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

Iron Hematoxylin 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 gray or blue-gray 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. This will maintain the staining solutions for a longer period of time.
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.
For staining large numbers of slides, the working hematoxylin solution may be diluted and affect the quality of the stain. If dilution occurs, discard the working solution and prepare a fresh working solution.
The shelf life of the stock hematoxylin solutions may be extended extended by keeping the solutions in the refrigerator at 4°C. Because of crystal formation in the working solutions, it may be necessary to filter them before preparing a new working solution.
In the final stages of dehydration (steps 9 to 11), the 100% ethanol and the xylenes 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 iron hematoxylin stain for 8 min and complete the staining procedure.