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Validation of dipslides as a tool for environmental sampling in a real-life

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Article  in  European Journal of Clinical Microbiology · November 2013

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Eur J Clin Microbiol Infect Dis
DOI 10.1007/s10096-013-2018-2
ARTICLE
Validation of dipslides as a tool for environmental sampling
in a real-life hospital setting
T. Ibfelt & C. Foged & L. P. Andersen
Received: 14 October 2013 / Accepted: 6 November 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Environmental sampling in hospitals is becoming
increasingly important because of the rise in nosocomial in-
fections. In order to monitor and track these infections and
optimize cleaning and disinfection, we need to be able to
locate the fomites with the highest amount of microorganisms,
but the optimal method for this is not clear. The aim of this
study was to evaluate which of four different dipslides or a
standard TSA contact plate were best at recovering human
bacteria from the environment. We tested four different
dipslides with selective and non-selective agars versus a stan-
dard TSA contact plate in order to find the best sampling
media. Two hundred sites in a children’s medical ward in
Copenhagen University hospital were sampled in autumn
2012. There was no difference in total bacteria count between
the TSA contact plate and the dipslides. Faecal indicator
bacteria recovery was the same for the dipslides and the
TSA contact plate. Dipslides may be equally well suited for
environmental sampling and hygiene assessment as TSA con-
tact plates. Dipslides have some advantages, such as better
sample security, easier sampling in confined spaces and longer
shelf life that may speak in favour of choosing these for
bacteria environmental sampling in hospitals, depending on
the task.
Introduction
Assessment of the general hygiene in hospital wards is impor-
tant in order to control outbreaks and nosocomial infections.
T. Ibfelt (*) : C. Foged : L. P. Andersen
Department of Infection Control (6901) , Copenhagen University
Hospital (Rigshospitalet), Juliane Maries Vej 28, 2100 Copenhagen,
OE, Denmark
e-mail: tobias@ibfelt.com
T. Ibfelt : C. Foged : L. P. Andersen
Department of Clinical Microbiology (9301), Copenhagen
University Hospital (Rigshospitalet), Copenhagen, Denmark
In this setting, environmental samples can help determine
contagious hotspots and maintain cleaning standards. Envi-
ronmental sampling in hospitals has two purposes, i.e. to
check the level of cleaning and to track pathogens and bacte-
rial hotspots in the hospital environment [1]. In the food
industry, sampling standards have been employed for a long
time, but in the clinical setting, there are currently no approved
standards for surface sampling. Different standards have been
proposed, depending on the purpose [2, 3]. To evaluate the
cleaning and general cleanliness, the recommended method is
a total count of colony forming units (CFU) on non-selective
agar contact plates [2, 3]. Identification of pathogens, howev-
er, is more complicated. Different methods have been used,
such as agar contact plates, swapping and dipslides, but the
evidence to support one method in favour of another is not
clear [4–8]. If the aim is to locate a specific pathogen, for
example, in relation to an outbreak, the best method is prob-
ably the use of a sponge or related technique where a greater
area is covered, thus increasing the chances of finding the
specific pathogen [1–3].
Most contact plates consist of tryptic soy agar (TSA),
which is a non-selective medium used to assess total bacteria
growth and to isolate potential pathogens. Dipslides have two
sides, one normally containing a non-selective medium (i.e.
TSA), and the other containing a selective agar such as a
McConkey or a violet red blood glucose (VRBG) agar. Fur-
thermore, the addition of neutralizers to the agar should im-
prove bacteria growth and thus pathogen recovery [9]. Neu-
tralizers are compounds that are designed to neutralize the
most common detergents and disinfectants used in the normal
cleaning routine in order to increase growth on the plates/
dipslides [10, 11].
Dipslides have some advantages over standard contact
plates. First, the square agar design with a hinge allows sam-
pling from spots where a contact plate cannot be used. Second,
dipslides are enclosed in a plastic container, which not only
reduces the risk of contamination during transport, but also
increases shelf life and postpones the drying out of the agars.

The sampling technique may also matter when using con-
tact plates or dipslides. Few studies have investigated whether
contact time and applied pressure affect bacteria recovery, and
there is not consensus about the optimal time or pressure.
Some studies proposed a contact time of up to 30 minutes or
more while others recommend a more realistic time of contact
for 5–15 seconds [4, 12–15].
Most comparative tests have not shown any difference
between dipslide and contact plate sampling techniques. How-
ever, most of the tests done on this subject have been done in
controlled laboratory environments with a known inoculum
and one or few bacterial species [4, 7, 8, 16–18]. It is therefore
difficult to say which method is best in the real hospital
environment where the bacterial flora consists of a multitude
of different bacteria, most of which originate from soil and
skin surfaces (i.e. Bacillus sp. and Coagulase-negative staph-
ylococci [CoNS]).
The aim of this study was to evaluate whether one of four
different dipslides or a TSA contact plate were better at
recovering bacteria from the environment in clinical depart-
ments. The main endpoints were total bacteria count as well a
recovery of specific human indicator bacteria.
For testing, we chose two dipslides with a TSA agar on one
side and a McConkey or a VRBG agar on the other side in
order to increase the chance of isolating faecal bacteria. These
two different dipslides were also tested with and without
neutralizer to evaluate the effect of added neutralizers on
bacteria recovery. Standard TSA contact plates were used as
references.
Methods
Sampling sites
Samples were taken from five sample points in 40 rooms in
autumn 2012 at a children’s internal medicine ward at the
Copenhagen University Hospital. These five points were the
bathroom floor, bathroom sink, faucet handle, toilet seat and
the bed headboard. The sites were selected based on a pilot
study done earlier where these sites produced the highest
bacterial count. Each sample point was sampled with four
different dipslides and a TSA contact plate. Twenty samples
were collected in each sample day for a total of ten non-
Table 1 Agar composition of the
dipslides and contact plate Medium Side 1
TSA contact plate TSA (total count)
PCR Dipslide TSA (total count)
SC2 Dipslide TSA (total count)
SCN Dipslide TSA (total count)
TSA tryptic soy agar, VRBG vi- TV Dipslide TSA (total count)
olet red blood glucose
Eur J Clin Microbiol Infect Dis
consecutive days. Sampling was done around noon and only
in rooms occupied by patients. The normal cleaning routine
for the rooms were done in the morning or afternoon and the
sampling time was therefore adjusted so the samples were not
taken immediately after cleaning. The cleaning staff used
detergents and no disinfectants for routine cleaning. Rooms
with patients in isolation because of infections or rooms being
cleaned with disinfectants were excluded from the study.
Sampling methods
Sampling was done using aseptic techniques. All sample sites
were sampled with a round TSA contact plate (Ø55 mm,
Oxoid, Denmark) and four different dipslides: PCV, SC2,
SCN and TV (3M, Denmark). The agar composition of the
dipslides are depicted in Table 1. Each sample site was sam-
pled using each of the four dipslides and the TSA contact plate
in random order from the same sampling site. When possible,
the five agars were pressed on adjacent surfaces, but because
of limited space in some sites, the same point was sampled
with all five agars. The agar was firmly pressed against the
surface for 5 seconds. When the surfaces were not flat, the
agar was rolled against the surface in order to get the largest
area of the agar in contact with the surface.
Incubation and identification
The dipslides were incubated for 48 hours at 35–37 °C. Fol-
lowing incubation, total bacteria count was determined and
the different species identified using conventional identifica-
tion and matrix-assisted laser desorption/ionisation-time of
flight (Maldi-Tof) (Bruker, Denmark). Maldi-Tof was used
for potential pathogens and Enterobacteriaceae. Total bacteria
count was determined using visual comparison with the sup-
plied culture chart supplied by the manufacturer and given as
CFU/cm2. Furthermore, all potential pathogens (all faecal
bacteria and S. aureus) were identified using Maldi-Tof, and
susceptibility testing was performed using disc diffusion
(Neo-Sensitabs, Rosco Diagnostica, Denmark).
Data management and statistical analysis
Total growth was read from the TSA side of all dipslides.
Testing of differences in total growth on the TSA agar was
Side 2 Neutralizer
- Yes
VRBG (Enterobacteriaceae) No
McConkey (Coliforms) No
VRBG (Enterobacteriaceae) Yes
McConkey (Coliforms) Yes
Eur J Clin Microbiol Infect Dis

Fig. 1 Total bacterial growth with different sampling methods on the

TSA sides of the dipslides and on the TSA contact plate. Numbers are Fig. 2 Faecal indicator bacteria growth with different sampling methods.
given as means ± standard error of the mean (SEM) Percentages are given as positive samples / total samples

done with a paired, two-tailed T-test. As for bacterial species
presence, the results from both sides of the dipslides were
pooled and given as binary results depending on whether the
specific bacteria were found or not. The TSA contact plate
was used as reference. The difference between the reference
and each dipslide was analysed using 2×2 tables and signif-
icance tested with McNemars test using IBM SPSS software
version 20. All results with a P value of less than 0.05 were
considered significant.
Sampling sites
Five points were sampled from the patient rooms. The highest
total count was found on the bathroom floor which had an
average bacterial count of 47 CFU/cm2, followed by the
bathroom sink, the toilet and the water faucet. The lowest total
bacteria count was found on the bed headboard. As for faecal
indicator bacteria, the most contaminated site was the toilet
and the bathroom floor, followed by the bed headboard and
the toilet sink. The lowest number of faecal bacteria was found
on the faucet handle.

Results
Bacterial species
The most prevalent bacteria group was skin bacteria (present
in 72 % of the samples), largely dominated by CoNS, follow-
ed by soil bacteria (Acinetobacter sp., Bacillus sp. and
mould), which was present in 31 % of the samples. No
Candida sp. were found among the mould. Water bacteria
was present in 8 % of the samples, dominated by Pseudomo-
nas-like bacteria. Faecal indicator bacteria E. coli and Entero-
coccus spp. was found in 14 % of all samples. No vancomycin
resistant Enterococci (VRE), Carbapenem-resistant Entero-
bacteriaceae (CRE) or extended-spectrum beta-lactamase
(ESBL) producing bacteria were found in the samples.
Comparison of plates
There was no difference in overall bacterial growth between
the TSA contact plate and the TSA side of any of the dipslides
(Fig. 1). In addition, there was no difference between the
different dipslides when comparing them individually. Sam-
pling with the TSA contact plate produced a significantly
higher recovery of CoNS, Acinetobacter sp. and Pseudomo-
nas-like sp. than any of the four different dipslides (Table 2).
There was no difference in recovery of either faecal indicator
bacteria, Enterobacteriaceae or S. aureus between the differ-
ent dipslides and the TSA contact plate (Fig. 2).
When comparing dipslides with identical agar composi-
tions, with and without neutralizer (SC2 vs. SCN and PCV vs.

Table 2 Bacterial recovery of the

dipslides and tryptic soy agar Species Total positive samples TSA contact plate PCV SC2 SCN TV
(TSA) contact plate n % % % % %

S. aureus +CoNS 199 99 77b 75b 76b 78b

b b b
Percentages are given as positive Acinetobacter sp. + 94 83 48 51 56 59b
samples / total positive samplesa Pseudomonas-like sp.
E. coli +Enterococcus spp.b Enterobacteriaceae 30 47 53 70 63 77
Significantly different from Faecal indicatorsa 29 52 45 38 66 45
TSA contact plate

TV), there was no difference except for faecal bacteria indi-
cator recovery, where the dipslides containing neutralizers
(SCN and TV) produced a significantly higher recovery than
the dipslides without neutralizers (PCV and SC2).
Discussion
The purpose of this study was to evaluate whether dipslides
with different agar compositions were better suited for environ-
mental sampling than the standard TSA contact plates. Except
for skin and water bacteria recovery, there was no difference in
environmental sampling performance between the TSA contact
plate and the tested dipslides. This is in accordance with previ-
ous studies evaluating dipslides versus other sampling tech-
niques, although these have almost solely been based on labo-
ratory experiments with a single bacterial species. Salo et al.
have published two works, both of which were unable to show
a difference in bacterial counts and recovery between contact
plates and dipslides [7, 8]. Kircheis et al. found that TSA
contact plates yielded a higher CFU count than 19 different
dipslides on samples from reprocessed endoscopes, although
no significant difference was reported [19]. Mueller et al. did a
comparison of dipslides, petrifilm and contact plates in water
samples from cooling towers but were unable to find any
significant difference in yield and recovery [20].
As for total bacterial count, most of the samples were
higher than the 5 CFU/cm2 recommended as cleaning stan-
dards [2, 3]. These standards are based on samples from hand-
touch surfaces and not from sites such as the floor or toilet
seat. Moreover, the samples were taken in the middle of the
day in rooms occupied by at least one patient and cleaning was
done either in the morning or in the evening. Hence, the
patients in the rooms had plenty of time to contaminate the
surfaces in the bathroom and on the bead headrest. It is
therefore not surprising that the values found were much
higher than cleaning standard values.
Agar plates with neutralizers did not show a higher recov-
ery of bacteria, except for faecal indicators. The evidence from
in vitro studies done by Dey and Engley have found signifi-
cantly higher recovery rates of S. aureus from sanitized sur-
faces with the use of neutralizers [11]. However, normal
cleaning in the sampled hospital rooms is not based on disin-
fectants, only in cases of outbreaks and isolated patients, both
of which were excluded from this study. This can explain that
we did not see a significant effect of the added neutralizers.
The sampling technique may be a weak point because we
had to sample the same spot with five different agars. This can
be done in two ways, that is, either by sampling five adjacent
spots at the sites or by sampling the exact same point five
times. There are pros and cons to both methods, but we chose
to sample five adjacent sites where possible. This may have
affected the results since the bacteria may not be present at
Eur J Clin Microbiol Infect Dis
each sample spot. We tried to avoid this by sampling in
random order with each agar and by doing a large amount of
samples, but it is possible that this sampling method may have
affected the obtained results. There are, however, also cons to
sampling from the exact same spot because the first agar
removes some of the bacteria from the spot, leaving less
bacteria for the subsequent sampling media. All in all there
is no sampling method without flaws and we decided to use
the technique which we believed to give the highest agree-
ment between the samples.
The combination of a non-selective agar and a selective
agar on the other side offers, at least theoretically, a bigger
chance of findings pathogens. Even though the dipslides had a
selective agar for Enterobacteriaceae or coliforms, they did
not produce a better recovery of these bacteria in our study.
However, as mentioned in the introduction, there are probably
better ways of sampling for these, i.e. large area sampling
using a swab, sponge or cloth technique, if the goal is to
determine the presence of a specific pathogen although the
evidence is not clear [1, 4, 21].
In summary, dipslides and TSA contact plates perform
equally well for assessment of total bacteria count which
reflects the general cleanliness and hygiene standards. We
only saw a better performance of the TSA contact plate in
recovery of CoNS, Acinetobacter sp. and Pseudomonas-like
spp.. Considering the other advantages of dipslides such as
easier sampling in small spaces, better sample protection
during transport and longer shelf life, dipslides may be used
as an alternative to standard TSA plates, especially when
sampling in confined and hard to reach spaces, and if trans-
portation to the laboratory is long.
Acknowledgments The authors wish to thank 3M Denmark for the
donation of all dipslides.
Funding This study was a part of the SiB (Sundhed i
Børneinstitutioner) innovation consortium, funded by The Danish Coun-
cil for Technology and Innovation under the Ministry of Science, Inno-
vation and Higher Education.
Conflict of interests None of the authors has conflicts of interest, and
3M had no influence on the design of the study, nor on the analysis and
interpretation of the results.
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