Permanent Stains

Permanent stains of fecal smears are most needed for the detection and identification of protozoan trophozoites, but they are also used for the identification of cysts. Wet mounts of fresh feces, even with stains such as methylene blue, are not as sensitive for trophozoites and therefore do not substitute for permanent stains. It is sometimes difficult to identify cysts which are detected in wet mounts thus, for each specimen, regardless of consistency, it may be worthwhile to fix a portion in PVA fixative or to prepare two fecal films fixed in Schaudinn fixative so that permanent stains can be performed if needed. Permanent stains also provide a permanent record and are easily referred to consultants if there are questions on identification. 

A number of staining procedures have been described. Some stains, such as chlorazol black, require fresh specimens and are not widely used. A variety of stains for fecal smears preserved by Schaudinn or PVA fixative have been described, including various hematoxylin stains. The stain most widely used in the United States is the Wheatley trichrome stain, which is the only permanent stain described in this chapter. The trichrome staining procedure uses reagents with a relatively long shelf life and is easy to perform. Note that there are differences in staining times depending on whether the specimen is fixed in Schaudinn or PVA fixative, as penetration is slower in the latter. 

Preparation of smears, (i) Unpreserved specimens with Schaudinn fixative. 

To prepare thin, uniform smears, place a drop of saline on a glass slide (1 by 3 in. [ca. 2.5 by 7.5 cm]). With an applicator stick, transfer a small, representative portion of the specimen to the drop of saline, and mix the two. Spread the solution into a film by rolling the applicator stick along the surface. Remove any lumps. 

Before watery specimens are smeared, apply an adhesive such 

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Thus, W,W,W W -tetraphenylhydra2ine [632-52-0] 5,10-dihydro-5,10-diphenylphena2ine [3665-72-3] and W,W,Ar-triphenyl-l,2-ben2enediamine [29325-54-0] are oxidation products of DPA. The permanent stain caused by these compounds is in part due to the formation of the following highly conjugated, polymeric products which are insoluble ... 

The Z-track method of IM injection is used when a drug is highly irritating to SC tissues or has tiie ability to permanently stain tiie skin. The nurse should adhere to the following procedure when using tiie Z-track technique (Fig. 2-8) ... 

RIFAMPIN The patient is informed about the reddish-orange or reddish-brown discoloration of body fluids (eg, tears, sweat, sputum, saliva). Advise the patient not to wear soft contact lenses during therapy because they may be permanently stained. 

The three principal microscopic examinations performed on stool specimens are direct wet mount, wet mount after concentration, and permanent stain. Although each examination can contribute to diagnosis, the yield of some methods is small with certain kinds of specimens. As a minimum, formed specimens should be examined by a concentration procedure. Soft specimens should be examined by concentration and permanent stain, and, if submitted fresh, by direct wet mount. Loose and watery specimens should be examined by wet mount and permanent stain. If specimens are received in fixative and the consistency is not known, concentration and permanent stain should be performed. Other examinations may be helpful. Special procedures which may assist in the diagnosis of specific parasites are noted below in discussions of the parasites. 

Type of specimen Direct wet mount Method Concen- tration Permanent stain... 

Permanently stained slides may be mounted with a cover slip or may be air dried and examined after oil is added. Slides should be examined at a magnification of x400 to X500 or greater after they are scanned under lower power to find optimal areas. A x50 oil immersion objective is particularly helpful, as it allows the easy use... 

Permanently stained slides should be kept for 2 years. 

The last material aspirated is most likely to contain amebae. Material may be examined microscopically in wet mounts and permanent stains, and in addition, it can be cultured for amebae if bacteria are also added to the culture as described below. Abscess material is often thick and difficult to examine. It may be treated with streptokinase and streptodonase enzymes to liquefy the specimen. 

The sediment may be used for microscopic examinations for amebae (wet mounts and permanent stains) and for the culture of amebae. 

Permanent stains can be prepared by the fixation of sediment in PVA fixative, with the subsequent preparation of smears and staining. 

Urine, feces, saliva, sputum, sweat, and tears may be colored red-orange. Soft contact lenses may be permanently stained. Advise patients of these possibilities. 

Red discoioration of body fluids Rifapentine may produce a predominately red-orange discoloration of body tissues or fluids (eg, skin, teeth, tongue, urine, feces, saliva, sputum, tears, sweat, cerebrospinal fluid). Contact lenses may become permanently stained. 

Methylrosanilinum chloride (Gentian violet) is a desinfectant with antifungal activity against yeasts. As a aqueous solution it is used topically to treat Candida infections. Adverse effects include severe irritation, temporary staining of skin, permanent staining of fabrics. Animal carcinogenicity has been described (restricted use in some countries). 

May discolor bodily secretions brown-orange, soft contact lenses may be permanently stained... 

Owing to their liquid nature at room temperature, essential oils are called as such. They should not be confused with fixed oils or fatty oils, which are composed of a naturally occurring mixture of lipids which may not necessarily be volatile. Therefore, essential oils differ entirely both in chemical and in physical properties from fatty oils. Essential oil evaporates completely when dropped on filter paper however, fixed oil leaves a permanent stain which does not evaporate even when heated. 

Tetracycline Moderate Possibility of permanent staining of developing teeth in the infant. Should be avoided during lactation. 

Polynucleotides, including RNA and DNA Acridine orange Pyronine Y (or G) Stained product can be assessed quantitatively Gives a permanent staining, so electrophoretogram can be stored for several weeks... 

The importance of avoiding permanent staining of the teeth cannot be over-emphasized, as many patients with acne are already prone to negative psychological effects. Minocycline should therefore be prescribed only with great care. 

Some cities add fluorides to their water supply. By doing so, they hope to improve the dental health of everyone living in the city. Young people, whose teeth are still developing, benefit the most. The process of adding fluorides to public water supplies is called fluoridation. Too much fluorine in the water leads to a light brown and permanent staining of teeth. 

A silver stain has been introduced that is 100 times more sensitive for proteins than Coomassie Blue, and 4 times more sensitive for nucleic acids than ethidium bromide.14 This stain is based on the reduction of Ag+ by thiol, tyrosine and amine functional groups in proteins and by the purine bases in DNA. The solid silver formed by reduction precipitates in the gel forming a permanent stain in the protein-nucleic acid zones, and is not reduced by bulfer or gel materials, so that... 

Rifampin s potent induction of hepatic enzymes, especially cytochrome P450 (CYP) 3A4, may enhance the ehmination of many other drugs, most notably the protease inhibitors used to treat HIV (Table 110-7). HIV-positive patients may beneht from the use of rifabutin instead of rifampin (see below). ° Also, women who use oral contraceptives must use another form of contraception during therapy because increased clearance of the hormones may lead to unexpected pregnancies. Patient records should be reviewed for potential drug interactions before dispensing rifampin. Rifampin may turn urine and other secretions orange-red and may permanently stain some types of contact lenses. 

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