Tolonium Chloride

The tolonium chloride is selectively and rapidly absorbed into the liposomes in the bacterial cell walls, as indicated by the small blue circles within the liposome.

From: Contemporary Esthetic Dentistry , 2012

Examination of the Sexual Assault Victim

James R. Roberts MD, FACEP, FAAEM, FACMT , in Roberts and Hedges' Clinical Procedures in Emergency Medicine and Acute Care , 2019

Perineal Toluidine Blue Dye Staining

Toluidine blue dye is a nuclear stain, also used for cancer detection and mast cell staining, that highlights areas of injury. It adheres to areas denuded by abrasions and lacerations where the epidermal layer of nonnucleated cells has been removed (Fig. 58.10). The underlying nucleated cells take up the dye. Although it is not a uniform standard of care and is unavailable in many EDs, the dye can enhance the examiner's ability to visualize and photographically document more subtle genital injuries (Fig. 58.11; also seeFig. 58.6B ). Genital lacerations may provide corroborating evidence of nonconsensual intercourse, or at least sexual activity. To outline injuries, apply a 1% aqueous solution of toluidine blue dye to the perineum and wipe the excess dye off with a cotton ball moistened with lubricating jelly. A swab containing the dye is commercially available from several distributors (http://nfni.org/products.html). After the excess dye is removed, any areas that retain the stain signify a disruption in the epidermis, most likely injury. Separate any folds of the area and carefully examine them to avoid missing injuries. Ideally, apply the dye before speculum examination to eliminate the possibility of iatrogenic injury. The procedure is described inFig. 58.10 andBox 58.2. In one study, the use of toluidine blue dye increased the injury detection rate from 16% to 40% in women, without the use of colposcopy 19 ; however, injuries detected with the aid of toluidine blue dye are not 100% specific for sexual assault because such injuries have also been found after consensual intercourse, especially in adolescents. 20

Collect external genital samples before the application of toluidine blue to avoid washing away potential DNA evidence. The use of toluidine blue dye itself does not interfere with DNA evidence from vaginal specimens, and it has proved safe for mucosal application. 21,22

Advances in oral cancer detection

Debolina Chakraborty , ... Amitava Mukherjee , in Advances in Clinical Chemistry, 2019

5.1 Toluidine blue staining method

TB staining method has been practiced since 1980s and is considered to be inexpensive and moderately sensitive [44]. Application of 1% toluidine blue dye to the suspected oral mucosa of the patient for a period 30   s helps in differentiating normal tissue from the malignant lesions [14]. Reports suggest that TB stain can only penetrate three to four layers of epithelial cells; thus, early dysplastic cells that remain covered by top epithelial layer do not get stained [45]. In a study conducted by Zhang et al., it was seen that TB staining had direct correlation with oral premalignant lesions (OPLs) and could predict cancer development. The study points out that 33% of TB-positive OPLs (with or without any dysplastic cells) progressed into OSCC [46]. However, clinicians cannot completely rely on this method as false-negative reports as high as 58% for epithelial dysplasia and up to 42% for in situ carcinoma have been reported [47]. It can be suggested that TB staining method should only be used as an additional adjunctive before resection and should not be considered as an easy alternative for biopsy [44].

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Sexual Assault

Ron M. Walls MD , in Rosen's Emergency Medicine: Concepts and Clinical Practice , 2018

Toluidine Blue Dye

Toluidine blue dye (TBD) is a stain that adheres to nuclei in damaged epithelial cells and has not been shown to interfere with DNA testing. Zink and colleagues have demonstrated that more tears were identified with TBD enhancement by direct visualization and colposcopy. 45 The dye should be applied prior to speculum insertion because the speculum may introduce genital injury. 46 The dye is applied to the external genitalia and then gently wiped with surgical lubricant, 1% acetic acid solution, or baby wipes to remove excess solution (Figs. 58.14 and58.15), which can lead to false-positive findings.

A White Patch On the Tongue

Helen McParland , Edward Odell , in Odell's Clinical Problem Solving in Dentistry (Fourth Edition), 2021

Toluidine Blue Staining

Toluidine blue (tolonium chloride) staining was described decades ago but its use has recently been revived in the form of new commercial test kits. The dye can be applied to the lesion by dabbing or in a mouthwash, and it is claimed that lesions that retain the blue stain indicate a higher risk of dysplasia or carcinoma compared with those that do not. The predictive value of the test remains unclear, with better results shown in high-risk populations. False-positive reactions are common because the dye binds to ulcer slough, staining ulcers, lichen planus and other benign lesions.

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Histology

James S. Lowe BMedSci, BMBS, DM, FRCPath , in Stevens & Lowe's Human Histology , 2020

Toluidine Blue Stain

Toluidine blue is used to demonstrate cells and fibres in very thin epoxy resin sections. Toluidine blue is one of the very few dyes that will penetrate the dense epoxy resin to stain the tissue section. It provides considerable cellular detail, staining the various components of the cells and fibres in the shades of blue in a way that represents their relative electron density; hence the resulting blue picture closely resembles a low-power electron micrograph but is blue instead of black. Prior to preparation of ultrathin sections for electron microscopy, a toluidine blue–stained survey section is obtained from the epoxy resin block to enable the microscopist to trim down the appropriate area of tissue for ultrathin sectioning.

Erythrocyte and Leucocyte Cytochemistry

Barbara J. Bain , in Dacie and Lewis Practical Haematology (Twelfth Edition), 2017

Toluidine blue stain

Toluidine blue staining is useful for the enumeration of basophils and mast cells. It binds strongly to the granules in these cells and is particularly useful in pathological states in which the cells may not be easily identifiable on Romanowsky stains. In AML and in CML and other MPN, basophils may be dysplastic and poorly granular, as may the mast cells in systemic mastocytosis.

Reagents

•

Toluidine blue 1%   w/v in methanol. Add 1   g of toluidine blue (BDH 34077) to 100   ml methanol and mix for 24   h on a roller or with a magnetic flea. The stain is stable indefinitely at room temperature. Keep tightly stoppered.

Method

1.

Place air-dried smears on a staining rack and flood with the toluidine blue solution.

2.

Incubate for 5–10   min.

3.

Rinse briefly in gently running tap water until clear and air dry.

Results and interpretation

The granules of basophils and mast cells stain a bright red/purple and are discrete and distinct (Fig. 15-15). Nuclei stain blue and cells with abundant RNA may show a blue tint to the cytoplasm. Although toluidine blue is said to be specific for these granules, with >   10   min incubation, the primary granules of promyelocytes are stained red/purple. However, these are smaller and finer than the mast cell or basophil granules and easily distinguished.

Toluidine blue staining does not distinguish between basophils and mast cells. This can be achieved by immunophenotyping (e.g. by identifying expression of mast cell tryptase).

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Lipids

M. Lamar Jones , in Theory and Practice of Histological Techniques (Sixth Edition), 2008

Sulfatides

These sulfate esters of cerebroside are the only lipids sufficiently acidic to induce a metachromatic shift in a variety of basic aniline dyes which have been used to demonstrate the lipid deposits in sulfatide storage disease, consequently termed metachromatic leucodystrophy. With cresyl violet, for example (Hirsch & Peiffer 1957), sulfatide appears orange in contrast to the orthochromatic color of other, less acidic, myelin lipids, but trivial metachromasia may be difficult to appreciate against a strong purple background. Distinction can be improved if the stained preparation is viewed in polarized light so that the sulfatide displays a green dichroism (Dayan 1967).

Toluidine blue, the standard dye exhibiting metachromasia with acidic polymers, shows sulfatides in metachromatic leucodystrophy as yellow, brown, or purple deposits. An acetone dehydration step is used to eliminate metachromasia induced by less polar groups (Bodian & Lake 1963).

The research method of choice for sulfatide (Kahlke 1967; Pearse 1968; P. Sourander, personal communication, 1969) is Holländer's (1963) adaptation of an acriflavine staining reaction for sulfated mucopolysaccharides (Takeuchi 1961, 1962). Holländer used a reagent sufficiently acidic that only sulfatide would be stained, with the exception of mast cell granules which can be distinguished by their persistence in a chloroform–methanol- extracted control section. The acriflavine–sulfatide complex fluoresces orange in ultraviolet light, or alternatively the reaction product can be converted to a stable red pigment with p-dimethylaminobenzaldehyde, giving excellent localization of stained sulfatide against a clear ground. Another method for sulfated mucins that can be successfully adapted to frozen sections for staining sulfatide is the high iron diamine method (Spicer 1965) given in Chapter 11, when the sulfatide stains purple and the background pale blue. Among lipids the staining reaction is confined to sulfatide, and the possibility of interference from cross-reacting mucins can be excluded by comparison with a de-lipidized control section.

Toluidine blue–acetone method for sulfatide (Bodian & Lake 1963)

Fixation and sections

Post-fixed cryostat sections; formal calcium-fixed frozen sections.

Reagents

0.01% toluidine blue in phosphate–citrate buffer at pH 4.7.

Buffer solution
0.2 M Na2HPO4 96 ml
0.1 M citric acid 104 ml

Method

1.

Mount sections onto slides.

2.

Stain for 16–18 hours in buffered toluidine blue.

3.

Wash in water.

4.

Dehydrate with acetone for 5 minutes.

5.

Mount in DPX.

Result

Sulfatide deposits metachromatic red–brown or yellow

Acriflavine–DMAB method for sulfatide (Holländer 1963)

Fixation and sections

Post-fixed cryostat sections; formal calcium-fixed frozen sections.

Preparation of reagents

a. Acriflavine stock solution
Acriflavine 100mg
Distilled water at 80°C 20 ml

Store in the dark at 4°C.

b. Acriflavine working solution
0.1 M citrate–HCl buffer pH 2.5 99 ml
Stock acriflavine solution 1 ml
c. DMAB solution
p-dimethylaminobenzaldehyde 0.6 g
20% hydrochloric acid 30 ml
Isopropanol 70 ml

Method

1.

Mount sections onto slides.

2.

Stain for 6 minutes in acriflavine solution.

3.

Differentiate for 1 minute in two changes of 70% isopropanol.

4.

Treat with DMAB reagent for 30–45 seconds.

5.

Rinse in distilled water for 2–3 minutes.

6.

Counterstain nuclei in Mayer's or Carazzi's hematoxylin.

7.

Blue in tap water, rinse in distilled water and mount sections in glycerin jelly.

Results

Notes

a.

Mast cell granules appear red but resist extraction in chloroform–methanol.

b.

Alternative: after stage 3 dehydrate sections in isopropanol, clear in xylene, and mount in a fluorescence-free medium.

c.

Sulfatide appears orange in ultraviolet light against a green ground.

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Expression, Function and Regulation of Mast Cell Granule Chymases During Mucosal Allergic Responses

Pamela A. Knight , ... Hugh R.P. Miller , in Mast Cells and Basophils, 2000

Altered mast cell kinetics

Toluidine blue-stained mast cells in Carnoy's-fixed jejunum were significantly (p < 0.03, Mann–Whitney) more abundant in mMCP-1 –/– mice (median 17.1 (range 5.5–72.8) IMMC per villus/crypt unit (VCU); n = 10) than in control mMCP-1 +/+ mice (median 9.8 (range 2.7–13.6) IMMC per VCU; n = 9). In contrast, esterase-positive mast cells in paraformaldehyde-fixed jejunum were generally less abundant in mMCP-1 –/– mice (median 7.05 (range 1.6–12.9) IMMC per VCU; n = 10) than in control mMCP-1 +/+ mice (median 10.5 (range 4.9–17.6) IMMC per VCU; n = 9).

When toluidine blue-positive mast cells were counted per mm2 in the crypts and basal lamina propria (in order to minimize errors due to intervillous spaces), mean values of 1505 (SE ± 257) IMMC in –/– mice and of 542 (SE ± 97) /mm2 (p < 0.004, Mann–Whitney) in +/+ mice were obtained (Table II). In both groups, the cells were predominantly (> 95%) intraepithelial, and a higher proportion of the IMMC in –/– mice were located basally in the crypts when compared with +/+ controls, where the cells were more abundant at the crypt/villus junction. In uninfected control mice, IMMC were so rare that it was not feasible to count per unit area, and even when counted per VCU median values of 0.03 IMMC per VCU were obtained for both mMCP-1 –/– (range 0–0.07) and mMCP-1   +/+ (range 0–0.04) groups of mice (n = 8 for both groups). The few cells that were detected were again intraepithelial in both groups, but it was not possible to determine whether there was the distinctive pattern of localization in the infected groups. These values are approximately 570- and 303-fold lower than the medians for infected –/– and +/+ groups, respectively. These data are consistent with published data (9), which showed that, at the time of worm expulsion, toluidine blue-positive IMMC were significantly more abundant in mMCP-1 –/– mice than in controls.

Table II. Summary of the Results (mean ± SE) from mMCP-1 –/– (n = 10) and mMCP-1 +/+ (n = 9) Mice for Samples Taken on Day 8 Following Infection with 400 Mouse-adapted N. brasiliensis L3

mMCP–/– mMCP   +/+ % difference -/vs +/+ P value
Mast cells (mm-   2) 1505 ±   257 542 ± 97 280 0.004
mMCP-1 (μgnf-   1) 0 220 ± 20 NA NA
Villus height (μm) 261 ±   23 352 ± 26 35 0.03
Crypt depth (μm) 177 ± 12 127 ± 28 28 0.005
Worm burden 42 ± 19 20 ± 13 NA NS
RT-PCR
mMCP-1 - +++
mMCP-2 ++ ++
mMCP-4 + +
mMCP-5 + +

Egg counts had fallen from a maximum 11,000 (day 6) to 0 e.p.g. (day 8). Mast cells are predominantly (&gt;   90%) intraepithelial in both groups, but in the mMCP-1 –/– group the granules are smaller, stain weakly for esterase and not at all for mMCP-1; the mast cells are concentrated in the crypts. By contrast, the mMCP-1 +/+ mast cells are intensely esterasepositive and mMCP-l+ with larger granules, and are located predominantly at the crypưvillus junctions.

Other features that were consistent with previous work (9) included the smaller granule size and lack of esterase staining of IMMC in parasitized –/– mice when compared with +/+ controls, although the expression of other chymases (mMCP-2, -4 and -5) in the gut mucosa was comparable in both groups of infected mice as judged by semiquantitative RT-PCR (9) (Table II). At the ultrastructural level, the granules in the –/– mice lacked the stellate outlines and internal crystalline structures (Fig. 1B) that are typically present in wild-type IMMC (Fig. 1A) (20,21). Instead, the –/– granules were oval, and a proportion (~   30%) had unusual intragranular divisions with dense, unstructured cores separated into several segments (Fig. 1B) as described previously (9). These data suggest that the mMCP-1 –/– mice generated IMMC as efficiently as wild-type mice but that there were substantial effects on granule morphology and biochemistry.

Fig. 1. Electron micrographs showing typical IMMC from +/+ (A) and –/– (B) jejunal sections, from day 8 post-infection. The crystalline bodies, clearly visible in A, are only found in +/+ IMMC, whereas granules containing internal divisions (B) were exclusive to the –/– IMMC. Original magnifications: ×   16,000(A) and ×   14,000(B).

One of the more interesting observations described in Table II, and confirmed in the study by Wastling et al. (9), was the increased numbers of IMMC in the parasitized gut of mMCP-1–/– mice when compared with +/+ controls. It is possible that this observation is due to lack of mMCP-1 in the granules and the failure of the IMMC to secrete this highly soluble chymase into the lateral spaces between the epithelial cells. Previous evidence from studies with RMCP-II indicate that soluble β-chymases may alter epithelial permeability by disrupting the epithelial tight junctions (22,23) (see discussion section). We have speculated that this could allow IMMC to migrate into the gut lumen (9), for which there is a precedent in parasitized sheep (24).

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Acute Basophilic Leukemia (AML, NOS)

In Diagnostic Pathology: Blood and Bone Marrow (Second Edition), 2018

Acute Mast Cell Leukemia

•

Toluidine blue with metachromatic staining in both mast cell leukemia and basophilic leukemia

•

MPO usually negative in both mast cell leukemia and basophilic leukemia

•

Elevated serum tryptase and strong tryptase expression by leukemic cells in mast cell leukemia only

•

α-naphthyl chloracetate esterase cytochemical stain positive in mast cell granules

•

CD117 strongly positive in mast cell leukemia, negative in acute basophilic leukemia

•

θ granules on electron microscopy in acute basophilic leukemia

•

D816V KIT mutation may be seen in mast cell leukemia

•

Rarely, mast cell leukemia may coexist with acute basophilic leukemia

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Ribonucleases - Part A

Kerstin Korn , ... Ulrich Hahn , in Methods in Enzymology, 2001

Principle

The dye toluidine blue O (TB) shows an absorption maximum at 625   nm. As a cationic compound it interacts with different polyanions, leading to complexes of different absorption maxima (metachromatic effect). Depending on the kind of polyanion and the concentration of each partner a bathochromic or hypsochromic shift in absorption occurs. If TB is added to molten bacterial growth media consisting of nutrient containing agar that contains sulfate esters it forms a red complex after solidification of the agar. If high molecular weight RNA (another polyanion) is additionally present in TB agar plates, the plate color is blue. This is due to the fact that TB forms a blue complex with the RNA. These solid bacterial growth media are ideal to serve as ribonuclease indicator plates: Colonies grown on these plates that (over)produce and/or secrete an RNase form red or pink halos, in contrast to controls that do not produce significant amounts of these enzymes (Fig. 1). 9,10 The smaller (oligo-)nucleotides resulting from RNase-mediated digestion are not capable for forming the blue complex. A similar assay has been described for DNase test agar systems. 11,12

Fig. 1. RNase indicator plate containing LB agar, RNA, and toluidine blue O. The colonies consist of Escherichia coli DH5α cells overproducing and secreting RNase T1 wild type (a) or variants thereof with lowered activities (c–e). As a control untransformed E. coli (b) was inoculated.

 From O. Landt, J. Thölke, H.-P. Grunert, W. Saenger, and U. Hahn, Biol. Chem. 378, 553 (1997).

We have used these TB indicator plates to identify RNase T1 overproducing and secreting Escherichia coli clones 13 or M13 phages presenting RNase A or T1 on their surfaces. 14,15 The plates are also convenient for the detection of RNase T1 variants obtained after oligonucleotide-directed specific or random mutagenesis. 16,17

Furthermore, holes punched into the solid medium with the aid of a pipette tip and filled with aliquots of column chromatography fractions show red halos after short incubation at 37°, if RNases are present.

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