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Microencapsulation with various materials has been used as an efficient method to improve the viability of probiotic bacteria in multiple food products and the human gastrointestinal tract. Although plate count agar is the most commonly used method for evaluating the viability of encapsulated bacteria, it is still far from providing reliable information about the intermediate state between viable and dead bacteria. This study optimized a tetrazolium salt-based colorimetric method for the detection of viable but non-culturable state within encapsulated Lactobacillus rhamnosus and Lactobacillus plantarum probiotic strains. The viability of encapsulated bacteria was assessed after different spray-drying conditions and also during two months of storage at room temperature. The ability to reduce tetrazolium salts of two lactic acid bacteria was verified and calibrated according to the experimental conditions (strains, incubation time, and microencapsulation material). The loss of bacterial cultivability was species-specific and more problematic throughout the processing than during the storage period. An outlet temperature of 73-75 °C yielded a higher viable but non-culturable state level than at 68-69 °C, especially in maltodextrin and trehalose powders. Whey protein was statistically the best carrier in preserving viable and culturable encapsulated bacteria after spray-drying and during storage, as compared to sugar-based carriers. The tetrazolium-optimized method was more sensitive and accurate for the evaluation of viable bacteria in microcapsules as compared to the conventional plate count methods available. It showed the high variability of CFU counts on Man–Rogosa–Sharpe (MRS) agar. This colorimetric technique could be considered a real-time, simple, cost-effective, and reliable alternative to culture-based methods in evaluating probiotic microencapsulation efficiency.

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Tamarind (Tamarindus indica) is a fruit tree of African origin cultivated in more than 50 countries, including Colombia, mainly in the Colombian Caribbean departments, where its harvest generates significant income in rural communities. Its establishment presents a difficulty because the sexual seed presents physical dormancy and poor germination. The objective of this study was to evaluate Tamarind seed viability by using the tetrazolium test and to reduce the time needed for the determination of its physiological quality. Ripe and healthy fruits were collected from patio trees, from which their seeds were extracted, disinfected with 1% sodium hypochlorite, and washed with plenty of water before use. Subsequently, they were scarified with No. 100 sandpaper by the edges, except in the area of the micropyle. The completely random design was used in a 3x3 bifactorial arrangement, with four repetitions. The first factor, tetrazolium concentration (%), was tested at levels of 0.50, 0.75 and 1.00%, and the second factor, immersion time, at 2, 4, and 6 hours. The analysis of variance did not show significance for the tetrazolium concentration, while it did for the immersion time (p≤0.01), and the tetrazolium-time interaction (p≤0.05). Therefore, the concentration of 0.50% tetrazolium for six hours of immersion is a reliable alternative to determine the physiological quality of tamarind seeds compared to conventional germination testing due to its low cost and execution time. Likewise, the percentages of germination and germination speed index were higher when the seed was scarified with sandpaper than the conventional method.

To ensure clean drinking water, viable pathogens in water must be rapidly and efficiently screened. The traditional culture or spread-plate process—the conventional standard for bacterial detection—is laborious, time-consuming, and unsuitable for rapid detection. Therefore, we developed a colorimetric assay for rapid microorganism detection using a metabolism-based approach. The reaction between a viable microorganism and the combination of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8) and 1-methoxy-5-methylphenazinium methyl sulfate (mPMS) results in a color change. In combination with a microplate reader, WST-8-mPMS reactivity was leveraged to develop a colorimetric assay for the rapid detection of various bacteria. The detection limit of the WST-8-mPMS assay for both gram-negative and gram-positive bacteria was evaluated. This WST-8-mPMS assay can be used to perform colorimetrical semi-quantitative detection of various bacterial strains in buffers or culture media within 1 h without incubation before the reaction. The easy-to-use, robust, rapid, and sensitive nature of this novel assay demonstrates its potential for practical and medical use for microorganism detection. [ABSTRACT FROM AUTHOR]

Background and Aims: Survival of periodontal ligament (PDL) cells after avulsion is an important factor in treatment prognosis. Grapefruit Seed Extract (GSE) can be a proper environment for preserving periodontal ligament cells. The objective of this study was to analyze the effect of different concentrations of GSE on the proliferation of fibroblast PDL cells. Materials and Methods: In this study, the undifferentiated PDL fibroblasts were obtained from two human premolars teeth and cultured in Dulbecco’s modified Eagle’s medium (DMEM). The cultured cells were exposed to different concentrations of GSE. The positive and negative control groups were cultured in fetal bovine serum (FBS) 10% and in a medium without FBS 10%, respectively. The plates were incubated for 1, 6, 12, 24, and 48 hrs. The PDL cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Statistical analysis of data was accomplished using repeated measure ANOVA with Post HOC Tukey, P0.05). Furthermore, all the samples were similar to the positive control group in three of the five timeperiods (P>0.05). Conclusion: GSE was more effective in fewer concentration and longer periods and it had no toxic effect on PDL cells. Therefore, GSE can be considred as a promoting medium in PDL regeneration of avulsed permanent teeth in the future.

The ethanol extracts of 155 different foodstuffs containing medicinal plants were investigated for their biofilm eradication activities against pathogenic bacteria. A combined method of a colorimetric microbial viability assay based on reduction of a tetrazolium salt (WST-8) and a biofilm formation technique on the 96-pins of a microtiter plate lid was used to screen the biofilm eradication activities of foodstuffs. The ethanol extracts of licorice (Glycyrrhiza glabra) showed potent biofilm eradication activities against Streptococcus mutans, Staphylococcus aureus, and Porphyromonas gingivalis. Among the antimicrobial constituents in licorice, glabridin had the most potent eradication activities against microbial biofilms. The minimum biofilm eradication concentration of glabridin was 25–50 μg/ml. Furthermore, the combination of glabridin with ɛ-poly-l-lysine, a food additive, could result in broad biofilm eradication activities towards a wide variety of bacteria associated with infection, including Escherichia coli and Pseudomonas aeruginosa. [ABSTRACT FROM AUTHOR]

In this work, a method for the measurement of one-dimensional (1D) UV radiation dose is described. It comprises a new tablet dosimeter that measures the dose using reflectance spectrophotometry. The tablet dosimeter elaborated is a solid structure with a cylindrical form and has been manufactured with polycaprolactone (PCL) doped with a representative of tetrazolium salts: 2,3,5−triphenyltetrazolium chloride (TTC). The PCL used makes the dosimeter biodegradable and therefore proecological. The TTC dopant is distributed uniformly in the whole PCL tablet, and the whole tablet changes color to red under UVC irradiation. The intensity of this color increases if the PCL–TTC tablet absorbs higher doses. The color of the tablet is stable for at least 30 days after irradiation. It is proposed that the PCL-TTC tablet be used for measurement with reflectance spectrophotometry in order to determine the reflectance of light versus the absorbed dose in a fast and easy manner. On this basis, the PCL-TTC tablet could be characterized by providing information on its dose range, which amounted to 0–2 J/cm2. Moreover, other parameters were derived, such as dose sensitivity, quasilinear dose range, and dose threshold. The morphology of the tablets studied using scanning electron microscopy revealed their high porosity, which however did not influence the reflectance measurements with the aid of the chosen instrument. UVC irradiation at a dose (15 J/cm2) much above the PCL-TTC tablets’ dose range did not alter the morphology of the tablets. The PCL-TTC tablet read with reflectance spectrophotometry is shown to be a promising and fast method for 1D UV dose measurements.

To ensure clean drinking water, viable pathogens in water must be rapidly and efficiently screened. The traditional culture or spread-plate process—the conventional standard for bacterial detection—is laborious, time-consuming, and unsuitable for rapid detection. Therefore, we developed a colorimetric assay for rapid microorganism detection using a metabolism-based approach. The reaction between a viable microorganism and the combination of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8) and 1-methoxy-5-methylphenazinium methyl sulfate (mPMS) results in a color change. In combination with a microplate reader, WST-8-mPMS reactivity was leveraged to develop a colorimetric assay for the rapid detection of various bacteria. The detection limit of the WST-8-mPMS assay for both gram-negative and gram-positive bacteria was evaluated. This WST-8-mPMS assay can be used to perform colorimetrical semi-quantitative detection of various bacterial strains in buffers or culture media within 1 h without incubation before the reaction. The easy-to-use, robust, rapid, and sensitive nature of this novel assay demonstrates its potential for practical and medical use for microorganism detection.

Background and Aims: Survival of periodontal ligament (PDL) cells after avulsion is an important factor in treatment prognosis. Grapefruit Seed Extract (GSE) can be a proper environment for preserving periodontal ligament cells. The objective of this study was to analyze the effect of different concentrations of GSE on the proliferation of fibroblast PDL cells. Materials and Methods: In this study, the undifferentiated PDL fibroblasts were obtained from two human premolars teeth and cultured in Dulbecco’s modified Eagle’s medium (DMEM). The cultured cells were exposed to different concentrations of GSE. The positive and negative control groups were cultured in fetal bovine serum (FBS) 10% and in a medium without FBS 10%, respectively. The plates were incubated for 1, 6, 12, 24, and 48 hrs. The PDL cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Statistical analysis of data was accomplished using repeated measure ANOVA with Post HOC Tukey, P<0.05 was considered statistically significant. Results: We found out that among different concentrations of GSE, 1:128 had the most impact on undifferentiated PDL fibroblasts. Although, the cell vitality was higher in the twelfth hour, 1:128 GSE and in the forty-eighth hour, 1:1024 GSE than the positive control group but they were not statistically significant (P>0.05). Furthermore, all the samples were similar to the positive control group in three of the five timeperiods (P>0.05). Conclusion: GSE was more effective in fewer concentration and longer periods and it had no toxic effect on PDL cells. Therefore, GSE can be considred as a promoting medium in PDL regeneration of avulsed permanent teeth in the future. [ABSTRACT FROM AUTHOR]

Salvia hispanica L. is an annual herbaceous plant, belonging to the Lamiaceae family, it stands out as the natural resource of plant origin with the highest content of fatty acids known so far. To obtain success in the production of seeds it is necessary to use lots of high quality, which can be evaluated, through the vigor of the same, at present, one of the main requirements for the evaluation of vigor refers to the obtaining of reliable results in a relatively short period of time. The tetrazolium test stands out for being fast and reliable. However, the methodology for the genus Salvia is not referenced within the standards of the International Seed Testing Association (ISTA), considering these facts, it becomes important to carry out the experiment for the development of a protocol that allows this analysis. The research was carried out in the Seed Laboratory of the Federal University of Pelotas, six batches of black chia seeds were used and four concentrations of tetrazolium salt (0.075%, 0.1%, 0.5% and 1%) were tested to evaluate the seed viability. The experimental delineation was in randomized blocks, submitted to the analysis of variance through the F test and subsequently the means compared to each other by the Tukey test, at 5% probability, for comparison of the means. The tetrazolium test conducted in the concentration of 0.075% is efficient to evaluate the viability of the seeds of S. hispanica L., as well as to differentiate lots with different physiological quality. [ABSTRACT FROM AUTHOR]

Summary: Silver(I)-assisted reduction of Blue tetrazolium chloride (3,3'-(3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)-bis(2,5-diphenyl-2H-tetrazolium) dichloride, BTC) by semicarbazide hydrochloride (SCH) was investigated in water-chloroform medium. The obtained colored products can be used for the visual detection and sensitive liquid-liquid extraction- spectrophotometric determination of Ag(I). Under the optimum conditions the calibration curve (which can be best approximated by a third-order polynomial; R2=0.9990) has two linear segments. For Ag(I) concentrations up to 0.011 g/ml, the linear regression equation had an intercept that was statistically indistinguishable from zero. The limit of detection, limit of quantitation, and molar absorptivity coefficient at λmax=573 nm were 0.6 ng/ml, 2 ng/ml and 1.2×106 l/(molcm), respectively. The regression equation of the second linear segment (0.021- 0.028 g/ml) was A = 200γ - 3.5 (R2=0.9989), where γ is the concentration in µg/ml. The relative standard deviation at the 22 ng/ml level (n = 5) was 3.8%. The effect of concomitant ions was studied, and the analysis of real samples tested the applicability of the developed procedure. [ABSTRACT FROM AUTHOR]

A liquid-liquid extraction-chromogenic system for vanadium(IV, V) containing 2,3-dihydroxynaphtahlene (DN), 2,3,5-triphenyl-2H-tetrazolium chloride (TTC), water and chloroform was studied in detail. When the vanadium is in the oxidation state of IV, the extracted species are aggregates containing three 1:2:1 (V:DN:TTC) ion-pair units composed of triphenyltetrazolium cations (TT+) and chelate anions {[VIVO(DN)(DNH)]− (I) and/or [VIV(OH)(DN)2]− (II)}. When the initial oxidation state of vanadium is V and the DN concentration is high, vanadium(V) is reduced by DN to a lower oxidation state, V(IV). However, at low DN concentration, vanadium(V) can enter the organic phase as a part of an ion-pair consisting of TT+ and [VVO2(DN)]− (III). The ground-state equilibrium geometries of the anions I, II, and III were optimized by quantum chemical calculations using BLYP/6-31++G⋆. The following characteristics were determined under the optimum conditions for VIV extraction: absorption maximum λmax = 333 nm, molar absorptivity ε333= 2.1x104 dm3 mol−1 cm−1, Sandell’s sensitivity SS = 2.4 ng cm−2, and fraction extracted E = 98%. The conditional extraction constant was calculated by two independent methods. The calibration graph was linear in the range 0.1-3.1 μg cm−3 (R2=0.9994) and the limit of detection was 0.03 μg cm−3.

The complex formation in the vanadium(V) / 4-(2-thiazolylazo)orcinol (TAO) / 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) liquid-liquid extraction-chromogenic system was studied. The chloroform-extracted complex has a composition of 2:2:2 under the optimum conditions (pH 4.8–5.2, extraction time 3 min, concentration of TAO 3.4´10–4 mol dm–3, and concentration of TTC 9.4´10–4 mol dm–3) and could be regarded as a dimer (D) of two 1:1:1 species (S) presented by the formula (TT+)[VO2(TAO)]. The constant of extraction was calculated by two methods and some analytical characteristics were determined. The wavelength of maximum absorption (lmax), molar absorptivity (el) and fraction extracted (E) were found to be l = 545 nm, e545 = 1.97´104 dm3 mol–1 cm–1, and E = 97.9 %. The ground-state equilibrium geometries of the complexes S and D were optimized by quantum chemical Hartree-Fock calculations using 3-21G* basis functions. The bonding and interaction energies were calculated as well.

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay is a popular tool in estimating the metabolic activity of living cells. The test is based on enzymatic reduction of the lightly colored tetrazolium salt to its formazan of intense purple-blue color, which can be quantified spectrophotometrically. Under properly optimized conditions the obtained absorbance value is directly proportional to the number of living cells. Originally, the MTT assay was devised for use in eukaryotic cells lines and later applied for bacteria and fungi. As the mechanism of MTT reduction was studied in detail mostly considering eukaryotic cells, the lack of information resulted in generating a vast variety of MTT based protocols for bacterial enzymatic activity evaluation. In the presented article the main aspects of the MTT assay applicability in bacterial research were summarized, with special emphasis on sources of inaccuracies and misinterpretation of the test results. [ABSTRACT FROM AUTHOR]

The MTT assay was the first widely accepted method to assess cytotoxicity and cell viability. However, there is controversy on whether this indicator is a useful tool. In this work we intend to expand the interpretability of the MTT study by its combination with widely used cellular biology techniques. We propose complementary approaches to the colorimetric assay, based on the use of measurements in three different settings: confocal microscopy, multi-well plate assay and flow cytometry. Using confocal microscopy, we confirmed that MTT uptake and reduction by cells is a time-dependent process, and that formazan accumulates in round-shaped organelles. Quantitative measurements with a multi-well fluorimeter combined with nuclear staining result in a useful method, yielding a ratio between formazan production and cell number that informs about the average cell metabolic state. We also found that flow cytometry is a suitable technique to measure MTT reduction in large cell populations. When assaying the effect of an oxidizing agent such as paraquat (PQ), this approach allows for the distinction of subpopulations of cells with different reducing power. Finally, we prove that it is feasible to monitor MTT reduction in an in vivo model, the Drosophila larvae, without affecting its survival rate. Formazan accumulates exclusively in the larval fat body, confirming its lipid solubility. The methods explored in this work expand the MTT potential as a useful tool to provide information of the physiological state of cells and organisms. [ABSTRACT FROM AUTHOR]

There is scientific evidence that beach sands are a significant contributor to the pathogen load to which visitors are exposed. To develop beach quality guidelines all beach zones must be included in microbiological evaluations, but monitoring methods for beach sand quality are relatively longstanding, expensive, laborious and require moderate laboratory infrastructure. This paper aimed to evaluate the microorganism activity in different beach zones applying and comparing a classical method of membrane filtration (MF) with two colorimetric screening methods based on fluorescein (FDA) and tetrazolium (TTC) salt biotransformation to evaluate a new rapid and low-cost method for beach sand microbiological contamination assessments. The colorimetric results can help beach managers to evaluate rapidly and at low cost the microbiological quality of different beach zones in order to decide whether remedial actions need to be adopted to prevent exposure of the public to microbes due to beach sand and/or water contamination. [ABSTRACT FROM AUTHOR]

Rapid and simple method for the determination of minimum biofilm eradication concentration (MBEC) using a colorimetric microbial viability assay based on reduction of a tetrazolium salt WST-8 and the biofilm formation method on 96-pegs on the lid of a microtiter plate was developed. The biofilms formed on the 96 pegs were challenged by antibiotics, and the MBEC was then determined from the microbial viability of the biofilms formed on the 96 pegs, assessed by the WST-8 colorimetric assay. The MBECs obtained by the proposed and conventional methods favorably agreed. The most effective inhibitors of S. aureus and P. aeruginosa biofilms were vancomycin and ciprofloxacin, respectively. In addition, we clarified that Staphylococcus aureus biofilm was maximally suppressed by a combination of vancomycin, daptomycin, and teicoplanin. The proposed method yields similar performance to conventional methods, but is faster and more easily implemented. Therefore, the proposed method alleviates the tediousness and time-consuming nature of conventional biofilm susceptibility assay. [ABSTRACT FROM AUTHOR]

The complex formation in the vanadium(V) / 4-(2-thiazolylazo)orcinol (TAO) / 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) liquid-liquid extraction-chromogenic system was studied. The chloroform-extracted complex has a composition of 2:2:2 under the optimum conditions (pH 4.8-5.2, extraction time 3 min, concentration of TAO 3.4 x 10-4 mol dm-3, and concentration of TTC 9.4 x 10-4 mol dm-3) and could be regarded as a dimer (D) of two 1:1:1 species (S) presented by the formula (TT+)[VO2(TAO)]. The constant of extraction was calculated by two methods and some analytical characteristics were determined. The wavelength of maximum absorption (λmax), molar absorptivity (ελ) and fraction extracted (E) were found to be λ = 545 nm, ε545 = 1.97 x 104 dm³ mol-1 cm-1, and E = 97.9%. The ground-state equilibrium geometries of the complexes S and D were optimized by quantum chemical Hartree-Fock calculations using 3-21G* basis functions. The bonding and interaction energies were calculated as well. [ABSTRACT FROM AUTHOR]

[Display omitted] • Neuro-2a cells were immobilised on electrodes of different materials. • Their viability was assessed using electrochemical methods. • Carbon and carbon/polyaniline electrodes provided the best results. • Light microscopy proved the presence of immobilised and living cells on electrodes. • The system was able to detect toxicity in fish extracts. Marine toxins are potent toxic compounds that may reach humans and poison them. Therefore, their detection in seafood is crucial to prevent intoxication cases. Colorimetric cell-based assays (CBAs) have been developed to analyse marine neurotoxins, such as ciguatoxins (CTXs) and tetrodotoxins (TTXs), and are based on the toxicological effect of these toxins on the cells. Cell viability can be quantified by measuring the mitochondrial activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). With the purpose of moving forward in the development of cell-based biosensors (CBBs) for neurotoxins, Neuro-2a cells were immobilised on electrodes of different materials (carbon, carbon/polyaniline, carbon/poly- l -lysine, carbon/poly(3,4-ethylenedioxythiophene) and gold) and their presence and viability were assessed by the detection of MTT formazan crystals with cyclic voltammetry (CV). Best results in terms of oxidation potential and current intensity were achieved with carbon and carbon/polyaniline electrodes. Light microscopy also proved the presence of immobilised and living cells on electrodes. Cell density, incubation time and MTT concentration were optimised. Appropriate electrochemical responses were obtained incubating 100,000 cells/electrode for 2 h and using 0.86 mg/mL MTT. The system was able to detect toxicity when exposed to CTX1B and TTX standard solutions as well as Seriola dumerili and Lagocephalus sceleratus fish extracts containing these toxins. [ABSTRACT FROM AUTHOR]

The possibility of application of the ternary ion-association complex of gallium(III), 4-(2-pyridyl azo)resorcinol (PAR) and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) for extraction-spectrophotometric determination of gallium(III) was studied. The liquid-liquid extraction system Ga(III)-PAR-TTC-HO-CHCl was applied for this purpose. The effect of the foreign ions on the extraction was investigated. Based on the obtained results, a sensitive, relatively simple, and inexpensive method for determination of gallium(III) in a model mixture was developed, which can be implemented in industrial, biological, medical, and pharmaceutical samples. [ABSTRACT FROM AUTHOR]

Extracorporeal photopheresis (ECP) is accepted as a second-line therapy for the treatment of acute and chronic steroid-refractory graft versus host disease (GvHD), cutaneous T-cell lymphoma and solid organ transplantation. ECP should be validated: we compared in parallel apoptosis and proliferation analysis of patient lymphocytes treated with 8-MOP ECP using respectively Annexin V/7-aminoactinomycin D (7-AAD) and CFSE with a tetrazolium salt (WST-1) method. Using WST-1 assay we found a significant decrement (p < 0.01) of metabolic activity at 4 days between ECP-treated and untreated cells. This finding was confirmed by the significant decrease of cell proliferation and increase of cell death observed by CFSE and 7AAD-Annexin V, respectively. Accordingly, once validated against a reference method, WST-1 could represent a rapid and easy assay for routinely quality control of ECP. [ABSTRACT FROM AUTHOR]

Although the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is widely used to measure the growth of animal cells, several interfering factors and obstacles for analyzing bacterial growth and for solubilizing bacterial-formazan have been reported. In this study, growth of Gram-negative Escherichia coli and Gram-positive Leuconostoc mesenteroides were analyzed using the MTT assay, and different solvents, specifically dimethyl sulfoxide, 1% Triton X-100, 1% Bile bovine, 10% sodium dodecyl sulfate, and sodium hydroxide (NaOH) were evaluated for solubilizing bacterial-formazan. MTT formazan that had been reduced by L. mesenteroides had fully developed color within 15 min in all the solvents tested. The solvent with the highest peak absorbance was 1 N NaOH. MTT formazan did not have a fully developed color response in the 0.001–0.1 N NaOH range, and its color intensity was not further enhanced at concentrations higher than 1 N. For all solvents, the color intensity of dissolved formazan was significantly higher in E. coli than in L. mesenteroides. The MTT assay showed sufficient sensitivity to detect bacterial cells during early growth phase that could not be detected by the plate counting method. These results suggest that NaOH (1 N) is a suitable solvent for analyzing bacterial growth in MTT assays. • The MTT-based assays was used for rapid and convenient bacterial growth evaluation. • Different solvents were tested, and MTT-based assay conditions were optimized. • 1–2 N NaOH was optimal for dissolving the bacterial formazan formed in cells. • MTT colorimetric response showed a highly accurate correlation of viable cell number. • Bacterial growth was also directly measured without solvent dissolution. [ABSTRACT FROM AUTHOR]

Background: Determining cerebral infarction volume is an important part of preclinical studies to determine the benefit of potential therapies on stroke outcome. A well-known problem in determining the actual infarction volume of rodent models is the presence of edema. Because of this, algorithms must be utilized to obtain the edema-adjusted ( E A )-infarct volume. Different methods based on 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining have been published describing algorithms to determine the E A -infarct volume., Materials and Methods: Simulated models of infarction and corresponding swelling were employed to determine which absolute method of calculation (Lin et al., Reglodi et al., or Belayev et al.) is the most accurate in calculating the absolute E A -infarct volume., Results: The Reglodi and Belayev methods were statistically more accurate in measuring E A -infarct volume than Lin's method, p = 0.0078. Though there was no significant difference between Reglodi's and Belayev's methods for the E A -infarction volume calculation, Reglodi's approach was closer to the ground-truth infarct volume while also being simpler and more straightforward to use., Conclusion: We recommend that Reglodi's method, that is E A -infarct volume = infarct volume × (contralateral hemisphere/ipsilateral hemisphere), to be used in calculating E A -infarct volume in TTC stained rodent brains. Further, factors such as inhomogeneous infarction distribution in a given brain slice can also contribute to the error in volume calculation. Therefore, the average of the infarct area obtained from anterior and posterior views of a given slice should be used to account for the variation. Considering different factors, we have provided a summary recommendation for calculating the infarction volume.