Z-5 matches B-5 for histological quality.

It is easy to formulate a fixative that has few if any of the disadvantages of B-5, as evidenced by the many solutions in the literature and the marketplace. Getting a fixative to produce the same quality results on a histological level obviously is much more difficult. Until now, no substitute has been capable of creating identical morphological images in a short period of time.

Written descriptions aside, the photomicrographs here clearly show how good Z-5 really is. The bone marrow biopsy (above left) shows beautifully detailed chromatin patterns and brightly differentiated eosinophils. It was fixed for 2 hours, then decalcified in DeltaCal (equivalent to RDO) for 0.5 hours before routine processing. Identical results were obtained after 1.5 hours of exposure to this rapid decalcifier. Interestingly, 22 hours of decalcification merely lessened the intensity of hematoxylin staining slightly. Z-5 protects specimens from acid hydrolysis during excessive decalcification.

Lymph nodes are equally spectacular after fixation in Z-5. Architecture is perfectly preserved (above right) and nuclear detail is unsurpassed (below left).

Using Z-5 is easy

Z-5 is sold as an aqueous concentrate in an innovative package comprised of two partially filled bottles. Each bottle contains enough concentrate to make 820 ml of diluted fixative. Fill the bottle up to the mark with alcohol, cap it and shake briefly to mix. You may use anhydrous or 95% ethanol, reagent alcohol or denatured ethanol. Once made, the solution is stable for at least a year (the concentrate is stable for at least two years).

The concentrate contains an indicator that is pink. When diluted as directed with alcohol, the color disappears. If diluted improperly with water, the pink color remains. You will always know if it is ready to use and has been diluted properly.

Use your B-5 fixation times as a guide in determining minimum exposures with Z-5. In all likelihood, you will be able to reduce fixation time from there. Specimens may be left in Z-5 for several days without harm.

If tissues have been fixed initially in neutral buffered formalin, rinse them briefly in water to remove phosphate salts before placing them into Z-5. Subsequent exposure to Z-5 will usually improve the morphology of these specimens, but they will not be comparable to those fixed only in Z-5 unless exposure to NBF has been brief.

Once fixed with Z-5, tissues can be placed directly into Z-Fix or Prefer on the processor. If you have neutral buffered formalin on the processor, rinse specimens briefly with water first to minimize precipitation of zinc phosphate. In any case, process with your standard schedule.

Because tissues do not become brittle in Z-5, getting perfect sections is noticeably easier than after B-5 fixation. Wrinkles, knife chatter, drying and shrinkage artifacts that are so common with B-5 will be eliminated with Z-5 (providing a reasonable processing schedule is used). Low power photomicrographs document these claims (figures below).

Using mercuric fixatives is a sure way to contaminate wastewater systems, even if you currently save all of your waste B-5 for proper disposal. Let's follow a specimen through the lab to pinpoint all of the spots where mercury leaves its deadly trail. Remember, mercury is not only in the tissue itself, it also contaminates the fluid that is trapped within the cassette.

To rinse or not to rinse.

If specimens are rinsed briefly in tap water after fixation, mercury goes directly down the drain. If not, it simply means more will come out in the first station on your tissue processor.

Station #1 on the tissue processor.

Even if this fluid is used for only one run of the processor, it will be heavily contaminated with mercury that has leached out of the specimen. That in turn will be carried over into the next station, and so on all the way up the sequence. But let's stay with the first station for a while. What is its fate?

Do you discard it down the drain after one use?

Do you discard it down the drain after more than one use? If so, it will contain even more mercury.

Is it recycled via distillation? If so, all of the mercury is left in the still bottoms and these must be disposed by a licensed waste hauler as mercury waste.

Do you detoxify your waste formalin? If so, you cannot pour the resultant solution down the drain, or put the gelled solid in the trash. It, too, must be hauled away as mercury waste, so there was little point in detoxifying it.

All other stations on the processor.

After a single run with just a few B-5 specimens, the first several stations will have measureable levels of mercury. Each run carries the contamination further up the line. All contaminated solutions must be considered mercury waste. If you have a 4 liter processor that is changed every Friday, you generate 40 gallons of mercury waste each week (not counting possibly contaminated paraffin)! There is no way to get rid of this legally except with a licensed waste hauler.

De-Zenkerization.

However you do it, removing mercury precipitate from sections is just that: removing mercury. The iodine converts the mercurous chloride pigment to insoluble mercuric iodide, so neither the iodine solution nor the rinse immediately following should be contaminated. Sodium thiosulfate solubilizes mercuric iodide, so this and its rinse will contain mercury. Are these going down the drain?

Now that most of the sources of contamination have been identified, do you see the extent and hopelessness of the problem? But how much is really involved here? Is it significant? Keep in mind that wastewater officials and hazardous waste authorities consider mercury a serious problem in concentrations down in the parts per billion or trillion levels. You are starting with a fixative containing nearly 40,000 ppm (parts per million). This equates to 40,000,000 ppb (parts per billion) and 40,000,000,000 ppt (parts per trillion)! A single drop (0.02 ml) contaminating the first station of a 4 liter processor would create a gallon of waste containing over 200 ppb. That is significant.