Improved Formazan Dissolution for Bacterial MTT Assay

ABSTRACT The MTT assay, based on the enzymatic reduction of the water-soluble, yellowish tetrazolium salt 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) to purple formazan, is commonly used for assessment of cell viability and proliferation. Accurate performance by the MTT assay depends on complete solubilization of cells and formazan and stability of the colored solution. Comparison of different solubilization solutions revealed that dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), buffered with ammonia buffer, pH 10, and containing 5% SDS, produced the best results. These two solvents provided rapid and complete solubilization of formazan and cells, with minimal background absorbance at 700 nm, good reproducibility (low interassay coefficient of variation), high sensitivity, and color stability for at least 24 h. A linear relationship between viable-cell number and formazan absorbance was preserved for cell densities up to ∼1 × 109 cells/mL for Gram-negative and Gram-positive microorganisms. Since MTT can be reduced by medium components in the absence of cells, blanks containing all medium components but no cells should be run simultaneously. Measurements at two wavelengths, one corresponding to absorption peak of formazan (570 nm) and a background absorbance far from the peak (700 nm), are necessary to avoid artifacts due to incomplete solubilization and turbidity. IMPORTANCE Reduction of the water-soluble tetrazolium salt 3-(4,5-dimethylthiazol)-2,5 diphenyl-tetrazolium bromide (MTT) to purple, water-insoluble formazan is commonly used for assessment of cell viability and proliferation. Spectrophotometric detection of formazan requires its solubilization. The solubilization solvent has a strong influence on data acquisition and often introduces artifacts, leading to misreading of results. This study offers a choice of solvents that minimize solubilization artifacts when the MTT test is applied to microbiological cultures.

7. Discussion of the paper has to be modified using with some recent references and some excessive explanations have to be removed. 8. Excessive supplementary data should be summarized and removed. 9. Line 97: Methods (inoculation, centrifugation,,,) and the sequence of experiments should be described in more detail.

Reviewer #2 (Comments for the Author):
In this manuscript the authors explore new methods for solubilization of formazan formed from MTT in an effort to improve the accuracy of laboratory procedures using MTT as an indication of viability. Major comments: 1) The authors measured absorbace at 570 and 600 with the 600 reading serving as a background measurement outside of the maxmum of formazon peak absorbance. However, all graphs depict only the AB570 measurement and do not adjust for the 600 nm background measurement. The authors even recomend in the conclusion on page 19 that measurements at two wave lengths be conducted (570 and 600 nm). For this reason, I believe the presented data should include deduction of the background. 2. The experiment measuring MTT conversion in culture of Staphlococcus aureus differs in that the graphs do not show measurements at 1, 2 and 24 hr but only 1 measurement. Text and figure legend do not provide the information on the incubation time for this set of data. 3. One might argue whether statistical analysis would be beneficial or not for this set of data. However, particularly in Fig 4, I believe there would be benefits for statistical analysis comparing OD with the different solubilization solutions. For example was OD for the ammonia-DMSO greater than the other 2 solutions? Because of the nature of the curve generated for the assay it may be difficult to develop a regression analysis that would allow comparison of different treatments. However, I would suggest the authors consider the possibility of doing this type of analysis. Figures 1, 2, and 3 get rather tedious. Would it be possible to prepare a graph presenting only 1 incubation time for each compound and statistically compare the OD at different concentrations (of formazan or bacteria). The presentation of the data in the figure could be more comparative, and information on stability over time in incubation, turbidity, and other data could then be discussed in the text. This suggestion is supported by the strong correlation in curves across incubations This might make the data more comprehensible to readers. This would also provide clarity on what constituted the most desired results for each experiments (most likely maximum 570 abs and minimum 600 nm absorbance). 4) In the methods on line 190-110, the 570 nm wavelength should be specifically stated. On line 115 I would suggest listing the dual wavelengths in parentheses so that the reader is clear on what wavelengths were used. 5) On line 119, I believe "decreed" should be "decreased". 6) Lines 257 to 258 are confusing to this reviewer. I'm unclear on how the methods would detect a shift in the formazan peak since the authors were only measuring at 570 nm. 7) Lastly, I think the readability of the paper would be improved if the authors articulated what constituted the best solvent for solubilization of formazan. I would think it would be maximal absorbance at 570 nm (for a particular formazan or bacterial concentration), minimal variance, and minimal absorbance at 600 nm.

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This research paper is good and could provide that 5 % SDS in DMF or in DMSO are suitable solvents for solubilizing bacterial formazan crystal using MTT colorimetric method.
However, there are some overlapping information with the literature for some part of the paper.
1. Discuss the originality (contribution, addition of knowledge to scientific literature or field) of the manuscript.
2. The abstract of the MS is not well documented to represent the whole manuscript objectives. Thus, it should be rewritten again in order to represent the paper sufficiently.
3. It will be useful to convert figure data to one table since there are not table. 4. Statistical analysis have to be discussed comprehensively in the results and discussion section as well.
5. Line 43: Additional experiments on misreading results caused by interference factors (media, reagents, samples) are needed.
6. Clearly identify the strengths and weaknesses of the method described in the manuscript.
7. Discussion of the paper has to be modified using with some recent references and some excessive explanations have to be removed.
8. Excessive supplementary data should be summarized and removed. 9. Line 97: Methods (inoculation, centrifugation,,,) and the sequence of experiments should be described in more detail.

Response to reviewers
Reviewer #1 (Comments for the Author): I am grateful to Reviewer # 1 for the useful suggestions and recommendations for improving this manuscript. All suggestions were taken into consideration and suitable changes have been introduced in the revised version.
This research paper is good and could provide that 5 % SDS in DMF or in DMSO are suitable solvents for solubilizing bacterial formazan crystal using MTT colorimetric method. However, there are some overlapping information with the literature for some part of the paper. 1. Discuss the originality (contribution, addition of knowledge to scientific literature or field) of the manuscript.

-This has been done by describing the effect of buffering and addition of SDS to tested organic solvents (Results & Discussion section)
2. The abstract of the MS is not well documented to represent the whole manuscript objectives. Thus, it should be rewritten again in order to represent the paper sufficiently.

-The abstract has been rewritten
3. It will be useful to convert figure data to one table since there are not table.

-Due to the character of the data, attempt to convert figures to a table, resulted in a confusing set of numbers. A table, however, has been added, summarizing the results of the linear regression analysis (Suppl. Table 1).
4. Statistical analysis have to be discussed comprehensively in the results and discussion section as well.
-Additional statistical analyses were performed (linear regression, and coefficient of variation), results were presented as Figs. 2 D and 3 D, Suppl. Fig. S7, and Suppl. Table 1. Details were given in the methodological section, and results were discussed in the Results and Discussion section. 5. Line 43: Additional experiments on misreading results caused by interference factors (media, reagents, samples) are needed.

-Distortion of results caused by interference factors, including precipitation, crystallization, low solubilization, shifts of absorption peaks, and instability of solutions have been demonstrated in the presented figures incorporated in the main text and in the supplementary figures, and have been discussed in the Results and Discussion section.
6. Clearly identify the strengths and weaknesses of the method described in the manuscript.

-The advantages and disadvantages of each tested solvent have been discussed at places
where data about the performance of the solvent were presented.
7. Discussion of the paper has to be modified using with some recent references and some excessive explanations have to be removed.

-Thorough literature search produced only two recent publications relevant to the topic of this study and they were added to the references list (Ref. 22 & 26)
8. Excessive supplementary data should be summarized and removed.
-Supplementary material has been shortened and excessive figures have been removed 2 9. Line 97: Methods (inoculation, centrifugation,,,) and the sequence of experiments should be described in more detail.

-Details about preparation and manipulation of samples have been added to the experiment section
. Reviewer #2 (Comments for the Author): I appreciate the time and efforts spent by Reviewer #2 for improving this manuscript. I am grateful for the criticism and believe all suggestions were properly addressed and suitable corrections were done.
In this manuscript the authors explore new methods for solubilization of formazan formed from MTT in an effort to improve the accuracy of laboratory procedures using MTT as an indication of viability. Major comments: 1) The authors measured absorbace at 570 and 600 with the 600 reading serving as a background measurement outside of the maxmum of formazon peak absorbance. However, all graphs depict only the AB570 measurement and do not adjust for the 600 nm background measurement. The authors even recomend in the conclusion on page 19 that measurements at two wave lengths be conducted (570 and 600 nm). For this reason, I believe the presented data should include deduction of the background.
-All figures now present OD at the formazan peak (570 nm for all solvents except ammonia DMSO which has a max. at 550 nm) with the background absorbance at 700 nm deducted.  Table 1. In addition, inter-assay coefficient of variation has been calculated. Results were presented as Suppl. Fig. S7 and discussed in the Results and Discussion section. Figures 1, 2, and 3 get rather tedious. Would it be possible to prepare a graph presenting only 1 incubation time for each compound and statistically compare the OD at different concentrations (of formazan or bacteria). The presentation of the data in the figure could be more comparative, and information on stability over time in incubation, turbidity, and other data could then be discussed in the text. This suggestion is supported by the strong correlation in curves across incubations This might make the data more comprehensible to readers. This would also provide clarity on what constituted the most desired results for each experiments (most likely maximum 570 abs and minimum 600 nm absorbance).

Descriptions of experiments presented in
-Results obtained at different incubation times are presented to give the reader a direct perception of the stability of formazan in different solvents, to show advantages and disadvantages of the tested solvents, and to demonstrate how artifacts distort the readings.

3
One set of figures (former Fig. 2

, A-F) has been removed and results described in the text. Figs 2 D and 3 D now present regression analysis lines of only one time point (1 hour).
4) In the methods on line 190-110, the 570 nm wavelength should be specifically stated. On line 115 I would suggest listing the dual wavelengths in parentheses so that the reader is clear on what wavelengths were used.
-Formazan absorption maximum wavelengths (570 nm for all solvents except ammonia DMSO, which shows a max. at 550 nm) have been added to the text. 5) On line 119, I believe "decreed" should be "decreased".
-The typo has been corrected 6) Lines 257 to 258 are confusing to this reviewer. I'm unclear on how the methods would detect a shift in the formazan peak since the authors were only measuring at 570 nm.
-A shift of formazan peak has been determined by recording and comparing absorption spectra. Selected spectra have been shown as supplementary figures. The paragraph mentioned above has been modified.

7)
Lastly, I think the readability of the paper would be improved if the authors articulated what constituted the best solvent for solubilization of formazan. I would think it would be maximal absorbance at 570 nm (for a particular formazan or bacterial concentration), minimal variance, and minimal absorbance at 600 nm.