this technique requires tagging of cells with a fluorophore , which is a molecule that will fluoresce when light at appropriate wavelengths is incident on it .
fluorophores should have good photostability and high quantum yield , and when used to image live cells they should be noncytotoxic and stable in vivo [ 2 , 3 ] .
they should also have low susceptibility to photobleaching without losing their sensitivity to image the cells . in order to overcome photon attenuation in living cells , fluorophores with long emission at the near - infrared ( nir ) region
the most commonly used fluorophore to image bacterial cell is fluorescein , but owing to a variety of factors it is not a good choice .
many other fluorophores are also in current use , and new fluorophores with high quantum yield and low cytotoxicity are also being continuously screened for imaging cells .  
porphyrin and its derivatives are nontoxic organic compounds whose fluorescent properties have been widely studied .
we have investigated the use of tetra ammonium tetrakis ( 4-sulphonato)phenyl porphyrin ( tpps ) ( figure 1 ) , a water - soluble anionic compound , with multiple excitation bands and wide excitation range , as a fluorescent dye for imaging bacterial cells .   in earlier studies , tpps has been used as a photosensitising agent for photo - dynamic therapy .
it was found to be moderately effective against gram - positive bacteria but had no cytotoxic effect on gram - negative bacteria , even after prolonged exposure to light at high frequency .
studies with tetra - cationic phthalocyanine as photosensitizer have also shown that the entry of photosensitizer into the cytoplasm increases brightness of cells , which was observed in images obtained from fluorescence microscope .
the uptake of photosensitizer was further found to be an important step in affecting cells ' survival rate .
these indicate that uptake of tpps into the cytoplasm by gram - positive bacteria is higher than that by gram - negative bacteria .  
we imaged gram - negative bacteria , pseudomonas aeruginosa , and gram - positive bacteria , bacillus cereus , on a fluorescent microscope using tpps as fluorophore and then analysed the brightness data obtained from those images .
tetraphenyl porphyrin was synthesized using the protocol described by gonsalves et al .   followed by sulphonation using chlorosulphonic acid as described by song et al . .
purity of the compounds synthesised is verified using thin - layer chromatography , nmr , uv visible spectroscopy , and mass spectrometry . 

eu732606 ) and bacillus cereus ( gram - positive bacteria ) used were isolated and cultured in our laboratory .
cultures of pseudomonas aeruginosa were grown overnight and inoculated in luria - bertani medium , incubated for 14 hours .
250  l of this culture was mixed with various concentrations of tpps and incubated in dark for 5 minutes .
the cells were then centrifuged for 5 minutes at 5000  rpm . the cell pellet was washed and then solubilised in pbs buffer before being imaged on an olympus x171 total internal reflection fluorescence microscope in fluorescence mode .
the sample was excited using light from a mercury - vapour lamp , which was passed through an olympus u - mwiba3 filter ( excitation filter 460  nm495  nm and emission filter 510  nm550  nm ) .
after that , a similar procedure as used for pseudomonas aeruginosa was followed and images were obtained .  
images collected from the andor ixon emccd camera were processed using andor - iq software version 1.8 .
all images were taken at an exposure time of 117.5  ms and real em gain of 44 .
another image processing software was used to measure the brightness of cells in different parts of the image .
brightness was 
 ( 1)v=0.299r+0.587g+0.114b , 
 where r , g , and b are the red , green , and blue pixels , respectively .
a plot was then constructed to analyse the variation of brightness with changes in concentration of tpps .
the cells with tpps fluoresced , when visualised under fluorescent microscope ( figure 2 ) with excitation at 488  nm wavelength , while control cells to which tpps was not added did not show any fluorescence .   cell viability , for the pseudomonas aeruginosa cells , as indicated by their motility , was not affected by the uptake of tpps ( supplementary material available online at doi:10.1155/2010/697528 ) .   image analysis revealed that the brightness increases with increasing concentrations of tpps for both pseudomonas aeruginosa and bacillus cereus before achieving saturation .
the concentration at which the brightness achieves saturation is about the same for both of the bacteria ( 0.150.25  mg / ml ) , but the brightness is consistently higher for bacillus cereus ( figure 3 ) .
this indicates that the uptake of tpps by the gram - positive bacteria is much higher than that of the gram - negative bacteria .
high quantum yield , high solubility in water , and its nontoxic nature are properties that confer considerable advantages to tpps over other dyes in current use . the technique developed has the added advantage in that it requires a very short preparatory time .
a variation in brightness was observed for the two different bacteria at the same concentrations of tpps . in conclusion
, tpps can be used as a stain for fluorescent imaging and analysis of various microorganisms and could be used for identifying pseudomonas aeruginosa and bacillus cereus .
 the use of tetraammonium tetrakis(4-sulphonato)phenyl porphyrin ( tpps ) , a water - soluble anionic compound , as a stain to analyse bacterial cells using fluorescent microscopy was investigated . 
 tpps was effectively used to analyse two different bacteria : pseudomonas aeruginosa and bacillus cereus . the variation in brightness with varying concentrations of tpps 
 was studied . 
 the patterns of variations for these bacteria were found to be the same , but with consistently higher brightness for bacillus cereus . 
1. Introduction
2. Experimental Section
3. Results and Discussions
4. Conclusions