i´m doing a research of marine bacteria and i want to estimate biomass, searching i found that the epiflouresnce method for counting bacteria is better but i don´t know which one i can use DAPI or Acridine orange.
Hola Vivian,
DAPI es mucho mas facil de manejar y mucho menos peligroso que AO, que es con alta probabilidad fuertemente cancerigenico (Acridina si lo es).
Por otro lado, para biomasas (Image Analysis), varios investigadores recomiendan AO, porque DAPI no tiñe toda la bacteria (es DNA-especifico, no como AO, que tiñe tambien las RNAs); por otro lado hay la correccion (Possch et al.)
Personalmente prefiero DAPI
Con cualquier duda, escribame a [email protected]
Un abrazo
Mirek Macek
I was actually waiting for a response as well and I do not know Spanish (?).
The Lady First!!!
I am fun of DAPI even though (some 1/4 od century ago) we were using AO.
Generally, several old fashion scientists, prefer to use AO for biomass measurements (by ImagaAnalysis); In fact, they are completely right: DAPI is DNA-specific meanwhile AO is staining also different RNAs and you can approximate the cell condition. In the recent decade, AO was used by medical researchers for apoptosis (but on the web you can see terrible recommendations, e.g., how to stain slides directly in staining vessels, Don't follow it!). Acridine is declared as a strong cancerogenic agent an even though AO does not, very special care must be apply (special filtration towers, special lab corner, etc.
Comparing to AO, DAPI is still without strong reports but as any NA-binding substance you must take care. I don't know your microscope, but not all epifluorescence microscopes posses UV filter set needed for DAPI (AO si working with blue-nearUV, the same as FITC)
As I am more protozoologist, I love the possibility to see protists (through nuclei; not all the cell is well stained for a long time) with chloroplasts and the possibility to distinguished them from cyanobacteria.
I recommend you to by 5mg (maximum 10mg), dissolve the whole content with filtered water 1ml/mg and fill the Eppendorfs, e.g., 2mL with aliquots of 200 /ul to be kept frozen. For using, 1800 /ul is added (10x); in the dark, the solution is stable at 4°C for weeks .
The DAPI amount differ water-to-water. For the best staining, we are adding 50 /ul to 1 to 2 ml of the sample (directly or partially concentrated) in the filtration appartus (black polycarbonate membrane 0.2 /um; Irgalan Blach or Chlorazol black is used; however, several last years Millipore (Hach) mebranes were not stainable), for 4 min. However, for some samples it is too much or is better to use lower amount. For some (protozoological) purposes, white membrane is OK but you should slow down to 10 /ul per sample (maybe 20 /ul to 10 ml of sample).
Good luck
Mirek Macek
Muchas gracias por su respuesta Miroslav Macek, me ha ayudado bastante. Si tengo más dudas le escribiré.
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