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July 1992, Vol 110, No. 7 >
ARTICLE | July 1992

What Is Actually Stained by Rose Bengal? FREE

Robert P. G. Feenstra; Scheffer C. G. Tseng, MD, PhD
[+] Author Affiliations

Accepted for publication September 4, 1991.

Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Fla, May 2, 1991.

Reprint requests to the Department of Ophthalmology, Bascom Palmer Eye Institute, PO Box 016880, Miami, FL 33101 (Dr Tseng).


Arch Ophthalmol. 1992;110(7):984-993. doi:10.1001/archopht.1992.01080190090035
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• It has been believed that 1% rose bengal does not stain normal, healthy cells but rather stains degenerated or dead cells and mucous strands. In contrast to this conventional knowledge, we discovered that both commercial additive-containing and additive-free rose bengal solutions stained four different types of healthy cultured cells, including rabbit corneal epithelial cells. Rose bengal staining was rapid, dose dependent, predominantly nuclear, and detectable with the naked eye at concentrations as low as 0.05% and 0.025% for the commercial additive-containing or additive-free solutions, respectively, and with the fluorescence microscope at a concentration of 0.001%. It is surprising to discover that rose bengal is not a vital dye; after staining, cells actually lost vitality, as evidenced by instant morphologic changes, subsequent loss of cellular motility, cell detachment, and cell death. Such an intrinsic toxic effect was augmented by light exposure. The rose bengal staining of live as well as detergent-treated (Triton X-100) cells could be blocked by such tear components as mucin and albumin, suggesting that normally negative rose bengal staining is due to the protective function of the preocular tear film, ie, staining is not dictated by lack of cell vitality. These data indicate that rose bengal staining ensues whenever there is poor protection of surface epithelium by the preocular tear film; this represents a new interpretation for rose bengal stains seen in various ocular surface disorders.

REFERENCES

1 +
Ehrlich P.  Über die Methylenblaureaction der lebenden Nervensubstanz . Biol Centralblatt . 1886-1887;;6:214-224.
2 +
Marx E.  Über vitale Färbungen am Auge und an den Lidern, I: Über Anatomie, Physiologie und Pathologie des Augenlidrandes und der Tränenpunkte . Albrecht Graefe Arch Ophthalmol . 1924;;114:465-482.
3 +
Sjögren H.  Zur Kenntnis der Keratoconjunctivits sicca (Keratitis filiformis bei Hypofunktion der Tränendrüsen) . Acta Ophthalmol . 1933;; (suppl 2) .
4 +
Passmore JW, King JH Jr.  Vital staining of conjunctiva and cornea: review of literature and critical study of certain dyes . Arch Ophthalmol . 1955;;153:568-574.
5 +
Norn MS.  Vital staining of cornea and conjunctiva . Acta Ophthalmol . 1962;;40:389-401.
6 +
Norn MS.  Specific double vital staining of the cornea and conjunctiva with rose bengal and alcian blue . Acta Ophthalmol . 1964;;42:84-96.
7 +
Norn MS.  Trypan blue: vital staining of cornea and conjunctiva . Acta Ophthalmol . 1967;;45:380-389.
8 +
Norn MS.  Dead, degenerate, and living cells in conjunctival fluid and mucous thread . Acta Ophthalmol . 1969;;47:1102-1115.
9 +
Norn MS.  Vital staining of the cornea and conjunctiva with a mixture of fluorescein and rose bengal . Am J Ophthalmol . 1967;;64:1078-1080.
10 +
Norn MS.  Rose bengal vital staining: staining of cornea and conjunctiva by 10% rose bengal, compared with 1% . Acta Ophthalmol . 1970;;48:546-559.
11 +
Norn MS.  Vital staining of cornea and conjunctiva: fluorescein—rose bengal mixture and tetrazolium-alcian blue mixture . Acta Ophthalmol . 1972;;( (suppl) ):113.
12 +
O'Day K.  Bengal rose as an aid in the diagnosis of'kerato-conjunctivitis sicca' (Sjögren's syndrome) . Med J Aust . 1951;;2:708-709.
13 +
Van Bijsterveld OP.  Diagnostic tests in the sicca syndrome . Arch Ophthalmol . 1969;;82:10-14.
14 +
Forster HW Jr.  Rose bengal test in diagnosis of deficient tear formation . Arch Ophthalmol . 1951;;45:419-424.
15 +
Lansche RK.  Vital staining in normal eyes and in keratoconjunctivitis sicca . Am J Ophthalmol . 1965;;60:520-525.
16 +
Bloch KJ, Buchanan WW, Wohl MJ, Bunim JJ.  Sjögren syndrome: a clinical, pathological, and serological study of sixty-two cases . Medicine . 1965;;44:187-231.
17 +
Manthorpe R, Frost-Larsen K, Isager H, Prause JU.  Sjögren syndrome: a review with emphasis on immunological features . Allergy . 1981;;36:139-153.
18 +
Fox RI, Howell FV, Bone RC, Michelson P.  Primary Sjögren syndrome: clinical and immunopathologic features . Semin Arthritis Rheum . 1984;;14:77-105.
19 +
Marsh RJ, Fraunfelder FT, McGill JI.  Herpetic corneal epithelial disease . Arch Ophthalmol . 1976;;94:1899-1902.
20 +
Wilson FM II.  Rose bengal staining of epibulbar squamous neoplasms . Ophthalmic Surg . 1976;;7:21-23.
21 +
In: Pavan-Langston D, ed. Manual of Ocular Diagnosis and Therapy . 2nd ed. Boston, Mass: Little Brown & Co Inc; 1985;:76-77.
22 +
Watson BD, Dietrich WD, Busto R, Wachtel MS, Ginsberg MD.  Induction of reproducible brain infarction by photochemically initiated thrombosis . Ann Neurol . 1985;;17:497-504.
23 +
Nanda SK, Hatchell DL, Tiedeman JS, Dutton JJ, Hatchell MC, McAdoo T.  A new method for vascular occlusion: photochemical initiation of thrombosis . Arch Ophthalmol . 1987;;105:1121-1124.
24 +
Huang AJW, Watson BD, Hernandez E, Tseng SCG.  Photothrombosis of corneal neovascularization by intravenous rose bengal and argon laser irradiation . Arch Ophthalmol . 1988;;106:680-685.
25 +
Feenstra RPG, Chen CF, Watson BD, Tseng SCG.  Photodynamic effect of rose bengal on cultured cells . Invest Ophthalmol Vis Sci . 1990;;31 ( (suppl) ):478. Association for Research in Vision and Ophthalmology Abstract.
26 +
Valenzeno DP.  Photomodification of biological membranes with emphasis on singlet oxygen mechanisms . Photochem Photobiol . 1987;;46:147-160.
27 +
Banks JG, Board RG, Carter J, Dodge AD.  The cytotoxic and photodynamic inactivation of micro-organisms by rose bengal . J Appl Bacteriol . 1985;;58:391-400.
28 +
Dahl TA, Midden WR, Neckers DC.  Comparison of photodynamic action by rose bengal in gram-positive and gram-negative bacteria . Photochem Photobiol . 1988;;48:607-612.
29 +
Roat MI, Romanowski E, Araullo-Cruz T, Gordon YJ.  The antiviral effects of rose bengal and fluorescein . Arch Ophthalmol . 1987;;105:1415-1417.
30 +
Hull DS, Strickland EC, Green K.  Photodynamically induced alteration of cornea endothelial cell function . Invest Ophthalmol Vis Sci . 1979;;18:1226-1231.
31 +
Menon IA, Basu PK, Persad SD, Rosatone S, Wiltshire JD.  A study on the sequence of phototoxic effects of rose bengal using retinal pigment epithelial cells in vitro . Exp Eye Res . 1989;;49:67-73.
32 +
Lambert DW.  Physiology of the tear film . In: Smolin G, Thoft RA, eds. The Cornea: Scientific Foundations and Clinical Practice . 2nd ed. Boston, Mass: Little Brown & Co Inc; 1987;:38-52.
33 +
Nichols BA, Chiappino ML, Dawson CR.  Demonstration of the mucous layer of the tear film by electron microscopy . Invest Ophthalmol Vis Sci . 1985;;26:464-473.
34 +
Tseng SCG.  Staging of conjunctival squamous metaplasia by impression cytology . Ophthalmology . 1985;;92:728-733.
35 +
Pflugfelder SC, Huang AJW, Feuer W, Chuchovski PT, Pereira IC, Tseng SCG.  Conjunctival cytologic features of primary Sjögren's syndrome . Ophthalmology . 1990;;97:985-991.
36 +
Tseng SCG.  Topical tretinoin treatment for dry-eye disorders . Int Ophthalmol Clin . 1987;;27:47-53.
37 +
Fullard RJ, Snyder C.  Protein levels in nonstimulated and stimulated tears of normal human subjects . Invest Ophthalmol Vis Sci . 1990;;31:1119-1126.
38 +
Meurman L.  Affinity of rose bengal for various blood components in vitro: on the distribution and kinetics of injected I131 rose bengal . Acta Med Scand . 1960;;167( (suppl 354) ):18-20.
39 +
Carlstedt I, Lindgren H, Sheehan JK.  The macromolecular structure of human cervicalmucus glycoproteins: studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion . Biochem J . 1983;;213:427-435.
40 +
Chao CW, Stuebben Am, Butala SM.  Characterization of ocular mucus extracts by crossed immunoelectrophoretic techniques . Invest Ophthalmol Vis Sci . 1990;;31:1127-1135.
41 +
Tseng SCG, Huang AJW, Sutter D.  Purification and characterization of rabbit ocular mucin . Invest Ophthalmol Vis Sci . 1987;;28:1473-1482.
42 +
Feenstra RPG, Tseng SCG. Comparison of fluorscein and rose bengal staining. Ophthalmology. In press.

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1 +
Ehrlich P.  Über die Methylenblaureaction der lebenden Nervensubstanz . Biol Centralblatt . 1886-1887;;6:214-224.
2 +
Marx E.  Über vitale Färbungen am Auge und an den Lidern, I: Über Anatomie, Physiologie und Pathologie des Augenlidrandes und der Tränenpunkte . Albrecht Graefe Arch Ophthalmol . 1924;;114:465-482.
3 +
Sjögren H.  Zur Kenntnis der Keratoconjunctivits sicca (Keratitis filiformis bei Hypofunktion der Tränendrüsen) . Acta Ophthalmol . 1933;; (suppl 2) .
4 +
Passmore JW, King JH Jr.  Vital staining of conjunctiva and cornea: review of literature and critical study of certain dyes . Arch Ophthalmol . 1955;;153:568-574.
5 +
Norn MS.  Vital staining of cornea and conjunctiva . Acta Ophthalmol . 1962;;40:389-401.
6 +
Norn MS.  Specific double vital staining of the cornea and conjunctiva with rose bengal and alcian blue . Acta Ophthalmol . 1964;;42:84-96.
7 +
Norn MS.  Trypan blue: vital staining of cornea and conjunctiva . Acta Ophthalmol . 1967;;45:380-389.
8 +
Norn MS.  Dead, degenerate, and living cells in conjunctival fluid and mucous thread . Acta Ophthalmol . 1969;;47:1102-1115.
9 +
Norn MS.  Vital staining of the cornea and conjunctiva with a mixture of fluorescein and rose bengal . Am J Ophthalmol . 1967;;64:1078-1080.
10 +
Norn MS.  Rose bengal vital staining: staining of cornea and conjunctiva by 10% rose bengal, compared with 1% . Acta Ophthalmol . 1970;;48:546-559.
11 +
Norn MS.  Vital staining of cornea and conjunctiva: fluorescein—rose bengal mixture and tetrazolium-alcian blue mixture . Acta Ophthalmol . 1972;;( (suppl) ):113.
12 +
O'Day K.  Bengal rose as an aid in the diagnosis of'kerato-conjunctivitis sicca' (Sjögren's syndrome) . Med J Aust . 1951;;2:708-709.
13 +
Van Bijsterveld OP.  Diagnostic tests in the sicca syndrome . Arch Ophthalmol . 1969;;82:10-14.
14 +
Forster HW Jr.  Rose bengal test in diagnosis of deficient tear formation . Arch Ophthalmol . 1951;;45:419-424.
15 +
Lansche RK.  Vital staining in normal eyes and in keratoconjunctivitis sicca . Am J Ophthalmol . 1965;;60:520-525.
16 +
Bloch KJ, Buchanan WW, Wohl MJ, Bunim JJ.  Sjögren syndrome: a clinical, pathological, and serological study of sixty-two cases . Medicine . 1965;;44:187-231.
17 +
Manthorpe R, Frost-Larsen K, Isager H, Prause JU.  Sjögren syndrome: a review with emphasis on immunological features . Allergy . 1981;;36:139-153.
18 +
Fox RI, Howell FV, Bone RC, Michelson P.  Primary Sjögren syndrome: clinical and immunopathologic features . Semin Arthritis Rheum . 1984;;14:77-105.
19 +
Marsh RJ, Fraunfelder FT, McGill JI.  Herpetic corneal epithelial disease . Arch Ophthalmol . 1976;;94:1899-1902.
20 +
Wilson FM II.  Rose bengal staining of epibulbar squamous neoplasms . Ophthalmic Surg . 1976;;7:21-23.
21 +
In: Pavan-Langston D, ed. Manual of Ocular Diagnosis and Therapy . 2nd ed. Boston, Mass: Little Brown & Co Inc; 1985;:76-77.
22 +
Watson BD, Dietrich WD, Busto R, Wachtel MS, Ginsberg MD.  Induction of reproducible brain infarction by photochemically initiated thrombosis . Ann Neurol . 1985;;17:497-504.
23 +
Nanda SK, Hatchell DL, Tiedeman JS, Dutton JJ, Hatchell MC, McAdoo T.  A new method for vascular occlusion: photochemical initiation of thrombosis . Arch Ophthalmol . 1987;;105:1121-1124.
24 +
Huang AJW, Watson BD, Hernandez E, Tseng SCG.  Photothrombosis of corneal neovascularization by intravenous rose bengal and argon laser irradiation . Arch Ophthalmol . 1988;;106:680-685.
25 +
Feenstra RPG, Chen CF, Watson BD, Tseng SCG.  Photodynamic effect of rose bengal on cultured cells . Invest Ophthalmol Vis Sci . 1990;;31 ( (suppl) ):478. Association for Research in Vision and Ophthalmology Abstract.
26 +
Valenzeno DP.  Photomodification of biological membranes with emphasis on singlet oxygen mechanisms . Photochem Photobiol . 1987;;46:147-160.
27 +
Banks JG, Board RG, Carter J, Dodge AD.  The cytotoxic and photodynamic inactivation of micro-organisms by rose bengal . J Appl Bacteriol . 1985;;58:391-400.
28 +
Dahl TA, Midden WR, Neckers DC.  Comparison of photodynamic action by rose bengal in gram-positive and gram-negative bacteria . Photochem Photobiol . 1988;;48:607-612.
29 +
Roat MI, Romanowski E, Araullo-Cruz T, Gordon YJ.  The antiviral effects of rose bengal and fluorescein . Arch Ophthalmol . 1987;;105:1415-1417.
30 +
Hull DS, Strickland EC, Green K.  Photodynamically induced alteration of cornea endothelial cell function . Invest Ophthalmol Vis Sci . 1979;;18:1226-1231.
31 +
Menon IA, Basu PK, Persad SD, Rosatone S, Wiltshire JD.  A study on the sequence of phototoxic effects of rose bengal using retinal pigment epithelial cells in vitro . Exp Eye Res . 1989;;49:67-73.
32 +
Lambert DW.  Physiology of the tear film . In: Smolin G, Thoft RA, eds. The Cornea: Scientific Foundations and Clinical Practice . 2nd ed. Boston, Mass: Little Brown & Co Inc; 1987;:38-52.
33 +
Nichols BA, Chiappino ML, Dawson CR.  Demonstration of the mucous layer of the tear film by electron microscopy . Invest Ophthalmol Vis Sci . 1985;;26:464-473.
34 +
Tseng SCG.  Staging of conjunctival squamous metaplasia by impression cytology . Ophthalmology . 1985;;92:728-733.
35 +
Pflugfelder SC, Huang AJW, Feuer W, Chuchovski PT, Pereira IC, Tseng SCG.  Conjunctival cytologic features of primary Sjögren's syndrome . Ophthalmology . 1990;;97:985-991.
36 +
Tseng SCG.  Topical tretinoin treatment for dry-eye disorders . Int Ophthalmol Clin . 1987;;27:47-53.
37 +
Fullard RJ, Snyder C.  Protein levels in nonstimulated and stimulated tears of normal human subjects . Invest Ophthalmol Vis Sci . 1990;;31:1119-1126.
38 +
Meurman L.  Affinity of rose bengal for various blood components in vitro: on the distribution and kinetics of injected I131 rose bengal . Acta Med Scand . 1960;;167( (suppl 354) ):18-20.
39 +
Carlstedt I, Lindgren H, Sheehan JK.  The macromolecular structure of human cervicalmucus glycoproteins: studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion . Biochem J . 1983;;213:427-435.
40 +
Chao CW, Stuebben Am, Butala SM.  Characterization of ocular mucus extracts by crossed immunoelectrophoretic techniques . Invest Ophthalmol Vis Sci . 1990;;31:1127-1135.
41 +
Tseng SCG, Huang AJW, Sutter D.  Purification and characterization of rabbit ocular mucin . Invest Ophthalmol Vis Sci . 1987;;28:1473-1482.
42 +
Feenstra RPG, Tseng SCG. Comparison of fluorscein and rose bengal staining. Ophthalmology. In press.

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