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In vitro Antimicrobial Activity Assessment of Zymox Otic Solution Against a Broad Range of Microbial Organisms

Rajvinder Atwal, PhD*

 

*Laclede Inc., Rancho Dominguez, California

 

This research was performed in the Laboratory Services Division, TherImmune Research Corporation, Gaithersburg, Maryland.

 

KEY WORDS: Zymox Otic solution, antimicrobial activity, bacteria, microorganisms

 

Abstract

The goal of this study was to assess the in vitro antimicrobial activity of Zymox Otic solution against a broad panel of microbial organisms. Serial twofold dilutions of Zymox Otic were used in microbroth dilution assays against 3 Gram-negative bacteria (Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa), 4 Gram-positive bacteria (Staphylococcus aureus, Streptococcus intermedius, Streptococcus pyogenes, and Corynebacterium diphtheriae), and 2 fungi (Candida albicans and Malassezia pachydermatis). The results of these assays showed that Zymox Otic inhibited the growth of every organism tested at various test compound dilutions. Zymox Otic had the greatest amount of antimicrobial activity against S. pyogenes exhibiting a minimal inhibitory concentration (MIC) at a 1:32 dilution of the test compound. A 1:16 dilution of Zymox Otic effectively inhibited the growth of S. aureus, E. coli, S. intermedius, C. albicans, P. aeruginosa, C. diphtheriae, and M. pachydermatis; and a 1:8 dilution of Zymox Otic effectively inhibited the growth of P. mirabilis.

Materials and Methods

The materials and reagents were used in the study are shown in Table 1.

The minimal inhibitory concentration (MIC) of Zymox Otic against each microorganism was determined using microbroth dilution analysis. Bacterial and fungal strains were obtained from American Type Culture Collection (ATCC) and cultured according to the manufacturer's specifications. For each organism tested, 10 mL of sterile liquid broth media was inoculated with a freshly plated, single-colony isolate and incubated overnight to establish logarithmic growth. The specific media and growth condition used for the individual organisms is detailed in Table 2. After overnight incubation, the bacteria or fungus was pelleted by centrifugation (3100 x g, 5 min), the supernatant discarded, and the pellet suspended in 0.9% sterile saline solution (9 g/L sodium chloride) to an optical density at 625 nm equal to 0.1. This density was assumed to be equivalent to 1 x 108 colony-forming units (CFU)/mL for dosing purposes. The suspended inoculum was diluted 1:100 in sterile culture media to a concentration of 1 x 106 CFU/mL. One hundred microliters of the diluted inoculum was added to triplicate wells of a 96-well plate containing 100 mL of freshly prepared Zymox Otic solution serially diluted twofold in the respective sterile culture media. One hundred microliters of the diluted inoculum was also added to triplicate wells containing 100 mL of serial twofold dilutions of a positive control antibiotic and to triplicate wells containing 100 mL of media only. This inoculation scheme yielded final concentrations of each microbial organism estimated to be 5 x 105 CFU/mL and Zymox Otic solution ranging from a high-test dilution of 1:2 (inoculum mixed with 100 mL of undiluted Zymox Otic) to a low test of 1:2048. The plates were incubated 16 to 48 hours at 370C, and the inhibition of bacterial growth was determined by measuring the optical density at 625 nm for of each well using a Spectramax 384 plate reader. The MIC for each compound was defined as the lowest compound dilution that completely inhibited growth. The results are presented as the percentage of growth relative to the no-compound control and were calculated using the formula:

 

100% x OD625X

OD625NC

 

where OD625X is the average OD625 of the triplicate test samples and OD625NC is the average OD625 of the triplicate no compound control samples.

Results and Discussion

The results of our analysis indicated that Zymox Otic had antimicrobial activity against all organisms tested (Table 3 and Figs. 1 through 9). Zymox Otic had the greatest amount of antimicrobial activity against Streptococcus pyogenes, exhibiting an MIC at a 1:32 dilution of the original test compound. A 1:16 dilution of Zymox Otic effectively inhibited the growth of Staphylococcus aureus, Escherichia coli, Streptococcus intermedius, Candida albicans, Pseudomonas aeruginosa, Corynebacterium diphtheriae, and Malassezia pachydermatis; and a 1:8 dilution of Zymox Otic effectively inhibited the growth of Proteus mirabilis. All of the control antimicrobial compounds had effective MICs within published ranges for the organisms tested. The Zymox Otic compound formed a gelatinous precipitate at the highest concentrations (1:2 and 1:4 dilution, Figs. 1 through 9 panel C) leading to an increase in the measured optical density at the high-test concentrations relative to the intermediate concentrations.  For Malassezia pachydermatis (Fig. 9, panels A and C), one well for each of the test concentration triplicates had a significantly higher OD625 than the other 2 replicates. Examination of these wells indicated that the fungus failed to evenly disperse during culturing and formed a mat on the top of the culture media. The presence of this mat inflated the optical density measured for these wells. Because this phenomenon was not observed in the no-compound control wells, the graph of the M. pachydermatis results that includes these data indicates an artificial enhancement of growth by the Zymox Otic compound at the higher concentrations (Fig. 9 panel C).

 

 

 

Table 1. Materials and Reagents Used in the Study

 

Staphylococcus aureus                                             ATCC                     29213

Escherichia coli                                                        ATCC                     25922

Streptococcus pyogenes                                           ATCC                     10389

Proteus mirabilis                                                       ATCC                     14153

Streptococcus intermedius                                       ATCC                     27335

Candida albicans                                                      ATCC                     10231

Pseudomonas aeruginosa                                        ATCC                     27853

Corynebacterium diphtheriae                                   ATCC                     14779

Malassezia pachydermatis                                       ATCC                     42756

96-well U-bottom plates                                             Falcon                    353227

TSA II 5% sheep blood agar plates                            Remel                     01202

Disposable bacterial spreaders                                 VWR                      WLS23286

Trypticase soy agar                                                   BBL                        211043

YM broth                                                                    Difco                      271120

YM agar                                                                     Difco                      271210

Sabouraud dextrose broth                                         Difco                      238220

Nutrient broth                                                             Difco                      233000

Brain heart infusion media                                         Remel                     452471

Sabouraud dextrose agar emmons                           Remel                     454471

Trypticase soy broth                                                  BD                          211768

Saline 0.9%                                                                LC234601              Fisher

Mueller Hinton II broth                                                 BD                          297310
(Cation adjusted)

Ampicillin                                                                    Fisher                     BP1760-25

Gentamycin                                                                Sigma                     G1522

Amphotericin B                                                           Fisher                     BP264520

Bifinazole                                                                   Fisher                     ICN15485601

Zymox Otic solution                                                   Laclede                 

 

Table 2. Growth Media and Culture Conditions Used to Culture and Assess the Antimicrobial Activity of Zymox Otic for Each Respective Organism

 

Staphylococcus aureus                            Trypticase soy broth                        37˚C/ambient air/16 hr

Escherichia coli                                       Trypticase soy broth                        37˚C/ambient air/16 hr

Streptococcus pyogenes           Mueller Hinton II broth (Cation adjusted)         37˚C/ambient air/16 hr

Proteus mirabilis                       Mueller Hinton II broth (Cation adjusted)         37˚C/ambient air/16 hr

Streptococcus intermedius                  Brain heart infusion media                      37˚C/5% CO2/16 hr

Candida albicans                                              YM broth                                 37˚C/ambient air/24 hr

Pseudomonas aeruginosa         Mueller Hinton II broth (Cation adjusted)         37˚C/ambient air/16 hr

Corynebacterium diphtheriae                  Trypticase soy broth                        37˚C/ambient air/24 hr

Malassezia pachydermatis                  Sabouraud dextrose broth                   37˚C/ambient air/48 hr

 

 

 

Table 3. Minimal Inhibitory Concentration (MIC) of Zymox Otic and Control Compounds

Staphylococcus aureus                              Ampicillin (mg/mL)                                                1

                                                                      Zymox (dilution)                                               1:16

Escherichia coli                                          Ampicillin (mg/mL)                                               16

                                                                      Zymox (dilution)                                               1:16

Streptococcus pyogenes                            Ampicillin (mg/mL)                                              0.04

                                                                      Zymox (dilution)                                               1:32

Proteus mirabilis                                        Ampicillin (mg/mL)                                                2

                                                                      Zymox (dilution)                                                1:8

Streptococcus intermedius                        Ampicillin (mg/mL)                                               25

                                                                      Zymox (dilution)                                               1:16

Candida albicans                                   Amphotericin B (mg/mL)                                         1.56

                                                                      Zymox (dilution)                                               1:16

Pseudomonas aeruginosa                        Gentamicin (mg/mL)                                            0.78

                                                                      Zymox (dilution)                                               1:16

Corynebacterium diphtheriae                     Ampicillin (mg/mL)                                              0.39

                                                                      Zymox (dilution)                                               1:16

Malassezia pachydermatis                       Bifonazole (mg/mL)                                             0.1

                                                                      Zymox (dilution)                                               1:16

 

The listed MIC for each of the control compounds is given in µg/mL and the MIC for Zymox is listed as a dilution of the original starting material in the respective growth media

 

Figure 1. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (C) against Staphylococcus aureus.

 

Figure 1A

Ampicillin (µg/mL)  256          128          64            32            16            8              4              2              1              0.5           0.25         0

Sample 1  0.043       0.042       0.045       0.049       0.042       0.199       0.583       1.053       1.206       1.235       1.234       1.279

Sample 2  0.043       0.042       0.045       0.046       0.047       0.199       0.648       1.042       1.166       1.196       1.203       1.236

Sample 3  0.042       0.044       0.052       0.067       0.05         0.147       0.606       1.028       1.175       1.193       1.204       1.229

Average   0.043       0.043       0.047       0.054       0.046       0.182       0.612       1.041       1.182       1.208       1.214       1.248

% Control                3.4           3.4           3.8           4.3           3.7           14.6         49            83            95            97            100          100

                                                                                                                                                                                               

Zymox (dilution)     1:2           1:4           1:8           1:16         1:32         1:64         1:128       1:256       1:512       1:1024     1:2048     0

Sample 1  0.271       0.054       0.066       0.057       1.119       1.09         0.985       0.95         0.926       0.939       1.024       1.223

Sample 2  0.108       0.256       0.117       0.062       1.054       1.092       0.995       0.95         0.952       0.942       1.042       1.217

Sample 3  0.177       0.066       0.062       0.061       1.074       1.096       0.996       0.979       0.967       0.983       1.058       1.272

Average   0.185       0.125       0.082       0.060       1.082       1.093       0.992       0.960       0.948       0.955       1.041       1.237

% Control                15.0         10.0         6.6           5.8           87.0         88.0         80.0         78.0         77.0         77.0         84            100

 

Figure 1C

 

Figure 1B

 

Figure 2A.

Ampicillin (µg/mL)  256          128          64            32            16            8              4              2              1              0.5           0.25         0

Sample 1  0.043       0.042       0.045       0.049       0.042       0.199       0.583       1.053       1.206       1.235       1.234       1.279

Sample 2  0.043       0.042       0.045       0.046       0.047       0.199       0.648       1.042       1.166       1.196       1.203       1.236

Sample 3  0.042       0.044       0.052       0.067       0.05         0.147       0.606       1.028       1.175       1.193       1.204       1.229

Average   0.043       0.043       0.047       0.054       0.046       0.182       0.612       1.041       1.182       1.208       1.214       1.248

% Control                3.4           3.4           3.8           4.3           3.7           14.6         49.1         83.4         94.7         96.8         97.2         100

                                                                                                                                                                                               

Zymox (dilution)     1:2           1:4           1:8           1:16         1:32         1:64         1:128       1:256       1:512       1:1024     1:2048     0

Sample 1  0.271       0.054       0.066       0.057       1.119       1.09         0.985       0.95         0.926       0.939       1.024       1.223

Sample 2  0.108       0.256       0.117       0.062       1.054       1.092       0.995       0.95         0.952       0.942       1.042       1.217

Sample 3  0.177       0.066       0.062       0.061       1.074       1.096       0.996       0.979       0.967       0.983       1.058       1.272

Average   0.185       0.125       0.082       0.060       1.082       1.093       0.992       0.960       0.948       0.955       1.041       1.237

% Control                15.0         10.1         6.6           4.8           87.5         88.3         80.2         77.6         76.6         77.2         84.2         100

 

Figure 2. Determination of minimal inhibitory concentration of ampicillin (B)  and Zymox Otic (C) against Escherichia coli.

 

Figure 2C

 

Figure 2B

 

Figure 3. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (C) against Streptococcus pyogenes.

 

Figure 3A.

Ampicillin (µg/mL)  5              2.5           1.25         0.625       0.31         0.156       0.078       0.039       0.019       0.009       0.005       0

Sample 1  0.309       0.307       0.306       0.308       0.304       0.307       0.303       0.306       0.699       0.942       1.008       1.054

Sample 2  0.311       0.304       0.306       0.306       0.31         0.307       0.303       0.304       0.819       0.996       1.059       1.097

Sample 3  0.309       0.308       0.306       0.306       0.307       0.305       0.305       0.306       0.81         0.998       1.071       1.119

Average   0.310       0.306       0.306       0.307       0.307       0.306       0.304       0.305       0.776       0.979       1.046       1.090

% Control                28.4         28.1         28.1         28.1         28.2         28.1         27.9         28.0         71.2         89.8         96.0         100.0

                                                                                                                                                                                               

Zymox (dilution)     1:2           1:4           1:8           1:16         1:32         1:64         1:128       1:256       1:512       1:1024     1:2048     0

Sample 1  1.15         1.282       0.387       0.329       0.327       0.99         1.267       1.262       1.233       1.206       1.12         1.124

Sample 2  0.934       1.297       0.404       0.339       0.328       0.869       1.085       1.085       1.112       1.099       1.073       1.098

Sample 3  1.034       0.576       0.365       0.342       0.342       0.916       1.051       1.112       1.077       1.035       1.05         1.053

Average   1.039       1.052       0.385       0.337       0.332       0.925       1.134       1.153       1.141       1.113       1.081       1.092

%Control 95.2         96.3         35.3         30.8         30.4         84.7         103.9       105.6       104.5       102.0       99.0         100.0

 

Figure 3C

 

Figure 3B

 

Figure 4. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (C) against Proteus mirabilis.

 

Figure 4A.

Ampicillin (µg/mL)  100          50            25            12.5         6.25         3.125       1.56         0.78         0.39         0.195       0.09         0

Sample 1  0.057       0.046       0.045       0.046       0.049       0.054       0.1           0.958       1.104       1.063       1.041       0.966

Sample 2  0.045       0.043       0.048       0.046       0.048       0.05         0.091       0.935       1.154       1.115       1.075       0.881

Sample 3  0.046       0.043       0.048       0.045       0.045       0.05         0.095       0.931       1.136       1.067       1.052       0.837

Average   0.049       0.044       0.047       0.046       0.047       0.051       0.095       0.941       1.131       1.082       1.056       0.895

%Control 5.5           4.9           5.3           5.1           5.3           5.7           10.7         105.2       126.5       120.9       118.0       100

                                                                                                                                                                                               

Zymox (dilution)     1:2           1:4           1:8           1:16         1:32         1:64         1:128       1:256       1:512       1:1024     1:2048     0

Sample 1  0.265       0.276       0.071       0.331       0.877       1.006       1.026       0.988       0.957       0.931       0.906       0.875

Sample 2  0.081       0.381       0.078       0.279       0.862       0.941       0.964       0.916       0.878       0.845       0.849       0.82

Sample 3  0.102       0.306       0.075       0.44         0.855       1.058       1.005       0.983       0.883       0.91         0.922       0.947

Average   0.149       0.321       0.075       0.350       0.865       1.002       0.998       0.962       0.906       0.895       0.892       0.881

%Control 17.0         36.4         8.5           39.7         98.2         113.7       113.4       109.3       102.9       101.7       101.3       100.0

 

Figure 4C

 

Figure 4B

 

Figure 5. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (C) against Streptococcus intermedius.

 

Figure 5A.

Ampicillin (µg/mL)  100          50            25            12.5         6.25         3.125       1.56         0.78         0.39         0.195       0.098       0

Sample 1  0.059       0.068       0.13         0.208       1.497       1.62         1.654       1.611       1.554       1.629       1.612       1.633

Sample 2  0.065       0.068       0.071       0.152       1.347       1.595       1.525       1.512       1.508       1.519       1.525       1.531

Sample 3  0.063       0.065       0.069       0.166       1.271       1.508       1.556       1.53         1.521       1.524       1.527       1.615

Average   0.062       0.067       0.090       0.175       1.372       1.574       1.578       1.551       1.528       1.557       1.555       1.593

% Control                3.9           4.2           5.6           11.0         86.1         98.8         99.1         97.4         95.9         97.8         97.6         100

                                                                                                                                                                                               

Zymox (dilution)     1:2           1:4           1:8           1:16         1:32         1:64         1:128       1:256       1:512       1:1024     1:2048     0

Sample 1  0.157       0.197       0.192       0.21         1.52         1.339       1.318       1.324       1.329       1.35         1.434       1.633

Sample 2  0.094       0.249       0.21         0.105       1.48         1.372       1.348       1.358       1.374       1.362       1.426       1.531

Sample 3  0.124       0.157       0.179       0.127       1.559       1.406       1.392       1.411       1.404       1.42         1.515       1.615

Average   0.125       0.201       0.194       0.147       1.520       1.372       1.353       1.364       1.369       1.377       1.458       1.593

% Control                7.8           12.6         12.2         9.2           95.4         86.1         84.9         85.6         85.9         86.5         91.5         100.0

 

Figure 5C

 

Figure 5B

 

Figure 6. Determination of minimal inhibitory concentration of amphotericin B (B) and Zymox Otic (C) against Candida albicans.

 

Figure 6A.

Amphotericin B (µg/mL)          100          50            25            12.5         6.25         3.125       1.56         0.78         0.39         0.195       0.098       0

Sample 1  0.05         0.046       0.044       0.044       0.045       0.047       0.045       0.713       0.925       0.974       1.024       1.107

Sample 2  0.056       0.061       0.047       0.059       0.052       0.051       0.046       0.855       0.947       0.92         1.013       1.153

Sample 3  0.062       0.051       0.048       0.046       0.043       0.047       0.044       1.121       0.818       1.201       1.145       1.111

Average   0.056       0.053       0.046       0.050       0.047       0.048       0.045       0.896       0.897       1.032       1.061       1.124

%Control 5.0           4.7           4.1           4.4           4.2           4.3           4.0           79.8         79.8         91.8         94.4         100

                                                                                                                                                                                               

Zymox (dilution)     1:2           1:4           1:8           1:16         1:32         1:64         1:128       1:256       1:512       1:1024     1:2048     0

Sample 1  0.107       0.087       0.071       0.055       1.146       1.33         1.317       1.189       1.142       1.086       1.202       1.107

Sample 2  0.103       0.087       0.065       0.057       0.942       1.177       1.139       1.141       1.125       1.049       1.003       1.153

Sample 3  0.091       0.084       0.057       0.056       1.02         1.241       1.138       1.241       1.091       0.971       1.039       1.111

Average   0.100       0.086       0.064       0.056       1.036       1.249       1.198       1.190       1.119       1.035       1.081       1.124

%Control 8.9           7.7           5.7           5.0           92.2         111.2       106.6       105.9       99.6         92.1         96.2         100.0

 

 

Figure 6C

 

Figure 6B

 

Figure 7. Determination of minimal inhibitory concentration of gentamicin (B) and Zymox Otic (C) against Pseudomonas aeruginosa.

 

Figure 7A.

Gentamicin (µg/mL) 100          50            25            12.5         6.25         3.125       1.56         0.78         0.39         0.195       0.098       0

Sample 1  0.042       0.041       0.041       0.041       0.041       0.042       0.042       0.045       0.446       1.268       1.289       1.412

Sample 2  0.044       0.042       0.043       0.041       0.042       0.042       0.042       0.046       0.478       1.182       1.241       1.343

Sample 3  0.048       0.042       0.053       0.042       0.04         0.039       0.039       0.055       0.399       1.136       1.225       1.394

Average   0.045       0.042       0.046       0.041       0.041       0.041       0.041       0.049       0.441       1.195       1.252       1.383

% Control                3.2           3.0           3.3           3.0           3.0           3.0           3.0           3.5           31.9         86.4         90.5         100

                                                                                                                                                                                               

Zymox (dilution)     1:2           1:4           1:8           1:16         1:32         1:64         1:128       1:256       1:512       1:1024     1:2048     0

Sample 1  0.197       0.251       0.068       0.077       1.303       1.146       1.278       1.328       1.279       1.26         1.278       1.412

Sample 2  0.093       0.278       0.081       0.074       1.194       1.121       1.233       1.259       1.25         1.284       1.264       1.343

Sample 3  0.094       0.215       0.072       0.071       1.275       1.163       1.299       1.337       1.304       1.282       1.3           1.394

Average   0.128       0.248       0.074       0.074       1.257       1.143       1.270       1.308       1.278       1.275       1.281       1.383

% Control                9.3           17.9         5.3           5.4           90.9         82.7         91.8         94.6         92.4         92.2         92.6         100.0

 

Figure 7C

 

Figure 7B

 

Figure 8. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (c) against Corynebacterium diphtheriae.

 

Figure 8A.

Ampicillin (µg/mL)  100          50            25            12.5         6.25         3.125       1.56         0.78         0.39         0.195       0.098       0

Sample 1  0.05         0.049       0.049       0.048       0.048       0.049       0.049       0.049       0.05         0.157       0.215       0.22

Sample 2  0.05         0.049       0.049       0.055       0.05         0.048       0.063       0.059       0.049       0.177       0.208       0.226

Sample 3  0.048       0.047       0.049       0.049       0.049       0.048       0.048       0.048       0.048       0.171       0.208       0.236

Average   0.049       0.048       0.049       0.051       0.049       0.048       0.053       0.052       0.049       0.168       0.210       0.227

% Control                21.7         21.3         21.6         22.3         21.6         21.3         23.5         22.9         21.6         74.0         92.5         100

                                                                                                                                                                                               

Zymox (dilution)     1:2           1:4           1:8           1:16         1:32         1:64         1:128       1:256       1:512       1:1024     1:2048     0

Sample 1  0.191       0.206       0.141       0.058       0.154       0.167       0.176       0.188       0.197       0.219       0.236       0.22

Sample 2  0.101       0.268       0.196       0.061       0.172       0.204       0.216       0.225       0.227       0.232       0.246       0.226

Sample 3  0.12         0.126       0.18         0.064       0.175       0.231       0.225       0.233       0.239       0.25         0.256       0.236

Average   0.137       0.200       0.172       0.061       0.167       0.201       0.206       0.215       0.221       0.234       0.246       0.227

%Control 60.4         88.0         75.8         26.8         73.5         88.3         90.5         94.7         97.2         102.8       108.2       100.0

 

Figure 8C

 

Figure 8B

 

Figure 9. Determination of minimal inhibitory concentration of Bifonazole (B) and Zymox Otic (C) against Malassezia pachydermatis.

 

Figure 9A.

Bifonazole (mg/mL) 25            12.5         6.25         3.125       1.56         0.781       0.39         0.195       0.0975     0.049       0.024       0

Sample 1  0.056       0.062       0.06         0.074       0.064       0.058       0.059       0.07         0.065       0.061       0.504       0.573

Sample 2  0.058       0.06         0.061       0.083       0.075       0.077       0.076       0.075       0.064       0.233       0.545       0.631

Sample 3  0.051       0.053       0.057       0.061       0.072       0.058       0.078       0.0650.078              0.075       0.064       0.162       0.544

Average   0.055       0.058       0.059       0.073       0.070       0.064       0.071       0.073       0.068       0.119       0.404       0.583

% Control                9.4           10.0         10.2         12.5         12.1         11.0         12.2         12.4         11.7         20.5         69.3         100

                                                                                                                                                                                               

Zymox (dilution)     1:2           1:4           1:8           1:16         1:32         1:64         1:128       1:256       1:512       1:1024     1:2048     0

Sample 1  0.282       0.2           0.159       0.07         0.18         1.743       1.747       0.428       1.954       1.894       1.954       0.573

Sample 2  0.509       0.231       0.149       0.068       0.061       0.584       0.5           0.377       0.435       0.583       0.462       0.631

Sample 3  0.497       0.193       0.177       0.065       0.16         0.416       0.496       1.894       0.5           0.527       0.536       0.544

Average   0.429       0.208       0.162       0.068       0.134       0.914       0.914       0.900       0.963       1.001       0.984       0.583

%Control 73.7         35.7         27.7         11.6         22.9         156.9       156.9       154.4       165.3       171.9       168.9       100.0

 

Figure 9C

 

Figure 9B

 

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