from a previous dish). After a few hours, fresh stock stain may be added to restore lost volume. Older, more concentrated stain produces more intense colors and may require slightly longer destaining times (an extra dip). Remember that PVA smears usually require a slightly longer staining time.
Although the trichrome stain is used essentially as a "progressive'' stain (that is, no destaining is necessary), best results are obtained by using the stain "regressively'' (destaining the smears briefly in acidified alcohol). Good differentiation is obtained by destaining for a very short time (two dips only, approximately 2 to 3 s); prolonged destaining results in poor differentiation.
It is essential to rinse the smears free of acid to prevent continued destaining. Since 90% alcohol will continue to leach trichrome stain from the smears, it is recommended that after the acid alcohol is used, the slides be quickly rinsed in 100% alcohol and then dehydrated through two additional changes of 100% alcohol.
In the final stages of dehydration (steps 9 to 11), the 100% ethanol and the xylenes (or xylene substitute) should be kept as free from water as possible. Coplin jars must have tight-fitting caps to prevent both evaporation of reagents and absorption of moisture. If the xylene becomes cloudy after addition of slides from the 100% ethanol, return the slides to fresh 100% ethanol and replace the xylene with fresh stock.
If the smears peel or flake off, the specimen might have been inadequately dried on the slide (in the case of PVA-fixed specimens), the smear may have been too thick, or the slide may have been greasy (fingerprints). However, slides generally do not have to be cleaned with alcohol prior to use.
If the stain, upon examination, appears unsatisfactory and it is not possible to obtain another slide to stain, the slide may be restained. Place the slide in xylene to remove the coverslip and reverse the dehydration steps, adding 50% ethanol as the last step. Destain the slide in 10% acetic acid for several hours; then wash it thoroughly first in water and then in 50 and 70% ethanol. Place the slide in the trichrome stain for 8 min and complete the staining procedure.