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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.


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

20
DETERMINATION OF THE CLEANING EFFICIENCY FOR GLASSWARE IN THE PHARMACEUTICAL MICROBIOLOGY LABORATORY 21

Detergents are specially formulated chemicals used to electrostatic forces (van der Waals and hydrophobic
clean equipment or surfaces by removing unwanted forces). If left unchecked and over a period of time,
matter (soil)4. Detergents generally work by penetrating extracellular polymeric substance production takes place
soil and reducing the surface tension (which adhere soil to leading to biofilm development14.
the surface) to allow its removal (in crude terms, a Further determinants of adhesion relate to the nature of
detergent increases the wettability of water). Many the surface itself. Rugosity of a surface increases surface
detergents are synthetic surfactants (an acronym for adhesion and hydrophobic surfaces are more likely to
surface active agents). Surfactants are schizophrenic facilitate microbial attachment. This means that a material
molecules, in that they have two sides to their nature. One like TeflonTM or plastic is more likely to be colonised by
part is solvent-loving or lyophilic (hydrophilic) and bacteria compared with a hydrophilic surface like glass or
another is solvent-hating or lyophobic (hydrophobic). metal. Nonetheless, glass can be prone to longer-term and
Surfactants remove particles from surfaces by either irreversible surface attachment by bacteria15. Rate of
capillary effects or electrostatic forces (many detergents attachment is also influenced by environmental factors
contain differently charged ions that can cause like temperature and pH16.
microorganisms to repel each other). There is a wide E. coli was also selected because it will grow easily
variety of detergents available with different and its pattern of growth is recognisable. It could be that
physicochemical properties5. In addition, there are other bacteria might react with any chemical residues in
differences in cleaning ability and residual levels68. different ways. The purpose here was to lay down the
The importance of the detergent is that should soil foundations of a study in which other bacteria can be
remain, as would occur with the absence of a detergent, used.
grease and other contaminating materials will prevent the
glass from becoming uniformly wetted.
In establishing a cleaning regime, it is, in the current
Method
climate, important to avoid the excessive use of energy To address the cleaning efficiency, a study was undertaken
and water in order to avoid wastage of resources. For this within a pharmaceutical microbiology laboratory with the
reason, the experiment evaluated the amount of water intention of establishing an effective and reproducible
used and, in selecting the optimal regime, considered the cleaning method.
quantities of water used. The study was designed to evaluate the efficiency of
While glassware can be visually inspected, the the cleaning process to effectively remove the detergent
effectiveness of the cleaning and assessment of whether residue on the cleaned glassware post-cleaning of the
there are any residues that might be inhibitory to microbial glassware using the detergent. The detergent used was 5%
growth can only be assessed through a challenge study. ExtranTM (Merck). The detergent is composed of anionic
Despite the importance of this being understood by many and non-ionic surfactants and phosphates with low
microbiologists, it is surprising, through literature review, concentrations of excipients. A 5% solution of the
how few studies there have been on this subject (such as detergent has a pH of 7.517. Although a propriety brand of
references 68). This paper describes a study where a detergent was used, there is nothing exceptional in the
standardised cleaning process was taken and the formulation and the detergent is generally similar to other
inhibitory presence of any chemical residues assessed neutral detergents with applicability for cleaning
post-cleaning and post-sterilisation through a bacterial glassware.
inoculation study. Four different manual cleaning methods were
The organism used for the study was Escherichia coli, evaluated with six items of glassware for each group. The
as a representative bacterium. E. coli is a Gram-negative, steps undertaken are summarised in Table 1.
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 Cleaning Group Cleaning procedure
standard culture media within a laboratory setting. method
E. coli was selected as the test organism because the number
adhesion of the organism to a variety of surfaces, 1 A Washed with 5% ExtranTM
including glass, has been observed by using several (detergent) then rinsed with potable
techniques. Atomic force microscopy suggests that rod- water and purified water, one time
shaped bacteria are particularly adept at surface
each, at 25oC (regular procedure)

attachment11. This appears to relate to bacterial cell 2 B Regular procedure (A above), plus
polarity, with nano-domains on the bacterial ends being rinsed 12 times with purified water
particularly important for adhesion (a combination of
at 25C.

peptidoglycan, proteins and lipids)12,13. The selection of an 3 C Washed with 5% ExtranTM


organism that would adhere to surfaces where chemical (detergent), no additional rinsing
residues may be found was deemed important. Not all 4 D Washed with potable water and
species of bacteria will adhere to surfaces as readily. For purified water at 25oC (with no
surface adhesion, the bacterium must overcome repulsive detergent used)
22 TIM SANDLE, RAVIKRISHNA SATYADA

The minimum volume of water was 50 mL for each rinse. soluble ball containing a precise number of
Water volumes were assessed using a liquid dispenser. microorganisms and they are frequently used in media
With the above groups, A was the established regime in growth promotion testing. The challenge inoculum was
the laboratory. Group B was a variation to see if additional confirmed by pour plate.
rinsing cycles are required (using purified water only). The bacterial inoculum covered a surface area of
Group C was run with detergent only to see if rinsing was approximately 0.5 cm2. The culture was spread inside the
necessary and Group D was a control group where no surface of the glass container evenly, with the far corners of
detergent was used. In practice, Group D would not be the surface covered. The bacterial inoculum was allowed to
effective for rendering glassware suitably clean. dry for 5 minutes underneath the unidirectional airflow
In the interpretation of the results, by running the four (dry was assessed by visual examination). In all, 24 items
different test conditions the following could be discerned. (six items of glassware per cleaning regime) were tested.
Once 5 minutes had elapsed, 10 mL of a 0.9% sterile
Difference in number of colonies of less than 15% on saline solution was added to each glass vial. The vial was
plates of Groups A, B, C and D would indicate that the then sealed by using a sterilised rubber stopper. Each vial
detergent has no toxicity or inhibitory characteristics was then gently swirled for 1 minute so that the entire saline
(that is, the detergent is not toxic or inhibitory). solution came into contact with the entire surface of the vial.
Differences between Groups B and D of greater than Following this, membrane filtration was performed for
15% would indicate an inhibitory residue (that is, the all the test vials using a 0.45 m membrane. The 10 mL of
cleaning procedure adopted in Groups B and A is not solution was filtered and the membranes were transferred
effective). to a pre-prepared soybean casein digest medium agar
Differences in counts of less than 15% between (SCDA) plate. During the test session, a negative control
Groups A and B and greater than 15% between Groups was performed by filtering 10 mL of 0.9% saline solution
A and C would indicate that the cleaning detergent has through the membrane.
inhibitory properties that are eliminated during routine All the test and negative control plates were incubated
washing (that is, the routine standard cleaning at 3035C for 3 days. This incubation time and
procedure, as described for Group A, is effective). temperature was considered sufficient for the recovery of
a laboratory culture of E. coli. The acceptance criteria
The washing process used either potable (mains) water, applied were as follows.
pharmacopoeial grade purified water (with a bioburden
limit of <100 colony forming units (CFU)/mL), both types a) Each test result from the groups must differ from the
of water, or only detergent. initial control count by no more than 50200%. A
The types of glassware cleaned were 100 mL glass factor of 2 is commonly applied to microbiological
vials closed with rubber stoppers and kept in place with testing undertaken using pour plate methodology.
seals. These were selected due to their size and shape. b) The negative control plates must be <1 CFU/mL.
They were used in the laboratory for the preparation and
sterilisation of culture media used in a sterility test.
Results
Previous experience had shown these types of vials are
hardest to clean in relation to other laboratory glassware The results from the four cleaning methods were
because of the deep crevices. evaluated. The results are presented in Table 2, and
To assess the effectiveness of the cleaning procedure a graphically in the series of accompanying figures (Figures
microbial inhibition study was conducted. After the 14). The initial count used in the study was 55 CFU/ 0.1
glassware had been cleaned and rinsed, representative sets mL. This is represented as the control count. The microbial
(each set containing 6 items of glassware) were wrapped recoveries for each group are shown in Tables 36.
in autoclave packaging and sterilised by moist heat using
an autoclave operating at 121C for 30 minutes. Table 2. Results of the four different cleaning regimes.
Following the sterilisation step, the glassware was
removed from the autoclave and unwrapped under a Results (CFU/10 mL)
Control
unidirectional airflow cabinet. Test plate Group Group Group Group count
Approximately not more than 100 CFU of the A B C `D
bacterium E. coli (American Type Culture Collection
(ATCC) 8739) was added to the inner surface of each glass
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 would lead to problems when that glassware is used for
test groups was similar, with the counts obtained not microbial testing.
differing by more than 15%. This level of consistency Group D acted as a control. Here, only water was used
gave the experiment a degree of robustness. 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
Discussion
chemicals from glassware. For practical and effective
The study has examined four different cleaning regimes cleaning, a detergent is required.
for laboratory glassware, with a focus on examining the The results obtained in Groups A and B were similar
efficacy of procedures to avoid glassware being presented (microbial recoveries at 73% and 76%, respectively). The
for testing with residues that could lead to the inhibition of microbial recoveries were within the acceptance criteria,
microbial growth. and, microbiologically, there is no significant difference
The data for Groups A, B and D showed no inhibition between 73% and 76%). This indicates that the
of microbial growth. Whereas, the process of using the established regime is suitable for cleaning glassware and
detergent alone (Group C) led to inhibition of the ensuring there are no detergent residues that could inhibit
microbial challenge. In this group procedure, a detergent microbial growth. While regime B could also be adopted,
wash was applied to the glassware with no additional in order to reduce water usage it would be sensible for the
water rinses. The experimental data suggests the detergent method set out in Group A to continue be followed.
alone has some inhibitory properties which is detrimental In terms of limitations, this study only looked at the
to the survival of the organisms. This infers that while a inhibitory effect on one type of bacterium. Although this
detergent is necessary to render laboratory glassware organism is representative of many others (and it is a
visually clean, failure to rinse the glassware adequately requirement to use this bacterium for many compendial
26 TIM SANDLE, RAVIKRISHNA SATYADA

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DETERMINATION OF THE CLEANING EFFICIENCY FOR GLASSWARE IN THE PHARMACEUTICAL MICROBIOLOGY LABORATORY 27

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