EJPPS – European Journal of Parenteral and Pharmaceutical
Sciences Volume 29 Issue 1
Evaluation of Three Different Contact Plate
Methods for Microbial Surface Sampling of
Naturally Occurring Human Borne Microbial
Contamination.
T Eaton (1) , K Capper (1), M Ward (1) and J Bright (1)
1 AstraZeneca, Macclesfield, UK
Corresponding Author: Tim Eaton, Sterile Manufacturing Specialist
AstraZeneca,
UK Operations,
Silk Road Business Park,
Macclesfield
Cheshire. SK10 2NA
England
Email: [Link]@[Link]
Telephone: +44(0) 1625 514916
Evaluation of Three Different Contact Plate Methods for Microbial Surface
Sampling of Naturally Occurring Human Borne Microbial Contamination
1
Summary
The ability of irradiated 55 mm diameter tryptone soya agar contact plates to recover naturally
occurring surface microbial contamination, using three different manual sampling procedures, was
investigated. The investigation was completed by sampling hard surfaces contaminated with
microbe-carrying particles (MCPs) dispersed from a person within heavily populated environments.
This is more representative of the contamination that is found within cleanrooms and avoids issues
resulting from the utilisation of standard commercial test organisms to distribute unicellular
microbes onto surfaces, which are likely to be transferred to the plate with different efficiencies
compared to the naturally occurring MCPs. It was determined that rolling the contact plate media
over the surface, using firm pressure for either 1 or 5 seconds recorded little differences in the mean
recovery efficiencies (53% and 48% respectively). However, both recorded significantly higher
efficiencies than just a single
1 second press of the media onto the surface with firm pressure (16%).
Key words: Microbiological surface contamination, contact plates, RODAC plates, microbe-
carrying particles (MCPs), environmental monitoring, recovery efficiency.
1. INTRODUCTION
For sterile products manufacturing, it is a requirement of Annex 1 of the European Union Guide to
Good Manufacturing Practice (EU GGMP) 1 that microbiological monitoring of cleanrooms includes
the use of 55 mm diameter contact plates for sampling defined surface locations. The Guide includes
limits to be applied to this monitoring and the expectation is that supporting data for the recovery
efficiency of the sampling method should be available.
Typically, circular RODAC (replicate organism detection and counting) plates (55 mm diameter,
24 cm2 surface area) containing nutrient agar (between 15.5 and 16 ml) are used for sampling
surfaces that are relatively flat. They are poured to give an agar meniscus that protrudes just above
the rim, and are based upon plates that were originally reported on in 1964 2. They are easy to use
and require minimal training and the full area of the nutrient agar is rolled over the surface to be
sampled, facilitated by the convex media profile, or they can be applied to the surface for a few
seconds ensuring uniform and steady pressure with no rolling action. Viable particles removed from
the surface adhere to the agar and the lidded plates are then incubated and examined and the
number of colony forming units (CFU) and types of micro-organisms recovered are reported and the
results expressed as the number of CFU per plate.
The recovery efficiency will be affected by many factors, including the sort of plate media used, the
type of micro-organisms present on the surface and the material and finish of the surfaces 3 to be
sampled. It will also be influenced by the contact area of the plate media with the surface being
sampled and may be influenced by the associated duration of this contact, both of which are
associated with the plate sampling method. To reduce variations associated with these two
parameters, there are commercially available plate holding devices which can be utilised for the
sampling that apply the same pressure each time and also have an indicator of the specified contact
time, that can be set for different durations. However, the surface sampling is typically a manual
procedure that does not utilise such devices and the application method and contact time are reliant
on the person undertaking the sampling and hence this is likely to be more variable.
In order to determine a convenient manual sampling procedure that can be routinely used to
provide more consistency of practice, three different methods were tested. Surfaces associated with
three different areas, each used on a daily basis by numerous people, were utilised. These surfaces
are continually contaminated with naturally occurring microbe-carrying particles (MCPs),
predominantly dispersed from personnel, in relatively large numbers, and are representative of the
majority of the microbes recovered from cleanroom environments. Actual cleanroom surfaces were
not utilised as the surface concentrations are too low and accurate results less likely be obtained.
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The use of these naturally occurring MCPs also avoids issues resulting from the utilisation of
standard commercial test organisms and a carrier medium to deposit suspensions of micro-
organisms onto the test surfaces. Upon evaporation of the carrier medium, sub-micron unicellular
microbes are distributed across the surfaces which are not representative of the larger MCPs
present in cleanrooms which have an average aerodynamic diameter of about 8 μm to 12 μm 4 ,5, 6.
On contact, these unicellular microbes may be transferred to the plate with different efficiencies
compared to the larger size naturally occurring MCPs, for which the efficiency is likely to be greater.
Bench and floor surfaces in laboratory and amenities areas, contaminated with MCPs, in sufficient
numbers (typically greater than 20 MCPs) that could be used for the comparison of the three
sampling methods, were identified from a preliminary study. From each of three adjacent but
separate positions at 20 different locations, two sequential samples were taken from the exact same
place using the three different sampling methods. Following incubation of the plates, the number of
recovered CFUs were counted and the recovery efficiencies for each sampling method determined
from the two samples, for each location, and an average efficiency then calculated. The resultant
data was reviewed in order to determine if the recovery of surface microorganisms was influenced
by the sampling method and the type of surfaces sampled and to also provide recommendations for
the most appropriate sampling procedure for routine use to help to ensure consistency of practice.
2. METHOD TO DETERMINE SURFACE MICROBIAL COLLECTION
EFFICIENCIES
Contact plates and incubation conditions
All plates utilised were Becton Dickenson, BD BBLTM IC-XT Trypticase™ Soy Agar medium with lecithin
and polysorbate 80 surface neutralising agents, 55mm diameter RODACTM LL. The plates have locking
lid features and are gamma irradiated and sealed in triplicate polythene bags, sourced from an
approved supplier and are routinely tested for their ability to recover microbial contamination.
Although for the purpose of this investigation, Beckton Dickinson plates were used, the results are
expected to be relevant to other similar contact plates.
Following sampling, all plates were immediately and simultaneously incubated, in the same
validated incubator, at 30- 350C for 5 days and the number of microbial colonies counted and
identification of the organisms from all plates completed.
Sampling methods
The following three different methods of contact plate sampling were used;
Method 1 - rolling the plate over the surface in a single motion, lasting 1 second, with firm force.
Method 2 - rolling the plate over the surface in a single motion, lasting 5 seconds, with firm force.
Method 3 - plate pressed onto the surface, with no rolling, with firm force
All of the sampling, for all three methods, was performed by the same person.
Sampling locations and surface materials
A total of 20 different surface locations, from tables, benches and floors associated with the
following three separate areas, were identified;
1. Microbiological testing laboratory
2. Microbiological samples receipt area
3. Amenities area used for personnel breaks and food consumption
The floor surfaces were vinyl and the bench and table surfaces were ‘Trespa’ (synthetic resin). The
sampling locations are described in table 1.
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Table 1 Sampling locations
Sampling Location Description
Location
Surface Room
Reference
1 Middle of back bench
2 Floor by cupboard
3 Floor between benches
4 Floor in front of instrument
5 Middle of right hand bench Microbiological
6 Middle of left hand bench testing laboratory
7 Floor between benches
8 Floor in front of fume cupboard
9 Floor in front of biosafety cabinet
10 Floor by entry door
11 Floor by entry door
12 Floor by exit door
13 Floor middle of room Microbiological
14 Floor in front of shelf samples receipt area
15 Floor in front of table
16 Table
17 Floor by entry door
18 Floor in front of microwaves
Amenities area
19 Floor in front of lockers
20 Table
Sampling procedure
From each of the 20 surfaces locations, three separate adjacent positions in close proximity to each
other were used. At the first position, a sample was taken using Method 1 (sample A), and a second
sample using the same method (sample B) was immediately taken at the exact same position. At the
second adjacent (non-sampled) position, the sampling was repeated using Method 2. Finally in the
third adjacent position, also not previously sampled, samples were taken in an identical manner
using Method 3.
Plates were labelled with the sampling method, the location from which the sample had been taken
and with the first (A) or second (B) sample reference. A total of 120 plates (40 for each sampling
method) were utilised.
Determination of recovery efficiency
A mathematical model is described which may be used to assess the efficiency and consistency of a
surface sampling method 7. This is based upon multiple and two stage sequential sampling and the
two stage sampling is a convenient method if the counts on the surface following the second
sampling are relatively low. The recovery efficiency for the two stage sampling can be determined
using equation 1 7.
Recovery efficiency (%) = [1 – (B /A)] x 100 Equation 1
Where
B = total count from second sample
A = total count from first sample
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3. TEST RESULTS
The results for the testing and the recovery efficiencies for each of the three different sampling
methods are shown in table 2. All contaminated plates were subject to Matrix-assisted laser
desorption/ionization-time of flight (MALDI-ToF) mass spectrometry identification and this
confirmed the majority of microbes to be the expected Gram positive skin microbes and typical
environmental microbes. Species of Staphylococcus, Micrococcus and Bacillus were most commonly
identified, along with fewer Microbacterium and single isolates of other organisms such as
Dermacoccus and Okibacterium. Shown in table 3 is a summary of the identification of the micro-
organisms recovered from the surfaces, from both the first and second plate samples.
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Table 2 Plate counts and recovery efficiencies for each sampling method
Plate Count (CFU)
Sampling Method 1 Sampling Method 2 Sampling Method 3
Sample
Reference Recovery Recovery Recovery
Bacteria Mould Efficiency Bacteria Mould Efficiency Bacteria Mould Efficiency
(%) (%) (%)
1A 23 0 41 0 57 0
87.0 73.2 71.9
1B 3 0 11 0 16 0
2A 45 2 31 1 41 0
59.6 34.4 31.7
2B 19 0 21 0 27 1
3A 41 1 29 1 42 0
47.6 26.7 50.0
3B 22 0 16 6 21 0
4A 44 0 31 2 23 0
61.4 72.7 -30.4
4B 15 2 8 1 29 1
5A 9 0 2 0 1 0
88.0 100.0c -100.0
5B 1 0 0 0 2 0
6A 5 1 7 0 9 0
83.3 57.1 -44.4
6B 1 0 3 0 13 0
7A 49 0 41 0 37 0
46.9 68.3 24.3
7B 26 0 13 0 24 4
8A 134 0 42 1 50 0
76.9 -48.8 48.0
8B 31 0 64 0 25 1
9A 59 1 104 0 42 0
40.0 58.7 45.2
9B 33 3 40 3 23 0
10A 44 0 41 3 32 0
34.1 81.8 34.4
10B 29 0 8 0 21 0
11A 17 0 40 1 33 1
52.9 46.3 2.9
11B 8 0 18 4 33 0
12A 29 0 46 8 20 3
13.8 64.8 56.5
12B 25 0 14 5 7 3
13A 33 2 42 2 24 3
74.3 65.9 -3.7
13B 9 0 15 0 26 2
14A 48 0 26 6 22 3
27.1 43.8 8.0
14B 30 5 16 2 23 0
15A 30 0 34 0 15 0
56.7 76.5 40.0
15B 12 1 8 0 9 0
16A 5 1 4 1 5 0
-16.7 80.0 60.0
16B 7 0 1 0 1 1
17A 50 1 35 0 21 0
27.5 -25.7 -19.0
17B 34 3 44 0 25 0
18A 33 0 30 0 13 1
66.7 40.0 -35.7
18B 11 0 18 0 19 0
19A 13 3 13 0 14 0
62.5 -7.7 42.9
19B 6 0 14 0 7 1
20A 17 0 12 1 6 1
64.7 46.2 42.9
20B 5 1 5 2 3 1
Total Aa 740 678 519
Total Ba 342 360 369
Total
1082 1038 888
A and Ba
Mean
Recovery 53% 48% 16%
Efficiencyb
Standard
26% 38% 44%
Deviation
6
Notes
a. Combined bacteria and mould counts
b. Mean of all of the individually calculated recoveries
c. The plate B count is 0 but there are counts with plate A and a 100% efficiency recorded
Table 3 Identification of recovered micro-organisms
Genus Species
Micrococcus luteus, antarcticus
hominis, epidermidis, haemolyticus, ureilyticus, warneri,
Staphylococcus
succinus, saprophyticus, equorum, saprophticus
Microbacterium saccharophilum
Corynebacterium tuberculostearicum, coyleae
myciodes, amyloliquefaciens, licheniformis,
Bacillus
amyloliquefaciens, cereus, thuringiensis
Mesobacillus thioparans, subterraneus
Cytobacillus kochii
Solibacillus silvestris
Paenibacillus taiwanensis, glucanolyticus
Brevundimonas vesicularis
Okibacterium fritillariae
Dermabacter hominis
Curtobacterium flaccumfaciens
Pantoea agglomerans
Rathayibacter rathayi
(pseudarthrobacter)- sulforivorans, equi, chlorophenolicus,
Arthrobacter
oryzae
Gordonia hongkongensis
Rothia kristinae kristinae
Kocuria arsenatis, iridica, palustris, rhizophila
Moraxella osloensis
4. DISCUSSION OF RESULTS
Combined bacteria and mould data
Table 2 shows the bacteria and mould counts and it can be seen from this table that the total count
for bacteria (2904) is much larger than for mould (104) and consequently, there are too few mould
counts to estimate the sampling efficiencies of the three methods for moulds alone. Although there
are likely to be differences in the recovery efficiency of moulds compared to bacteria, fundamentally
due to differences in their sizes, it is reasonable to combine all of the counts, as the low mould
counts are only a small proportion (3.5%) of the total counts. This small proportion will not have a
significant influence to the mean recovery efficiency calculations. Additionally, mould may also be
(rarely) recovered from the cleanroom environment, as well as a much higher relative number of
bacteria, and so the inclusion of these low mould counts will be more representative of actual
cleanroom environmental conditions. Table 3 shows the identification of the recovered micro-
organisms and confirms them to be human borne and general environmental isolates, typical of
what is recovered from pharmaceutical manufacturing cleanrooms.
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Overall number of MCPs reported for each sampling method
Shown in figure 1 are the total counts (plates A and B) at each location for each of the three
sampling methods. Method 1 showed the highest number of total counts (1082 and highest at 11 of
the 20 locations). Method 2 had a similar total number of counts (1038) and Method 3 had the
lowest number of counts (888 and lowest at 9 of the 20 locations). If the number of counts
associated with just the first samples (sample A) are considered, counts of 740, 678 and 519 were
recorded for Methods 1, 2 and 3 respectively. This is shown in figure 2 and shows a distribution
similar to the total counts shown in figure 1. In terms of initial recovery, this suggests that Method 1
is superior to Method 2 and that Method 2 is superior to Method 3. Application of a Friedman test
(adjusted for ties) gives a p-value of 0.009, which against the criterion of <0.05, is statistically
significant. It has however been assumed that the three adjacent sampling positions at each location
have similar microbial concentrations and that the higher counts associated with Methods 1 and 2,
compared with Method 3, are due to the superior effectiveness of the sampling procedures. This is a
reasonable assumption and the likelihood that all of the sampling locations associated with Method
3 were consistently lower than the adjacent locations is unlikely, as demonstrated by figures 1 and 2,
where the distributions are generally consistently reduced at all locations, for both the first (sample
A) and also the second (sample B) samples. Additionally, the two plate sampling procedure utilised,
with sequential samples at the exact same position, will also be useful to address any local variations
in surface concentrations associated with the different positions at the same sampling location. This
provides further confidence that the results can be utilised to provide an accurate reflection of the
actual recoveries associated with each sampling method.
Figure 1 Total counts (samples A and B) and sampling locations
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Figure 2 First sample counts (sample A) and associated sampling areas and their locations
Calculated recovery efficiencies
Shown in table 2 are the mean recovery efficiencies which are 53%, 48% and 16% for sampling
Methods 1, 2 and 3 respectively. These were determined for combined bacteria and mould counts
and to avoid bias from the samples that had the higher counts, the efficiencies were determined for
each location and the mean efficiency calculated from all of the individual efficiencies. Shown in
figure 3 are the individual recovery efficiencies, at each of the twenty surface locations, for each
sampling method. This shows that for some of the sampling (17%), the count recorded for the
second sample was higher than for the first sample and the calculated recovery efficiency is a value
less than zero, which is not possible. These ‘impossible efficiencies’ are likely to be related to the
acknowledged errors and inconsistencies that are associated with the microbiological testing
methods and become more significant when dealing with very low counts. They will have an
influence on the calculated recovery efficiencies but as all of the sampling is subject to the same
microbiological testing method, these samples have been retained and all results considered as a
collective set, and will provide a worst-case underestimate of the recovery efficiencies. It should also
be noted that the majority of the impossible efficiencies occurred with Method 3 (60%) with lesser
occurrences with Method 2 (30%) and Method 1 (10%).
One contributory reason for the reduced overall recovery efficiency of sampling Method 3 is likely to
relate to the reduced area of contact between the surface being sampled and the plate media, as
the procedure involves only pressing the media onto the surface with no rolling action that is utilised
with Methods 1 and 2. Simple experiments completed at AZ Macclesfield involving replicating both
sampling methods on coloured paper and determining the areas deposited by the wet media
surface, indicated that sampling Method 3 has only approximately 89% of the contact area
compared to Methods 1 and 2. However, even when this correction factor is applied to Method 3, it
does not account for all of the reduced recovery efficiency compared with the other two sampling
methods and other factors must therefore be applicable.
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Figure 3 Recovery efficiencies for each surface location and each sampling method
Recovery from different types of surfaces
It is also useful to determine if there is any distinction between the recovery from the different
surfaces. Table 2 confirms that the first plate counts are generally lower for the benches and tables
compared to the floors where locations 5 and 6 (both microbiology testing laboratory), location 16
(sample receipt area) and location 20 (amenities area) are the four locations with the lowest counts.
The bench at location 1 (microbiology testing laboratory) is an exception to this, with counts similar
to the floor samples from the microbiology testing laboratory (locations 2 to 4 and 7 to10). Shown in
figure 4 are the recovery efficiencies for each sampling method relevant to the surfaces that were
sampled. Although there were only 5 bench and table samples compared to 15 floor samples,
overall, these results suggest that the initial microbial concentrations are lower for the tables and
benches than for the floor samples. However, the recovery efficiency of Methods 1 and Method 2
for bench and table samples are similar to the corresponding recovery efficiency for floor samples.
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Figure 4 Recovery efficiency for bench, table and floor locations for each sampling methods
5. CONCLUSIONS
The recovery of naturally occurring surface microbial contamination with sterile tryptone soya agar
55 mm diameter RODAC plates, using three different manual sampling methods has been evaluated.
It was shown that the two sampling methods (Methods 1 and 2) that rolled the plate media over the
surface under consideration both had similar recovery efficiencies (53% and 48% respectively) and
were better than the sampling method (Method 3) that just pressed the plate media onto the
surface (16%). Method 3, as well as having a poorer recovery efficiency than the other methods, also
showed a more variable range of efficiencies. Although sampling Methods 1 and 2 both rolled the
plate media over the surface but with different contact times (1 second and 5 seconds, respectively)
there was little difference in the recovery efficiency. It was determined that the reduced counts
associated with sampling Method 3 were unlikely to be fully due to a reduced area of contact of the
plate media with the surface sampled compared with Methods 1 and 2 (a reduction of 89%).
Additionally, it was considered that there were no significant reductions in the surface microbial
concentrations that were sampled using Method 3 that would account for the reduced efficiency.
Issues associated with surface variations would, however, be offset by the two plate sampling
procedure utilised that identically and sequentially sampled from the exact same position, at each
sampling location. It was also noted that for the two different types of hard surfaces utilised (vinyl
and ‘Trespa’) for the evaluation, there were no differences in the recovery efficiencies. Overall, it is
concluded that there is little difference between Methods 1 and 2 but both are better sampling
procedures than Method 3.
For such routine manual surface sampling of the cleanroom areas, the single use of sampling
Method 1 would help to ensure consistency of practice. Due to known variations in the recovery
efficiencies associated with different surfaces 3, further investigation of the recovery associated with
different cleanroom surfaces, such as garment fabric, glove material and stainless steel should be
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evaluated. This could be similarly done utilising naturally occurring surface microbial contamination
and sampling Method 1 to provide comprehensive information regarding recovery efficiencies for
different cleanroom surfaces.
REFERENCES
1. The rules governing medicinal products in the European Union –Volume 4 EU guidelines to good
manufacturing practice - medicinal products for human and veterinary use - Annex 1-
Manufacture of sterile medicinal products. European Commission, Brussels, 2022.
2. Hall L.B and M.J. Hartnett Measurements of Bacteria contamination on surfaces in
Hospitals, Public Health Rep 79 (11):1021-1024 (1964).
3. Werner, H-P., Swinke, U. and Werner, G. Development of a new test method for surface
disinfection procedures. III. The impression method: influence of the test surface material and the
types of microorganisms on the recovery rate. Zentralbl Bakteriol Orig B. 165 (1):20-42 (1977).
4. T Eaton, C Davenport and W Whyte. Airborne microbial monitoring in an operational cleanroom
using an instantaneous detection system and high efficiency microbiological samplers. European
Journal of Parenteral & Pharmaceutical Sciences 2012; 17(2): 61-69.
5. Noble WC, Lidwell OM and Kingston D. The size distribution of airborne particles carrying
micro-organisms. Journal of Hygiene 1963; 61: 385-391.
6. Whyte W and Hejab M. Particle and microbial airborne dispersion from people. European
Journal of Parenteral and Pharmaceutical Science 2007; 12(2): 39-46.
7. W. Whyte, W. Carson and A. Hambraeus. Methods for calculating the efficiency of bacterial
surface sampling techniques. Journal of Hospital Infection (1989) 13, 3341.
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