Journal ofiipplied Bacteriology 1980, 49, 119-1 26

6941121/79

Changes in Preservative Sensitivity for the

USP Antimicrobial Agents Effectiveness
Test Micro-organisms
M. M. A. AL-HITI AND P. GILBERT
Department of Pharmacy, University of Manchester, Manchester M I 3 9PL, U.K.
Received 24 December 1979 and accepted 19 February 1980
Chemically defined and semi-defined media were designed for the preservative-effi
cacy testing micro-organisms designated by the United States Pharmacopoeia, in
which the organisms went into the stationary phase of growth at an optical density
(E470) of 1.0, because of depletion of a single carbon, nitrogen or phosphate source.
Aspergillus niger was grown on solid media containing concentrations of these
nutrients which limited the rates of mycelial development and sporulation density. The
ability of the micro-organisms to survive and grow in the presence of chlorhexidine
diacetate, benzalkonium chloride and thiomersal varied markedly with the nutrientdepletion of the inocula. No universal pattern of sensitivity emerged among microorganisms. Only A . niger showed little overall change in preservative sensitivity. These
results highlight the need to define more adequately growth media and conditions for
the production of inocula for antimicrobial challenge tests.

MICROBIAL
CHALLENGES form a useful basis for evaluating the biological availability
of preservatives in pharmaceutical and cosmetic products that are liable to microbial
spoilage (Anderson & Crompton 1967; Eriksen 1970; Norton et al. 1974). The United
States Pharmacopoeia1 (XIX) (USP) Antimicrobial Agents Effectiveness Test provides an officially recommended test for evaluating the effectivenessof preservatives in
medicinal products (Anon. 1975). The test was designed for use with ophthalmic, aural
and nasal preparations but is often used as a guideline for other products including
non-pharmaceutical materials. In many instances, however, products complying with
this test are subsequently found to be inadequately preserved (Moore 1978). Results of
the USP Test indicate whether the product will contend adequately with a defined
challenge by five designated strains of micro-organisms. The conditions specified by
the Pharmacopoeia, however, leave ample room for variation; indeed conflicting
results may often be obtained within a single laboratory (Moore 1978).
The bacterial envelope is remarkably flexible in its structure and composition. It is
highly responsive to changes in the nutritional environment, resulting in differences in
the sensitivity of cells towards drugs, through variation in permeability of the cell
envelope (Brown 1975). Thus Gram negative bacteria (Finch & Brown 1975; Gilbert &
Brown 1978a, b) and Gram positive bacteria (Gilbert & Brown 1980) and yeasts
(Johnson et al. 1978), have all been reported to vary in drug sensitivity according to
conditions of vegetative growth. It has been suggested that the organisms used in the
USP Test for antimicrobial effectiveness might also vary in a similiar fashion (Brown
1977; Hobbs et al. 1979). In addition a preserved system is unlikely to be challenged
during normal use with typical laboratory cultures grown in chemically rich media
designed to give optimal conditions for growth. Instead the micro-organisms would
have adapted to their own particular environments (Yablonski 1972). Thus the nutri01980 The Society for Applied Bacteriology
0021 -8847/80/040119+08$01.00/0
[I 191

120

M. M. A. AL-HIT1 AND P. GILBERT

tional status of the challenge inocula might influence the decision about the adequate
preservation of a product. As the USP Test is essentially a biological assay of
preservative availability, it is not absolutely necessary for it to mimic the in-use
situation; it must, however, be reproducible. Growth conditions of the challenge
inocula are inadequately specified in the USP and might therefore lead to results that
vary between laboratories.
This study therefore investigates the effects of depletion of carbon (C-dep), nitrogen
(N-dep) and phosphate (P-dep) during the growth of the USP Test organisms upon
their ability to grow subsequently in the presence of three typical preservatives. The
object was to improve the reliability of the test. A preliminary report of some of these
results has already been published (Al-Hiti & Gilbert 1979).

Materials and Methods

Organisms
Stuphylococcus aureus, ATCC 6538; Escherichia coli, ATCC 8739; Pseudomonas ueruginosa, ATCC 9027; Candida ulbicuns, ATCC 8739, and Aspergillus niger, ATCC
16404 were used throughout. Cultures were maintained on slopes of Nutrient Agar
(Oxoid, CM3) at room temperature after incubation overnight at 35C for the bacteria
and yeast and 7 d at 30C for the fungus. Slopes were replaced at 28 d intervals.

Chemicals
Chlorhexidine diacetate was obtained as Hibitane from ICI Ltd. (Macclesfield, Cheshire) and benzalkonium chloride and morpholinopropane-sulphonic acid (MOPS) from
Sigma Chemicals (Poole, Dorset). Microbiological media were supplied by Oxoid
except for the yeast extract which was purchased from Difco Laboratories. All other
reagents were obtained from BDH and were of the purest available grade.
Liquid media
Chemically defined or semi-defined simple-salts media were designed for the growth of
the micro-organisms, based initially on those of Gilbert & Brown (1 9786) for Esch. coli,
Vogel & Bonner ( 1956) for Ps. aeruginosa, Kobayashi et al. (1 964) for C. albicans and
supplemented with yeast extract and thiamine-HC1 for Staph. uureus. Although media
for the different organisms must inevitably be different quantitatively, reflecting their
different growth requirements, it was desirable that qualitatively they were as similar as
possible. Thus the constituents of these established simple salts media were rationalized as much as possible with respect to carbon, nitrogen and phosphate sources, buffer
systems and salts. The media were sterilized by autoclaving at 1 15Cfor 30 min. Biotin,
thiamine-HC1 and FeNH4(S0& were sterilized separately as concentrated solutions
by membrane filtration.
Cleated Erlenmeyer flasks (250 ml) containing 100 ml of the various media were
inoculated from overnight cultures grown in simple-salts liquid media and incubated in
an orbital shaker (Gallenkamp Ltd., London) at 100 osc./min and 35C. Growth was
monitored by optical density (Euo) using a Cecil CE303 spectrophotometer (Cecil

NUTRIENT-DEPLETION AND PRESERVATIVE SENSITIVITY

12 1

Instruments Ltd., Cambridge) and 1 cm glass cuvettes. Initially the concentrations of

either the carbon, nitrogen or phosphate source (Table 1) were varied and the optical
density of the cultures determined when they reached the stationary phase of growth
(Fig. 1). All remaining nutrients [Mg S04, 0.5 m~;FeNH~(S04)2,
0.03 mM, and for
Esch. coli and Staph. aureus KCl, 13.4m ~were
] present in excess. The media for Staph.
aureus were also supplemented with yeast extract (1 .O g/l), thiamine-HCl(1 .O mg/l) and
biotin (0.15 mg/l) and that for C. albicans with biotin (0.15 mg/l). pH was measured
before and after growth of the cultures to check the buffering capacity of the media (pH
TABLE1
Media composition for the growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans
Concentration (mM) to give stationary phase at an optical density (E470) of 1.O
Growth
limiting
nutrient
Glucose
Glycerol
Sodium citrate
K2HP04
(NH4)zS04

Escherichia
coli

Staphylococcus Psrudomonas Candida

aureus
arruginosa alhicans

6.0

3.0

Carbon-depleted*

12.5

8.5

0.13

1.2

Nutrient-depletion

0.05
2.5

9.0
0.15
2.0

0.05
2.5

Phosphate-depleted*
Nitrogen-depleted*

* If non-limiting added at five times these concentrations. t No added phosphate for phosphatedepleted, otherwise 2.0 mM. $ No added (NH4)2S04 for nitrogen-depleted, otherwise 5.0 mM.

7.2). The P-dep and all staphylococcal cultures were buffered using MOPS (200 mM);
the remainder were buffered with phosphates (KH*P04,28mM; K*HP04,72 mM). The
concentrations of the carbon, nitrogen and phosphate sources within the media
causing the cultures to enter their stationary phase of growth at an optical density of
1.0 were used in the design of the simple-salts media. The nutrient to be depleted was
supplied at this concentration, and the remainder at 5 times this level.
Solid media design

For A. niger the USP Test specifies that the challenge inocula is a spore suspension.
Solid media were therefore devised for the growth of this organism based on the
simple-salts liquid media of Kobayashi et al. (1964) and solidified with 1% (w/v)
bacteriological agar (Oxoid L1 1). Plates were inoculated centrally with an agar disc cut
from a 7 d simple-salts plate culture of A. niger, using a 4 mm flamed cork borer. Rates
of increase in colony size were determined directly by daily measurement over 7 d and
the density of spores within the colony was assessed daily for 7 d by flooding replicate
plates with water, agitating with a glass spreader and performing total spore counts on
the final suspension. Concentrations of carbon, nitrogen and phosphate sources were
varied as with the liquid media. Rates of increase in colony size altered linearly with
respect to limiting nutrient concentration. At very low phosphate concentrations,
however, although mycelial growth rate was limited by the phosphate concentration,

122

M. M. A. AL-HIT1 AND P. GILBERT

the density of sporulation within the colony was very much reduced. Choices of
limiting nutrient concentrations for the final media (Table 2) were therefore made
subjectively, selecting those that restricted the rate of colony development but allowed
a sufficient level of sporulation for harvesting and preparation of spore suspensions
after 7 d incubation. For C-dep and N-dep cultures these reduced the rate of increase in

0.5
O

0.2 0.4
06
0.8
poG3-concentration CO MI

Fig. 1 . Effect of phosphate concentration upon the stationary phase optical density (E470) of (a)
Psrudomonas aeruginosa grown in simple-salts media with glycerol ( 0 ) and sodium citrate as
carbon source ( 0 ) ; (b) Staphylococcus aureus grown in simple-salts media including yeast
extract (I g/l.) as a source of vitamins and amino acids.

colony diameter by 50% from that on complete media. For P-dep cultures, however,
where sporulation density varied with phosphate concentration, this was not possible
and that concentration giving a 50% reduction in spore density of the colony was
selected.

Sensitivity toward preservatives

Liquid cultures of the bacteria and yeast, depleted either in carbon, nitrogen or
phosphate source, were prepared in 250 ml cleated Erlenmeyer flasks containing 100 ml
of the appropriate media (Table l), grown overnight (16 h) at 35C in an orbital shaker
(100 osc./min). These had been inoculated from similarly grown cultures in identical

NUTRIENT-DEPLETION AND PRESERVATIVE SENSITIVITY

123

media. Aspergillus niger was grown on solid media (Table 2) for 7 d at 35C and the
spores harvested by flooding the plates with water and agitating using glass spreaders.
Cultures of micro-organisms so obtained were serially diluted in distilled water and 0.1
ml amounts, containing 1 x lo2, 1 x lo3 and 1 x lo4 viable cells, spread on to predried
nutrient agar plates containing varying concentrations of thiomersal, benzalkonium
chloride or chlorhexidine diacetate. The number of colony forming units (c.f.u.) was
TABLE
2
Media composition for the growth of
Aspergillus niger
Concentrations of growth limiting
nutrients (mM) for Aspergillus niger
in Bacteriological Agar (1% wiv)
wiv)
Carbon- Phosphatelimited
limited

Nutrient
Glucose
K2HP04
(NH4)2S04

3.0
72.0
15.0

Nitrogenlimited

45.0
0.05
15.0

45.0
72.0

0.0

Additives MgS04, 0.5 mM; FeS04, 0.03 mM; biotin,

0.15 mg/l; KH2P04, 30 mM (carbon and nitrogenlimiting media only); MOPS buffer, 200 mM (phosphate-limiting media only).

determined after incubation of the plates at 35C for 24 h for the bacteria and 48 h for
the yeast and fungi. Experiments were done in triplicate and results were expressed as
percentage reduction in c.f.u. relative to the controls.

Results and Discussion

Media design

Chemically defined and semi-defined liquid media were designed for the bacteria and
yeast in which logarithmic growth ceased at an optical density of 1.0 because of
depletion of one key nutrient, either a carbon, nitrogen or phosphate source (Table 1).
All non-limiting nutrients were available in excess. For the growth of Staph. aureus it
was necessary to supplement the simple-salts media with a source of vitamins and
amino acids. These also served to some extent as nitrogen and phosphate sources. Thus
no added nitrogen or phosphate were included in the media when these were the
required nutrients for depletion (Fig. lb).
For Ps. aeruginosa two carbon sources were used (sodium citrate or glycerol). This
organism utilizes citrate in preference to other carbon sources (Hamilton & Dawes
1959), it was therefore of interest to see whether the nature of the carbon source
influenced drug sensitivity. Curiously, not only were the molar requirements of the
organism different for the two carbon sources, but their phosphate requirement
increased threefold when sodium citrate replaced glycerol as the sole carbon source

124

M. M. A. AL-HIT1 AND P. GILBERT

(Table 1, Fig. 1). This obviously reflects a major change in the physiological status of
the cells.
Preservative sensitirity

The ability of the USP Test organisms, grown under C-dep, N-dep and P-dep conditions, to survive and grow in the presence of varying concentrations of preservatives
was assessed. Typical results are illustrated in Fig. 2, and collected results for all the

Benzoihonium chloride concentration

(% w/v

lo4)

Fig. 2. Effect of depletion of carbon (m), nitrogen (o), or phosphate ( 0 ) during the growth of
Staphylococcus aureus, upon its ability to survive and grow on agar plates containing various
concentrations of benzalkonium chloride.

micro-organisms, as that concentration of preservative reducing the c.f.u. by 90%, in

Table 3. With the exception of A . niger the preservative sensitivity of the microorganisms varied markedly with nutrient-depletion. Least variation in sensitivity was
observed towards thiomersal, and the greatest towards benzalkonium chloride. Notably Ps. aeruginosa was most resistant to all the agents, justifying its notoriety as an
organism particularly resistant to chemical inactivation (Brown 1975). Greatest variation in preservative sensitivity with nutrient-depletion was also observed for this
organism; the iso-effective concentrations for benzalkonium chloride, for example,
varied from 2.5 x
f><
w/v) for citrate-grown P-dep cells and 5 x 10 -z w/v) for
glycerol-grown C-dep cultures. The greatest changes in drug sensitivity for this
organism were observed for those cultures grown on different carbon sources, rather
than the other nutrient-depletions. Variation in preservative sensitivity for the remaining organisms was significant, but with the exception of the thiomersal sensitivity of
Staph. aureus never exceeded three times the minimum observed effective concentration. No universal pattern of nutrient-depletion and drug sensitivity evolved between

(x

NUTRIENT-DEPLETION AND PRESERVATIVE SENSITIVITY

125

species but general patterns emerged for the three preservatives for each organism.
Thus, for Esch. coli the sensitivity of C-dep > N-dep 3 P-dep; for Ps. aeruginosa,
P-dep > N-dep 3 C-dep and for Staph. aureus, N-dep > P-dep 3 C-dep.
From the results of this study and others (Hobbs et al. 1979) there appears to be no
rationale for the choice of a single nutrient-depletion, minimizing preservative sensitivity, for the growth of challenge test inocula. The results do, however, indicate that the
use of different media within different laboratories could be a primary cause of
interlaboratory variation, and influence the results of a challenge test for preservative
TABLE
3
Effect of various nutrient-depletions upon the ability of micro-organisms to grow in
the presence of preservative
Preservative concentration required to reduce the number of colony
forming units by 907,; (7"wjv x lo4)
Thiomersal
Organism
Escherichiu coli
Pseudomonus
ueruginosu
(grown on citrate)
(grown on glycerol)
Staphylococcus
uureus
Cundidu albicuns
Aspergillus niger

N-dep C-dep

Benzalkonium
chloride

Chlorhexidine

P-dep N-dep C-dep P-dep N-dep C-dep P-dep

0.53

0.37

0.60

24.0

13.0

20.0

100.0

65.0

1.90
5.00

1.40
5.00

0.25
3.70

28.0
62.0

24.0
45.0

30.0
41.0

500.0

500.0

25.0
180.0

0.12
0.009
0.025

0.54
0.009
0.025

0.14
0.013
0.025

8.0
10.5
2.3

10.5

10.5

11.0

10.0

2.3

2.3

0.5
52.0
2.6

0.6
45.0
2.5

0.6
35.0
2.6

100.0 450.0

82.0

N-dep, nitrogen depleted; C-dep, carbon depleted; P-dep, phosphate depleted.

efficacy, especially when the concentrations of preservative employed are just adequate. Growth conditions are inadequately specified within the USP Antimicrobial
Agents Effectiveness Test and allow such a situation to occur. There would appear,
therefore, to be justification in designating a single medium for the growth of each
organism to be used in this and similar tests. Insufficient data are available to decide
whether this medium shall be chemically-defined and of low complexity or undefined,
such as nutrient broth. There is, however, one major advantage in using chemicallydefined media in that they are unlikely to vary significantly between manufacturers.
Adoption of these measures would increase test reproducibility, but to increase
relevance to the in viuo state, additional testing must be done with organisms from an
unpreserved or inadequately preserved product (Yablonski 1972), and possibly also
with organisms isolated from the manufacturing environment and grown in the
unpreserved product.

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M. M. A. AL-HIT1 AND P. GILBERT

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