Dear K.Karthik Loeffler’s methylene blue dye: in my experience the procedure to realize the dye is: mix 0.2 ml of 1% KOH aqueous solution (dissolve 0.1 g KOH in 10 ml dH2O), 19.8 ml dH2O, and 6 ml of ethanolic methylene blue solution (dissolve 60 mg methylene blue in 6.0 ml ethanol 95% - dilute 5.7 ml absolute ethanol and 0.3 ml dH2O). Loeffler’s methylene blue reagent must be ‘ripened by oxidation,’ obtaining polychrome methylene blue. Normally, oxidation takes several months, but it can be hastened, ten days, by aerating the reagent: placing it in bottles filled no more than half-full and shake the bottle frequently. Loeffler’s methylene blue stain improves with age, and the shelf-life of this reagent is 5–10 years; thus, the reagent can be prepared in batches large enough to last for this time period. I used the dye, with observation in wet form, to highlight the cells extracted, by squeezing, in PBS of the vaginal swab (Image above)
Dear K.Karthik Loeffler’s methylene blue dye: in my experience the procedure to realize the dye is: mix 0.2 ml of 1% KOH aqueous solution (dissolve 0.1 g KOH in 10 ml dH2O), 19.8 ml dH2O, and 6 ml of ethanolic methylene blue solution (dissolve 60 mg methylene blue in 6.0 ml ethanol 95% - dilute 5.7 ml absolute ethanol and 0.3 ml dH2O). Loeffler’s methylene blue reagent must be ‘ripened by oxidation,’ obtaining polychrome methylene blue. Normally, oxidation takes several months, but it can be hastened, ten days, by aerating the reagent: placing it in bottles filled no more than half-full and shake the bottle frequently. Loeffler’s methylene blue stain improves with age, and the shelf-life of this reagent is 5–10 years; thus, the reagent can be prepared in batches large enough to last for this time period. I used the dye, with observation in wet form, to highlight the cells extracted, by squeezing, in PBS of the vaginal swab (Image above)
Dear Huss,
Thank you for the comments for the question i raised. It was helpful and informative
Dear Huss,
Thank you for the comments for the question i raised. It was helpful and informative
More
VOTE
Just to define: Polychrome methylene blue (there might be several recipes how to get <P.MB.>):
<the preferred stain for a smear when the presence of anthrax bacilli is suspected. A positive result is a McFadyean reaction*) of a purple capsule around a blue bacillus.
(Saunders Comprehensive Veterinary Dictionary, 3 ed. © 2007 Elsevier, Inc. All rights reserved)
*) McFadyean's reaction:
a special staining reaction, demonstrating a pink capsule around a blue cell, after staining with methylene blue, which is used as a presumptive diagnosis foranthrax in a blood smear. For an image see: http://textbookofbacteriology.net/Anthrax_3.html, Fig. 9.
(Saunders Comprehensive Veterinary Dictionary, 3 ed. © 2007 Elsevier, Inc. All rights reserved) and
<an alkaline solution of methylene blue that undergoes progressive oxidative demethylation with aging (ripening) to produce a mixture of methylene blue, azures, and methylene violet.
(Farlex Partner Medical Dictionary © Farlex 2012)
From wikipedia (http://en.wikipedia.org/wiki/Methylene_blue):
<<When methylene blue is "polychromed" (oxidized in solution or "ripened" by fungal metabolism, [28] as originally noted in the thesis of Dr D L Romanowsky in 1890's), it gets serially demethylated and forms all the tri, di, mono and non methyl intermediates - which are Azure B, Azure A, Azure C and thionine respectively.[29] This is the basis of the basophilic part of the spectrum of Romanowski-Giemsa effect. If only synthetic Azure B and Eosin Y is used, it may serve as a standardized Giemsa stain; but, without methylene blue, the normal neutrophilic granules tend to overstain and look like toxic granules. On the other hand, if methylene blue is used it might help to give the normal look of neutrophil granules and may additionally also enhances the staining of nucleoli and polychromatophilic RBCs (reticulocytes).[30] (Lit.References see wikipedia)
For an "old" reference see: http://pubs.acs.org/doi/pdf/10.1021/ja01414a031:
Chemical Studies on Polychrome Methyleneblue, by Ward J. MacNeal , John A. Killian, J. Am. Chem. Soc., 1926, 48 (3), pp 740–747 DOI: 10.1021/ja01414a031; Publication Date: March 1926.
Recent information on P-MB.: http://stainsfile.info/StainsFile/dyes/52015.htm
and also: P. N. Marshall: The composition of stains produced by the oxidation of Methylene Blue. The Histochemical Journal July 1976, Volume 8, Issue 4, pp 431-442 (only PPV: link.springer.com/article/10.1007%2FBF01003831). Perhaps a recipe you can use: http://www.ihcworld.com/_faq/histology-faq/stain/s12.htm:
<< McFaydean's stain for anthrax bacilli. Question: What is M'Faydean's stain?
Answer: [This has been put together from three replies to a question raised on the HistoNet newsgroup.]
M'Faydean's stain is a simple stain using any well polychromed methylene blue (e.g. aged Loefflers). It is applied to heat-fixed smears for 10-30 seconds.
Polychroming (demethylation) is traditionally achieved by exposure of Loeffler's soln. to light and air for several months until it acquires a purplish tinge. However the oxidation process can be accelerated by application of heat as in Unna's method. (G. Gurr, 1963 p. 88 & 91); also E. Gurr, 1960, pp. 264-268).
Loeffler's methylene blue:
Methylene blue 0.5 g
1% w/v Aq. KOH 1.0 ml
Ethanol 30.0 ml
D.water 70.0 ml
Warm the water to 50 C., stir in methylene blue and add other ingredients, cool and filter before use.
Polychrome methylene blue (Unna):
Methylene blue 1.0 g
Pot. carbonate 1.0 g
Ethanol 20.0 ml
D.water 100 ml
Dissolve methylene blue in water, add pot. carb. and alcohol, place in boiling water bath and evaporate to 100 ml.
Any other polychrome methylene blue formulation should work well also.
Results: Bacilli appear Navy Blue with Anthax showing a narrow area (capsule) around and between bacilli that is reddish purple (metachromatic). A strong word of warning: many species of bacillus may also be encapsulated, e.g. Cereus etc. If you produce any positives get them confirmed at a Reference Microbiology Lab. for Infectious Diseases, or try the Armed Forces Institite of Pathology. (.......) >>
A recipe for acid-polychromed methylene blue you can find in: http://jama.jamanetwork.com/article.aspx?articleid=443026: An acid Polychrome -Methylene Blue solution for routine and special staining, by E. W. Goodpasture, JAMA. 1917;LXIX(12)
Polychromed Methylene Blue solution should be available for purchase (e.g. Pro-Lab Diagnostics , http://www.pro-lab.com/msds/stains_pl7061_polychrome_methylene_blue_msds_eu.pdf, SIGMA-Aldrich @ http://www.sigmaaldrich.com/catalog/product/sigma/l6254: Leishman’s stain, syn.: Eosin-polychrome methylene blue ) and perhaps other sellers for Loeffler's Methylenblue solution.
Disclaimer: neither affiliation to any mentioned company nor financial interests.
Just to define: Polychrome methylene blue (there might be several recipes how to get
*) McFadyean's reaction:
a special staining reaction, demonstrating a pink capsule around a blue cell, after staining with methylene blue, which is used as a presumptive diagnosis foranthrax in a blood smear. For an image see: http://textbookofbacteriology.net/Anthrax_3.html, Fig. 9.
(Saunders Comprehensive Veterinary Dictionary, 3 ed. © 2007 Elsevier, Inc. All rights reserved) and
From wikipedia (http://en.wikipedia.org/wiki/Methylene_blue):
<
Chemical Studies on Polychrome Methyleneblue, by Ward J. MacNeal , John A. Killian, J. Am. Chem. Soc., 1926, 48 (3), pp 740–747 DOI: 10.1021/ja01414a031; Publication Date: March 1926.
Recent information on P-MB.: http://stainsfile.info/StainsFile/dyes/52015.htm
and also: P. N. Marshall: The composition of stains produced by the oxidation of Methylene Blue. The Histochemical Journal July 1976, Volume 8, Issue 4, pp 431-442 (only PPV: link.springer.com/article/10.1007%2FBF01003831). Perhaps a recipe you can use: http://www.ihcworld.com/_faq/histology-faq/stain/s12.htm:
<< McFaydean's stain for anthrax bacilli. Question: What is M'Faydean's stain?
Answer: [This has been put together from three replies to a question raised on the HistoNet newsgroup.]
M'Faydean's stain is a simple stain using any well polychromed methylene blue (e.g. aged Loefflers). It is applied to heat-fixed smears for 10-30 seconds.
Polychroming (demethylation) is traditionally achieved by exposure of Loeffler's soln. to light and air for several months until it acquires a purplish tinge. However the oxidation process can be accelerated by application of heat as in Unna's method. (G. Gurr, 1963 p. 88 & 91); also E. Gurr, 1960, pp. 264-268).
Loeffler's methylene blue:
Methylene blue 0.5 g
1% w/v Aq. KOH 1.0 ml
Ethanol 30.0 ml
D.water 70.0 ml
Warm the water to 50 C., stir in methylene blue and add other ingredients, cool and filter before use.
Polychrome methylene blue (Unna):
Methylene blue 1.0 g
Pot. carbonate 1.0 g
Ethanol 20.0 ml
D.water 100 ml
Dissolve methylene blue in water, add pot. carb. and alcohol, place in boiling water bath and evaporate to 100 ml.
Any other polychrome methylene blue formulation should work well also.
Results: Bacilli appear Navy Blue with Anthax showing a narrow area (capsule) around and between bacilli that is reddish purple (metachromatic). A strong word of warning: many species of bacillus may also be encapsulated, e.g. Cereus etc. If you produce any positives get them confirmed at a Reference Microbiology Lab. for Infectious Diseases, or try the Armed Forces Institite of Pathology. (.......) >>
A recipe for acid-polychromed methylene blue you can find in: http://jama.jamanetwork.com/article.aspx?articleid=443026: An acid Polychrome -Methylene Blue solution for routine and special staining, by E. W. Goodpasture, JAMA. 1917;LXIX(12)
Polychromed Methylene Blue solution should be available for purchase (e.g. Pro-Lab Diagnostics , http://www.pro-lab.com/msds/stains_pl7061_polychrome_methylene_blue_msds_eu.pdf, SIGMA-Aldrich @ http://www.sigmaaldrich.com/catalog/product/sigma/l6254: Leishman’s stain, syn.: Eosin-polychrome methylene blue ) and perhaps other sellers for Loeffler's Methylenblue solution.
Disclaimer: neither affiliation to any mentioned company nor financial interests.
More
VOTE
Dear K.Karthik
Loeffler’s methylene blue dye: in my experience the procedure to realize the dye is: mix 0.2 ml of 1% KOH aqueous solution (dissolve 0.1 g KOH in 10 ml dH2O), 19.8 ml dH2O, and 6 ml of ethanolic methylene blue solution (dissolve 60 mg methylene blue in 6.0 ml ethanol 95% - dilute 5.7 ml absolute ethanol and 0.3 ml dH2O). Loeffler’s methylene blue reagent must be ‘ripened by oxidation,’ obtaining polychrome methylene blue. Normally, oxidation takes several months, but it can be hastened, ten days, by aerating the reagent: placing it in bottles filled no more than half-full and shake the bottle frequently. Loeffler’s methylene blue stain improves with age, and the shelf-life of this reagent is 5–10 years; thus, the reagent can be prepared in batches large enough to last for this time period. I used the dye, with observation in wet form, to highlight the cells extracted, by squeezing, in PBS of the vaginal swab (Image above)
Dear K.Karthik
Loeffler’s methylene blue dye: in my experience the procedure to realize the dye is: mix 0.2 ml of 1% KOH aqueous solution (dissolve 0.1 g KOH in 10 ml dH2O), 19.8 ml dH2O, and 6 ml of ethanolic methylene blue solution (dissolve 60 mg methylene blue in 6.0 ml ethanol 95% - dilute 5.7 ml absolute ethanol and 0.3 ml dH2O). Loeffler’s methylene blue reagent must be ‘ripened by oxidation,’ obtaining polychrome methylene blue. Normally, oxidation takes several months, but it can be hastened, ten days, by aerating the reagent: placing it in bottles filled no more than half-full and shake the bottle frequently. Loeffler’s methylene blue stain improves with age, and the shelf-life of this reagent is 5–10 years; thus, the reagent can be prepared in batches large enough to last for this time period. I used the dye, with observation in wet form, to highlight the cells extracted, by squeezing, in PBS of the vaginal swab (Image above)
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VOTE
Dear K.Karthik,
you are welcome !
Best, Wolfgang
Dear K.Karthik,
you are welcome !
Best, Wolfgang
More
VOTE
Since my previous post has not been saved again after a few corrections by myself( the replier), I shall point you here only to:
A simple, reliable M'Fadyean stain for visualizing the Bacillus anthracis capsule
M.P. Owen, W. Schauwers, M.E. Hugh-Jones, J.A. Kiernan, P.C.B. Turnbull, W. Beyer in: Journal of Microbiological Methods 92 (2013) 264–269,
to be found in RG at:
http://www.researchgate.net/publication/235378616_A_simple_reliable_M'Fadyean_stain_for_visualizing_the_Bacillus_anthracis_capsule
Article A simple, reliable M'Fadyean stain for visualizing the Bacil...
Since my previous post has not been saved again after a few corrections by myself( the replier), I shall point you here only to:
A simple, reliable M'Fadyean stain for visualizing the Bacillus anthracis capsule
M.P. Owen, W. Schauwers, M.E. Hugh-Jones, J.A. Kiernan, P.C.B. Turnbull, W. Beyer in: Journal of Microbiological Methods 92 (2013) 264–269,
to be found in RG at:
http://www.researchgate.net/publication/235378616_A_simple_reliable_M'Fadyean_stain_for_visualizing_the_Bacillus_anthracis_capsule
Article A simple, reliable M'Fadyean stain for visualizing the Bacil...
More
VOTE
Just to define: Polychrome methylene blue (there might be several recipes how to get <P.MB.>):
<the preferred stain for a smear when the presence of anthrax bacilli is suspected. A positive result is a McFadyean reaction*) of a purple capsule around a blue bacillus.
(Saunders Comprehensive Veterinary Dictionary, 3 ed. © 2007 Elsevier, Inc. All rights reserved)
*) McFadyean's reaction:
a special staining reaction, demonstrating a pink capsule around a blue cell, after staining with methylene blue, which is used as a presumptive diagnosis foranthrax in a blood smear. For an image see: http://textbookofbacteriology.net/Anthrax_3.html, Fig. 9.
(Saunders Comprehensive Veterinary Dictionary, 3 ed. © 2007 Elsevier, Inc. All rights reserved) and
<an alkaline solution of methylene blue that undergoes progressive oxidative demethylation with aging (ripening) to produce a mixture of methylene blue, azures, and methylene violet.
(Farlex Partner Medical Dictionary © Farlex 2012)
From wikipedia (http://en.wikipedia.org/wiki/Methylene_blue):
<<When methylene blue is "polychromed" (oxidized in solution or "ripened" by fungal metabolism, [28] as originally noted in the thesis of Dr D L Romanowsky in 1890's), it gets serially demethylated and forms all the tri, di, mono and non methyl intermediates - which are Azure B, Azure A, Azure C and thionine respectively.[29] This is the basis of the basophilic part of the spectrum of Romanowski-Giemsa effect. If only synthetic Azure B and Eosin Y is used, it may serve as a standardized Giemsa stain; but, without methylene blue, the normal neutrophilic granules tend to overstain and look like toxic granules. On the other hand, if methylene blue is used it might help to give the normal look of neutrophil granules and may additionally also enhances the staining of nucleoli and polychromatophilic RBCs (reticulocytes).[30] (Lit.References see wikipedia)
For an "old" reference see: http://pubs.acs.org/doi/pdf/10.1021/ja01414a031:
Chemical Studies on Polychrome Methyleneblue, by Ward J. MacNeal , John A. Killian, J. Am. Chem. Soc., 1926, 48 (3), pp 740–747 DOI: 10.1021/ja01414a031; Publication Date: March 1926.
Recent information on P-MB.: http://stainsfile.info/StainsFile/dyes/52015.htm
and also: P. N. Marshall: The composition of stains produced by the oxidation of Methylene Blue. The Histochemical Journal July 1976, Volume 8, Issue 4, pp 431-442 (only PPV: link.springer.com/article/10.1007%2FBF01003831). Perhaps a recipe you can use: http://www.ihcworld.com/_faq/histology-faq/stain/s12.htm:
<< McFaydean's stain for anthrax bacilli. Question: What is M'Faydean's stain?
Answer: [This has been put together from three replies to a question raised on the HistoNet newsgroup.]
M'Faydean's stain is a simple stain using any well polychromed methylene blue (e.g. aged Loefflers). It is applied to heat-fixed smears for 10-30 seconds.
Polychroming (demethylation) is traditionally achieved by exposure of Loeffler's soln. to light and air for several months until it acquires a purplish tinge. However the oxidation process can be accelerated by application of heat as in Unna's method. (G. Gurr, 1963 p. 88 & 91); also E. Gurr, 1960, pp. 264-268).
Loeffler's methylene blue:
Methylene blue 0.5 g
1% w/v Aq. KOH 1.0 ml
Ethanol 30.0 ml
D.water 70.0 ml
Warm the water to 50 C., stir in methylene blue and add other ingredients, cool and filter before use.
Polychrome methylene blue (Unna):
Methylene blue 1.0 g
Pot. carbonate 1.0 g
Ethanol 20.0 ml
D.water 100 ml
Dissolve methylene blue in water, add pot. carb. and alcohol, place in boiling water bath and evaporate to 100 ml.
Any other polychrome methylene blue formulation should work well also.
Results: Bacilli appear Navy Blue with Anthax showing a narrow area (capsule) around and between bacilli that is reddish purple (metachromatic). A strong word of warning: many species of bacillus may also be encapsulated, e.g. Cereus etc. If you produce any positives get them confirmed at a Reference Microbiology Lab. for Infectious Diseases, or try the Armed Forces Institite of Pathology. (.......) >>
A recipe for acid-polychromed methylene blue you can find in: http://jama.jamanetwork.com/article.aspx?articleid=443026: An acid Polychrome -Methylene Blue solution for routine and special staining, by E. W. Goodpasture, JAMA. 1917;LXIX(12)
Polychromed Methylene Blue solution should be available for purchase (e.g. Pro-Lab Diagnostics , http://www.pro-lab.com/msds/stains_pl7061_polychrome_methylene_blue_msds_eu.pdf, SIGMA-Aldrich @ http://www.sigmaaldrich.com/catalog/product/sigma/l6254: Leishman’s stain, syn.: Eosin-polychrome methylene blue ) and perhaps other sellers for Loeffler's Methylenblue solution.
Disclaimer: neither affiliation to any mentioned company nor financial interests.
Just to define: Polychrome methylene blue (there might be several recipes how to get
*) McFadyean's reaction:
a special staining reaction, demonstrating a pink capsule around a blue cell, after staining with methylene blue, which is used as a presumptive diagnosis foranthrax in a blood smear. For an image see: http://textbookofbacteriology.net/Anthrax_3.html, Fig. 9.
(Saunders Comprehensive Veterinary Dictionary, 3 ed. © 2007 Elsevier, Inc. All rights reserved) and
From wikipedia (http://en.wikipedia.org/wiki/Methylene_blue):
<
Chemical Studies on Polychrome Methyleneblue, by Ward J. MacNeal , John A. Killian, J. Am. Chem. Soc., 1926, 48 (3), pp 740–747 DOI: 10.1021/ja01414a031; Publication Date: March 1926.
Recent information on P-MB.: http://stainsfile.info/StainsFile/dyes/52015.htm
and also: P. N. Marshall: The composition of stains produced by the oxidation of Methylene Blue. The Histochemical Journal July 1976, Volume 8, Issue 4, pp 431-442 (only PPV: link.springer.com/article/10.1007%2FBF01003831). Perhaps a recipe you can use: http://www.ihcworld.com/_faq/histology-faq/stain/s12.htm:
<< McFaydean's stain for anthrax bacilli. Question: What is M'Faydean's stain?
Answer: [This has been put together from three replies to a question raised on the HistoNet newsgroup.]
M'Faydean's stain is a simple stain using any well polychromed methylene blue (e.g. aged Loefflers). It is applied to heat-fixed smears for 10-30 seconds.
Polychroming (demethylation) is traditionally achieved by exposure of Loeffler's soln. to light and air for several months until it acquires a purplish tinge. However the oxidation process can be accelerated by application of heat as in Unna's method. (G. Gurr, 1963 p. 88 & 91); also E. Gurr, 1960, pp. 264-268).
Loeffler's methylene blue:
Methylene blue 0.5 g
1% w/v Aq. KOH 1.0 ml
Ethanol 30.0 ml
D.water 70.0 ml
Warm the water to 50 C., stir in methylene blue and add other ingredients, cool and filter before use.
Polychrome methylene blue (Unna):
Methylene blue 1.0 g
Pot. carbonate 1.0 g
Ethanol 20.0 ml
D.water 100 ml
Dissolve methylene blue in water, add pot. carb. and alcohol, place in boiling water bath and evaporate to 100 ml.
Any other polychrome methylene blue formulation should work well also.
Results: Bacilli appear Navy Blue with Anthax showing a narrow area (capsule) around and between bacilli that is reddish purple (metachromatic). A strong word of warning: many species of bacillus may also be encapsulated, e.g. Cereus etc. If you produce any positives get them confirmed at a Reference Microbiology Lab. for Infectious Diseases, or try the Armed Forces Institite of Pathology. (.......) >>
A recipe for acid-polychromed methylene blue you can find in: http://jama.jamanetwork.com/article.aspx?articleid=443026: An acid Polychrome -Methylene Blue solution for routine and special staining, by E. W. Goodpasture, JAMA. 1917;LXIX(12)
Polychromed Methylene Blue solution should be available for purchase (e.g. Pro-Lab Diagnostics , http://www.pro-lab.com/msds/stains_pl7061_polychrome_methylene_blue_msds_eu.pdf, SIGMA-Aldrich @ http://www.sigmaaldrich.com/catalog/product/sigma/l6254: Leishman’s stain, syn.: Eosin-polychrome methylene blue ) and perhaps other sellers for Loeffler's Methylenblue solution.
Disclaimer: neither affiliation to any mentioned company nor financial interests.
More
VOTE