Available via license: CC BY 4.0
Content may be subject to copyright.
Page 1/9
Development of a Novel Selective Medium for
Culture of Gram-negative Bacteria
Shooq Yousef Al-blooshi
RAK Medical and Health Sciences University: Ras Al Khaimah Medical and Health Sciences University
Mustafa Amir Abdul Latif
RAK Medical and Health Sciences University: Ras Al Khaimah Medical and Health Sciences University
Nour K. Sabaneh
RAK Medical and Health Sciences University: Ras Al Khaimah Medical and Health Sciences University
Michael Mgaogao
RAK Medical and Health Sciences University: Ras Al Khaimah Medical and Health Sciences University
Ashfaque Hossain ( ashfaque@rakmhsu.ac.ae )
RAK Medical and health Sciences University https://orcid.org/0000-0001-8574-2211
Research note
Keywords: Mueller Hinton Agar, Gram-Positive abcteria, Gram-Negative bacteria, Bacterial Culture Media,
Selective media, Clove
DOI: https://doi.org/10.21203/rs.3.rs-322259/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Page 2/9
Abstract
Objective: Although many bacterial culture media are commercially available, 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 the 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 modied to develop MHA-C15
(MHA containing15 % volume / volume water extract ofclove). Different Gram-negative bacterial
pathogens including
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 ndings show that MHA-C15 is a
newly developed selective media for culture of Gram-negative bacteria.
Introduction
Bacterial culture (growth) media contains nutrients necessary for their growth. All microorganisms cannot
grow in a single culture medium as their growth requirements 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 bacteria, particularly the
Enterobacteriaceae group of bacteria [3]. The use of selective media is essential in isolation of pathogens
from infection sites so that accurate pathogen identication and diagnosis can be made and treatment
can be initiated. Also, it is an essential rst step in microbiological investigation of environmental
samples. Although several selective media are commercially available, there is a constant effort to
develop newer media for more ecient isolation and identication of bacterial species [4]. Human body
harbors an astonishingly high number of bacterial species [5]. Studies have shown that we have
approximately 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 bacteria species in the environment also cannot be cultured due
to the lack of appropriate media which are needed for their growth [6]. This is the main driving force
behind the constant effort for the formulation and development of newer media for bacterial culture [8–
10]. Such new 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
avonoids which have been found to possess antimicrobial properties against wide variety of bacterial
pathogens. Since prehistoric times, traditional healers have used plants to prevent or cure infectious
conditions [11]. Cloves are aromatic ower buds of a tree,
Syzygium aromaticum
. They have been widely
known for their antibacterial, antiviral and antifungal properties and also for their use in herbal medicine
Page 3/9
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. Cloves have also been used in
treating dental infections and pain [12]. The extracts of clove exhibit antimicrobial effect on multidrug
resistant microorganisms as well as methicillin-resistant
Staphylococcus aureus, Bacillus subtilis,
Salmonella typhi
and
Serratia marcescens
. Various phytochemicals such as sesquiterpenes,
monoterpenes, hydrocarbon, and phenolic compounds 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
bacterial species. It is anticipated that the nding of this research project will stimulate exploration of
different plant materials for their suitability in developing selective and differential media for the growth
of different types of bacteria present in nature for which there exists no appropriate culture media.
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 minutes at 50° C. Then, the extracted material was ltered using
Whatman lter paper and stored at 4° C until used. Mueller Hinton agar (MHA) containing different
concentrations of clove extract (5–20%) was prepared by adding different volumes of the extract and
autoclaved. We followed the manufacturer’s instruction in preparation of the MHA plates and the volume
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 volume / volume); MHA-C10 (MHA containing 10 % extract volume / volume) and so on for other
concentrations.
Inoculation of different bacteria
Gram-positive bacterial species tested were
Staphylococcus aureus, Staphylococcus epidermis,
Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis
,
Bacillus subtilis
spp. and
Streptococcus mutants
. The Gram-negative bacterial species included in this study were
Escherichia coli,
Klebsiella pneumonia, Pseudomonas aeruginosa, Acinetobacter baumannii
and
Salmonella typhimurium
.
Bacterial strains were grown overnight in Mueller Hinton broth. A loop-full of bacteria was taken from
such cultures and was streaked onto the MHA-C agar plates (MHA-C5, MHA-C10, MHA-C15 and MHA-
C20) and incubated at 37° C for 24 hours. The plates were then observed for growth.
Page 4/9
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 and also as we were using this media in our initial
experiments to determine the antimicrobial activity of clove extract. We observed that modied MHA
(MHA containing clove extract) exhibited a differential property i.e. at certain concentration (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 containing 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 Gram-negative pathogen. 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 (Fig.1 and Fig.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 Gram positive and Gram
negative bacterial species. Even on MHA-C15, the growth of Gram negative was relatively less in
comparison to MHA-C10 (Fig.2), suggesting that clove extract has a concentration dependent inhibitory
effect on bacterial growth.
The count of bacteria grown on different MHA-C plates are shown in Fig.2. Counting of bacteria was
carried out for quantitative evaluation of bacterial growth as a function of the concentration of the clove
extract. Bacterial numbers decreased as the concentration of clove extract increased in the MHA plates
for both Gram-positive 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-negative 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 different antimicrobial agents because of the presence of highly
ecient eux systems which permits its growth in presence of different inhibitors [14]. This capacity
may have contributed to the ecient growth of this bacteria on MHA-C15. Other Gram-positive and Gram-
negative bacteria tested included
Bacillus subtilis
and
Streptococcus mutans
and
Salmonella
typhimurium.
These bacteria also followed the same pattern i.e. growth on MHA-C15 for Gram-negatives
and no growth for Gram-positives (data not shown). This nding demonstrates that the concentration of
Page 5/9
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 present in this medium inhibited growth of
these bacteria. Taken together these data demonstrate that MHA- C15 and MHA-C20 are the media that
does not allow growth of Gram-positive bacteria tested in this study but allows the growth of Gram-
negative bacteria. However, the number of colonies on MHA-C20 decreased for Gram-negative bacteria
compared to MHA-C15. Moreover, it demonstrates the inhibition effect of clove on Gram positive bacteria
as the concentration of the clove material increased. Taking all of this together, MHA-C15 may be
considered as an ideal media for Gram-negative. However, investigation with other Gram-negative
bacteria need to be done to determine whether the observation with the selected Gram-negative bacteria
also holds true for other Gram-negative bacterial species.
How clove extract selectively inhibits Gram-positive bacteria is unknown. The 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 antifungal activity. Antimicrobial activity of clove
also reported to be due to high tannin content (10–19%) [15–17]. The cell wall of Gram-positive bacteria
has a thick layer of peptidoglycan, which is much thinner in Gram-negative bacteria. This difference in
cell wall thickness is basis for differential susceptibility of many bacterial species to different type of
antibiotics and natural compounds. It may be speculated that the primary target for the growth inhibitory
compounds presents in clove is most likely bacterial cell wall [18].
In conclusion, after several trials with different concentrations of water extract of clove, we found that
MHA-C15, supported the growth of different Gram-negative 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 anticipated that this newly developed
media would prove useful in the selective culture of other Gram-negative bacterial species in both clinical
and environmental settings. 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 differential growth
of different species of Gram-negative bacteria.
Limitations
Limited number of pathogenic Gram-positive and Gram-negative bacterial species were tested.
List Of Abbreviations
Not applicable
Declarations
Author’s contribution
Page 6/9
SA, ML and NS performed all the experiments, MM provided technical assistance, AH obtained fund,
conceived and supervised the research. SA, ML and NS prepared the rst draft. AH edited and nalized
the manuscript.
Funding
The research was supported by the Department of Medical Microbiology and Central Research
Laboratory of RAK Medical and Health Sciences University.
Ethical approval and consent to participate
Not applicable
Availability of data and materials
Not applicable
Consent to publish
Not applicable
Conicts of interest
The authors declare no conict of interest
Acknowledgments
We thank RAKMHSU for supporting the research work and Think Science-Emirates Foundation for
supporting the project.
References
1. Stewart EJ. Growing uncultivable bacteria. J Bacteriol. 2012; 194: 4151-4160.
https://dx.doi.org/1128/JB.00345-12.
2. Bonnet M, Lagier J, Raoult, D, Khelaia S. 2020. Bacterial culture through selective and non-selective
conditions: the evolution of culture media in clinical microbiology. 2020. New Microbe New Infect.
2020; 34: 100622.
3. Lagier J, Edouard S, Pagnier I, Mediannikov O, Drancourt M, Raoult 2015. Current and past strategies
for bacterial culture in clinical microbiology. Clin Microb Rev. 2015; 28: 208 –236.
4. Madigan M, Martinko J (eds). Brock Biology of Microorganisms, 11th ed, Prentice Hall. 2005. ISBN 0-
13-144329-1.
5. Whitman WB, Coleman DC, Wiebe WJ. 1998. Prokaryotes: the unseen majority.Proceedings of the
National Academy of Sciences, USA95: 6578–6583.
Page 7/9
6. Sender R, Fuchs S, Milo R. 2016. Revised estimates for the number of human and bacterial cells in
the body. PLoS Biol. 2016; 14: e1002533. https://doi.org/10.1371/journal.pbio.1002533.
7. Achtman M, Wagner M. Microbial diversity and the genetic nature of microbial species. Nat Rev
Microbio.2008; 6: 431-440.
8. Gabunia K, Deslate HM, Garcia J. 2019. Effectiveness of Corn Husk Extract as an Alternative Culture
Media for the growth of Escherichia coli and Staphylococcus aureus. Int J Res Publi 2019; 39: 4
http://ijrp.org/paper-detail/758.
9. Shareef SA. 2019. Formulation of alternative culture media from natural plant protein sources for
cultivation of different bacteria and fungi. Zanco J Pure and Appl Sci 2019; 31
http://dx.doi.org/10.21271/Zjpas.31.4.7.
10. Cruz CH, Santos JB, Santos FP, Silva GB, Cruz EF, Ramos GL, Nascimento JDS. Texturized soy protein
as an alternative low-cost media for bacterial culture. Bac Empire. 2020; 3: 74-76.
https://doi.org/10.36547/be.2020.3.4.74-76.
11. Petrovska BB. Historical review of medical plants’ usage. Pharmacog Rev. 2012; 6: 1-5.
12. Kuranaswamy A. Multimodal management of dental pain with focus on alternative medicine.
Contem Clin Dentistr. 2026; 7: 131-139.
13. Abdallah Plants: An alternative source for antimicrobials. J. Appl, Pharma Sci. 2011; 1: 16-20.
14. Poole K.
Pseudomonas aeruginosa
: resistance to the max.Front Microbiol. 2011;2:65
https://doi.org/10.3389/fmicb.2011.00065.
15. Suresh P, Ingle VK, Vijayalakshmi V. Antibacterial activity of eugenol in comparison with other
antibiotics. J. Food Sci. Technol. 1992; 29: 256–257.
16. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol. Rev. 1999; 12: 564-582.
17. Nuñez L, D' Aquino M. Microbicide activity of clove essential oil (Eugenia caryophyllata). Brazilian J.
2012; 43: 55-1260.
18. AbdelFattah A, Aboelazab Y, Khallaf M, El-Kenany Y. Antimicrobial activity of ethanolic extracts of
clove and thyme. Arab Univ J Agri Sci 2019; 27: 491-499.
Figures
Page 8/9
Figure 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 9/9
Figure 2
Growth of Gram-positive and Gram-negative bacteria on MHA containing different concentrations of
clove extract. The data represents mean and standard error of mean of three independent experiments.
MHA-C5 contains 5 % of extract and so on (detailed in the materials and methods section). MHA-C15 had
the lowest concentration the extract, supporting the growth of the Gram-negative bacteria while
suppressing the growth of Gram-positive bacteria. EC- Escherichia coli, PA-Pseudomonas aeruginosa, KP-
Klebsiella pneumonia, SA-Staphylococcus aureus, EF-Enterococcus faecalis, SP- Streptococcus
pyogenes.
