International Journal of Microbiological Research 3 (3): 208-215, 2012

ISSN 2079-2093
© IDOSI Publications, 2012
DOI: 10.5829/idosi.ijmr.2012.3.3.64231

A Novel Improved Bioburden Recovery Method Using Swabbing Technique

Mostafa E.A. Eissa and Ahmed M. Mahmoud

Quality Control Supervisor, Hikma Pharmaceutical Company, 6th October City, Egypt

Abstract: The validation of surface-recovery methods is a pre-requisite for residual determination of cleaning
effectiveness in process validation studies. These methods should be challenged in the laboratory using
pilot-scale controlled conditions in order to evaluate the suitability for their intended use. Different swabbing
techniques have been studied and compared with rinse method for 7 microorganisms (Staphylococcus aureus,
Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Kocuria rosea, Candida albicans and
Aspergillus niger) on 5 different coupon surfaces (Stainless steel, Glass, Rubber, TEFLON and Plexiglass)
representing machine materials in production area. Only one swabbing technique gave good results in
comparison to both rinsing method and previously reported Nylon™ flocked QUANTISWAB™. The new
swabbing technique is based on ordinary Rayon™ fiber which is known of low microbial recovery using
traditional method. However simple modification in the recovery procedure using simple device improved the
bioburden recovery to become over 80%. Moreover this novel method has good flexibility to be customized
to enhance recovery according to cleaning validation condition and study.

Key words: Swabbing Technique Rinsing Method Rayon™ Fiber Bioburden Recovery Coupon

INTRODUCTION Validation of cleaning methods is required to

ensure that the equipment-cleaning cycle consistently
The objective of the cleaning validation is to provides results that meet acceptable levels of
verify the effectiveness of the cleaning procedure for cleanliness. Guidelines for cleaning requirements are
removal of product residues, degradation products, provided to the pharmaceutical industry in the cGMP
preservatives, excipients and/or cleaning agents as documents in the United States, Europe and other
well as the control of potential microbial contaminants. countries. Equipment cleaning requirements are also
In addition one needs to ensure there is no risk addressed in the Pharmaceutical Inspection Convention
associated with cross-contamination of active ingredients (PIC) recommendations and the Parenteral Drug
[1]. Association (PDA) Technical Report No. 29, Points to
It is therefore not surprising that cleaning validation Consider for Cleaning Validation [3].
undergoes extensive regulatory review in the Cleaning should be carried out as soon as practical
pharmaceutical industry during inspections. In fact, after the end of processing and should leave the plant in
during the past few years, cleaning validation has a suitable condition for next use. In developing the
ranked among the top 10 areas of concern in warning sampling plan for a validation study, it makes scientific
letters issued by the FDA. According to Kristen Evans, sense to incorporate an understanding of the limitations
leader of the Guidance and Policy Team in the Division of the sampling method relative to the surface to be
of Manufacturing and Product Quality in FDA’s Center sampled. The two methods of sampling generally
for Drug Evaluation and Research, equipment cleaning employed are swab and / or rinse sampling. The selection
and maintenance ranked second in warning letters and of either of these techniques must be consistent with
GMP citations for the fiscal year (FY) 2004-2005 [2]. sound scientific judgment and must support the objective
Although not all cleaning issues relate to microbial of the study [4].
contamination, controlling bioburden through adequate Equipment swabbing must be performed by qualified
cleaning processes is a regulatory expectation enforced personnel and sterile swabs made from materials that
by the FDA. do not interfere with the test should be used. There are

Corresponding Author: Mostafa E.A. Eissa, Quality Control Supervisor, Hikma Pharmaceutical Company, 6 th October City,
Egypt. Tel: +20224705461 - +201006154853.
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various types of swabs used to monitor flat or hard-to-reach surfaces such as the bottom of a tank, O-rings, traps,
transfer lines and U-bends. Swab sampling should be carried out wearing sterile gloves to minimize adventitious
contamination. For bioburden recovery, after swabbing is complete, the swab may be streaked onto an agar medium or
broken into a diluent, vortexed for about 30s and the liquid sample preparation is then tested by pour-plate or membrane
filtration method [5].
Equipment rinse is performed using a solvent that will not interfere with recovery. Sometimes placebo is used,
although this approach is not typical for collection of bioburden. Most rinse samples are collected using purified water
or water for injection (WFI). For bioburden recovery, after the rinse sample is collected, it is processed via the membrane
filtration technique [5]. Swabbing technique although has its special advantage over rinsing method yet its major
disadvantage is its low recovery of collected bioburden of microorganisms which is related to the swab fiber matrix that
hinders the release of microbial cells. In this work, a special but simple technique was looked for to release gathered
microbial cells from swab material. This new method was compared with ordinary, modified swabbing and rinse
techniques.
The importance of swabbing technique is extended to include other practical fields such as inanimate surfaces of
medical facility [6], medical instruments [7], clinical specimen [8] and food and veterinary industry [9, 10].

MATERIALS AND METHODS

Representative Challenge Organisms:

Challenge microorganism used for the validation studies Vendor (number) Organism Type
Staphylococcus aureus ATCC 6538 Gram-positive coccus
Bacillus subtilis ATCC 6633 Gram-positive spore-forming rod
Pseudomonas aeruginosa ATCC 9027 Non-fermenting, Gram-negative rod
Escherichia coli ATCC 8739 Fermenting, Gram-negative rod
Kocuria rosea EM isolate Gram-positive coccus
Candida albicans ATCC 10231 Yeast
Aspergillus niger ATCC 16404 Filamentous fungus

Sterilized coupons (small pieces of material media were centrifuged, the supernatant was removed and
representing equipment to be sampled e.g. Stainless steel, the microbial pellet was re-suspended in sterile USP saline
Glass, Rubber, TEFLON and Plexiglass). TS.
Bacterial suspensions were adjusted with the
Preparation of the Working Cultures: Fresh buffer diluent to an optical density of 0.1-0.3 at a
transfers using agar medium or culture broth were wavelength of 550 nm, using a spectrophotometer; yeast
prepared and the bacterial cultures were grown using suspensions were adjusted with the buffer diluent to a
Tryptone soya medium (agar or broth) and incubated 5.0 McFarland turbidity standard [11, 12]. As a guideline,
at 30-35°C for 18-24 h. Yeast cultures were grown a 1-mL aliquot of the 10 5 or 10 6 dilutions of these
using Sabouraud's dextrose agar (SDA) medium recommended standardized suspensions of bacteria and
(agar or broth) and incubated at 20-25°C for 2-3 d. a 1-mL aliquot of the 104 dilution of the recommended
Cultures of filamentous fungi (mold) were inoculated onto standardized suspension of yeasts would yield counts in
SDA and incubated at 20-25°C for 5-7 d or until good the range of 10-100 CFU.
sporulation was achieved. If the standardized inocula of bacteria and yeasts
Transfers were prepared on solid media, the bacteria weren’t used promptly (within 2 h), the suspensions
and yeast cultures were harvested by washing each slant should be stored under refrigeration. Suspensions of
or plate with approximately 2 mL of sterile USP Saline test vegetative organisms prepared in USP saline TS or a
solution (TS), pH 7.0 buffered sodium chloride solution or buffer solution remain viable and stable for 7-10 d if
pH 7.2 phosphate buffer. Transfers prepared in liquid maintained under refrigerated conditions.

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Spore Suspension Preparation: Mold spores were The swab was streaked onto an agar medium or
harvested by washing the agar surface with sterile USP broken into a diluent, vortexed for about 30 s and the
saline TS or a buffer solution containing 0.05% liquid sample preparation was processed by the membrane
polysorbate 80. A sterile inoculating loop or some sterile filtration method and inoculated onto TSA with
glass beads were used to loosen the spores and combine incubation conditions at 30-35°C for 3-5 d.
the washings in a sterile container. The bacterial spore Results were reported as number of CFU per swab
suspension (e.g., Bacillus subtilis), was prepared by and the percent recovery for each test organism was
harvesting the inoculated agar plates with sterile water calculated. Swab sampling was done in triplicate, for each
and heat shock the suspension for 15 min at 65-70°C, challenge organism.
starting the timing when the temperature reaches 65°C A test-negative control for each swab set was
[11, 12]. Then the suspension was cooled rapidly in an ice applied to verify aseptic manipulations by carrying out
bath (0-4°C) and stored under refrigeration. the procedure just described but with uninoculated
coupons. The inoculums level counts exceeded 100 CFU
Bioburden Recovery Methods for Cleaning Validation and microbial growth was recovered from the negative
Procedure: Bioburden recovery methods under study control samples.
were grouped into:
Recovery Study Using the Dry Method: The dry
Rinse method. method was used for spore-forming bacteria
Swab method (wet and dry) with different (e.g. Bacillus subtilis) only because vegetative cells
modification for comparison of best recovery: suffer desiccation and, therefore, would not be viable on
Direct swabbing technique: Usual normal method of dry surfaces [5, 15].
sampling. Each type of coupon surface was inoculated with
25-100 CFU of the inoculum's suspension prepared in
Agar Streaking Technique: After sampling swab was sterile saline solution using the micropipette, the inoculum
streaked on solid agar media. was allowed to evaporate to dryness under laminar flow
conditions.
Five Minutes Sonication after Swabbing: Tubes of swabs The swab method was used to recover the test
in diluents were sonicated before filtration. organisms. Using a wet swab the dried inoculum was
removed from the surface of the coupon.
Prolonged Vortexing: 2 minutes vortexing instead of The swab was streaked onto an agar medium or
30 seconds before filtration. broken into a diluent, vortexed for about 30 s and the
liquid sample preparation was processed by the membrane
Glycerol Swabbing: Normal swabbing technique except filtration method and inoculated onto TSA with
using glycerol as diluent for sampling. (at which the swab incubation conditions at 30-35°C for 3-5 d.
was immersed in liquid glycerol just prior sampling). Results were reported as number of CFU per
swab and the percent recovery for each challenge
Outward Flushing Technique: Swab after vortexing was spore former organism was calculated. Swab sampling
subjected to washing by flushing diluent from inside of was done in triplicate, for each challenge organism.
the swab hollow stick to outside. A test-negative control for each swab set was applied
to verify aseptic manipulations by carrying out the
Recovery Study Using the Wet Method: This procedure just described but with uninoculated
procedure was performed for vegetative cells coupons. Test should be repeated if the inoculums level
(bacteria and fungi) to prevent loss of viability due to counts exceeded 100 CFU or less than 25 CFU and
desiccation. microbial growth was recovered from the negative control
Each type of coupon surface was inoculated with samples.
25-100 CFU of the inoculum's suspension prepared in In Recovery study using the wet method by swab
sterile saline solution using micropipette and a contact technique,the inoculum's volume on the surface should
time of less than 1 min was allowed. The swab method not be less than 100 ul for Rayon sterile swab used in this
was used to recover the test organisms by using a dry study. Acceptance criteria for the bioburden recovery
swab to remove the liquid inoculum's from the surface of percent from coupon surface should not be less than
the coupon [5, 13, 14]. 50 % of the inoculum's control.

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Recovery Study Using the Rinse Method: The inoculated Results demonstrated in Figure 5 showed that in
coupon was placed in an aliquot of sterile diluents general all microorganisms failed to be recovered using
(e.g., purified water) contained in a sterile vessel, which 2 minutes vortexing swabbing technique except 89% for
would be used to rinse the given piece of equipment Aspergillus niger from stainless steel surface while the
[5, 16]. lowest was 5% for Staphylococcus aureus from
The vessel was shaked well and then the coupon was plexiglass. Results shown in Figure 6 indicated that all
removed aseptically. The rinse solution was filtered microorganisms failed to be recovered using glycerol
through a 0.45-µm membrane filter. Then, aseptically the swabbing method (Glycerol immersion) with exception of
filter was removed and placed onto a TSA agar plate. The Staphylococcus aureus and Eschericia coli from
prepared TSA plates were incubated at 30-35°C for 3-5 d. stainless steel surfaces. While Candida albicans,
The recovered colonies were enumerated and results were Kocuria rosea, Pseudomonas aeruginosa and Bacillus
reported. This procedure (rinse sampling) was performed subtilis gave 0% recovery from stainless steel surfaces.
in triplicate, for each challenge organism. A test-negative Results illustrated in Figure 7 revealed that all
control for each swab set was applied to verify aseptic microorganisms were recovered from all surfaces using
manipulations by carrying out the procedure just swab filtration - after outward flushing of swabs using
described but with uninoculated coupons. Test should be buffer pH 7 with Escherichia coli giving highest recovery
repeated if the inoculums level counts exceeded 100 CFU followed by Kocuria rosea from rubber surface material.
and microbial growth was recovered from the negative Pseudomonas aeruginosa followed by Bacillus subtilis
control samples. gave the lowest recovery from all surfaces if compared
with other microorganisms. The swabbing technique (with
RESULTS outward flushing) is valid to be used in cleaning
validation studies.
Results demonstrated in Figure 1 showed that
generally all microorganisms were recovered from all DISCUSSION
surfaces using the rinsing method; with observation that
Staphylococcus aureus and Bacillus subtilis showed the Validation studies are usually performed using
highest recovery while Escherichia coli followed by representative surfaces identified on the production areas.
Pseudomonas aeruginosa gave the lowest recoveries In the current study material of coupons was carefully
from all surface materials. Results illustrated in Figure 2 chosen to represent the majority of equipments and
revealed that; all microorganisms generally failed to be machines in the production area of pharmaceutical facility.
recovered using direct swabbing method, except for Among the six methods of swabbing technique only one
Aspergillus niger. Aspergillus niger although failed for method gave promising results. The remaining five
the rubber surface, yet passed for all other surfaces. methods failed -with few exceptions with some
The highest recovery was 78% for Aspergillus niger microorganisms on some surfaces- to achieve 50%
from glass surface while the lowest was 12% for recovery.
Candida albicans from glass surface material. Rinsing method was found to be superior in terms of
Modifications were done on the sampling or testing recovery if compared to traditional swabbing technique
techniques to increase the bioburden recovery from however new modification in swabbing process rendered
various surfaces using cotton swabs. Results shown both methods nearly equal (approximate overall recovery
in Figure 3 indicated that all microorganisms failed to 79% for rinsing versus about 80% for swabbing
be recovered using direct agar streaking method. technique). Both methods were nearly the same since in
The highest recovery was 25% for Aspergillus niger either technique a process of flushing with diluent was
from stainless steel surface while the lowest was 1% for done as method of extraction of microorganisms. The aim
Kocuria rosea from glass surface material. Results of using glycerol in one of the swabbing method was to
illustrated in Figure 4 revealed that in general all form a thin layer around swab material that can take
microorganisms failed to be recovered after 5 minutes microorganisms off the surface however results obtained
sonication swabbing technique. The highest recovery were variable in which Staphylococcus aureus and
was 71% for Aspergillus niger from stainless steel Escherichia coli gave the highest results among the
surface while the lowest was 0% for Staphylococcus studied group on stainless steel surface while the
aureus from stainless steel and glass surface material. remaining failed to give acceptable results.

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Fig. 1: The recovery percentage of bioburden from rinsing technique for all tested surfaces microorganisms

Fig. 2: The recovery percentage of bioburden from direct swabbing technique for all tested surfaces and all
microorganisms

Fig. 3: The recovery percentage of bioburden from agar streaking technique for all tested surfaces and all
microorganisms

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Fig. 4: The recovery percentage of bioburden from filtration after 5 minutes sonication swabbing technique for all tested
surfaces and all microorganisms

Fig. 5: The recovery percentage of bioburden from filtration after 2 minutes vortexing swabbing technique for all tested
surfaces and all microorganisms

Fig. 6: The recovery percentage of bioburden from glycerol swabbing technique for most of the tested surfaces and
microorganisms

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Fig. 7: The recovery percentage of bioburden from swab outward flushing -applying pressurized flushing by sterile
pumping device with buffer pH 7 at the internal side of the swab- criteria for all tested surfaces and all
microorganisms

It should be noted that Aspergillus niger spores difficulties to release viable micro-organisms. Our study
gave the highest recovery in comparison with some (unpublished data) revealed that Rayon™ swabs can pick
microorganisms with some surfaces. This can be up to about 96% of microorganisms from the surfaces
attributed to the large size of fungal spore which does used in the study but the recovery on the media was low
not allow for it to be trapped in microstructure of swab suggesting that the remaining cells were trapped in the
matrix. swab itself. Since number of microorganisms left on the
Vortexing for 30 seconds followed by 2 minutes was surfaces under study was very small it could be
found to be superior over sonication for 5 minutes and concluded that the role of cell to surface interaction plays
overall recovery for all microorganisms from all surfaces a minor role in swabbing process. Possible explanation for
was 32, 22 and 20% respectively. In the study of the high bioburden recovery from Rayon™ swabs using
Dalmaso et al. [17] the Nylon™ flocked outward flushing technique is that during the flow of the
QUANTISWAB™ was able to recover 60% of micro- diluent from inside of the swab to outside it dragged
organisms, which is considerably higher than traditional trapped microorganisms which were held between fibers
Rayon™ swabs with only 20% recovery. Our study is of the swab and mechanically carried them out through
in agreement in the part of using traditional Rayon™ fluid stream a process which could not be achieved by
swabs with ordinary technique which gave recovery other physical means such as vortexing and sonication.
about 8% using streaking on agar method and Finally, it could be concluded that, in general, the best
approximately 32% using membrane filtration technique method for microbial recovery is the rinse method and
for all microorganisms on all used surfaces while using swab-filtration method (internal flushing of swabs
modified swabbing with outward flushing technique gave using buffer pH 7) while direct filtration, agar streaking,
overall recovery above 80% for the seven microorganisms 5 minutes sonication, 2 minutes vortexing and swab-
used with the five surfaces. filtration method (Glycerol immersion) failed in giving
The advantage of this new technique is that it can acceptable recovery.
be modified by several ways to maximize the recovery. It is obvious that microbial recovery from Rayon™
This can be achieved with either change volume of swabs is dependent mainly on the direction of flow of
washing diluent, change number of washes or type of extraction diluents through swab matrix and the number of
diluent. Dalmaso et al. [17] suggested that Rayon™ washes which is not normally achieved using only simple
classical swabs can absorb microorganisms but have vortexing technique.

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