Journal

of Hospital

infection

(1991)

18 (Supplement

B), 13-22

Computerized
image analysis of full-hand
touch
plates: a method
for quantification
of surface
bacteria
on hands and the effect of antimicrobial
agents
J. J. Leyden,
K. J. McGinley,
M. S. Kaminer,
J. Bakel,
S. Nishijima*,
M. J. Grove7 and G. L. Grove?
University of Pennsylvania,
School of Medicine, Department of Dermatology,
of Dermatology,
Kansai Medical
Philadelphia,
PA 19104, Department
University, Moriguchi,
Osaka, Japan and tSimon Greenberg Foundation, 835
Sussex Blvd., Broomall, PA 19008, USA
Summary:
A method is described for quantification
of the bacterial flora on
the hand surface. Computer-assisted
image analysis of bacterial
growth of
large full-hand
touch plates provides a quantifiable
measure of the bacterial
flora on the hand surface. Image analysis pixel intensity
values showed a
significant
correlation
(P < 0.0001) with colony forming
unit values determined by the glove juice method. Image analysis of impressions
from hands
treated with various antimicrobial
agents in detergent bases showed that 4%
chlorhexidine
gluconate produces a-96% reduction
after a 30 s washing and
98% reduction
after a 3 min washing while 7.5% povidoneiodine
and 1%
triclosan
produce a 77% and 70% reduction
after 3 min respectively,
and
70% isopropanol
produces a 98% reduction
after a 30 s wash.

Keywords: Image analysis;

hands;

bacteria.

Introduction
Resident bacteria and transient
pathogens on the hands of health care
personnel
have been recognized
as potential
sources of nosocomial
infections.
Frequent
handwashing,
usually with antimicrobial
agents in
detergent vehicles, is standard practice for health care personnel. Currently,
the methodologies
used to determine
the efficacy of antimicrobial
agents
primarily
involve variations of the glove juice test in which the number of
resident bacteria or inoculated
pathogens are recovered by removal with a
detergent inside a sterile glove or by utilization
of touch plates methods
before and after treatment
with a test agent.14 Many investigators
have
demonstrated
that antimicrobial
agents in detergent
vehicles
reduced
bacteria to a greater degree than a plain detergent. The magnitude
of the
effect on the resident
bacteria
is low, particularly
in view of the
demonstrated
greater effect for theseagents on large populations
of bacteria
on other body sites. Results from testing the effect of chlorhexidine
0195%6701/91/06BO13+10

0 1991 The Hospital

SOS.OO/O

13

Infection

Smety

14

J. J. Leyden

et al.

gluconate and povidone-iodine

usually show approximately
a one logarithm
reduction
after a 3 min scrubbing.
Application
to higher densities of
bacteria on other body sites demonstrates
reduction
of several orders of
magnitude.
Recently, we demonstrated
that the subungual spaces contain
large populations
of bacteria and that these bacteria contribute
significantly
to the number
of organisms
recovered
by the glove juice test.6*7 For
example, use of. 4% chlorhexidine
and 10% povidone-iodine
on hands in
which the subungual
spaces have been obliterated
by cyanoacrylate
glue
results in up to a four logarithm
reduction compared to hands in which the
subungual
spaces are not occluded
which show only a one logarithm
reduction.7
These studies demonstrate
the need for a methodology
other
than the glove juice method for evaluating
the degerming
efficacy of an
agent intended to rapidly reduce bacteria on the surface of the hand, e.g. the
hand of a nurse or physician
between patient-patient
contact. The glove
juice method seems more appropriate
for evaluating
the degerming effect
for surgeons who have hands inside gloves for prolonged
periods of time
during which time subungual bacteria can find their way into the glove and
potentially
to the patient.
Subungual
bacteria
are unlikely
to be of
significance
in the casual, brief contact which commonly
occurs between
health care personnel and patients.
In this work, we describe a methodology
for quantifying
the effect of
antimicrobial
substances on the surface flora and demonstrate
the relative
efficacy of commonly
used antimicrobial
agents for both brief washing and
standard preoperative
surgical scrubbing.
Methods

Subjects
Healthy adults who were not using any topical antimicrobial
agent for at
least 1 month served as test subjects after giving informed consent. Potential
subjects were screened and only those showing
a pattern of bacterial
colonization
of the hands and finger surface were used (Figure 1). Subjects
with dry, chapped hands commonly have very low numbers of bacteria and
are not acceptable for this procedure. In our studies, we avoided health care
personnel
because of their frequent
use of antimicrobial
agents. Our
subjects were primarily
university
students and non-health
care employees
of the University.
Cultures and hand surface-hand stamping
Cultures of the surface bacterial flora were obtained by pressing the palmar
surface on 245 x 245 x 20 mm plates containing
200 ml of trypticase Soy
agar and broth
(BBL-Becton
Dickinson,
Cockysville,
MD)
which
contained 2% polysorbate-80,
0.1% lecithin and 0.1% sodium thiosulphate
as neutralizers,
and 4% added sodium chloride to give a total of 4.5% to
prevent spreading of aerobic spore-forming
organisms and Gram-negative

Image

analysis

of hand bacteria

Figure 1. Large touch plate of hand demonstrating

colony
digitized
by computer.
Left panel shows pre-treatment
reduction
after treatment
with triclosan.

forming units as seen after being

and right panel shows a 65%

bacteria, and 0.04% cycloheximide

to prevent fungal contamination.
The
addition
of 4% sodium
chloride
was done after preliminary
studies
demonstrated
no difference
in the density of colony forming
units of
bacteria from plates not containing
sodium chloride. The suitability
of the
neutralizing
agents was tested by washing hands with 4% chlorhexidine
gluconate and immediately
applying the hand to large touch plates seeded
with a lawn of M~crococcus Zuteus (Figure 2). Effective neutralization
was
demonstrated
for all antimicrobial
test agents. The reproducibility
of serial
imprints of the hand was determined in 20 subjects who were sampled three

Figure
luteus;

2. Left panel shows carry over of chlorhexidine

inhibiting
right panel shows effective neutralization
of potential
carry

growth
of Micrococcus
over effect.

16

J. J. Leyden

et al.

times in succession. In 60 subjects, the right and left hands were compared.
In 20 subjects the composition
and density of microorganisms
on the
hands was determined by immersing each hand in a sterile glove containing
50 ml of 0.1% Tween 80 and scrubbing the palmar surface for 1 min. The
subungual
spaces were obliterated
with cyanoacrylate
glue in order to
prevent sampling of that reservoir of organisms. The number of bacteria
recovered by the glove juice procedure, expressed as log,, colony forming
units (cfu) from these 20 subjects (40 hands), was compared to the image
analysis data obtained from large hand touch plates of these subjects.
Image analysis
The image analysis system consisted of a central computer (Dell Computer
PCs Limited
Systems 200), colour video monitor
(Mitsubishi),
video
Inc.), and densitometry
software
camera (Southern
Micro Instruments,
package (Southern Micro Instruments,
Inc.). Images of the full-hand
touch
plates (FHTP)
were transmitted
(digitized)
onto a video monitor
in
black/white
mode. The system employed grey scale values (GSV) from 0 to
255 (0 = black; 255 = white), with the range between black and white
sub-divided
based on relative shades of grey. Working
area on the video
screen was 466 X 466 pixels (which represented
cm2 after calibration),
generating 217 156 pixels per field, The system applied a GSV to each pixel.
Maximum
cumulative
GSV for each field was 55.6 X 106, minimum
was
zero (black). Pseudocolour
enhancement
was then performed.
In brief,
the computer assigned each GSV a colour (0 = dark purple --, 255 = light
pink) based on established
relationships
of the colour spectrum.
The
computer then changed the image on the video monitor so that the GSV
were represented
by colours rather than shades of grey. Pseudocolour
served as a visual enhancement
to aid in defining borders of objects of
interest on the video screen.
The image of the FHTP was then re-acquired
in the pseudocolour
mode
to allow for background
calibration.
Background
of the FHTP
was
calibrated by adjusting the gain control (S/N ratio) on the video camera. To
ensure reproducibility
and allow comparison between subjects, FHTP were
calibrated so that all colonies were between dark purple and light pink. The
bacterial colonies that were closest to the white end of the grey scale in the
black/white
mode were calibrated to be light pink, taking care to eliminate
white (in the pseudocolour
mode) from the colonies of interest. This was
done because white is assigned a value greater than 255, and represents the
limit of the systems ability to analyse grey scale variations.
By calibrating
the colonies so those that appeared closest to white were light pink in
pseudocolour
(as close to white as the softwares resolution
allowed), we
maximized
the use of grey scale values between
black and white.
Background
colour was normalized
to a value of 0 intensity units (i.u.) by
editing the pseudocolour
spectrum.
Following
background calibration,
area and intensity
calculations were

Image

analysis

17

of hand bacteria
14.

IO
9

12.

a
IO

6
;
:
o

5
4
3
2
I
0

Ir
2000

4000

:
2

8
6

IILL100

6000

81

Arithmetic

Figure 3. Distribution
normally
distributed.

0 10000

12000

14

intensity

values

indicates

-t

Logarithmic

scale

of grey level pixel

=i---

scale

that their

values

are log

performed.
Area refers to the area of objects selected for data analysis, and
intensity is the summation
of the intensity value (pseudocolour
correlate of
GSV) for each pixel in the objects selected for data analysis. Objects of
interest (bacterial colonies) were identified
by selection of pseudocolours
that correspond to the colonies on the plate. Pseudocolour
selection allowed
refinement
of colony
analysis by choosing
only those pseudocolours
that corresponded
to bacterial
colonies,
so eliminating
background
contamination.
Area and intensity
measurements
were displayed on the
video monitor upon completion
of this process.
Hand area for each subject was determined
by outlining
the hand on
paper, acquiring
the hand image onto the video screen, tracing the hand
image on the video screen and calculating
the area.

Effect of antimicrobial

agents

The effect of antimicrobial

agents and a plain detergent were determined for
both a 30 s handwashing
and a 3 min scrub. In the former, hands were
wetted with warm water, 2 ml of the test agent was added and the hands
washed vigorously
for 30 s. Hand scrubbing
was done by wetting hands,
applying 2 ml of the test agent then scrubbing
the hands for 1 min with a
sterile brush, Following
this the debris from the subungual
areas was
removed by a technician
and then 2 ml of the test agent was applied, the
hands scrubbed for 1 min, rinsed and the process repeated again. Cultures
of the hand were obtained prior to washing and after air drying of the hands
following
use of test agents. Carry over of antimicrobial
agents could be
demonstrated
by scrubbing for 3 min, air drying for 5 min and then placing
the hand on a plate seeded with M. Zuteus (A.T.C.C.27141).
Plates
containing
the neutralizing
agents described above did not show carry over
effects for any of the antimicrobial
agents tested. The test agents used were:

J. J. Leyden

18

et al.

DE)-4%
Hibiclens
(Stuart
Pharmaceuticals,
Wilmington,
chlorhexidine
gluconate in detergent base;
Hibistat
(Stuart
Pharmaceuticals,
Wilmington,
DE)-0.5%
chlorhexidine
gluconate with 70% isopropyl alcohol;
SoftCide
(Stahman,
Weston & Co., Inc., N. Hampton,
NH)-1%
chloroxylenol
(PCMX)
in detergent base;
Phisoderm
(Winthrop
Consumer
Products,
New
York,
NY)-non-medicated
detergent;
Betadine
(Purdue
Frederick
Co.,
Norwalk,
CT)-7.5%
povidone-iodine
surgical scrub;
70% Isopropyl
alcohol;
Bat Down
(Decon Laboratories
Inc. Wayne Pa.) 1% Triclosan
in
detergent base.
Results

The results of the qualitative

and quantitative
analysis of microorganisms
recovered from the hand are found in Table I. The dominant
organisms
were staphylococci
which
composed
88.5%
of the total flora and
coryneforms
which made up 11% of the total flora. Gram-negative
bacteria
and aerobic spore-forming
organisms were frequently
found (72% and 90%
respectively)
but were present in very low numbers.
The use of 4.5%
sodium chloride prevented
swarming of these organisms and addition of
0.04% cycloheximide
prevented fungal overgrowth,
all of which would have
inhibited
image analysis.
No significant
difference was found between the grey level intensity
of
colony forming
units of bacteria on the right and the left hand of 60
subjects, a correlation
coefficient
of O-82, P < 0~0003. Serial sampling
showed no significant
differences
in the area covered by bacteria on
successive samples with logarithm
means of 4.39 f 0.10, 4.44 f 0.13 and
4.38 f 0.12 log intensity cmp2. There was a significant
correlation
between
the log cfu and the log pixel grey level intensity
on image analysis
(P < O*OOOl; Figure 4).
The effects of a 30 s washing and a 3 min scrubbing with various agents
Table

I. Microflora

on hand surface

Prevalence
Staphylococci
Coryneforms
Gram negative rods
Bacillus spp.
Fungi (other than yeasts)
Yeasts
Others
*Logarithmic
subjects).

mean

and standard

100
100
72
90

88.5
11.0
0.1
0.1
0.02
0.1
0.1

z:
70
deviation,

Composition

post 5-hour

soap wash

(%)

Mean

?c SD*

5.1 f 0.5
3.8fl.O
0.7f0.9
1.4fl.O
0.9 f 0.9
1.3*1.0
0.5 f0.9
(A = 40, left and right

hands,

20

Image

analysis

19

of hand bacteria

r2= 0.44,

0.5

I.5

2
Log

Figure
density

2.5
CFU

Detergents
4% Chlorhexidine
gluconate
7.5% Povidone-iodine
1% Triclosan
1% Chloroxylenol
Non-medicated

II. Eficacy

4.5

cm-

4. Correlation
of pixel grey intensity
(INT)
determined
by the glove juice procedure.
Table

3-5

P=0.0001

of antimicrobial

cme2 with

the colony

forming

unit

detergents

3 min scrub

30 s wash

P value

***97.9 f 2.3
***76,9f
13.2
***70.1 -f 14.2
32.9 AL22.5
31~9zt30~1

***92.6 2~ 6.3
**so.2 * 25.2
*48.3 rk 30.2
25.0f21.9
21.9f25.4

NS
***
***

Panel of 10 subjects; N = 20 for each time period. Results expressed

as % reduction
pre/post
image
analysis intensity
cm-*. P value is for the difference
between 30 s and 3 min washing. Asterisks
indicate
statistical
difference
between detergent
base and test agents: *P i 0.05; **P < 0.01; ***P < O-001.

are seen in Table II. Washing

with a plain detergent
resulted
in a
non-significant
reduction
of 3 1.9% reduction
after a 3 min scrub;
povidone-iodine
produced
a 50.2% reduction
after 30 s and 76.9%
reduction after a 3 min scrub both of which were significant
(P < 0.01 and
P < 0.001, ANOVA),
while chlorhexidine
gluconate
produced
a 92.6%
reduction
after 30 s and 97.9% reduction
after a 3 min scrub (P < 0.001).
Both the 30 s and 3 min washings with 1% triclosan produced significant
reductions
(P < 0.05 and P < 0.001, ANOVA),
while PCMX
failed to
produce
a significant
reduction
at both time points. The use of 4%
chlorhexidine
gluconate,
7.5% povidone-iodine
and 1% triclosan
in
detergent bases produced a significantly
greater reduction in bacteria than
use of the plain detergent
alone after both 30 s and 3 min washings
(P -=E0.001).
Chlorhexidine
used for 30 s produced a greater reduction
than a 3 min
scrub with povidone-iodine
(P < 0.01) and triclosan (P < 0.001). Three min

J. J. Leyden

20
Table

III.

Test

agent

Effect of antimicrobial

et al.

solutions on surfaceflora

% Reduction
on
30 s exposure (3 ml)

70% Isopropyl
alcohol
0.5% Chlorhexidine
and 70% isopropyl
alcohol
Panel of 10 subjects; N = 20 for each testing.
reduction of pre/post intensity cme2 as determined
hand touch

of the hand

98.7zk2.7
97.9 * 3.4
Results expressed as %
by image

analysis

of full

plates.

scrubbing with chlorhexidine

was more effective than 3 min scrubbing with
povidone-iodine
(P < 0.01) and triclosan (P < 0.001).
Isopropanol
produced
a 98.7%
reduction
after
30 s and 0.5%
chlorhexidine
in 70% isopropanol produced comparable results (Table III).
Discussion

In this study we have demonstrated

that computer-assisted
image analysis
of large touch plates provides a reproducible,
quantifiable
measure of the
surface bacterial population
on the hand. This method provides results
which correlate with recovery
of bacteria by the glove juice method
provided that the subungual regions are obliterated with cyanoacrylate
glue
to ensure that only bacteria from the hand surface are being harvested.
Using this methodology,
we have found that 4% chlorhexidine
gluconate
in a detergent base produces a profound reduction both after a 30 s wash and
a 3 min scrubbing.
The level of reduction
after a 3 min scrub was
comparable
to that found
with
70% isopropanol
applied
for 30 s.
Povidone-iodine
and triclosan produced significant reductions at both time
points while PCMX was no better than the detergent alone. In the case of
topical 70% isopropanol
we have found interesting
differences in efficacy
between
use alone and following
a pre-alcohol
hand wash with
a
non-medicated
detergent
(unpublished
observations).
The
latter
is
associated with a significant reduction in the effect of alcohol suggesting that
deposition of fatty acids or other ingredients
of the detergent interfere with
the efficacy of alcohols. These interesting
results indicate the need for
further work in view of the use of alcohols following
handwashing
with
plain detergents as preoperative
degerming techniques in some countries.
The need to degerm the hand is most frequently
expressed in terms of the
surgeon whose hands are washed before operating and health care personnel
who wash between handling
of patients. The former is referred to as
surgical handwashing
where the aim is to reduce both the resident bacterial
flora as well as any transient pathogenic organisms such as Staphylococcus
aureus, Gram-negative
bacteria and yeasts. The latter is usually referred to
as hygienic handwashing
in which the primary need is to eradicate transient
pathogenic bacteria in order to minimize
nosocomial infections.
However,

Image

analysis

of hand bacteria

21

in hygienic
handwashing
it is also desirable to reduce the resident flora
which can cause infection, particularly
in the immunocompromised
host. In
the case of surgical handwashing,
variations of the glove juice or sterile bag
technique
are commonly
employed.
Hygienic
handwashing
is frequently
evaluated by determining
the ability of a test agent to reduce pathogenic
bacteria such as Escherichia coli or other Gram-negative
bacteria including
Acinetobacter
spp. and Enterobacter spp. 2,3 All of these procedures provide
relevant data on the in-vivo
antimicrobial
activity of agents designed to
produce a degerming effect on hands. The methodology
described in this
work evolved from our observation
that the glove juice, sterile bag
technique samples both the bacterial flora of the surface of the hand as well
as the subungual
spaces. We demonstrated
that the subungual
spaces
contribute
significantly
to the recovery of bacteria in the glove juice test and
that currently
available
antimicrobial
detergents
fail to eradicate that
population
of bacteria. Furthermore,
interpretation
of results obtained by
the glove juice method are complicated
in terms of discerning
the relative
effects of a test agent on the surface flora and the subungual space.7
As a result of these studies, we feel that the evaluation
of degerming
agents on the hand flora should take into account the finding that the surface
flora is much more accessible and more easily reduced than subungual
bacteria. Health care personnel who move from patient to patient and have
brief contact need to minimize
transfer of bacteria from the hand surface.
The need to minimize
transfer of bacteria encompasses not only transient
pathogens but also the recent bacterial flora. Surgeons or others performing
longer procedures, particularly
when operating within the body, need to be
more concerned with the subungual
bacterial population
in addition
to
surface bacteria. This population
can find its way into the glove and thus
pose a potential hazard to the patient. The evaluation of the efficacy of an
antimicrobial
agent designed for use on the hands needs to be tailored to the
setting in which that agent will be used. For health care personnel who have
brief patient contact, evaluation of the effect of an agent on the surface flora
and transient
pathogens is critical while agents designed to be used by
surgeons should be evaluated for their effort on both the surface and
subungual flora.
We acknowledge

the support

of Simon

Greenberg

Foundation.

References
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RN, McGrath
MB, Goss WA. Applications
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model for
multiparameter
measurements
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1986; 96: 27-37.
Ayliffe GAJ, Babb JR, Quoraishi
AH. A test for hygienic hand disinfection.
J Clin Pathol
1978; 31: 923-928.

22

J. J. Leyden

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et al.

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