Intravacuolar stains for yeast and other fungi

Claim 1

A method of forming distinctive intravacuolar structures in fungal cells comprising: a) combining a sample that contains fungal cells with a staining solution comprising a dye of the formula ##STR10## wherein each R 1 is independently H; or an alkyl group having from 1-6 carbons; or a trifluoromethyl; or a halogen; or --OR 6 , --SR 6 or --NR 6 R 7 where R 6 and R 7 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic, heteroalicyclic, aromatic or heteroaromatic rings, containing 1-6 heteroatoms, wherein the heteroatoms are O, N or S; or R 6 and R.sup. 7 taken in combination are --(CH 2 ) 2 --M--(CH 2 ) 2 -- where M=a single bond, --O--, --CH 2 --, or --NR 8 --where R 8 is H or an alkyl group having 1-6 carbons; and t=1-4; R 2 is an alkyl group having 1-6 carbons; X is O, S, Se or NR 9 , where R 9 is H or an alkyl group having 1-6 carbons; or X is CR 10 R 11 where R 10 and R 11 , which may be the same or different, are independently alkyl groups having 1-6 carbons, or R 10 and R 11 taken in combination complete a five or six membered saturated ring; R 3 , R 4 and R 5 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); or R 4 and R 5 , taken in combination, have the formula ##STR11## forming a quinolinium ring system; wherein R 12 , R 13 , R 14 and R 15 are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); Z - is a biologically compatible counterion; L is Cl, Br, I, F, or --OSO 2 R 16 where R 16 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; and OMEGA is a cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the heteroatoms are O, N or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, or alkoxy, having 1-6 carbons; where the dye is present in an amount sufficient to give a distinctive intravacuolar structure or structures; and b) allowing sufficient time for the dye to combine with one or more cells in the sample and form the distinctive intravacuolar structure or structures.

Claim 2

A method of forming distinctive intravacuolar structures in fungal cells comprising: a) combining a sample that contains fungal cells with a staining solution comprising a dye of the formula ##STR10## wherein each R 1 is independently H; or an alkyl group having from 1-6 carbons; or a trifluoromethyl; or a halogen; or --OR 6 , --SR 6 or --NR 6 R 7 where R 6 and R 7 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic, heteroalicyclic, aromatic or heteroaromatic rings, containing 1-6 heteroatoms, wherein the heteroatoms are O, N or S; or R 6 and R.sup. 7 taken in combination are --(CH 2 ) 2 --M--(CH 2 ) 2 -- where M=a single bond, --O--, --CH 2 --, or --NR 8 --where R 8 is H or an alkyl group having 1-6 carbons; and t=1-4; R 2 is an alkyl group having 1-6 carbons; X is O, S, Se or NR 9 , where R 9 is H or an alkyl group having 1-6 carbons; or X is CR 10 R 11 where R 10 and R 11 , which may be the same or different, are independently alkyl groups having 1-6 carbons, or R 10 and R 11 taken in combination complete a five or six membered saturated ring; R 3 , R 4 and R 5 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); or R 4 and R 5 , taken in combination, have the formula ##STR11## forming a quinolinium ring system; wherein R 12 , R 13 , R 14 and R 15 are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); Z - is a biologically compatible counterion; L is Cl, Br, I, F, or --OSO 2 R 16 where R 16 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; and OMEGA is a cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the heteroatoms are O, N or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, or alkoxy, having 1-6 carbons; where the dye is present in an amount sufficient to give a distinctive intravacuolar structure or structures; and b) allowing sufficient time for the dye to combine with one or more cells in the sample and form the distinctive intravacuolar structure or structures.

Claim 14

A method of analyzing the metabolic activity of fungal cells, comprising: a) combining a sample containing fungal cells with a staining solution comprising a dye of the formula ##STR14## wherein each R 1 is independently H; or an alkyl group having from 1-6 carbons; or a trifluoromethyl; or a halogen; or --OR 6 , --SR 6 , or --NR 6 R 7 where R 6 and R 7 , which are the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic, heteroalicyclic, aromatic or heteroaromatic rings, containing 1-4 heteroatoms, wherein the heteroatoms are O, N or S; or R 6 and R 7 taken in combination are --(CH 2 ) 2 --M--(CH 2 ) 2 -- where M=a single bond, --O--, --CH 2 --, or --NR 8 -- where R 8 is H or an alkyl group having 1-6 carbons; and t=1-4; R 2 is an alkyl group having 1-6 carbons; X is O or S; R 3 , R 4 and R 5 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); or R 4 and R 5 , taken in combination, have the formula ##STR15## forming a quinolinium ring system; wherein R 12 , R 13 , R 14 and R 15 are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); or R 4 and R 5 , taken in combination, have the formula ##STR16## forming a quinolinium ring system; wherein R 12 , R 13 , R 14 and R 15 are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 ; --SR 6 or --(NR 6 R 7 ); Z - is a biologically compatible counterion; L is Cl, Br, I, F, or --0S0 2 R 16 where R 16 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; and OMEGA is a cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the heteroatoms are O, N or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, or alkoxy having 1-6 carbons; where the dye is present in an amount sufficient to give a distinctive intravacuolar structure or structures; b) allowing sufficient time for the dye to combine with one or more cells in the sample and form the distinctive intravacuolar structure or structures; c) illuminating the sample; d) detecting the presence of a distinctive intravacuolar structure or structures in the fungal cells; and e) correlating presence or magnitude of the distinctive intravacuolar structure or structures with metabolic activity of the fungal cells.

Claim 15

A method of analyzing the metabolic activity of fungal cells, comprising: a) combining a sample containing fungal cells with a staining solution comprising a dye of the formula ##STR14## wherein each R 1 is independently H; or an alkyl group having from 1-6 carbons; or a trifluoromethyl; or a halogen; or --OR 6 , --SR 6 , or --NR 6 R 7 where R 6 and R 7 , which are the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic, heteroalicyclic, aromatic or heteroaromatic rings, containing 1-4 heteroatoms, wherein the heteroatoms are O, N or S; or R 6 and R 7 taken in combination are --(CH 2 ) 2 --M--(CH 2 ) 2 -- where M=a single bond, --O--, --CH 2 --, or --NR 8 -- where R 8 is H or an alkyl group having 1-6 carbons; and t=1-4; R 2 is an alkyl group having 1-6 carbons; X is O or S; R 3 , R 4 and R 5 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); or R 4 and R 5 , taken in combination, have the formula ##STR15## forming a quinolinium ring system; wherein R 12 , R 13 , R 14 and R 15 are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); or R 4 and R 5 , taken in combination, have the formula ##STR16## forming a quinolinium ring system; wherein R 12 , R 13 , R 14 and R 15 are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 ; --SR 6 or --(NR 6 R 7 ); Z - is a biologically compatible counterion; L is Cl, Br, I, F, or --0S0 2 R 16 where R 16 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; and OMEGA is a cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the heteroatoms are O, N or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, or alkoxy having 1-6 carbons; where the dye is present in an amount sufficient to give a distinctive intravacuolar structure or structures; b) allowing sufficient time for the dye to combine with one or more cells in the sample and form the distinctive intravacuolar structure or structures; c) illuminating the sample; d) detecting the presence of a distinctive intravacuolar structure or structures in the fungal cells; and e) correlating presence or magnitude of the distinctive intravacuolar structure or structures with metabolic activity of the fungal cells.

Claim 27

A method of detecting fungal cells, comprising a) combining a sample thought to contain one or more fungal cells with a staining solution comprising a dye of the formula ##STR19## wherein each R 1 is independently H; or an alkyl group having from 1-6 carbons; or a trifluoromethyl; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ) where R 6 and R 7 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic, heteroalicyclic, aromatic or heteroaromatic rings, containing 1-6 heteroatoms, wherein the heteroatoms are O, N or S; or R 6 and R 7 taken in combination are --(CH 2 ) 2 --M--(CH 2 ) 2 -- where M=a single bond, --O--, --CH 2 , or --NR 8 -- where R 8 is H or an alkyl group having 1-6 carbons; and t=1-4; R 2 is an alkyl group having 1-6 carbons; X is O, S, Se or NR 9 , where R 9 is H or an alkyl group having 1-6 carbons; or X is CR 10 R 11 where R 10 and R 11 , which may be the same or different, are independently alkyl groups having 1-6 carbons, or R 10 and R 11 taken in combination complete a five or six membered saturated ring; R 3 , R 4 and R 5 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); or R 4 and R 5 , taken in combination, have the formula ##STR20## ps forming a quinolinium ring system; wherein R 12 , R 13 , R 14 and R 15 are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); Z - is a biologically compatible counterion; L is Cl, Br, I, F, or --OSO 2 R 16 where R 16 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; and OMEGA is cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms (wherein the heteroatoms are O, N or S), that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, or alkoxy having 1-6 carbons; where the dye is present in an amount sufficient to give a distinctive intravacuolar structure or structures; b) allowing sufficient time for the dye to combine with said fungal cells in the sample and form the distinctive intravacuolar structure or structures; c) illuminating the sample; and d) detecting the presence of cells containing said distinctive intravacuolar structure or structures.

Claim 28

A method of detecting fungal cells, comprising a) combining a sample thought to contain one or more fungal cells with a staining solution comprising a dye of the formula ##STR19## wherein each R 1 is independently H; or an alkyl group having from 1-6 carbons; or a trifluoromethyl; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ) where R 6 and R 7 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic, heteroalicyclic, aromatic or heteroaromatic rings, containing 1-6 heteroatoms, wherein the heteroatoms are O, N or S; or R 6 and R 7 taken in combination are --(CH 2 ) 2 --M--(CH 2 ) 2 -- where M=a single bond, --O--, --CH 2 , or --NR 8 -- where R 8 is H or an alkyl group having 1-6 carbons; and t=1-4; R 2 is an alkyl group having 1-6 carbons; X is O, S, Se or NR 9 , where R 9 is H or an alkyl group having 1-6 carbons; or X is CR 10 R 11 where R 10 and R 11 , which may be the same or different, are independently alkyl groups having 1-6 carbons, or R 10 and R 11 taken in combination complete a five or six membered saturated ring; R 3 , R 4 and R 5 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); or R 4 and R 5 , taken in combination, have the formula ##STR20## ps forming a quinolinium ring system; wherein R 12 , R 13 , R 14 and R 15 are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or --OR 6 , --SR 6 or --(NR 6 R 7 ); Z - is a biologically compatible counterion; L is Cl, Br, I, F, or --OSO 2 R 16 where R 16 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; and OMEGA is cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms (wherein the heteroatoms are O, N or S), that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, or alkoxy having 1-6 carbons; where the dye is present in an amount sufficient to give a distinctive intravacuolar structure or structures; b) allowing sufficient time for the dye to combine with said fungal cells in the sample and form the distinctive intravacuolar structure or structures; c) illuminating the sample; and d) detecting the presence of cells containing said distinctive intravacuolar structure or structures.