Dental Research Journal

Original Article
Comparative assessment of antimicrobial efficacy of different hand
sanitizers: An in vitro study
Vardhaman Mulchand Jain1, Gundabaktha Nagappa Karibasappa1, Arun Suresh Dodamani1, Vishwakarma K. Prashanth1,
Gaurao Vasant Mali1
1
Department of Public Health Dentistry, ACPM Dental College, Dhule, Maharashtra, India

ABSTRACT
Background: To evaluate the antimicrobial efficacy of four different hand sanitizers against
Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and
Enterococcus faecalis as well as to assess and compare the antimicrobial effectiveness among four
different hand sanitizers.
Materials and Methods: The present study is an in vitro study to evaluate antimicrobial efficacy
of Dettol, Lifebuoy, PureHands, and Sterillium hand sanitizers against clinical isolates of the
aforementioned test organisms. The well variant of agar disk diffusion test using Mueller‑Hinton
agar was used for evaluating the antimicrobial efficacy of hand sanitizers. McFarland 0.5 turbidity
standard was taken as reference to adjust the turbidity of bacterial suspensions. Fifty microliters
of the hand sanitizer was introduced into each of the 4 wells while the 5th well incorporated with
sterile water served as a control. This was done for all the test organisms and plates were incubated
Received: February 2016 in an incubator for 24 h at 37°C. After incubation, antimicrobial effectiveness was determined using
Accepted: June 2016 digital caliper (mm) by measuring the zone of inhibition.
Results: The mean diameters of zones of inhibition (in mm) observed in Group A (Sterillium),
Address for correspondence:
Dr. Vardhaman Mulchand
Group B (PureHands), Group C (Lifebuoy), and Group D (Dettol) were 22 ± 6, 7.5 ± 0.5, 9.5 ± 1.5,
Jain, and 8 ± 1, respectively. Maximum inhibition was found with Group A against all the tested
D‑1, Dongare Maharaj organisms. Data were statistically analyzed using analysis of variance, followed by post hoc test for
Nagar, Opposite Cotton group‑wise comparisons.The difference in the values of different sanitizers was statistically significant
Market, Parola Road,
Dhule ‑ 424 001,
at P < 0.001.
Maharashtra, India. Conclusion: Sterillium was the most effective hand sanitizer to maintain the hand hygiene.
E‑mail: dr.vardhaman.jain@
gmail.com Key Words: Anti infective agent, hand sanitizers, hygiene, organisms, test

INTRODUCTION important to prevent many communicable diseases.

The word “hygiene” is derived from the ancient
Hospital and community‑acquired infections are Greek goddess “Hygeia” that means “goddess of
escalating and pose a serious public health problem healing.” The importance of hygiene is universally
worldwide.[1] Hands are considered to be the primary recognized and evidence‑based. It is well known
route for transmitting microbes and infections to the
individuals.[2] Personal as well as hand hygiene is This is an open access article distributed under the terms of the Creative
Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which
allows others to remix, tweak, and build upon the work non‑commercially,
Access this article online
as long as the author is credited and the new creations are licensed under
the identical terms.

Website: www.drj.ir For reprints contact: [email protected]

www.drjjournal.net
www.ncbi.nlm.nih.gov/pmc/journals/1480 How to cite this article: Jain VM, Karibasappa GN, Dodamani AS,
Prashanth VK, Mali GV. Comparative assessment of antimicrobial efficacy
of different hand sanitizers: An in vitro study. Dent Res J 2016;13:424-31.

424 © 2016 Dental Research Journal | Published by Wolters Kluwer ‑ Medknow

 Jain, et al.: Antimicrobial efficacy of hand sanitizers

that hand hygiene is crucial to prevent and minimize Many hand sanitizers are available in the market
healthcare‑associated infections.[3] The Centers for with varying degree of effectiveness that are
Disease Control and Prevention, the World Health registered in the National Agency for Food and Drugs
Organization, and many other health experts promote Administration and Control. Moreover, in outreach
hand hygiene as the single most important measure programs, screening procedures in day‑to‑day practice,
in the prevention of hospital‑acquired infections. water scarcity areas, and bed‑side and chair‑side
Several studies have shown the importance of proper clinical examination, hand sanitizers could be an
hand hygiene in reducing the incidence of nosocomial alternative to achieve asepsis. However, clinicians and
infections.[4‑8] It is estimated that at any one time, more common man face the dilemma while choosing the
than 1.4 million people worldwide are suffering from best among the lot.
infections acquired in hospitals. These nosocomial
Some products marketed to the public as antimicrobial
infections are also, in most cases, the result of poor
hand sanitizers are not effective in reducing bacterial
hand hygiene.[9]
counts on hands. In fact, despite a label claim of
At present, washing hands with appropriate soap reducing “germs and harmful bacteria” by 99.9%,
followed by applying hand antiseptics are two some studies have observed an apparent increase in
important hand hygiene method in clinical practice. the concentration of bacteria in handprints impressed
Hand sanitizers significantly increase the chance of on agar plates after cleansing.[20] Hence, there still
maintaining the hands clean and aseptic. exists a need for verification of these claims by
the regulatory authorities for the enforcement of
Traditionally, microbes habitation on hands is
good‑quality measures. To overcome this ambiguity,
divided into resident and transient floras. Involved
the present study was carried out to assess the
resident floras are commonly Staphylococcus aureus,
antimicrobial effectiveness of four different hand
Staphylococcus epidermidis, and Enterococcus
sanitizers against the test organisms.
faecalis that colonize the deeper skin layers and
are resistant to mechanical removal. The transient
MATERIALS AND METHODS
floras consists of S. aureus, Escherichia coli, and
Pseudomonas aeruginosa that colonize the superficial
The present study is an in vitro study conducted
layers of skin in a short period of time.[10] Therefore,
at the Department of Microbiology, Government
we selected these organisms to determine their
Medical College, Dhule, Maharashtra, India.
susceptibility to different hand sanitizers tested in this
Ethical clearance for the study was obtained from
study.
the Institutional Ethical Review Committee. Four
Scientific studies have shown that after hand different brands of hand sanitizers were selected
washing, as many as 80% of individuals retain out of many available in the market based on their
some pathogenic bacteria on their hands.[11] Hand popularity and maximum usage in Dhule City.
washing removes body’s own fatty acids from the Selected hand sanitizers to test their antimicrobial
skin, which may result in cracked skin that provides efficacy were Sterillium  (Bode Chemie, Hartmann
an entry portal for pathogens.[12,13] To overcome the Group, Germany), PureHands (Himalaya Drugs
limitations of plain hand washing, hand sanitizers Company, India), Dettol  (Reckitt Benckiser, UK),
were introduced claiming to be effective against and Lifebuoy (Hindustan Unilever Pvt. Ltd.,
those pathogenic micro-organisms as well as to India) [Figure 1]. Recently manufactured and packed
improve skin condition due to the addition of sanitizers have been purchased based on their
emollients in it.[14] popularity from the local retail outlet. The study was
conducted over a period of 10 days. The composition
Hand sanitizers were also effective in reducing
of various hand sanitizers is shown in Table 1.
gastrointestinal illnesses in households,[15] respiratory
tract infections, and skin infections,[16] in curbing The culture media used in the present study were
absentee rates in elementary schools,[17] and in reducing Mueller‑Hinton agar for agar diffusion method while
illnesses in university dormitories.[18] Furthermore, to nutrient broth and nutrient agar medium for bacterial
reduce infections in healthcare settings, alcohol‑based isolate preservation. The clinical isolates of S. aureus,
hand sanitizers are recommended as a component of S. epidermidis, E. faecalis, E. coli, and P. aeruginosa
hand hygiene.[19] were obtained from the culture plates of the respective

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 Jain, et al.: Antimicrobial efficacy of hand sanitizers

micro-organisms preserved on the nutrient agar of antimicrobial efficacy of hand sanitizers.[24,25]

slants and were stored at 4°C in the Department of Sterile Mueller‑Hinton agar plates were inoculated
Microbiology, Government Medical College, Dhule, with standardized test organisms [Figure 2]. A sterile
Maharashtra, India. cotton swab was dipped into a test tube containing the
Preparation of McFarland standards and inoculum and was rotated properly to allow maximum
standardization of test organisms contact. Excess inoculum was removed by pressing
Bacterial suspensions varied in the turbidity and rotating the swab firmly against the inside wall
and could cause potential bias in the result, to of the tube above the liquid level. The swab was
overcome this and standardize the microbial testing; then streaked over the surface of the medium three
McFarland standards was taken as a reference to times while rotating the plate at 60° angle after each
adjust the turbidity of bacterial suspensions. The application. The swab was also passed around the edge
McFarland 0.5 turbidity standard was prepared of the agar surface. The inoculum was left to dry for a
by adding 0.5  ml of 1.175%  w/v barium chloride few minutes at room temperature with the lid closed.
dihydrate (BaCl2·2H2O) solution to 99.5  ml of With the aid of a sterile 6 mm cork borer, 4 equally
15  w/v sulfuric acid  (H2SO4).[21] This was mixed spaced holes were bored in the agar plate with a fifth
well and then aliquoted into test tubes identical to hole in the center of the plate. The agar plugs were
the ones used in preparing inoculum suspensions of discarded using a sterile needle [Figure 3]. Fifty
the test organisms. The accuracy of the density of microliters of the hand sanitizer was then introduced
the standard was verified using a spectrophotometer. into each of the 4 wells while the central well was
The absorbance of the 0.5 McFarland standards at filled with an equal volume of sterile water to serve
wavelength of 625  nm was 0.08–0.10. The tubes as control [Figure 4]. This was done for all the test
were stored in a well‑sealed container in the dark at organisms and hand sanitizers. The plates were
room temperature until when required.[22] incubated for 24 h at 37°C in an upright position. They
A sterile loop was used to pick a loopful of inoculum were then examined for zones of inhibition which
from a pure culture of the test organism. This was indicated the degree of susceptibility or resistance of
then transferred and suspended into a tube containing the test organism to the antibacterial agent [Figure  5].
sterile normal saline (NaCl 8.5 g, distilled water 1 L). The point of abrupt diminution growth which
The tube was compared with the turbidity standard,
and the density of the organism was adjusted
by adding more bacteria or sterile saline until
standardization was attained.[23]
Agar diffusion test (well variant) to determine
susceptibility of test organisms to hand sanitizers
Disk agar diffusion technique described by Bauer
et al. and Valgas et al. was used for the evaluation

Table 1: Hand sanitizers used in the study and their Figure 1: Different hand sanitizers used in the study.
Ingredients
Hand Ingredients
Sanitizer
Sterillium Propan‑2‑ol, Propan‑1‑ol, Mecetronium ethyl sulfate,
Glycerol, Tetradecan‑1‑ol, fragrances, Patent blue V,
Purified water
PureHands Hrivera, Coriander, Lime, Ushira, Neem
Dettol Denatured Alcohol‑ 69.4% w/w, Water PEG/PPG‑17/6
copolymer, Propylene glycol, Acrylate/C10‑30
alkyl acrylate, cross polymer, Tetrahydroxpropyl
ethylenediamine, Perfume.
Lifebuoy Ethyl alcohol 95% v/v IP 55% w/w, Isopropyl alcohol
10% IP w/w, Tocopheryl acetate IP 0.05% w/w, Figure 2: Sterilized Mueller-Hinton agar plates inoculated with
Perfumed gel base: qs to 100% w/w
standardized test organisms.

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 Jain, et al.: Antimicrobial efficacy of hand sanitizers

Statistical analysis
Data were statistically analyzed with analysis of
variance followed by post hoc test for group‑wise
comparisons. All statistical procedures were performed
using Statistical Package for Social Sciences (SPSS)
version 21.0 software (IBM, Armonk, NY, USA).
The data exhibited a normal and homogeneous
distribution; thus, zone of inhibition (in mm) was
analyzed using the mean of all the readings obtained,
Figure 3: Four equally spaced holes in the agar plate with the and the difference in the values of different hand
5th hole in the center. sanitizers was statistically significant at P < 0.001.

RESULTS

Hand sanitizers were effective against all the test

organisms. The antimicrobial effectiveness was
assessed by measuring the zone of inhibition against
the particular test organism. Maximum inhibition
(in mm) was seen in Group A (Sterillium), i.e.,
27  ±  1.414 and minimum in Group  B  (pure hands),
i.e.,  3.5  ±  4.95 against S. aureus. The difference in
Figure 4: Fifty microliters of the hand sanitizer used was the values of the different sanitizers was statistically
introduced into each of the 4 wells and central well filled with significant [P < 0.001, Table 2].
an equal volume of sterile water.
Group A showed the highest antimicrobial effectiveness
corresponds to the complete growth inhibition was followed by Group C, Group D, and Group B
taken as the zone edge.[24] The test was carried out five respectively, against all the different test organisms used
times, and the average of all readings was taken as the in the study. Group A could inhibit all the bacteria either
zone of inhibition in each case. Inhibition zones were Gram‑positive or Gram‑negative effectively, whereas
measured with the aid of a digital caliper (mm). other sanitizers showed a limited action [Graph 1].
Schematic representation of agar diffusion When subjected to post hoc test analysis, statistically
test (well variant) to determine susceptibility of high significant difference was observed against all the
test organisms to hand sanitizers bacterial isolates when Group A was compared with
any other group. However, there was no statistically
Different hand sanitizers used and sterilized Mueller-Hinton agar plates were significant difference when Group  B, Group  C, and
inoculated with standardized test organisms [Figures 1 and 2]
Group D were compared with each other against all
test organisms. The mean difference is statistically
Sterile cotton swab dipped into tube containing inoculum and excess
removed by rotating firmly against inside wall of the tube above the liquid level significant at the 0.05 level [Tables 3‑5].
Swab was streaked over agar surface three times while rotating the agar
plate at an angle of 60° after each application
DISCUSSION

Inoculum left to dry for few minutes at room temperature with the lid closed
Infection with environmental microbes is increasing
alarmingly. Normal human skin always harbors
Sterile 6 mm cork borer used to prepare 4 equally spaced holes in agar plate with the 5th bacteria (102 and 106 CFU/cm2). The transfer of
hole in the center of the plate and agar plugs discarded using sterile needle [Figure 3]
bacteria from the hands to food, objects, or people
Fifty microliters of the hand sanitizer was then introduced into each of the 4 wells
plays an important role in the spread of many
while the central well filled with an equal volume of sterile water to serve as control [Figure 4] communicable diseases.[26] The critical density of
micro-organisms on the hands needed for the spread
This was done for all the test organisms and plates were incubated in
incubator for 24 h at 37°C in an upright position of pathogens remains unknown, and it may depend
on the type and duration of contact, the type of
Analysis of zone of inhibition to evaluate antimicrobial efficacy of different hand sanitizers micro-organism, the patient’s resident flora, and their
indicating susceptibility of the respective test organism [Figure 5]
colonization resistance.[2]

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 Jain, et al.: Antimicrobial efficacy of hand sanitizers

To overcome the negative impact of microbial disruption of tissue membranes, and dissolution of
contamination in health‑care settings, hand sanitizers several lipids.[27] Alcohol has increasing effectiveness
are recommended as an adjunct to plain hand from 60% to 90% with 1‑propanol being most
washing.[19] Most commonly and easily available effective followed by 2‑propanol and finally by
hand sanitizers in the Indian market were selected ethanol, whereas Coriander, Lime, and Neem were
for the study. Among the four hand sanitizers used the active ingredients responsible for antimicrobial
in this study, Sterillium, Dettol, and Lifebuoy were activity in PureHands herbal hand sanitizer.
alcohol‑based and PureHands was herbal, i.e.,
Many studies have been conducted to assess the
non-alcohol‑based hand sanitizer.
antimicrobial effectiveness of hand sanitizers alone,
Alcohol was the main active ingredient in but very few literature are available to assess the
alcohol‑based hand sanitizer which exerts difference between various disinfectants and hand
antimicrobial activity by causing protein denaturation, sanitizers. Disinfectants are chemical agents with
an immediate and sustained activity which destroys
micro-organisms to such a level mandated for hygienic
and surgical indications. Sanitizers, on the other hand,
are agents with an immediate activity that reduce the
number of micro-organisms to a safe level to meet the
public health requirements. Disinfectant uses a better
form of alcohol (propanol) to achieve more bacterial
reduction as compared to sanitizers (ethanol). Both can
achieve bacterial reduction on contact (in 15–30 s).
Figure 5: Analysis of zone of inhibition to evaluate antimicrobial Traditionally, agar diffusion method and agar dilution
efficacy of different hand sanitizers. Labelling on the side of method are commonly employed for assessment of
respective zone of inhibition as A, B, C, D etc. the antimicrobial activity of any material. In the
present study, the Kirby–Bauer method  (Agar disk
30 diffusion method) was chosen instead of the agar
27 S. Aureus
S. Epidermidis
dilution method. The disadvantage of the agar dilution
25
method is being technique‑sensitive and can alter
Zone of Inhibition (in mm)

P. Aeruginosa
22
20 19.5
E. Coli
E. Faecalis
some properties of the sanitizer being tested, and few
16.5
15.5 sanitizers could not be homogeneously dissolved. The
15
advantages of agar disk diffusion method are chemical
10.5
10 8.5 8.5
9.5
8.5 8.5 8.5
7.5
properties of the sanitizer remains unchanged, an
7 7.5 7.5 7 7.5 7.5

5
easy and less technique sensitive method.[28,29] It
3.5
allows direct comparison of all groups of sanitizers,
0
Group A Group B Group C Group D
indicating which group has the maximum potential to
Different Hand Sanitizers eliminate that particular test organism.
Graph 1: Zone of inhibition (in mm) measured at the end of Sterillium was the most effective disinfectant among
24 h of different hand sanitizers against all the test organisms. all the hand sanitizers against all the bacteria used

Table 2: Zone of inhibition (in mm) measured at the end of 24 h of different hand sanitizers against particular
test organism
Test Organism (Mean±SD) ANOVA P
Group A Group B Group C Group D
S. aureus 27±1.414 3.5±4.95 8.5±0.707 7.5±0.707 F=31.921 0.003
S. epidermidis 22±1.414 7±0.0 8.5±0.707 7.5±0.707 F=138.0 0.001
P. aeruginosa 19.5±0.707 7.5±0.707 10.5±0.707 8.5±0.707 F=120.0 0.001
E. coli 15.5±0.707 7.5±0.707 9.5±0.707 8.5±0.707 F=51.667 0.001
E. faecalis 16.5±0.707 7±0.0 8.5±0.707 7.5±0.707 F=106.11 0.001
Group A‑Sterillium; Group B: PureHands; Group C: Dettol; Group D: Lifebuoy ** P<0.001, HS

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 Jain, et al.: Antimicrobial efficacy of hand sanitizers

Table 3: Comparison between different groups of Table 4: Comparison between different groups of
Hand sanitizers in relation to the S. aureus and Hand sanitizers in relation to the P. aeruginosa and
S. epidermidis by Post hoc test E. coli by Post hoc test
Bacteria Group Other Mean Significance Bacteria Group Other Mean Significance
groups Difference (P) groups Difference (P)
S. aureus Group A Group B 23.500* 0.005 P. aeruginosa Group A Group B 12.000* 0.000
Group C 18.500* 0.013 Group C 9.000* 0.001
Group D 19.500* 0.010 Group D 11.000* 0.001
Group B Group A −23.500* 0.005 Group B Group A −12.000* 0.000
Group C −5.000 0.775 Group C −3.000 0.079
Group D −4.000 1.000 Group D −1.000 1.000
Group C Group A −18.500* 0.013 Group C Group A −9.000* 0.001
Group B 5.000 0.775 Group B 3.000 0.079
Group D 1.000 1.000 Group D 2.000 0.285
Group D Group A −19.500* 0.010 Group D Group A −11.000* 0.001
Group B 4.000 1.000 Group B 1.000 1.000
Group C −1.000 1.000 Group C −2.000 0.285
S. epidermidis Group A Group B 15.000* 0.000 E. coli Group A Group B 8.000* 0.002
Group C 13.500* 0.001 Group C 6.000* 0.006
Group D 14.500* 0.000 Group D 7.000* 0.004
Group B Group A −15.000* 0.000 Group B Group A −8.000* 0.002
Group C −1.500 0.950 Group C −2.000 0.285
Group D −0.500 1.000 Group D −1.000 1.000
Group C Group A −13.500* 0.001 Group C Group A −6.000* 0.006
Group B 1.500 0.950 Group B 2.000 0.285
Group D 1.000 1.000 Group D 1.000 1.000
Group D Group A −14.500* 0.000 Group D Group A −7.000* 0.004
Group B 0.500 1.000 Group B 1.000 1.000
Group C −1.000 1.000 Group C −1.000 1.000
*.The mean difference is significant at the 0.05 level *.The mean difference is significant at the 0.05 level

in the present study. This might be attributed to the Furthermore, a study conducted among school
presence of alcohol (propanol 75%) in liquid form that children showed significantly high efficacy of hand
soaks and penetrates the skin creases and nail folds sanitizers in reducing microflora on hand.[17,34]
unlike the gel form sanitizers that glide over and coat A study conducted by Mondal and Kolhapure showed
the skin. It also contains Mecetronium ethyl sulfate to that PureHands, the herbal hand sanitizer, was effective
contribute for residual effect for approximately 3–5 h against E. coli, Proteus mirabilis, Shigella sonnei,
which is lacking among other hand sanitizers.[30] S. aureus, and S. epidermidis.[2] The present study also
The results of the present study are similar to the showed antimicrobial efficacy of PureHands against
findings of Reena Rajkumari, where sterillium was tested organisms; however, it was the least effective
more effective against Candida albicans, E. coli, among all the hand sanitizers which may be probably
and Klebsiella pneumoniae.[31] A study conducted by due to low antimicrobial potency of Coriander, Lime,
Oke et al. revealed that Dettol hand sanitizer was and Neem present in it. Further studies are required to
effective only against P. aeruginosa whereas it was find the exact cause of least effectiveness of PureHands
not effective against S. aureus and E. coli.[32] Lifebuoy herbal hand sanitizer against the tested organisms.
hand sanitizer also showed antimicrobial activity
against the tested organisms; however, the exact and CONCLUSION
valid comparison could not be done with other studies
Sterillium possessed maximum antimicrobial effect
due to lack of scientific literature.
against all the Gram‑positive as well as Gram‑negative
Similar to the present study, several studies reported bacteria used in the study, followed by Dettol, Lifebuoy,
significantly better antimicrobial efficacy of hand and PureHands respectively. Despite the claims of
sanitizers, as well as a decrease in nosocomial efficacy and 99.9% bacterial reduction by hand sanitizer
infection rates as compared to hand washing.[9,33] manufacturers, there still exists a need for verification of

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 Jain, et al.: Antimicrobial efficacy of hand sanitizers

Table 5: Comparison between different groups REFERENCES

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