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Determination of the cleaning efficiency for

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European Journal of Parenteral & Pharmaceutical Sciences 2015; 21(1): XX
2016 Pharmaceutical and Healthcare Sciences Society

Determination of the cleaning efficiency for

glassware in the pharmaceutical microbiology
laboratory
Tim Sandle1* and Ravikrishna Satyada2
1
Bio Products Laboratory, Elstree, Hertfordshire, UK
2
Microbiology, Gland Pharma Limited, Hyderabad, India

The cleaning of glassware is an important preliminary step prior to sterilisation in the microbiology
laboratory. If insufficient cleaning takes place, residues of culture media and chemicals can remain
and these could potentially have an impact upon microbial growth during various microbiological
qualitative and quantitative tests. This paper outlines a study where four different manual cleaning
regimes were used to evaluate glassware cleaning. It was found that a 5% solution of neutral
detergent, followed by two rinses was the most efficient method. The experiment is put forward as a
case study for other laboratories to replicate as necessary.

Key words: Chemical cleaning, cleaning, laboratory glassware, microbiological analysis, microbiology,
detergent, water.

Introduction residues removed prior to the glassware being sterilised.

While single-use, sterile disposable items are of growing
demand in many pharmaceutical processing areas and within
laboratories, the use of glassware remains commonplace.
Such items include beakers, pipettes, volumetrics and flasks,
Erlenmeyer flasks, Petri dishes, glass bottles, test tubes and so
on. Such glassware is commonly cleaned, sterilised and re-
used.
For the pharmaceutical microbiology laboratory, it is
important that newly purchased and recycled glassware is free
from any interfering residues and microorganisms. While
microorganisms can be destroyed or inactivated by
sterilisation methods, such as heat, chemical residues can still
remain in and on the glassware that are used for various
microbiological tests. Such residues can lead to invalid test
results1. For example, residues can inhibit culture growth,
cross-contaminate batches, and cause non-reproducible
results. An intimate knowledge of the physical and chemical
attributes of the contaminants should be understood by the
laboratory.
To avoid these scenarios from occurring, laboratory
glassware must be cleaned thoroughly and any interfering
*Corresponding author: Tim Sandle, Head of Microbiology, Bio Products
Laboratory, Dagger Lane, Elstree, Hertfordshire, UK. Email:
timsandle@btinternet.com
Residues include traces of laboratory culture media and
chemicals.
Special glass laboratory apparatus can be washed by hand
in a soaking bath or by machine in a laboratory dishwasher.
However, to effectively achieve the necessary level of
cleanliness, an appropriate laboratory detergent must be
selected along with an effective cleaning method. Cleaning
can be defined as the process of removing residues and soil
(such as dirt, grease, protein residues and so on) from
surfaces to the extent that they are visually clean. This
involves defined methods of application and the use of a
detergent2. The requirement for defined methods is
recommended by the World Health Organizations Good
Practices for Pharmaceutical Microbiology Laboratories,
WHO Technical Report Series No. 9613. The best way to
ensure residues are removed is to clean glassware as soon as
possible after use.
Different cleaning methods will produce different levels
of efficiency. This will depend upon the detergent selected,
the type of water used for rinsing, the rinse process (and
number of rinses), and the drying time. Although acids can be
used to clean glassware (such as sulphuric or perchloric
acids), the use of such chemicals carries considerable safety
risks and their use in a microbiology laboratory is
uncommon. For this reason, the focus is on the use of a
detergent.

20
DETERMINATION OF THE CLEANING EFFICIENCY FOR GLASSWARE IN THE PHARMACEUTICAL MICROBIOLOGY LABORATORY
Detergents are specially formulated chemicals used to
clean equipment or surfaces by removing unwanted
matter (soil)4. Detergents generally work by penetrating
soil and reducing the surface tension (which adhere soil to
the surface) to allow its removal (in crude terms, a
detergent increases the wettability of water). Many
detergents are synthetic surfactants (an acronym for
surface active agents). Surfactants are schizophrenic
molecules, in that they have two sides to their nature. One
part is solvent-loving or lyophilic (hydrophilic) and
another is solvent-hating or lyophobic (hydrophobic).
Surfactants remove particles from surfaces by either
capillary effects or electrostatic forces (many detergents
contain differently charged ions that can cause
microorganisms to repel each other). There is a wide
variety of detergents available with different
physicochemical properties5. In addition, there are
differences in cleaning ability and residual levels68.
The importance of the detergent is that should soil
remain, as would occur with the absence of a detergent,
grease and other contaminating materials will prevent the
glass from becoming uniformly wetted.
In establishing a cleaning regime, it is, in the current
climate, important to avoid the excessive use of energy
and water in order to avoid wastage of resources. For this
reason, the experiment evaluated the amount of water
used and, in selecting the optimal regime, considered the
quantities of water used.
While glassware can be visually inspected, the
effectiveness of the cleaning and assessment of whether
there are any residues that might be inhibitory to microbial
growth can only be assessed through a challenge study.
Despite the importance of this being understood by many
microbiologists, it is surprising, through literature review,
how few studies there have been on this subject (such as
references 68). This paper describes a study where a
standardised cleaning process was taken and the
inhibitory presence of any chemical residues assessed
post-cleaning and post-sterilisation through a bacterial
inoculation study.
The organism used for the study was Escherichia coli,
as a representative bacterium. E. coli is a Gram-negative,
facultatively anaerobic, rod-shaped bacterium9. Bacterial
cells are rod-shaped, approximately 2.0 m in length and
0.251.0 m in diameter; the cell volume is 0.60.7 m3 Table 1. Summary of cleaning procedure adopted.
10
. The organism can be grown and cultured readily on
standard culture media within a laboratory setting.
E. coli was selected as the test organism because the
adhesion of the organism to a variety of surfaces,
including glass, has been observed by using several
techniques. Atomic force microscopy suggests that rod-
shaped bacteria are particularly adept at surface
attachment11. This appears to relate to bacterial cell
polarity, with nano-domains on the bacterial ends being
particularly important for adhesion (a combination of
peptidoglycan, proteins and lipids)12,13. The selection of an
organism that would adhere to surfaces where chemical
residues may be found was deemed important. Not all
species of bacteria will adhere to surfaces as readily. For
surface adhesion, the bacterium must overcome repulsive
21
electrostatic forces (van der Waals and hydrophobic
forces). If left unchecked and over a period of time,
extracellular polymeric substance production takes place
leading to biofilm development14.
Further determinants of adhesion relate to the nature of
the surface itself. Rugosity of a surface increases surface
adhesion and hydrophobic surfaces are more likely to
facilitate microbial attachment. This means that a material
like TeflonTM or plastic is more likely to be colonised by
bacteria compared with a hydrophilic surface like glass or
metal. Nonetheless, glass can be prone to longer-term and
irreversible surface attachment by bacteria15. Rate of
attachment is also influenced by environmental factors
like temperature and pH16.
E. coli was also selected because it will grow easily
and its pattern of growth is recognisable. It could be that
other bacteria might react with any chemical residues in
different ways. The purpose here was to lay down the
foundations of a study in which other bacteria can be
used.
Method
To address the cleaning efficiency, a study was undertaken
within a pharmaceutical microbiology laboratory with the
intention of establishing an effective and reproducible
cleaning method.
The study was designed to evaluate the efficiency of
the cleaning process to effectively remove the detergent
residue on the cleaned glassware post-cleaning of the
glassware using the detergent. The detergent used was 5%
ExtranTM (Merck). The detergent is composed of anionic
and non-ionic surfactants and phosphates with low
concentrations of excipients. A 5% solution of the
detergent has a pH of 7.517. Although a propriety brand of
detergent was used, there is nothing exceptional in the
formulation and the detergent is generally similar to other
neutral detergents with applicability for cleaning
glassware.
Four different manual cleaning methods were
evaluated with six items of glassware for each group. The
steps undertaken are summarised in Table 1.
Cleaning Group Cleaning procedure
method
number
1 A Washed with 5% ExtranTM
(detergent) then rinsed with potable
water and purified water, one time
each, at 25oC (regular procedure)
2 B Regular procedure (A above), plus
rinsed 12 times with purified water
at 25C.
3 C Washed with 5% ExtranTM
(detergent), no additional rinsing
4 D Washed with potable water and
purified water at 25oC (with no
detergent used)
22 TIM SANDLE, RAVIKRISHNA SATYADA

The minimum volume of water was 50 mL for each rinse.
Water volumes were assessed using a liquid dispenser.
With the above groups, A was the established regime in
the laboratory. Group B was a variation to see if additional
rinsing cycles are required (using purified water only).
Group C was run with detergent only to see if rinsing was
necessary and Group D was a control group where no
detergent was used. In practice, Group D would not be
effective for rendering glassware suitably clean.
In the interpretation of the results, by running the four
different test conditions the following could be discerned.
Difference in number of colonies of less than 15% on
plates of Groups A, B, C and D would indicate that the
detergent has no toxicity or inhibitory characteristics
(that is, the detergent is not toxic or inhibitory).
Differences between Groups B and D of greater than
15% would indicate an inhibitory residue (that is, the
cleaning procedure adopted in Groups B and A is not
effective).
Differences in counts of less than 15% between
Groups A and B and greater than 15% between Groups
A and C would indicate that the cleaning detergent has
inhibitory properties that are eliminated during routine
washing (that is, the routine standard cleaning
procedure, as described for Group A, is effective).
The washing process used either potable (mains) water,
pharmacopoeial grade purified water (with a bioburden
limit of <100 colony forming units (CFU)/mL), both types
of water, or only detergent.
The types of glassware cleaned were 100 mL glass
vials closed with rubber stoppers and kept in place with
seals. These were selected due to their size and shape.
They were used in the laboratory for the preparation and
sterilisation of culture media used in a sterility test.
Previous experience had shown these types of vials are
hardest to clean in relation to other laboratory glassware
because of the deep crevices.
To assess the effectiveness of the cleaning procedure a
microbial inhibition study was conducted. After the
glassware had been cleaned and rinsed, representative sets
(each set containing 6 items of glassware) were wrapped
in autoclave packaging and sterilised by moist heat using
an autoclave operating at 121C for 30 minutes.
Following the sterilisation step, the glassware was
removed from the autoclave and unwrapped under a
unidirectional airflow cabinet.
Approximately not more than 100 CFU of the
bacterium E. coli (American Type Culture Collection
(ATCC) 8739) was added to the inner surface of each glass
soluble ball containing a precise number of
microorganisms and they are frequently used in media
growth promotion testing. The challenge inoculum was
confirmed by pour plate.
The bacterial inoculum covered a surface area of
approximately 0.5 cm2. The culture was spread inside the
surface of the glass container evenly, with the far corners of
the surface covered. The bacterial inoculum was allowed to
dry for 5 minutes underneath the unidirectional airflow
(dry was assessed by visual examination). In all, 24 items
(six items of glassware per cleaning regime) were tested.
Once 5 minutes had elapsed, 10 mL of a 0.9% sterile
saline solution was added to each glass vial. The vial was
then sealed by using a sterilised rubber stopper. Each vial
was then gently swirled for 1 minute so that the entire saline
solution came into contact with the entire surface of the vial.
Following this, membrane filtration was performed for
all the test vials using a 0.45 m membrane. The 10 mL of
solution was filtered and the membranes were transferred
to a pre-prepared soybean casein digest medium agar
(SCDA) plate. During the test session, a negative control
was performed by filtering 10 mL of 0.9% saline solution
through the membrane.
All the test and negative control plates were incubated
at 3035C for 3 days. This incubation time and
temperature was considered sufficient for the recovery of
a laboratory culture of E. coli. The acceptance criteria
applied were as follows.
a) Each test result from the groups must differ from the
initial control count by no more than 50200%. A
factor of 2 is commonly applied to microbiological
testing undertaken using pour plate methodology.
b) The negative control plates must be <1 CFU/mL.
Results
The results from the four cleaning methods were
evaluated. The results are presented in Table 2, and
graphically in the series of accompanying figures (Figures
14). The initial count used in the study was 55 CFU/ 0.1
mL. This is represented as the control count. The microbial
recoveries for each group are shown in Tables 36.
Table 2. Results of the four different cleaning regimes.
Results (CFU/10 mL)
Control
Test plate Group Group Group Group count
A B C `D
Test plate 1 39 35 1 36 55

vial, via the use of a micropipette. This strain of E. coli is Test plate 2 32 43 0 39 55
frequently used for assays to assess antimicrobial
preservatives, for assessing antimicrobial handwashing
Test plate 3 41 44 0 40 55

formulations, and for media growth promotion testing. The Test plate 4 44 40 1 38 55
strain was isolated from animal faeces and deposited into
the ATCC in 1941. The strain is relatively safe to handle
Test plate 5 43 43 1 41 55

within the laboratory, requiring a Biosafety Level of 1. Test plate 6 42 46 3 44 55

The microbial suspension was prepared using
BioBallsTM (bioMerieux). A BioBallTM is a small water
Mean 40 42 1 40 55
DETERMINATION OF THE CLEANING EFFICIENCY FOR GLASSWARE IN THE PHARMACEUTICAL MICROBIOLOGY LABORATORY 23

Microbial recoveries for each group

Group A

Table 3. Group A test results.

Test plate Count Control count Percentage recovery Pass /fail

(CFU) [A] (CFU) [B] [A B x 100]

1 39 55 71% Pass
2 32 55 58% Pass
3 41 55 75% Pass
4 44 55 80% Pass
5 43 55 78% Pass
6 42 55 76% Pass
Mean recovery 73%

E.coli
Mean
Test Plate 6
Observed Growth

Test Plate 5
Test Plate 4
Test Plat 3
Test Plat 2
Test Plate 1

0 10 20 30 40 50 60 70 80 90 100
CFU
Control Group A

Figure 1. Group A microbial recoveries.

Group B

Table 4. Group B test results.

Test plate Count Control count Percentage recovery Pass /fail

(CFU) [A] (CFU) [B] [A B x 100]
1 35 55 63% Pass
2 43 55 78% Pass
3 44 55 80% Pass
4 40 55 73% Pass
5 43 55 78% Pass
6 46 55 84% Pass
Mean recovery 76%
24 TIM SANDLE, RAVIKRISHNA SATYADA

E.coli

Mean
an
Observed Growth

Test Plate 6
Test Plate 5
Test Plate 4
Test Plat 3
Test Plat 2
Test Plate 1

0 10 20 30 40 50 60 70 80 90 100
CFU
Control Group B

Figure 2. Group B microbial recoveries.

Group C

Table 5. Group C test results.

Test plate Count Control count Percentage recovery Pass /fail

(CFU) [A] (CFU) [B] [A B x 100]

1 1 55 2% Fail
2 0 55 0% Fail
3 0 55 0% Fail
4 1 55 2% Fail
5 1 55 2% Fail
6 3 55 5% Fail
Mean recovery 2%

E.coli
Mean
n
Test Plate 6
Observed Growth

Test Plate 5
Test Plate 4
Test Plat 3
Test Plat 2
Test Plate 1
0 10 20 30 40 50 60 70 80 90 100
CFU
Control Group C

Figure 3. Group C microbial recoveries.

DETERMINATION OF THE CLEANING EFFICIENCY FOR GLASSWARE IN THE PHARMACEUTICAL MICROBIOLOGY LABORATORY 25

Group D

Table 6. Group D test results.

Test plate Count Control count Percentage recovery Pass /fail

(CFU) [A] (CFU) [B] [A B x 100]
1 36 55 66% Pass
2 39 55 71% Pass
3 40 55 73% Pass
4 38 55 69% Pass
5 41 55 75% Pass
44 46 55 84% Pass
Mean recovery 73%

E.coli
Mean
n
Test Plate 6
Observed Growth

Test Plate 5
Test Plate 4
Test Plat 3
Test Plat 2
Test Plate 1

0 10 20 30 40 50 60 70 80 90 100
CFU
Control Group D

Figure 4. Group D microbial recoveries.

The degree of variation between the results of the different
test groups was similar, with the counts obtained not
differing by more than 15%. This level of consistency
gave the experiment a degree of robustness.
Discussion
The study has examined four different cleaning regimes
for laboratory glassware, with a focus on examining the
efficacy of procedures to avoid glassware being presented
for testing with residues that could lead to the inhibition of
microbial growth.
The data for Groups A, B and D showed no inhibition
of microbial growth. Whereas, the process of using the
detergent alone (Group C) led to inhibition of the
microbial challenge. In this group procedure, a detergent
wash was applied to the glassware with no additional
water rinses. The experimental data suggests the detergent
alone has some inhibitory properties which is detrimental
to the survival of the organisms. This infers that while a
detergent is necessary to render laboratory glassware
visually clean, failure to rinse the glassware adequately
would lead to problems when that glassware is used for
microbial testing.
Group D acted as a control. Here, only water was used
and, as would be expected, no inhibition of microbial
growth was observed. However, this method would not be
recommended to remove all residues of culture media and
chemicals from glassware. For practical and effective
cleaning, a detergent is required.
The results obtained in Groups A and B were similar
(microbial recoveries at 73% and 76%, respectively). The
microbial recoveries were within the acceptance criteria,
and, microbiologically, there is no significant difference
between 73% and 76%). This indicates that the
established regime is suitable for cleaning glassware and
ensuring there are no detergent residues that could inhibit
microbial growth. While regime B could also be adopted,
in order to reduce water usage it would be sensible for the
method set out in Group A to continue be followed.
In terms of limitations, this study only looked at the
inhibitory effect on one type of bacterium. Although this
organism is representative of many others (and it is a
requirement to use this bacterium for many compendial
26
tests, such as the Microbial Limits Test), other organisms
may respond differently to chemical residues.
Another limitation is that, while the organism used in
the test is not inhibited by any chemical residues that
might have remained behind on the surface of the
glassware, it is unknown whether any chemical traces
were indeed present. This could be confirmed through
further study, such as taking swabs for total organic
carbon analysis.
While the study only considered one type of detergent,
the detergent selected was representative of the type used
in many laboratories and it would be expected that similar
results would be obtained when using other neutral
detergents recommended for the cleaning of glassware.
Conclusion
The data has shown that laboratory detergents are
potentially inhibitory for microbial growth and, where
glassware is recycled through washing and later
sterilisation, residues of detergents must be effectively
removed. The study has shown how an established
cleaning regime washing with a detergent, then rinsing
with potable water and purified water is effective. The
method discussed is intended as a case study;
microbiology laboratories that re-use glassware are
encouraged to conduct a similar exercise.
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 DETERMINATION OF THE CLEANING EFFICIENCY FOR GLASSWARE IN THE PHARMACEUTICAL MICROBIOLOGY LABORATORY 27

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