prestoblue and alamar blue reagents are resazurin based , membrane permeable solutions that upon reduction form resorufin , a red fluorescent compound which can be quantitatively measured to determine viability . initially developed as a cell viability indicator , prestoblue has been indicated for use on nonmammalian cells , such as bacteria , yeast , and eukaryotic cells . the variable reading methods of prestoblue makes this reagent an attractive alternative in cellular and microbiology . prestoblue can be measured either visually , using absorbance or utilising the fluorescent outputs of the reduced resorufin . p - iodonitrotetrazolium violet ( int ) is a tetrazolium dye precursor that once reduced forms a purple formazan dye ( sigma - aldrich ) . sodium 3-[1-(phenyl amino - carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro ] benzene sulfonic acid hydrate ( xtt ) is also a tetrazolium based reagents which in the presence of metabolically active cells reduces the tetrazolium salt to an orange coloured formazan compound . the reduction of the tetrazolium salt is due to the activity of the mitochondria enzyme in the active cells . the intensity of the formazan compound can be measured using absorbance where the intensity of the compound is directly proportional to the number of metabolically active cells . the aim of this study was to validate the use of prestoblue as a growth indicator and cell viability reagent by comparing it to other similar commercially available reagents . all microbial strains were purchased from american type culture collection ( atcc ) , md , usa . the a431 cell line and hela cell line were purchased from ecacc and highveld biological ( pty ) ltd . prestoblue and alamar blue were both purchased from invitrogen corporation , san diego , usa . the cell culture medium , trypsin - edta , fetal bovine serum ( fbs ) , phosphate buffer saline ( pbs ) , and antibiotics were supplied by highveld biological ( pty ) ltd . the microorganisms used in this study included prevotella intermedia ( atcc 25611 ) , streptococcus mutans ( atcc 25175 ) , and propionibacterium acnes ( atcc 11827 ) . s. mutans and p. intermedia were grown on casein - peptone soymeal - peptone agar medium ( caso ) enriched with 1% sucrose ( merck chemicals ( pty ) ltd . , wadeville , south africa ) under anaerobic conditions in an anaerobic jar with anaerocult a ( merck kgaa , darmstadt , germany ) at 37c for 48  hrs . inoculants were prepared by suspending bacterial test organisms in quarter strength sterile ringer solution ( merck kgaa , darmstadt , germany ) until turbidity was compatible with mcfarland standard 1 ( merck chemicals ( pty ) ltd . , wadeville , south africa ) . furthermore a bacterial concentration of 4  10 ( colony forming units ) cfu / ml for p. intermedia and 3  10  cfu / ml for s. mutans was used . p. acnes was cultured on tryptone soy agar ( merck chemicals ( pty ) ltd . , wadeville , south africa ) under anaerobic conditions in an anaerobic jar with anaerocult a at 37c for 72  hrs . subculturing was done every 4th week as the doubling time of the bacterium is slow . inoculants were prepared by suspending the bacterial colonies in nutrient broth ( merck chemicals ( pty ) ltd . , wadeville , south africa ) until turbidity was compatible with a mcfarland standard 0.5 at a bacterial concentration of 1.5  10  cfu / ml . the microdilution technique using 96-well microtitre plates , as described by eloff , was used to obtain the mic values of the positive drug controls against the various microorganisms . the positive controls for oral bacteria , chlorhexidine gluconate ( chx ) ( dental warehouse , sandton , south africa ) at 12.5  g / ml , and tetracycline ( sigma - aldrich , 3050 spruce street , st . louis ) at 200  g / ml for p. acnes were serially diluted in relevant broth medium adding 48  h old oral bacteria and 72  h old p. acnes and were incubated at 37c in anaerobic conditions . the final concentration of chx , ranged 3.13  g / ml2.44  10  g / ml and tetracycline ranged 100  g / ml0.781  g / ml . s. mutans and p. intermedia were incubated for 24  hrs and p. acnes for 72  hrs at 37c . to indicate bacterial growth , 40  l of ( 0.2  mg / ml ) int , 20  l prestoblue , and 20  l alamar blue was added to microplate wells and reincubated until a colour change occurred . the mic was defined as the lowest concentration that inhibited the growth of the bacteria . for the antimycobacterial assay the h37rv ( atcc 27264 ) the bacteria were cultured on lwenstein - jensen ( lj ) medium ( sa medical research council , pretoria ) for three weeks ( 37c , 5% co2 ) . one colony was transferred under sterile conditions to 50  ml of 7h9 broth media ( sigma - aldrich , south africa ) , 10% oadc ( sigma - aldrich , south africa ) and 2% panta ( becton , dickinson and company , usa ) . after the final incubation period , the bacteria were adjusted in 7h9 broth media to a turbidity of a mcfarland standard 1 . bacterial suspensions were further diluted 1  :  25 to obtain a concentration of 2  10  cfu / ml . the microtitre alamar blue assay ( maba ) and microtitre prestoblue assay ( mpba ) were used to determine the mic of positive drug controls on the bacteria . standard antituberculosis agents , inh ( sigma - aldrich , becton , dickinson and company ) , and rif ( sigma - aldrich , becton , dickinson and company ) were used . stock solutions were diluted in 7h9 broth media to a final assay concentrations which ranged 5  g / ml0.078  g / ml and 6  g / ml0.1875  g / ml for inh and rif , respectively . a 2% dimethyl sulfoxide ( dmso ) solvent control , media control , and bacterial control was included in the assay . one hundred microlitres of bacteria was added to the inner wells of the 96-well plates . the outer perimeter wells were used to compensate for evaporation by adding 200  l of dh2o . the plates were incubated for 5 to 7 days at 37c , 5% co2 . after 5 days 20  l of alamar blue / prestoblue indicator solution if a colour change was observed , all the subsequent wells received 20  l of indicator solution . if no colour change was observed the plates were incubated for further 24  hrs . the hela and a431 cell lines were maintained in eagle 's minimum essential medium supplemented with 10% fbs and 1% antibiotics ( 100  u / ml penicillin , 100  g / ml streptomycin ) and 250  g / ml fungizone . cells were subcultured by treating them with trypsin - edta ( 0.25% trypsin containing 0.01% edta ) for 10 minutes . the method described by zheng et al .   was used to perform the assay . both cell lines were seeded in a 96-well microtitre plate at a concentration of 1  10  cells / ml . the cells were exposed to the positive drug control actinomycin d ( sigma - aldrich , south africa ) with concentrations ranging between 0.5  g / ml and 0.002  g / ml . the microtitre plate was incubated for further 72  hrs and thereafter 50  l xtt was added to a final concentration of 0.3  mg / ml to one set of plates and 20  l prestoblue was added to another set of plates . the plates were incubated for further 2  hrs where after the absorbance of the colour complex was read at 490  nm with a reference wavelength set at 690  nm for xtt and at 570  nm with a reference wavelength set at 600  nm for prestoblue , using a bio - tek power - wave xs multiwell plate reader . the colour change of the growth indicators int , prestoblue , and alamar blue for s. mutans and p. intermedia is depicted in figure 1 . the development time for each of the oral bacteria differed . with the addition of int , p. intermedia developed within 40 minutes ; however , s. mutans development took over two hours and it was difficult to distinguish where the mic occurred . conversely with prestoblue or alamar blue , s. mutans developed immediately and the mic was easily distinguishable , with pink indicating viable bacteria and blue indicating nonviable bacteria . p. intermedia developed within 20 minutes , although the colour change took slightly longer with alamar blue . the mic values obtained for the inhibition of s. mutans and p. intermedia by chx showed the same values using both prestoblue and alamar blue at 4.88  10  g / ml and 0.391  g / ml , respectively . p. intermedia showed a slightly higher mic with the development of int ( at 0.78  g / ml ) . it was almost impossible to determine the mic value for the inhibition of s. mutans using int . there appeared to be a distinctive colour change for each of the bacteria whether using prestoblue or alamar blue . the colour change of the growth indicators int and prestoblue for p. acnes were observed after addition of the reagents as seen in figure 2 . the comparison between int and prestoblue for determination of mic values for bacterial cells treated with tetracycline the p. acnes bacteria had a tendency to coagulate at the bottom of the wells during the incubation period and this was where the int was reduced to the violet colour . the prestoblue provided an advantage in that the entire well was coloured , making visualisation of mic values easier . tetracycline treated cells in the presence of int showed no colour change as seen in figure 2(a ) . however in the presence of prestoblue , there was a clear distinction between wells with metabolically active bacterial cells ( pink ) and those which had been inhibited ( blue ) by the tetracycline control drug . although the incubation period of the prestoblue plates was slightly longer ( 3  hrs ) compared with int ( 1  h ) the determination of mic values was much clearer . the mic of tetracycline was determined to be 3.125  g / ml with prestoblue and could not be visually determined with int . the health risk involved in working with m. tuberculosis meant no clear pictures of the colour changes could be obtained . due to the slow growth rate of m. tuberculosis , the conversion of the alamar blue and prestoblue into a red colour took up to 24  hrs . although a colour change could be observed after 3  hrs , the longer incubation period resulted in a more distinguishable separation between viable pink bacterial wells and blue inhibited wells . there were no differences observed between the mic values of the positive controls when comparing alamar blue and prestoblue . inh showed an mic of 0.13  g / ml whereas rifampicin showed an mic of 0.75  g / ml for both prestoblue and alamar blue . the prestoblue had a slightly faster conversion rate compared to the alamar blue , but at 24  hr incubation the readings of the mics were the same . xtt and prestoblue were used to determine the antiproliferative effect of actinomycin d on hela cells and a431 cells as seen in figure 3 . for both growth indicators a colour change was observed after approximately 2  hrs . the ic50 values observed when comparing xtt to prestoblue were similar . on the hela cells the positive control showed ic50 values of 0.005  2  10  g / ml and 0.006  1  10  g / ml for xtt and prestoblue , respectively , whereas on the a431 cells the positive control showed ic50 values of  0.011  2  10  g / ml for both growth indicators . the colour change was observed at approximately the same time and both growth indicators showed a distinguishable colour change . prestoblue showed great potential as a growth indicator for various microorganisms as well as a cell viability . it has minor limitations such as light sensitivity and the varied time taken for colour development , which is dependent on the metabolic rates of various bacteria and cell cultures . prestoblue and alamar blue give clear and easily determined visual mic values for s. mutans , p. intermedia , p. acnes , and m. tuberculosis . however , s. mutans , p. intermedia , and m. tuberculosis developed a colour change more rapidly in prestoblue than in alamar blue . furthermore alamar blue and prestoblue as growth indicator reagents have an advantage over other reagents , such as int , in that the whole assay well changes colour instead of only the bacteria . therefore prestoblue may be the better indicator for these microorganisms . comparing the reagents xtt and prestoblue as cell viability indicators it was observed that both growth indicators showed clear distinction between viable cells and nonviable cells and the ic50 values obtained from both reagents were similar .
this study compared different commercially available viability reagents . 
 the growth indicator reagents include p - iodonitrotetrazolium violet ( int ) , prestoblue , and alamar blue which were used for antimicrobial analysis against streptococcus mutans , prevotella intermedia , propionibacterium acnes , and mycobacterium tuberculosis . 
 prestoblue and alamar blue are resazurin based reagents that resulted in a quick and easily distinguishable colour change that allowed for visual readings . 
 int and sodium 3-[1-(phenyl amino - carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro ] benzene sulfonic acid hydrate ( xtt ) are tetrazolium based reagents which are converted to a formazan dye in the presence of metabolically active mitochondria enzyme . for cell viability analysis , 
 reagents xtt and prestoblue were compared . 
 prestoblue was able to clearly indicate the minimum inhibitory concentration ( mic ) of various positive drug controls on various microbial strains . 
 prestoblue was also a good indicator of the 50% inhibitory concentration ( ic50 ) of positive drug controls on various cell lines .