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Formulation of Alternative Culture Media from Natural Plant Protein Sources for Cultivation of Different Bacteria and Fungi

Authors:
ZANCO Journal of Pure and Applied Sciences
The official scientific journal of Salahaddin University-Erbil
https://zancojournals.su.edu.krd/index.php/JPAS
ISSN (print ):2218-0230, ISSN (online): 2412-3986, DOI: http://dx.doi.org/10.21271/zjpas
RESEARCH
PAPER
Formulation of Alternative Culture Media from Natural Plant Protein
Sources for Cultivation of Different Bacteria and Fungi
Susan A. Shareef
Department of General Science, College of Basic Education, SalahaddinUniversity-Erbil, Kurdistan Region, Iraq.
A B S T R A C T
This study was carried out to find out some semisynthetic new media for routine   in
microbiology laboratories, with cheap natural protein source. Agar culture media were formulated using natural plant
sources such as cowpea, lentil, split pea, chickpea, soy protein, mung beans and rice and used for cultivation of
Staphylococcus aureus, Escherichia coli, B. cereus, Pseudomonas aeruginosa, Penicillium sp. and Aspergillus sp. All
tested microorganisms grew well on most plant sources except on rice. B. cereus showed the best growth and large
colonies on formulated media. E. coli and P. aeruginosa also showed good growth, and Pigmentation of P. aeruginosa
was not affected. S. aureus, P. aeruginosa and B. cereus produced beta hemolysis on all formulated media. Each
bacteria exhibit the same diameter of inhibition of antimicrobials used zone on all formulated media. Results showed
that most protein sources used were suitable for cultivation, hemolysis detection and antimicrobial susceptibility test.
KEY
WO
R
D
S:
Alternative media; formulated; protein source; bacteria cultivation.
DOI: http://dx.doi.org/10.21271/ZJPAS.31.4.7
ZJPAS (2019) , 31(4);61-69 .
1. INTRODUCTION
Generally microorganisms grow naturally
in almost all types of habitats. They need
essential sources of nutrients to maintain
growth and reproduction. Microorganisms
generally, need sources of energy, carbon,
nitrogen, phosphorus, sulfur and various
minerals (Harvey et al., 2007). Culture media
is a nutrient material prepared for the growth
of microorganisms in a laboratory. Nutrient
agar medium is commonly used as general
purpose medium for the cultivation of broad
range of bacteria.
It is a basic medium composed of peptic
digest of animal tissue, beef extract, yeast
extract, sodium chloride and agar.
Microbiological researches are carried out at
high cost and scarcity of culture media
(Adesemoye and Adedire, 2005). It is one of the
serious problems for developing country.
Microbial researches are hindered by high cost
of culture media (Mekala et al., 2016).
The search for alternative, cheap media for
use in laboratory agents for routine
microbiological experiments is going on.
Recent research has been focused on replacing
the culture media with alternatives cheap
materials (Mateen et al., 2012; Abbott and
Chapman, 1981; Babbar and Jain, 1998;
Babbar and Jain, 2004; Bromke and Furiga,
1991). Legume seeds serve as a good protein
source and they are very cheap and widely
available materials.
* Corresponding Author:
Susan A. Shareef
E-mail: suzan.sharif@su.edu.krd
Article History:
Received: 07/03/2018
Accepted: 26/05/2019
Published: 10/09 /2019
Shareef. S. /ZJPAS: 2019, 31 (4): 61-69
62
ZANCO Journal of Pure and Applied Sciences 2019
This study is aimed to find a cheap solid
culture media used in routine microbiological
experiments derived from plant products once
the commercial ones are expensive and in
most cases (most of the times) are not
available.
2. MATERIALS AND METHODS
2.1. Culture Media Preparation
The tested alternative nutrient samples such
as cowpea (Vigna), lentil (Lens culinaris),
split pea (Pisum sativum), Chickpea (Cicer
arietinum), soy protein, mung beans (Vigna
radiata) and rice (Oryza sativa) were
obtained from Erbil local markets.
The samples were ground into powder
using electric blender (Tefal) and filtered by
sieve and stored dry in air tight containers.
Three grams of each sample and 2 gm of
agar were dissolved in 100 ml of distilled
water. The pH of the media were measured,
which was between 6.4-6.8. Culture media
were sterilized by exposing to microwaves
(Gosonic- Model No. GOM-423) for 3 minutes
(Shareef et al., 2019) and poured into
sterilized petri dish. The appearance and the
color of each prepared alternative media
were reported. Nutrient agar (NA), Mueller-
Hinton, blood agar and potato dextrose agar
(PDA) were prepared and used as a control.
2.2. Collection of Tested Microorganisms
The tested bacteria were: Staphylococcus
aureus, Escherichia coli, Bacillus cereus and
Pseudomonas aeruginosa, while the tested
fungi were: Penicillium sp. and Aspergillus
sp. All tested bacteria and fungi were
obtained from the Department of Biology /
College of Science.
2.3. Inoculation of tested microorganisms
2.3.1. Bacteria
Serial dilution was done according to
standard method to obtain a final
concentration of 1.5x108 cell/ml. Both NA and
formulated media was inoculated in triplicate
with 0.1 ml of a bacterial suspension broth.
Solid media was inoculated on to the center
and was spread uniformly by a sterile glass
rod. All inoculated plates were incubated at
37oC for 24h.
2.3.2. Fungi
Pure fungal cultures of Penicillium sp.
and Aspergillus sp. were used. A fungal disc
was cut by using sterile cork borer, placed
upside down on the center surface of the
formulated culture media. Another prepared
fungal disc were inoculated on PDA media as
control. All inoculated plates were incubated
at 28±3 °C for 5 days.
After incubation fungal mycelia diameter
was measured using a ruler in 3 directions
and then average diameter was calculated for
each fungus.
2.4. Blood Hemolysis test
Five ml of blood was added to 95 ml of
alternative culture media to determine blood
hemolysis. Blood base agar (LAB28) was
used to prepare blood agar that used as
control.
2.5. Antimicrobial susceptibility test
Antimicrobial susceptibility for four types
of antibiotics (Amoxicillin, Amikacin,
Gentamicin and Cloxacillin (Bioanalyse)) was
tested for all species of tested bacteria on
formulated culture media. The inoculum of
each species of tested bacteria were, the
suspensions broth were adjusted to an optical
density of 0.5 McFarland. The antibiotic
susceptibility testing was determined by using
the KirbyBauer diffusion technique
(Cheesbrough, 1987). Plates of formulated
media and Mueller-Hinton were inoculated
using
sterile cotton swabs. Tested antibiotic
discs were placed on the inoculated media
surfaces and incubated for 24 hours at 37oC, the
diameter of zone of inhibition were measured after
incubation period and compared with the
inhibition zone on Mueller-Hinton medium.
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ZANCO Journal of Pure and Applied Sciences 2019
2.6. Statistical data analysis
A two way ANOVA was used to analyze the
results.
3. RESULTS
Since the pH of different formulated culture
media were ranged between 6.4-6.8, they were
used without adjusting the pH.
The characteristics of formulated culture
media were shown in the table (1), the color
of most of the prepared media was white to
creamy except for mung beans was light
green and cowpea was light brown. The
appearance of all prepared media was turbid
and some contains particles, except that of
protein soy agar was clear and transparent.
Table 1. Characteristics of formulated culture
media
Color
Particles
Turbidity
Light brown
+
T
Creamy
-
ST
Creamy
-
ST
Light amber
-
C
Light green
+
T
Creamy
-
ST
White
-
ST
T=Turbid, ST=Slightly Turbid, C=Clear
All tested bacteria showed significantly
(p>0.05) high growth rate on formulated
media except rice. Among all tested bacteria;
B. cereus showed the best growth on all
formulated culture media (figure 1), there was
no considerable differences in colony growth
rates between the alternative media and NA.
They exhibit swarming motility on chickpea
and covered the surface of culture media,
mean swarming was less on mung beans,
cowpea, split pea and lentil. Swarming was
controlled by increasing agar rate to 3% in
the media (figure 2-4).
Fig 1. Bacterial growth on different
formulated media
Fig 2. B. cereus colonies on Chickpea
0
10
20
30
40
50
60
70
80
Cowpea Lentil Chickpea Mung
beans Soy
protein Split pea Rice NA
Bacterial colony number
Plant Protein Sources
Staphylococcus aureus
Escherichia coli
Bacillus cereus
Pseudomonas aeruginosa
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ZANCO Journal of Pure and Applied Sciences 2019
Fig 3. B. cereus growth on Soy protein agar
Fig 4. B. cereus growth on Cowpea agar
P. aeruginosa produced large opaque
irregular colonies with sweet odor and
characteristic pigments (figure 5). Pigmentation
on formulated media was less obvious on soy
protein agar. E. coli grew well on all
formulated media except on rice, while S.
aureus produced smaller colony size than on
NA.
Fig 5. Fungal growth on formulated media
Both Penicillium and Aspergillus grew well
on most prepared media except on rice (figure
6), while they produced small colonies on soy
protein in comparison to their growth on PDA
agar. Although these fungi showed better
growth on PDA, the formulated media were
suitable for routine culture of tested fungi
(figure 7,8).
Fig 6. Penicillium colony on chickpeas
0
0.5
1
1.5
2
2.5
3
3.5
Colony diameter (mm)
Plant protein sources
Penicillium sp.
Aspergillus sp.
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ZANCO Journal of Pure and Applied Sciences 2019
Fig. 7. Aspergillus and Penicillium colony on
Split pea
Fig 8. Pigmentation produced by P. aeruginosa on
Lentil agar
taphyllococcus aureus, B. cereus and P.
aeruginosa showed beta hemolysis on
formulated blood agar. Hemolysis reactions of
tested bacteria on formulated blood agar were
shown in figure (9,10).
Fig 9. Beta hemolysis by B. cereus on Lentil
agar
Fig 10. Beta hemolysis by P. aeruginosa on
split pea agar
Staphyllococcus aureus hemolysis reaction
on formulated blood agar media was less
obvious than on blood agar while B. cereus
and P. aeruginosa produced clear zone.
The susceptibility of bacteria against 4
antibiotics was tested on formulated media.
Inhibition zone of each bacteria against
antibiotics was measured and compared with
inhibition zone of the same one on Mueller-
Hinton agar as shown in table (2-4). All
tested bacteria exhibited approximately the
same inhibition zone against each antimicrobial
agent on all formulated media. There were no
significant differences between the inhibition
zones on these media comparing with
inhibition zones on Mueller-Hinton agar.
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ZANCO Journal of Pure and Applied Sciences 2019
Table 2. Antibiotic activity against S. aureus on different formulated media
(Inhibition zone (mm))
Antibiotic discs
Cowpea
Lentil
Chickpea
Soy
protein
Mung
beans
Mueller-
Hinton
Amoxicillin
R
R
R
R
R
R
Amikacin
30
31
32
30
30
30
Gentamicin
33
34
36
36
40
36
Cloxacillin
R
R
R
R
R
R
R= resistant
Table 3. Antibiotic activity against P. aeruginosa on different formulated media
(Inhibition zone (mm))
Antibiotic discs
Cowpea
Lentil
Chickpea
Soy
protein
Mung
beans
Mueller-
Hinton
Amoxicillin
R
R
R
R
R
R
Amikacin
R
R
R
R
R
R
Gentamicin
R
R
R
R
R
R
Cloxacillin
R
R
R
R
R
R
Table 4. Antibiotic activity against B. cereus on different formulated media
(Inhibition zone (mm))
Antibiotic discs
Cowpea
Lentil
Chickpea
Soy
protein
Mung
beans
Mueller-
Hinton
Amoxicillin
16
15
14
13
13
14
Amikacin
34
33
36
35
36
36
Gentamicin
32
32
32
30
32
30
Cloxacillin
R
R
R
R
R
R
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ZANCO Journal of Pure and Applied Sciences 2019
4. DISCUSSION
Because the pH of different formulated
culture media were ranged between 6.4-6.8
they were used without adjusting the pH. This
may be helpful in the laboratories that may
not have pH meter especially in developing
countries. In a study by Ravathie et al the
pH of formulated culture media was between
(6.12 - 6.89) (Ravathie et al., 2012), they
found no significant change in the number of
colonies whether pH was adjusted to 7.0 or
went on with the experiments as such.
The properties and appearance of all
prepared media supported them to be suitable
to use in microbiology laboratories.
The results showed that the formulated
media contained sufficient amount of nutrients
to support the growth of the tested bacteria
(S. aureus, E. coli, P. aeruginosa B. cereus )
as well as fungi (Penicillium sp and
Aspergillus sp.) except for rice. The
composition of culture media is an important
factor for growth of microorganism. The
alternative culture media such as cowpea,
lentil, chickpea, soy protein, mung beans and
split pea contain protein and other nutrient
sources that microorganism need for growth.
Agar has no nutrient value, it was used as
solidifying agent to allow carrying out of
common techniques such as streaking out
cultures easy and then estimate microbial
growth on solid media (Sambali and Mehrotra,
2010).
Many studies have been carried out to
replace nutrient agar with alternative
formulated culture media from natural protein
source. In a study, Sago replaced nutrient agar
for the growth of different bacteria (Kapilan
and Thavaranjit, 2008). In another study by
Annan Prah et al in 2010., cowpea agar was
effectively replaced with NA. Berde and
Berde (2015) used vegetable waste as
alternative microbiological media. The legume
seeds were also used as alternative culture
media (Ravathie et al., 2012).
Pseudomonas aeruginosa produced large
opaque irregular colonies with sweet odor and
characteristic pigments due to blue colored
pyocyanin which spreads out over the
formulated culture media. Pigmentation on
formulated media was less obvious on soy
protein agar. Pseudomonas aeruginosa
produces many extracellular pigments that are
excreted into the culture medium surrounding
the colonies. Pigment production is depends
on the composition of the medium (Garibaldi,
1967).
Pigment formation and elaboration rely on
a dynamic metabolic equilibrium provided by
medium constituents such as peptones,
minerals, and various ions (Daly et al, 1984).
Pigmentation by P. aeruginosa was not
affected by the new media.
Although the tested fungi showed better
growth on PDA, the formulated media were
suitable for routine culture. In a study by
Mekala et al (2016), when culture media were
formulated from rice, chickpea, corn, dhal,
thinai, natural soy flour and processed soy
flour, they found that Sclerotium sp.,
Aspergillus sp., Fusarium sp. and Penicillium
sp. showed significantly (p>0.05) high growth
rate in PDA than other alternative culture
media. Fungi require carbohydrate rich media
to grow well and PDA contains large amount
of carbohydrate that required for fungal
growth and it also dextrose that serves as a
growth stimulant (Beever and Bollard, 1970).
Hemolysis is the ability of bacteria to
breakdown the red blood cells by the action
of hemolysins, erythrocyte-lysing enzymes, that
it produce. Colonies to induce hemolysis when
grown on blood agar is used to classify
certain microorganisms. Blood hemolysis
detection requires a culture medium for
bacterial growth and a source of blood as a
culture medium supplement. In this study
formulated media were used instead of blood
base agar and supplemented with blood to
obtain hemolysis.
There was no significant differences
between the inhibition zone on these media in
comparison to inhibition zone on Mueller-
Hinton agar as shown in table (2-4). Based on
this finding, formulated media can be used for
antimicrobial susceptibility test for student
experiment.
Results showed that all the plant natural
protein sources used are an acceptable
alternatives for the routine culture and
subculture, hemolysis detection, antimicrobial
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ZANCO Journal of Pure and Applied Sciences 2019
susceptibility test and fungi cultivation instead
of NA, blood base agar, Mueller-Hinton agar
and PDA, respectively for different species of
bacteria and fungi specially for routine student
experiments in practical microbiology.
In developing countries, with high costs of
manufacturing dehydrated media, the use of
protein formulated media is more practical and
cheaper compared with commercial ones that
are widely used in laboratories. Beside they
can be prepared immediately.
There are a number of studies concentrated
on alternative source of culture media to be
used instead of NA, but there was no
previous studies about using them for more
purposes such as hemolysis detection and
antimicrobial susceptibility test.
5. CONCLUSION
Many protein sources such as legume seeds
were used in formulation of culture media for
cultivation of different microorganisms
including bacteria and fungi. Most of
formulated media (except rice) were good and
suitable for microbial growth. Nutrient agar,
blood base agar, Mueller-Hinton agar and
potato dextrose agar can be replaced with one
of these cheap alternative media for routine
experiments in microbiology schools specially
in developing countries for culture, hemolysis
detection and antimicrobial susceptibility tests
in addition of fungi cultivation.
For this purpose only a bottle of agar that
cost 30$ with 600g of one of the alternative
nutrients that costs ~1.00$ is needed to
prepare 20 litters of four important and
essential culture media that used widely in
microbiology laboratory.
Therefore the use of different natural plant
protein sources as culture media in
laboratories is very much possible and cheaper
when compared to commercially prepared
culture media. Although plant protein sources
can be prepared easily, rapidly and they can
be stored in air tight containers for a long
time at room temperature.
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