ascarid nematode larvae. Two sequences of the internal transcribed spacer (ITS) regions of the rRNA gene of the ascarid parasites (ITS1 and ITS2) were compared with those registered in GenBank. PCR amplification of the ITS regions was sensitive enough to detect a single larva of Ascaris suum mixed with porcine liver tissue. These results suggest that even a single larva embedded in tissues from patients with larva migrans could be identified by sequencing the ITS regions (7).
Although formalin fixation is the most common storage, transportation, and preservation method for stool samples, it dramatically reduces the ability to extract DNA from stool samples for PCR-based diagnostic tests. Apparently, the deleterious effects of formalin are both time and concentration dependent and may result from fragmentation of fixed DNA during its purification. This has been seen in studies of the effect of formalin fixation on PCR of Entamoeba histolytica (8). The TOTAL-FIX stool collection kit is a single-vial system that provides a standardized method for untrained personnel to properly collect and preserve stool specimens for the detection of helminth larvae and eggs, protozoan trophozoites and cysts, coccidian oocysts, and microsporidian spores. Concentrations, permanent stains, most fecal immunoassays, and some molecular methods can be performed from a TOTAL-FIX preserved specimen. TOTAL-FIX is a mercury-, formalin-, and PVA-free fixative that preserves parasite morphology and helps with disposal and monitoring problems encountered by laboratories (www.med-chem.com; accessed 7/24/13). TOTAL-FIX is similar to UNIFIX and Zinc PVA (Z-PVA), commonly used fixatives that have been commercially available and used in many laboratories since 1992 (9, 10).
Parasites found in feces can be preserved in one of two ways (9, 10). One procedure involves thorough mixing of the fecal specimen directly in the fixative. This mixture can then be stored at room temperature without any additional processing. There are several disadvantages of this method: (i) if mixing is not complete, some of the organisms will not be well preserved and will disintegrate during storage; (ii) if the number of organisms is small, a random aliquot may not reveal the parasites; and (iii) the proper ratio of fixative to specimen may not be correct when one is working with larger volumes of material.
The second approach is to clean and separate the organisms from excess debris and concentrate them before fixation. The specimens can be mixed with a large volume of water, passed through a series of screens (large to small pore size), and finally allowed to sediment in a conical container (pilsner glass). After approximately 1 h, the sediment can be washed several times, resedimented, and finally fixed with hot (60 to 63°C) fixative to quickly preserve and stop any further development of certain helminth (Ascaris and Trichuris) eggs. The main disadvantage of this method is extensive washing and manipulation, which may lead to some organism distortion or destruction before fixation. Also, this approach is not very practical unless specimens are being processed for teaching/holding purposes.
The adult forms of most helminths must be relaxed prior to fixation. If this preliminary step is not performed, the worms often contract and curl up when they come in contact with the fixative, thus preventing visual examination of some of the key morphologic characteristics. Several methods, including those involving tap water, physiologic saline, and dilute menthol, are available for this purpose.
Definitive morphology needed for identification of protozoan trophozoites and cysts is best seen on the permanent stained smear, which can be prepared from fresh fecal specimens or from stool that has been submitted to the laboratory in fixative. No washing techniques should be used before bulk fixation, because the trophozoites will be destroyed. Immediately after collection or submission to the laboratory, the specimens should be thoroughly mixed with the fixative of choice. The ratio of fixative to stool should be at least 3 parts fixative to 1 part stool, and the fixation time should be at least 4 h (or less, depending on the amount of specimen used). The normal collection vial systems usually require a fixation time of at least 30 min. If an entire fecal specimen is used, 4 h is recommended. Individual slides can then be prepared from bulk-fixed material by a technique described by Scholten and Yang (11). This method involves centrifugation and smear preparation from the sediment. Although formalin-preserved specimens (except for sodium acetate-acetic acid-formalin-preserved fecal material) are not recommended for the preparation of permanent stained smears, formalin fixation for cyst preservation is recommended for the preservation of teaching specimens that will be examined as wet mounts only. The morphology of cysts can often be seen in formalinized wet mounts sufficiently well to identify organisms to the species level or certainly to be highly suggestive.
Streck tissue fixative has also been tested as a substitute for formalin and polyvinyl alcohol in fecal preservation. Stool samples were examined microscopically as follows: (i) in wet mounts (by bright-field and epifluorescence microscopy), (ii) in modified acid-fast-, trichrome-, and safranin-stained smears, and (iii) with two commercial test kits. Specific results showed that Cyclospora oocysts retained full fluorescence, modified acid-fast- and safranin-stained smears of Cryptosporidium and Cyclospora oocysts were equal in staining quality, and results were comparable in the immunofluorescence assay and enzyme immunoassay commercial kits. However, stool fixed in Streck tissue fixative and stained with trichrome showed unacceptable staining quality compared with stool fixed in preservatives, many of which contained PVA (fecal adhesive). Thus, Streck fixative is an excellent substitute for formalin; however, modifications to the trichrome procedure will be required to improve the staining characteristics of protozoan parasites (12). With the introduction of the Universal Fixative, TOTAL-FIX, formalin is no longer a consideration.
Formalin-Saline Solution
Although formalin is generally used at room temperature for routine diagnostic work, hot (60 to 63°C) 5% formalin in a ratio of 3 parts fixative to 1 part stool is recommended for bulk specimens containing intestinal protozoa. Long-term storage of protozoa is enhanced with buffered formalin; the solution should be replaced every 6 months. Acceptable morphology for teaching purposes can be maintained for 6 months to several years, depending on the organism and the lag time between specimen collection and fixation. The organism cytoplasm will tend to become glassy or granular with very poor nuclear definition. Cysts of Entamoeba coli and Giardia lamblia (G. duodenalis, G. intestinalis) tend to maintain their morphologic characteristics for years, whereas cysts of E. histolytica and E. hartmanni do not preserve well (Fig. 9.1). All of these cysts retain their ability to take up iodine in a wet, direct smear.
Figure 9.1 (Left) Entamoeba coli cyst (unstained). (Right) Giardia lamblia cyst (unstained). doi:10.1128/9781555819002.ch9.f1
The standard 10% formalin will fix protozoan cysts; however, it does not preserve morphology as well as does the buffered 5% formalin.
Formalin-Saline Solution
Formaldehyde is normally purchased as a 37% HCHO solution; however, for dilution, it should be considered to be 100%.
Buffered Formalin-Saline Solution
