a sterile 6 mm cork borer, 4 equally spaced holes were bored in the agar plate with a fifth hole in the center of
the plate. Fifty microliters (50 µL) of the hand sanitizer was then introduced into each of the 4 wells while the
central well was filled with an equal volume of sterile water to serve as a control. This was done for all the test
organisms and hand sanitizers. The plates were incubated for 24 hours at 37ºC in an upright position. They
were then examined for zones of inhibition. The test was carried out in duplicates and the average of two
readings was taken as the zone of inhibition in each case. Inhibition zones were measured with the aid of a ruler
and all the measurement was taken as millimeter .
Determination of minimum inhibitory concentration (MIC)
MIC was carried out to determine the lowest concentration of test substances needed to prevent the
growth of a given organism in vitro . Various concentrations of the sanitizers were prepared in ascending
order (40%, 60%, 80%, and 100%). In case of iodine solution, a formulation of 0.5%, 1%, 1.5%, and 2% of iodine
solutions were used. The tubes were incubated for 24 hours at 37˚C and examined for visible growth or
turbidity. The concentration of the sanitizer at which no visible growth was observed compared with the
controls, was regarded as the MIC .
Determination of minimum bactericidal concentration (MBC)
MBC is the lowest concentration of a specific antimicrobial that kills 99.9% of cells of a given bacterial
strain . MBC was determined by assaying for live organisms in the tubes from the MIC tests which have
shown no visible growth. A loop full of inoculums from the MIC tubes was streaked on fresh nutrient agar
plates without the hand sanitizer incorporated into them. The plates were observed for growth after incubated
at 37ºC for 24 hours. Absence of growth indicated a bactericidal effect of the sanitizers at that concentration
which is the MBC.
Determination of efficacy of hand sanitizers in reducing viable counts of bacteria on the hands of
All the three commercial hand sanitizers and Lugol's iodine were further evaluated for their efficacy in
reducing baseline bacterial counts of resident flora on the hands of subjects. Twenty individual volunteers were
randomly selected for the study and verbal permission was obtained from all participating volunteers prior to
the experiment. Before starting this procedure, the volunteers were well educated about correct hand
disinfection procedure according to WHO . The five randomly selected subjects hand were examined for
baseline bacterial count reduction with each sample. Sterile nutrient agar plates were serially numbered and
marked as with sanitizer and without sanitizer. At first, the test was carried out with unwashed hands of the
subjects. Subjects' left hands were gently used to make a finger impression on the agar by pressing and rolling
the finger on the agar in the plate marked as without sanitizer. After that, three milliliters of the sanitizer was
applied to the hand and then rubbed thoroughly on the palm, hands, and fingernails until the hands became dry.
Further the finger impression was repeated on the plate marked with sanitizer for all subjects. The plates were
incubated for 24 hours at 37ºC and after 24 hours the number of colonies was counted with a colony counter.
The reduction in colony-forming unit (CFU) percentage was calculated to evaluate the efficacy of different hand
sanitizers. The CFU percent reduction was determined by the following simple formula.
CFU percent reduction
Where A is the viable counts of microorganism before treatment
Where B is the viable counts of microorganism after treatment
Agar diffusion test
In the susceptibility test, all the test products exhibited inhibitory activity against the test isolates (Table
2), except Sepnil against S. typhi. There was no inhibition zone for Sepnil against S. typhi (Figure 1), and also had
lowest inhibition zone against S. flexneri, S. aureus, E.coli, and S. pneumoniae, which were 6.63 mm, 9.63 mm, 10.13
mm, and 8.30 mm respectively. Thus Sepnil was the least effective hand sanitizer to kill bacteria in agar
diffusion test. Handirub gave better agar diffusion test result against S. aureus, E. coli, S. flexneri, and S. typhi by
comparing with Hexisol and Sepnil. It showed the maximum diameter of the inhibition zone against S. typhi (27
Citation: Rahman Md.N, Abdullah-Al-Shoeb M, Huq S and Abul Kalam Azad M. Assessment of antibacterial efficacy of Lugol's iodine compared with commercial