REAGENTS

(R1) Formalin

Formalin is the aqueous solution of the gas formaldehyde. It is commercially available at a concentration of 37%-40%; for dilution purposes it should be considered a pure solution. The commercially available product contains a variable quantity of formic acid, which hinders the fixation of parasites, so it is best to use neutralized or buffered formalin.

Neutralized formalin

Procedure

-     Neutralize the formic acid (in the formalin) by adding 10 N NaOH until the pH becomes neutral (check with pH paper or on a pH meter).

-     To fix stool specimens, a 10% neutralized formalin solution is generally used.  To fix protozoa, a 5% solution is recommended.

Neutralized formalin solution (10%)

Procedure

-     Combine 100 ml neutralized formalin with 900 ml distilled water or saline;

-     Mix well.  Store in a sealed container.

Neutralized formalin solution (5%)

Procedure

-     Combine 50 ml neutralized formalin with 950 ml distilled water or saline;

-     Mix well.  Store in a sealed container.

Buffered formalin (10%)

Composition

-     Sodium phosphate, monobasic (NaH2PO4)                4.0 g

-     Sodium phosphate, dibasic (Na2HPO4)                      6.5 g

-     Commercial formalin (37%-40%)                          100 ml

-     Distilled water or saline                                        900 ml

Procedure

-     Dissolve the salts in distilled water;

-     Add formalin and mix well;

-     Store in a sealed container.

Buffered formalin (5%)

Composition

-     Sodium phosphate, monobasic (NaH2PO4)                4.0 g

-     Sodium phosphate, dibasic (Na2HPO4)                      6.5 g

-     Commercial formalin (37%-40%)                            50 ml

-     Distilled water or saline                                         950 ml

Procedure

-     Dissolve the salts in distilled water;

-     Add formalin and mix well;

-     Store in a sealed container.

(R2) Physiological saline

Composition

-     NaCl                                                                     8.5 g

-     Distilled water                                                    1000 ml

Procedure

-     Dissolve the sodium chloride in distilled water, and mix well;

-     Store in a sealed container.

The quantity of salt and water can be proportionately reduced. For optimum storage:

-       Distribute 10 ml aliquots in sealable glass test tubes;

-       Sterilize in an autoclave at 121 °C for 15 minutes and allow to cool;

-       Store at 4 °C until use.

Physiological saline should be renewed frequently and stored in sealed test tubes to prevent accidental contamination with plant spores, pollen, mould or free-living airborne amoeba. These contaminants, during microscopic examination, can be mistaken for intestinal protozoa.

(R3) Sucrose solution 

Procedure

-     Dissolve 500 g sucrose in 320 ml distilled water;

-     Heat the solution until it becomes transparent (do not boil);

-     Carefully add 6.5 g phenol crystals (under a fume hood);

-     Mix well.  Store in a sealed container.

The solution is stable.

(R4) SAF (Sodium acetate-acetic acid-formalin) 

Composition

-     Anhydrous sodium acetate (NaC2H3O2)                       9 g

-     Glacial acetic acid                                                  20 ml

-     Commercially available formalin (37%-40%)             40 ml

-     Distilled water                                                     925 ml

In alternative to 9 g anhydrous sodium acetate, 15 g sodium acetate trihydrate (CH3CO2Na ∙ 3H2O) can be used. 

Procedure

-     Dissolve the salt in distilled water;

-     Add formalin and glacial acetic acid, and mix well;

-     Adjust to pH 4.15, if necessary.

This solution is stable, in a sealed glass bottle, for at least one year.

If just one fixing agent needs to be chosen from those commercially available, SAF is recommended for the following reasons:

·      Does not contain mercuric compounds and therefore is safer, less corrosive and damaging to the environment;

·      Maintains morphology of cysts, helminthic eggs and larvae, coccidian oocysts and microsporidia spores but, above all, is excellent for the morphological differentiation of the nucleus of protozoan trophozoites;

·      Enables the use of concentration procedures with formalin-ether or formalin-ethyl acetate and flotation with zinc sulfate;

·      Allows the search for parasites coproantigens and staining with fluorochromes;

·      Enables permanent staining with iron-hematoxylin (excellent results) and trichrome staining;

·      Allows modified trichrome staining for microsporidia;

·      Is inexpensive and can be easily prepared in the laboratory.

However, there are some disadvantages of SAF:

-     SAF-fixed specimens stain poorly with trichrome compared to specimens fixed with Schaudinn's solution or PVA solution;

-     Due to the low formalin concentration, a ratio of 1 part feces to at least 3 parts fixative must be used to avoid fecal specimen fermentation.

(R5) Schaudinn’s fixative

Saturated solution of mercuric chloride (HgCl2)

Procedure

-     Dissolve 10 g HgCl2 in 100 ml warm (not boiling) distilled water;

-     Leave to cool (mercuric chloride crystals deposit);

-     Filter off the clear supernatant;

-     Store in a sealed glass bottle until use.

Schaudinn's stock solution

Composition

-     Saturated mercuric chloride solution                  2 volumes

-     95% ethanol                                                     1 volume

-     Mix well.

Schaudinn’s working solution

-       Immediately before use, add  5.0 ml glacial acetic acid to 95 ml of stock solution

-       Mix well.

 

(R6) PVA solution (polyvinyl alcohol – mercuric chloride)

Polyvinyl alcohol is a synthetic polymer of vinyl alcohol. PVA-fixed specimens smeared onto a slide, once dried, form a very adhesive transparent plastic film that withstands alcohol, ether, xylene and acetone. The true fixative is mercury chloride, an energetic oxidizing agent that precipitates proteins. This action is enhanced by acetic acid.

PVA solution 

Composition

-     Schaudinn's stock solution                                   93.5 ml

-     Glycerol                                                              1.5 ml

-     Glacial acetic acid                                                 5.0 ml

-     Polyvinyl alcohol 90-50 (low or medium viscosity)       5 g

Procedure

-     Combine the first three ingredients and heat the mixture in a water bath (75° C), making sure that the ethanol (in Schaudinn's solution) does not burn

-     Slowly add 5 g polyvinyl alcohol, stirring continuously; if possible, use a heating plate with a magnetic stirrer.

During cooling, the solution may become slightly cloudy but this can be ignored. The solution is stable for at least 1 year if stored in a sealed glass bottle.  If it becomes too viscous, whitish or opaque over time, it can no longer be used.

PVA solution is the best fixative for trichrome staining, but has some disadvantages:

-     Mercuric chloride is highly toxic to both man and the environment, and must be disposed of as toxic waste;

-     Difficult to prepare in the laboratory, also because it is not easy to find good quality polyvinyl alcohol. If possible, purchase the fixative ready-made from a commercial supplier;

-       Because of the corrosive power of mercuric chloride, smears, before staining, must not come into contact with metals, e.g. they must not be stored in aluminum foil, otherwise the specimen will flake off from the slide;

-       Can be used for concentration with formalin-ether or formalin-ethyl acetate, but the number of eggs and cysts identifiable is far lower than for specimens fixed with formalin. When the parasite load is low, the result may be false negative;

-       The addition of iodine for direct microscopic examination can sometimes cause precipitates, which make microscopic examination difficult.

Because of the toxic nature of mercuric chloride, attempts have been made to replace it with copper or zinc sulfate. Unfortunately, with this modification, the nuclear and cytoplasmic morphology is not comparable to that seen with mercuric chloride, and in some cases it is impossible to recognize the morphology of pathogenic protozoa.

(R7) MIF solution (merthiolate-iodine-formalin)

This solution enables simultaneous fixing (with formalin) of protozoa and their staining with two coloring agents (iodine and eosin). MIF solution is mainly used for in-the-field investigations.

Advantages:

-       Examination of fresh feces with MIF solution gives prompt diagnosis, showing the presence of any intestinal protozoa (trophozoites or cysts) or helminthic eggs and larvae. The fixed and stained stool specimens can be examined even weeks or months later.

Disadvantages:

-       Protozoa identification is often difficult; the microscopist must be experienced;

-       Permanent stains can be performed on MIF-fixed specimens but the morphology of organisms is not as good as that seen with Schaudinn's, PVA or SAF solutions;

-       The components of MIF solution must be stored separately and mixed only immediately before use;

-       Iodine solution is unstable and must be frequently renewed;

-       Concentration techniques are not always efficient;

-       Merthiolate tincture (containing eosin, 1/1000) it is not readily available commercially.

MIF solution for staining fresh stool specimens

Composition

-     Merthiolate tincture, 1/1000 Lilly®                        7.75 ml

-     Lugol's 5% iodine solution                                   1.00 ml

-     Formalin 37%-40%                                             1.25 ml

This solution is stable for up to 8 hours. The quantity of reagents may be reduced proportionately if needed. For use, see: “Microscopic examination with stains for temporary (wet mount) preparations”.

MIF solution for test tube staining or for fecal specimen storage

MF stock solution

Composition

-     Merthiolate tincture, 1/1000 Lilly®                         200 ml

-     Formalin 37%-40%                                                25 ml

-     Glycerol                                                                 5 ml

-     Distilled water                                                     250 ml

Mix well. Store in a sealed, brown glass bottle. The solution is stable for several months.

Lugol's 5% iodine solution

Composition

-     Iodine crystals                                                          5 g

-     Potassium iodide (KI) crystals                                  10 g

-     Distilled water                                                     100 ml

Procedure

-     Dissolve potassium iodide in 20-30 ml distilled water;

-     Slowly add iodine crystals to get a homogeneous solution;

-     Add the remaining water, mix well and filter.

-     Store in a tightly sealed brown bottle.  The solution is stable for several weeks.  

MIF working solution

Composition

-     MF stock solution                                               2.35 ml

-     Lugol's 5% iodine solution                                   0.15 ml

Immediately before use, add MF stock solution to Lugol's iodine solution, and not vice versa.   For use, see: “Microscopic examination with stains for temporary (wet mount) preparations”.

(R8) Lugol's 1% iodine solution for direct examination of fresh or formalin-ether/ethyl acetate concentrated feces

Composition

-     Iodine crystals                                                          1 g

-     Potassium iodide (KI) crystals                                    2 g

-     Distilled water                                                     100 ml

Procedure

-     Dissolve the potassium iodide in 10-20 ml distilled water;

-     Slowly add iodine crystals to get a homogeneous solution;

-     Add the remaining water, stir well and filter.

This solution is quite stable and keeps its staining properties for about one month if stored in a tightly sealed brown glass bottle sheltered from sunlight. A ready to use 1% iodine solution stabilized with PVP (polyvinyl-pyrrolidone) is commercially available and has superior stability (1 year). For use, see: “Microscopic examination with stains for temporary (wet mount) preparations”.

(R9) Lugol's 1% iodine solution with glycerol

Composition

-     1% Lugol's iodine (or 1% Lugol's iodine PVP) solution 100 ml

-     Glycerol                                                             0.25 ml

Stir well. For storage and stability, see (R8).

For use, see: “Microscopic examination with stains for temporary (wet mount) preparations”.

(R10) Bailenger's stain (fuchsin-crystal violet)

Composition

-     Crystal violet                                                          2.0 g

-     Basic fuchsin                                                       0.05 g

-     Ethyl alcohol 95%                                               10.0 ml

-     Crystallized phenol*                                            10.0 ml

* Melted in water bath at 56 °C under a fume hood.

Procedure

-     Mix the reagents thoroughly

-     Leave to stand overnight

-     Add 100 ml distilled water

-     Stir well and filter.

The solution must be sheltered from sunlight and stored in a tightly sealed brown glass bottle.  When used, do not shake the bottle, but take the top layer of the solution with a pipette.  For use, see: “Microscopic examination with stains for temporary (wet mount) preparations”.

(R11) Sargeaunt's solution (for staining of chromatoid bodies)

Composition

-     Malachite green                                                      0.2 g

-     Glacial acetic acid                                                 3.0 ml

-     Ethanol 95%                                                        3.0 ml

Procedure

-     Dissolve malachite green in alcohol;

-     Add acetic acid

-     Add distilled water to bring the volume to 100 ml.

The solution is quite stable if stored at room temperature.  For use, see:  Direct microscopic examination after concentration.

(R12) Merbromin solution

Composition

-     Merbromin                                                               2 g

-     Distilled water                                                     100 ml

Procedure

-     Dissolve merbromin in distilled water, and mix well.

-     Store at room temperature.

The solution is stable for 1 year.

(R13) Nigrosin solution

Composition

-     Nigrosin                                                                   1 g

-     Distilled water                                                     100 ml

Procedure

-     Dissolve nigrosin in distilled water, and mix well.

-     Store at room temperature.

The solution is stable for 1 year.

(R14) Ethanol 70% plus iodine

Add 5% iodine solution to the needed volume of 70% ethanol in order to achieve the color of strong tea.  This solution must be made fresh when the color fades. Replacement time will depend on the number of smears stained (1 to several weeks).

(R15) Trichrome stain

Composition

-     Chromotrope 2R                                                    0.6 g

-     SF light green                                                        0.3 g

-     Phosphotungstic acid                                              0.7 g

-     Glacial acetic acid                                                 1.0 ml

-     Distilled water                                                   100.0 ml

Procedure

-     Place the solids in a 250 ml glass beaker;

-     Add acetic acid to moisten the solids and let stand for 30 minutes;

-     Add distilled water and stir carefully.

-     Store in a dark glass bottle out of sunlight.

The solution should become deep purple. It is stable for at least 1 year if stored at room temperature.  A ready to use solution is commercially available.

(R16) Destaining solution

Composition

-     Glacial acetic acid                                                 0.5 ml

-     Ethyl alcohol 90%                                               99.5 ml

Do not substitute the 90% ethanol with 95% ethanol, which could result in excessive destaining.

(R17) Mayer's albumin 

Procedure

-     Mix equal parts of glycerol and fresh egg albumen

-     Filter over gauze

-     Add some thymol crystals as preservative.

-     Store at 4° C.

-     Mix well by inversion before use.

Stable at least one year.

(R18)  Heidenhain’s hematoxylin solution A

Composition

-     Hematoxylin crystals                                                10 g

-     95% ethanol                                                      1000 ml

Procedure

-     Dissolve hematoxylin crystals in alcohol in a clear glass container;

-     Seal and leave to oxidize (or “ripen”) in daylight (not direct sunlight) for at least 6-8 weeks. Frequent shaking hastens the ripening process. A longer exposure (months, up to two years) increases the staining properties of the solution.

(R19) Mordant iron alum solution B

Composition

-     Ferrous ammonium sulfate Fe(NH4)2(SO4)2 6H2O       10 g

-     Ferric ammonium sulfate FeNH4(SO4) 12H2O            10 g

-     Concentrated hydrochloric acid (37%)                     10 ml

-     Distilled water                                                    1000 ml

Procedure

-     Dissolve the two salts in distilled water;

-     Slowly add hydrochloric acid;

-     Store at room temperature (best, in a refrigerator) in a dark glass bottle.

(R20) Heidenhain’s hematoxylin working solution

Procedure

-     Mix equal parts of solutions A (R18) and B (R19), filter.

-     Let stand at room temperature for at least four hours (best, overnight) before use.

If the solution is used immediately after preparation, parasites will stain bright blue with little differentiation of nuclear details. The best results are obtained 3-4 days after preparation of the working solution.  

The working solution is stable for at least one week, but can be prolonged by storage in a sealed, dark glass flask. To check if the solution is still of good quality:

-       Add two drops of ammonium hydroxide in a test tube filled with tap water;

-       Add two drops of hematoxylin working solution;

-       If the resulting color is blue or blackish-blue, the working solution can be used, otherwise (if brown or brownish) the solution must be remade.

(R21) Destaining solution 1

Composition

-     Ethyl alcohol 95%                                                  70 ml

-     Distilled water                                                       30 ml

-     Concentrated hydrochloric acid                                 1 ml

Mix well.

(R22) Destaining solution 2

Composition

-     Mordant iron alum solution (R19)                              5 ml

-     Distilled water                                                       95 ml

Mix well.  This solution must be made fresh every time.

(R23) Mayer’s hematoxylin

Composition

-     Hematoxylin crystals                                                 1 g

-     Potassium alum                                                       50 g

-     Sodium iodate (NaIO3)                                          0.20 g

-     Chloral hydrate                                                        50 g

-     Distilled water                                                    1000 ml

Procedure

­       Melt (at ~90 °C) the hematoxylin and potassium alum in 500 ml distilled water, allow to cool;

­       Add the sodium iodate previously dissolved in 50 ml distilled water and the chloral hydrate dissolved in 450 ml distilled water;

­       Allow to “ripen” in an incubator for 48 hours at 48-50 °C, in a sealed glass container;

­       Remove from the incubator, uncover (or transfer to an unsealed container), and allow to cool, mixing slowly and evenly with a magnetic stirrer for about two hours.

­       Add 5 ml glacial acetic acid and stir well.

­       Filter before use.

Mayer's hematoxylin solution is stable for at least a year if stored in a dark glass bottle at room temperature (20-25 °C). 

Hematoxylin solutions (Heidenhain, Mayer) are commercially available, but are often not properly “ripened” (oxidized). If this is the case, the parasites are only weakly stained and nuclear details are poorly differentiated.

(R24) Aqueous ammonia solution

Procedure

-     To 100 ml distilled water, add 8 drops concentrated ammonia.

-     Mix well.

(R25) Ziehl-Neelsen modified acid-fast stain (hot method, for intestinal coccidia)

Solution A

-      Basic fuchsin                                                         0.3 g

-      Ethanol 95%                                                         10 ml

Dissolve fuchsin in ethanol, and mix well.

Solution B

-      Phenol crystals                                                       5.0 g

-      Distilled water                                                   100.0 ml

Dissolve phenol crystals in distilled water in a water bath at 56 °C under a fume hood.

Carbol-fuchsin working solution

Mix solutions A and B.  Store at room temperature. The solution is stable for at least 1 year.

(R26) Acid destaining solution

Composition

-      Concentrated hydrochloric acid (37%)                       3 ml

-      Distilled water                                                       97 ml

Add hydrochloric acid, drop to drop, to the water (not vice versa) and mix well.

(R27) Methylene blue solution (counter-stain)

Composition

-     Methylene blue                                                       0.3 g

-     Distilled water                                                     100 ml

Dissolve the stain in distilled water and mix well.

Store at room temperature. The solution is stable for 1 year.

(R28) Kinyoun’s stain (cold method, for coccidia)

Solution A

-     Basic fuchsin                                                            4 g

-     Ethanol 95%                                                         20 ml

Dissolve stain in ethanol and mix well.

Solution B

-     Phenol crystals melted in water bath (56? C)              8 ml

Procedure

-     Mix solutions A and B;

-     Add 100 ml distilled water, stir well;

-     Let stand for 1-2 days.

-     Filter and store in a dark glass bottle.

Stable for an indefinite period.

(R29) Acid destaining solution

Composition

-     Concentrated sulfuric acid                                        1 ml

-     Distilled water                                                       99 ml

Add acid, drop to drop, to the water (not vice versa) and mix well.

(R30) Methylene blue solution (counter-stain)

Composition

-     Methylene blue                                                       0.3 g

-     Distilled water                                                     100 ml

Dissolve the stain in distilled water and mix well.

Store at room temperature. The solution is stable for 1 year.

(R31) Modified trichrome stain for microsporidia

Composition

-     Chromotrope 2R                                                    6.0 g

-     Fast green FCF                                                    0.15 g

-     Phosphotungstic acid                                            0.70 g

-     Glacial acetic acid                                                 3.0 ml

-     Distilled water                                                   100.0 ml

Compared to the trichrome solution of Wheatley, the concentration of chromotrope 2R here is 10-times higher.  For the procedure, see Trichrome solution

(R32) Acid alcohol solution

Composition

-     Ethanol 90%                                                     995.5 ml

-     Glacial acetic acid                                                 4.5 ml

Mix well and store in a sealed container.

(R33) 10% Giemsa’s solution   

Composition

-     Giemsa stock solution                                            10 ml

-     Buffered water (pH 7.2)                                         90 ml

Mix well.

This solution is stable for 12 hours at room temperature.

Ready to use Giemsa stock solution is commercially available. Given its instability, it is recommended to purchase only small quantities (100-250 ml) and store at room temperature, sheltered from sunlight and properly sealed.

Do not shake the flask before use: the non-dissolved crystals might return into suspension and deposit on the preparation during staining.

Do not withdraw the stain solution using a plastic or glass pipette, but pour off a small quantity into a clean and dry container. Then use a graduated pipette to withdraw the needed amount of solution. Throw away the remainder; never put it back into the flask.

Buffered water (pH 7.2)

Buffer stock solution

-     KH2PO4                                                               2.69 g

-     Na2HPO4                                                             8.36 g

-     Distilled water                                                     100 ml

Dissolve the salts in distilled water. Mix well.

Procedure

-     Mix 20 ml buffer stock solution with 1000 ml distilled water

-     Check pH with pH paper or meter.

Mineral waters with different pH values are commercially available.  Buffered water can be replaced with commercially available still mineral water with a declared pH of 7.2.

(R34) Field stain

Field stain A

-     Methylene blue                                                     0.80 g

-     Azur 1                                                                 0.50 g

-     Na2HPO4                                                             5.00 g

-     KH2PO4                                                               6.25 g

-     Distilled water                                                     500 ml

Procedure

-     Dissolve salts in hot distilled water;

-     Let cool and add stains;

-     Let stand for 24 hours;

-     Filter over blotting paper;

-     Store at room temperature out of the sunlight.

Stable for about 12 months.

Field stain B

-     Eosin                                                                   1.00 g

-     Na2HPO4                                                             5.00 g

-     KH2PO4                                                               6.25 g

-     Distilled water                                                     500 ml

Procedure

-     Dissolve salts in hot distilled water;

-     Let cool and add stains;

-     Let stand for 24 hours;

-     Filter over blotting paper;

-     Store at room temperature out of the sunlight.

Stable for about 12 months.

Ready to use Field solutions A and B are commercially available.

(R35) Calculating the force of gravity (g)

g = r x (RPM)2 x 1.118 x 10-5

where r is the radius of the centrifuge in centimeters.