Development of a novel selective medium for culture of Gram-negative bacteria

Abstract

Objective:
Although many bacterial culture media are available commercially, there is a continuous effort to develop better selective media for bacteria, which cannot be grown on existing media. While exploring antibacterial properties of clove, we observed that it has the potential to selectively inhibit growth of certain types of bacteria. This led us to do the experiments, which resulted in developing a new media which selectively allowed the growth of only Gram-negative bacteria, while inhibiting the Gram-positive bacteria.

Results:
Mueller Hinton Agar (MHA) was used as the base media and was modified to develop MHA-C15 (MHA containing 15% volume/volume water extract of clove). Gram-negative bacterial pathogens Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Pseudomonas aeruginosa grew on MHA-C15. However, none of the major Gram-positive bacterial pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Bacillus spp. and Enterococcus spp. grew on it. Taken together, these findings show that MHA-C15 is a newly developed selective media for culture of Gram-negative bacteria.

Full text

Al‑blooshietal. BMC Res Notes (2021) 14:211
https://doi.org/10.1186/s13104‑021‑05628‑2
RESEARCH NOTE
Development ofanovel selective medium
forculture ofGram‑negative bacteria
Shooq Yousef Al‑blooshi1, Mustafa Amir Abdul Latif1, Nour K. Sabaneh1, Michael Mgaogao1,2 and
Ashfaque Hossain1,2*
Abstract
Objective: Although many bacterial culture media are available commercially, there is a continuous effort to develop
better selective media for bacteria, which cannot be grown on existing media. While exploring antibacterial proper‑
ties of clove, we observed that it has the potential to selectively inhibit growth of certain types of bacteria. This led
us to do the experiments, which resulted in developing a new media which selectively allowed the growth of only
Gram‑negative bacteria, while inhibiting the Gram‑positive bacteria.
Results: Mueller Hinton Agar (MHA) was used as the base media and was modified to develop MHA‑C15 (MHA
containing 15% volume/volume water extract of clove). Gram‑negative bacterial pathogens Escherichia coli, Klebsiella
pneumoniae, Salmonella typhimurium and Pseudomonas aeruginosa grew on MHA‑C15. However, none of the major
Gram‑positive bacterial pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes,
Streptococcus mutans, Bacillus spp. and Enterococcus spp. grew on it. Taken together, these findings show that MHA‑
C15 is a newly developed selective media for culture of Gram‑negative bacteria.
Keywords: Mueller Hinton agar, Gram‑positive bacteria, Gram‑negative bacteria, Bacterial culture media, Selective
media, Clove
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Introduction
Bacterial culture (growth) media contains nutrients
necessary for their growth. All microorganisms cannot
grow in a single culture medium as their growth require-
ments vary; while for many microorganisms, the growth
requirements are unknown [1, 2]. Selective media allows
the growth of one class of bacteria while inhibiting the
others. For example, MacConkey agar is a selective
media that inhibits the growth of many Gram-positive
bacteria and favors the growth of Gram-negative bacte-
ria, particularly the Enterobacteriaceae group of bacteria
[3]. e use of selective media is essential in isolation of
pathogens from infection sites so that accurate pathogen
identification and diagnosis can be made and treatment
can be initiated. In addition, it is an essential first step
in microbiological investigation of environmental sam-
ples. Although several selective media are commercially
available, there is a constant effort to develop newer
media for more efficient isolation and identification of
bacterial species [4]. Human body harbors an astonish-
ingly high number of bacterial species [5]. Studies have
shown that we have approximately the same or more
number of bacteria in and on our body as compared to
our own body cells [6]. Most of these bacterial species
cannot be cultured in the commercially available culture
media [7]. It is also predicted that a large number of bac-
teria species in the environment also cannot be cultured
due to the lack of appropriate media which are needed
for their growth [6]. is is the main driving force behind
the constant effort for the formulation and development
of newer media for culture of bacteria [8–10]. Such new
Open Access
BMC Research Notes
*Correspondence: ashfaque@rakmhsu.ac.ae
2 Central Research Laboratory, RAK Medical and Health Sciences
University, Ras Al Khaimah, United Arab Emirates
Full list of author information is available at the end of the article

Page 2 of 6
Al‑blooshietal. BMC Res Notes (2021) 14:211
media will allow scientists to grow and study currently
un-culturable bacterial species.
Plants are rich in a wide variety of chemical compounds
such as tannins, terpenoids, alkaloids, and flavonoids
which have been found to possess antimicrobial prop-
erties against wide variety of bacterial pathogens. Since
prehistoric times, traditional healers have used plants
to prevent or cure infectious conditions [11]. Cloves
are aromatic flower buds of the tree, Syzygium aromati-
cum. ey have been widely known for their antibacte-
rial, antiviral and antifungal properties and also for their
use in herbal medicine for centuries. Cloves are used as
a spice in cooking different types of food items and also
used in food preservations for its antimicrobial effect
with no known side effects. Clove essential oil (CEO) is
traditionally used in the treatment of burns and wounds,
and as a pain reliever in dental care as well as treating
tooth infections and toothache [12]. e extracts of clove
exhibit antimicrobial effect on multidrug resistant micro-
organisms as well as methicillin-resistant Staphylococcus
aureus, Bacillus subtilis, Salmonella typhi and Serratia
marcescens. Various phytochemicals such as sesquiter-
penes, monoterpenes, hydrocarbon, and phenolic com-
pounds are present in cloves. In clove oil, eugenyl acetate,
eugenol, and β-caryophyllene are the most important
phytochemicals exhibiting antibacterial activity [13].
Although different chemical compounds are being used
to develop different types of selective media, surveys of
existing literature showed that plant extracts have not
been investigated extensively as a component in bacterial
culture media. In this project, we used water extract of
the clove to develop a selective media for Gram-negative
bacterial species, which does not allow the growth of
Gram-positive bacteria. It is anticipated that the finding
of this research project will stimulate exploration of dif-
ferent plant materials for their suitability in developing
selective and differential media for the growth of differ-
ent types of bacteria present in nature for which there
exists no appropriate culture media at present.
Main text
Materials andmethods
Preparation ofclove extract
Dried and powdered clove (Al Faris Spices, Salmabad,
Bahrain) was used to prepare a water extract. A 20%
(weight/volume) clove powder suspension in hot distilled
water was prepared and mixed using a magnetic stirrer
hot plate for 30min at 50°C. en, the extracted mate-
rial was filtered using Whatman filter paper and stored
at 4°C until used. Mueller Hinton agar (MHA) contain-
ing different concentrations of clove extract (5–20%)
was prepared by adding different volumes of the clove
extract and autoclaved. We followed the manufacturer’s
instruction in preparation of the MHA plates and the vol-
ume of water to be added to the media to be prepared
was adjusted according to the volume of clove extract to
be added for each concentrations of extract. We labelled
the plates MHA-C5 (MHA containing 5% extract vol-
ume/volume); MHA-C10 (MHA containing 10% extract
volume/volume) and so on for other concentrations.
Inoculation ofdierent bacteria
Gram-positive bacterial species tested were Staphylo-
coccus aureus, Staphylococcus epidermis, Streptococcus
pneumoniae, Streptococcus pyogenes, Enterococcus fae-
calis, Bacillus subtilis spp. and Streptococcus mutants.
e Gram-negative bacterial species included in this
study were Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa and Salmonella typhimurium.
Bacterial strains were grown overnight in Mueller Hin-
ton broth. A loop-full of bacteria were taken from such
cultures and streaked onto the MHA plates and on MHA
containing different concentrations of extracts i.e., MHA-
C5, MHA-C10, MHA-C15 and MHA-C20 and incu-
bated at 37°C for 24h. e plates were then observed for
growth.
Results anddiscussion
In a study to determine antibacterial effect of clove
extract on different bacteria, we serendipitously decided
to incorporate clove extract into agar media. We used
MHA in this study as this agar media is recommended
for antibacterial susceptibility assays. Moreover, we were
using this media in our initial experiments to determine
the antimicrobial activity of clove extract. We observed
that modified MHA (MHA containing clove extract)
exhibited a differential property i.e., at certain concen-
tration (20%) it inhibited the growth of Gram-positive
bacteria S. aureus, but had no effect on the growth of
Gram-negative bacteria, E. coli. We then tried a series of
different MHA-clove plates with 5% increment in clove
concentration (0%, 5%, 10%, 15% and 20%) in parallel to
determine the minimum concentration of clove extract
exhibiting this differential effect, i.e., inhibiting the
growth of S. aureus but allowing the growth of E. coli. We
observed from this experiment that MHA-C15 (MHA
containing 15% clove extract) was the agar plate contain-
ing minimum concentration of clove extract exhibiting
this differential effect i.e., allowing growth of E. coli but
suppressing the growth of S. aureus. MHA-Clove-10 and
MHA-Clove-5 supported the growth of both S. aureus
and E. coli.
After standardizing the concentration of incorporated
clove extract needed to differentiate between major
Gram-positive and Gram-negative bacterial species, we
extended our study to include other Gram-positive and

Page 3 of 6
Al‑blooshietal. BMC Res Notes (2021) 14:211
Gram-negative pathogens. To our great satisfaction, we
observed that MHA-C15 which differentiated between
growth of S. aureus and E. coli, also differentiated other
Gram-positive and Gram-negative bacterial species
(Figs. 1, 2). We tried MHA-C25 and observed that it
inhibited the growth of both Gram positive and Gram-
negative bacterial species. It is possible that at 25%, clove
extract reaches toxic levels for both Gram-positive and
Gram-negative bacterial species. Even on MHA-C15, the
growth of Gram-negative bacteria was relatively less in
comparison to MHA-C10 (Fig.2), suggesting that clove
extract has a concentration dependent inhibitory effect
on bacterial growth.
e count of bacteria grown on different MHA-C
plates are shown in Fig.2. Counting of bacteria was car-
ried out for quantitative evaluation of bacterial growth as
a function of the concentration of the clove extract. Bac-
terial numbers decreased as the concentration of clove
extract increased in the MHA plates for both Gram-pos-
itive and Gram-negative bacteria. However, on MHA-
C15, the count of Gram- positive bacteria dropped to
zero, but Gram-negative organisms continued to grow.
Comparison of relative growth of three major Gram-neg-
ative bacteria on MHA-C15 showed that Pseudomonas
aeruginosa grew best, which was followed by Escherichia
coli and Klebsiella pneumoniae. Opportunistic bacteria
Pseudomonas aeruginosa is intrinsically resistant to dif-
ferent antimicrobial agents because of the presence of
highly efficient efflux systems which permits its growth
in presence of different inhibitors [14]. is capacity may
have contributed to its efficient growth on MHA-C15.
We carried out Gram staining and antibiotic sensitiv-
ity testing (by disk diffusion method) of bacteria grown
MHA and MHA-C15 grown in parallel. No changes were
noticed either in Gram staining or in antibiotic sensitivity
of the organisms, indicating that MHA-C15 can be used
to cultivate bacteria with no apparent changes in these
vital properties of the bacteria tested (Additional file 1:
FiguresS1, S2, S3, S4).
Other Gram-positive and Gram-negative bacteria
tested included Bacillus and Streptococcus mutans and
Salmonella typhimurium. ese bacteria also followed
the same pattern i.e., growth on MHA-C15 for Gram-
negatives and no growth for Gram-positives (data not
shown). is finding demonstrates that the concentration
of the clove present in these two media (MHA-C5 and
MHA-C10) was not inhibitory and allowed the growth of
all the bacterial species tested. However, no growth was
observed on MHA-C15 and MHA-C20 for all the Gram-
positive bacterial species, indicating that the clove pre-
sent in this medium inhibited growth of these bacteria.
ese data demonstrate that MHA-C15 and MHA-C20
are the media that do not allow growth of Gram-positive
bacteria tested in this study but allows the growth of
Gram-negative bacteria. However, the number of colo-
nies on MHA-C20 decreased for Gram-negative bacte-
ria compared to MHA-C15. So, MHA-C15 was media
containing the lowest concentration of extract which
supported the growth of Gram-negative bacteria but
inhibited the growth of Gram-positive bacteria. Taken
together, MHA-C15 may be considered as an ideal media
for Gram-negative. However, investigation with other
Gram-negative bacteria needs to be done to determine
whether the observation with the selected Gram-neg-
ative bacteria also holds true for other bacterial species
belonging to this group of bacteria.
How clove extract selectively inhibits Gram-positive
bacteria is unknown. e antimicrobial activity of clove
extract is reported to be associated with Eugenol (2
methoxy-4 allyl-phenol), the main component of clove
oil, which is known to exhibit antibacterial and antifun-
gal activity. Antimicrobial activity of clove also reported
to be due to high tannin content (10–19%) [15–17]. e
cell wall of Gram-positive bacteria has a thick layer of
peptidoglycan, which is much thinner in Gram-negative
bacteria. is difference in cell wall thickness is basis
for differential susceptibility of many bacterial species
to different type of antibiotics and natural compounds.
So, it may be speculated that the primary target for the
growth inhibitory compounds presents in clove is most
likely bacterial cell wall [18]. e culture of microorgan-
isms is a prerequisite for any study with them. Bacterial
community in environment or in clinical settings is usu-
ally polymicrobial, consisting of both Gram-positive and
Gram-negative species. It is often challenging to differ-
entiate the different types of bacteria and grow them in
pure culture. As this newly developed MHA-C15 selec-
tive medium differentiates between Gram-positive and
Gram-negative bacterial species, it will serve as a useful
adjunct to the currently available bacterial culture media.
In conclusion, after several trials with different con-
centrations of water extract of clove, we found that
(See figure on next page.)
Fig. 1 Growth of Gram‑positive and Gram‑negative bacteria on MHA and MHH containing different concentrations of extract (5%; MHA‑C5
through 20%; MHA‑C20). Gram‑positive bacteria Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pyogenes grew on MHA‑C5 and
MHA‑C10 but not on MHA‑C15 and MHA‑C20. Gram‑negative bacteria Escherichia, Pseudomonas aeruginosa and Klebsiella pneumoniae grew on
MHA containing all the concentrations of extract tested

Page 4 of 6
Al‑blooshietal. BMC Res Notes (2021) 14:211
Gram Positive
Bacteria MHAMHA-C5MHA-C10 MHA-C15MHA-C20
Staphylococcus
aureus
Streptococcus
pyogenes
Enterococcus
faecalis
Gram negative
Bacteria MHAMHA-C5MHA-C10 MHA-C15MHA-C20
Escherichiacoli
Gram negative
Bacteria MHAMHA-C5MHA-C10 MHA-C15MHA-C20
Klebsiella
pneumoniae
Pseudomonas
aeruginosa

Page 5 of 6
Al‑blooshietal. BMC Res Notes (2021) 14:211
MHA-C15, supported the growth of different Gram-neg-
ative bacterial species and at the same time inhibited the
growth of all the Gram-positive bacterial species tested.
So, MHA-C15 can be described as a culture media for
selective growth of Gram-negative bacteria. It is antici-
pated that this newly developed media would prove
useful in the selective culture of other Gram-negative
bacterial species in both clinical and environmental set-
tings. Our future goal is to use graded concentration of
clove extract with and without other plant materials to
formulate bacterial growth medium which will allow dif-
ferential growth of different species of Gram-negative
bacteria.
Limitations
Limited number of pathogenic Gram-positive and Gram-
negative bacterial species were tested.
Abbreviations
EC: Escherichia coli; KP: Klebsiella pneumoniae; PA: Pseudomonas aeruginosa; SA:
Staphylococcus aureus; MHA: Mueller Hinton agar; MHA‑C15: MHA containing
15% clove extract; CEO: Clove essential oil.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13104‑ 021‑ 05628‑2.
Additional le1: Figure S1. Gram staining of Escherichia coli (EC) grown
on (a) MHA, (b) MHA‑C15; antibiotic sensitivity test of EC grown on (c)
MHA and (d) MHA‑C15. Figure S2. Gram staining of Klebsiella pneumo-
niae (KP) grown on (a) MHA, (b) MHA‑C15; antibiotic sensitivity test of
KP grown on (c) MHA and (d) MHA‑C15. Figure S3. Gram staining of
Pseudomonas aeruginosa (PA) grown on (a) MHA, (b) MHA‑C15; antibiotic
sensitivity test of PA grown on (c) MHA and (d) MHA‑C15. Figure S4. Gram
staining of Staphylococcus aureus (SA) grown on (a) MHA, (b) MHA‑C15;
antibiotic sensitivity test of SA grown on (c) MHA and (d) MHA‑C15.
Acknowledgements
We thank RAKMHSU for supporting the research work and Think Science‑
Emirates Foundation for supporting the project.
Authors’ contributions
SA, ML and NS performed all the experiments, MM provided technical assis‑
tance, AH obtained fund, conceived and supervised the research. SA, ML and
NS prepared the first draft. AH edited and finalized the manuscript. All authors
read and approved the final manuscript.
Funding
The research was supported by the Department of Medical Microbiology and
Central Research Laboratory of RAK Medical and Health Sciences University.
Availability of data and materials
Additional files available. The datasets used and/or analysed during the cur‑
rent study available from the corresponding author (ashfaque@rakmhsu.ac.ae)
on reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no conflict of interest.
Author details
1 Department of Medical Microbiology and Immunology, RAK Medical
and Health Sciences University, Ras Al Khaimah, United Arab Emirates. 2 Central
Research Laboratory, RAK Medical and Health Sciences University, Ras Al
Khaimah, United Arab Emirates.
Received: 23 February 2021 Accepted: 21 May 2021
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