Special Stain Procedures
There is a large number of apparatus used for staining depending on laboratory volume and the
stain protocol. High volume laboratories use an autostainer similar to the Shandon Autostainer.
An autostainer has the advantage of hands off operation and achieves stain consistency from batch to batch.
Regardless of the staining method, quality control is checked on a per batch basis with
Dientamoeba fragilis and Cryptosporidium parvum control slides. D. fragilis used to check the
Iron Haematoxylin (I.H.) stain has 1 - 2 nuclei containing 3 - 5 granules. If the stain is adequate
individual granules should be visible. If the laboratory incorporates an acid fast stain with
the I.H. a control slide of C. parvum is run to check the modified acid fast Kinyoun stain. Phenol
is added to the Kinyoun eliminating the need for heat (modified). The acid fast stain
should reveal individual sporozoites of C. parvum.
Trichrome staining will be discussed under Microsporidium.
The above photo of a Shandon Autostainer slide holder illustrates the high volume capacity of this machine.
I will not get into the actual stain protocol for the Acid Fast/Iron Haematoxylin Stain here except to outline the steps in general and explain their purpose. For the complete stain protocol click Iron Haematoxylin.
Step Twelve
In "Step Six", you added a drop of Mayer's Albumin to a clean, labeled glass slide. Mayer's Albumin is a mixture of egg albumin and glycerin mixed at a ratio of 1:1. The glycerin prevents the slide from drying completely and the egg albumin, a protein, is denatured when immersed in 70% ethanol and fixes the stool to the glass slide.
A short water bath precedes the Modified Kinyoun Stain (Acid Fast). Here organisms such as Cryptosporidium parvum, Isospora belli and Cyclospora cayetanensis are stained. The slides are rinsed in water to remove excess stain then decolorized in acid alcohol. In this case decolorizing removes the acid fast stain from organisms or debris that should not be stained (red).
There is a water rinse then the slides are immersed in the Iron Haematoxylin. Haematoxylin is obtained from the logwood tree. Protein is stained blue. Nuclear material is stained darker almost black with the rest of the cell stained light blue to dark blue. Picric acid is used to decolorize. The slides emerge from the picric acid with a yellow/brown hue.
The slides are then rinsed in a water bath containing ammonium hydroxide to aid in removing the picric acid. A running water bath follows for an extended period to completely remove any trace of picric acid. Not only will the presence of picric acid interfere (alter color) with the examination of the slide but it will reduce the permanence of the slide.
A number of alcohol dehydration steps are used to remove any trace of water from the slide. Xylene substitute is not misable with water so if the slides are coverslipped with a trace of water present they will appear cloudy and would have to be dehydrated again. Although this is not a big problem it can be a tedious undertaking especially if the lab is busy or if the coverslips have been in place for some time. After dehydration the slides are cleared with xylene or xylene substitute and then coverslipped using a mounting fluid such as permount.
The staining is complete and these slides are ready for coverslipping.
Coverslipping requires patience and practice to keep the formation of bubbles to a minimum. Taking care to avoid the more granular bits of debris when making the smear will pay big dividends towards this end. Slides properly prepared and stained can be kept for years without any discoloration.