MOJ Biology and Medicine

Research Article Open Access

Investigation of microorganisms associated with the

spoilage of onions around dutsinma metropolis
Abstract Volume 2 Issue 4 - 2017

The study was conducted to investigate the microorganisms associated with the
Orpin JB,Yusuf Z, Mzungu I, Orpin CA
spoilage of onion bulbs around Dutsinma metropolis. A total of 100 samples were Department of Biological Sciences, Federal University, Nigeria
collected from Hayin Gada and Wednesday Markets. Culture, Gram staining
and characterization were done for analyses. The bacteria species isolated were Correspondence: Orpin JB, Department of Biological Sciences
Staphylococcus sp, Bacillus sp, Pseudomonas sp, and Esherichia coli, while the fungi Federal University, Dutsinma, PMB 5001, Dutsinma, Nigeria,
were Aspergillus sp, Mucor and Yeast. Esherichia coli had the highest frequency Email [email protected]
of occurrence (25.00%) and the lowest was Pseudomonas sp (9.38%) whereas
Aspergillus sp had the highest frequency of occurrence (18.75%) and the lowest was Received: October 06, 2017 | Published: December 20, 2017
Mucor (6.25%). The biochemical test revealed that Bacillus sp and Staphylococcus sp
were Catalase and Coagulase positive while Pseudomonas sp and Esherichia coli were
Coagulase negative. Sensitivity test confirmed that Staphylococcus sp was resistant
to Chloramphenicol, Septrin, Amoxicillin, Gentamicin, Streptomycin, Tarivid,
Augmentin, Pefloxacin, Sparfloxacine, and Ciprofloxacin, antibiotics while Bacillus
sp, Esherichia coli and Pseudomonas sp were sensitive to the antibiotics

Keywords: investigation, microorganisms, spoilage, onions, dutsinma

Introduction and jute bags. These packaging materials come from palm, bamboo
and fibrous jute trees. However these materials provide no barriers
Total or since ancient times, onions (Allium cepa, L.) have been of dust, and can easily be crushed which might lead to damage of the
an important dietary resource and have also been of interest for onions. During storage, some losses occur due to sprouting, drying
medical purposes.1 Traditionally, onions and plants belonging to the and rotting.14 Bulb rots are a common cause of onion loss during
Allium genus have been used as an herbal remedy for a wide range storage. They are caused by microorganisms particularly fungi. The
of ailments, due to their association with many pharmacological black mould disease caused by Aspergillus niger is a limiting factor
effects.2,3 Biological effects attributed to onions have been commonly in onion production worldwide.15 Aspergillus niger has been reported
ascribed to the volatile sulfur-containing compounds, such as to survive between onion crops as a soil saprophyte in or on bulbs
thiosulfinates, mainly responsible for the characteristic taste, aroma in field or storage and is ubiquitous in nature. The fungus invades
and lachrymatory effects.4 These compounds are formed from cysteine bulbs of onions in field or storage whenever they find injured tissues
sulfoxide precursors and the effect of the enzyme alliinase which is by producing various enzymes or toxins.16 Association of Aspergillus
released from cell vacuoles when tissues are damaged.5 However, these niger with onion seeds produced in hot climates and their transmission
volatile products are highly unstable and recently, attention has been from soil and naturally-contaminated seeds to onion seedlings cause
focused on the effects of phenolic compounds, such as flavonoids, 30-80% loss or spoilage of onion bulbs.17
which are more stable.6 Onion is known for being a good natural
source of flavonoids mainly represented by the flavonols - quercetin Aim and objectives
and kaempferol, which are present as their glycosides.7 Protection of
food from pathogens and spoilage organisms has been traditionally The aim of this research work is to investigate the Microorganisms
achieved by chemical methods, but during recent years there has been associated with the spoilage of onions around Dutsinma metropolis
an increase in consumer interest in developing Onion which contain with the following objectives:
a low level or are free of chemical preservatives.8 The emergence i) To isolate the Microorganisms responsible for causing onions
of pathogens which are resistant to classical preservatives has also spoilage
created an urgent necessity to find alternative antimicrobial agents.9
In consequence, the food industry is interested in developing natural ii) To identify the Microorganisms responsible for causing onions
components for the partial replacement of synthetic antimicrobials.10 spoilage
Onions can be considered as a good source of natural additives to iii) To characterize the Microorganisms responsible for the
retard food deterioration.11 Numerous benefits have been attributed to spoilage of onions using biochemical test.
onions including prevention of cancer and cardiovascular disorders,
reduction in the blood levels of cholesterol, reduction in osteoporosis.12 Justification
Reduction in stomach ulcers, inhibition of the proliferation of cultured
This is an investigation of public health significance by determining
ovarian, breast and colon cancer cells, inhibition of platelets-mediated
the risk factor posed to the consumers of spoilt onions, as to suggest
thrombosis, prevention of inflammatory processes associated with
appropriate control measures and possible solutions in countering
asthma, treatment of fever, common cold, cough, sore throat and its
spoilage and storage.
use as an antimicrobial agent.13 Onions are packed locally in baskets

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© 2017 Orpin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and build upon your work non-commercially.
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Statement of the research problem 1hour (60 minutes). The materials are petridishes, test tubes, conical
flask, a beaker and universal bottles.
This research is going to identify the problems which are
encountered when spoilage occurs in onion. Everyday vegetables are Media preparation
being consumed in massive amounts. The onion that is affected by
microbes or contaminated by microbes when consumed leads to very The media used for the isolation and characterization are
severe health problems. These microbes that enhance this spoilage are Sabouraud dextrose agar , nutrient agar, nutrient broth, MacCkonkey,
Streptococcus pyrogenes, Esherishia coli, Pseudomonas aeroginosa, Eosin methycylene blue, Pepton water, Urea agar, Plate count,
Morgorella morgonii, Staphylococcus aureus, Staphylococcus Citrate agar, Agar agar, Triple sugar iron agar, Methyl red and Voges
saprophyticus, Bacillus pumilis, Bacillus lensus, Staphylococcus prosker respectively, and was prepared according to manufacturer’s
capilis, Streptococcus sciuri, Bacilus lensus, bacillus antracis, bacillus instructions.
subtilis, streptococcus equines, Mucor, Yeast, Aspergillus niger,
Aspergillus flavus, Fusarium oxysporum, Penicillium digitatum. The
Isolation of bacteria and fungi
proper storage of onions makes it available all year that is why this Serial dilution and sample preparation
research is being carried out to minimize the spoilage of onions if
possible. 70% of alcohol was used to wipe the surface of the spoiled onion
bulb. About 1g of the spoiled onion fruit was cut using a sterile razor
Material and method blade from each of the sample and it was transferred separately into
a sterile motor and pestle , sterile distilled water was added into the
The study was conducted in Dutsinma Local Government Area, motor, it was then crushed, 1ml of the sample was added into a 9ml
Katsina State. It is located on Latitude 12o 27’18”N and longitude 7o of the sterile Nutrient broth in a Universal bottles for each sample,
29’29”E and has its headquarters in the town of Dutsinma. It has an then the samples was allowed to stay for 15min. 1ml from each of the
estimated area of 527km2 (203sqkm) and a population of 169,671 as at test tube was used to carried out serial dilution for each sample from
2006 census.18 The population and activities in the local government 101-105.
area have increased in the last 3 years which may be due to the
establishment of the new Federal University .The Local Government Inoculation and incubation
is bounded by Kurfi and Charanchi Local Governments to the North,
For bacteria, about 0.5ml of the aliquot from serial dilution of
Kankia Local Government to the East, Safana and Dan- Musa Local
the sample was added in to the media (i.e. Nutrient, MacCkonkey
Governments to the West, and Matazu Local Government to the
and Eosin methycylene blue Agar respectively), and each media
Southeast. The people are predominantly farmers, cattle rearers and
was poured in to sterile Petri dish using sterile storage. It was then
traders (Figure 1).
allowed to solidify. The sample procedure was repeated for the other
sample. Then the inoculated plates were incubated inverted at 370C
for 24hrs. For fungi, antibiotic (Chloramphenicol) was added to the
media before it cools and solidifies to prevent the growth of bacteria,
the same procedure with the bacteria was used. The plates was then be
incubated at room temperature for 5 to 7 days.

Microbial colony count

Bacterial and fungal count was carried out on each of the plate
to determine the number of bacterial and fungal growth. This was
obtained by counting the whole plate and using the number obtained
to multiply by the dilution factor.

Macroscopy and colonial identification

Characterization and identification of the colony isolates was
achieved by initial morphological examination of the colonies in the
Figure 1 Map of Katsina locating Dutsinma at the centre.
plate (macroscopy) for colonial appearance, size, elevation, form,
Sample collection edge, consistency, colour, opacity, heamolysis and pigmentation hence
result was recorded. The isolates were identified and characterized
The onion bulbs were randomly selected from two different based on their cultural characteristics, gram reaction and biochemical
markets which include; Wednesday market, and Hayin gada market, reaction as follows:
packaged in different sterile polythene bags and transported to the
Microbiology Laboratory of the Department of Biological Sciences Identification of bacteria and fungi (Gram reaction):
Federal University Dutsinma for analysis.
This was carried out to differentiate gram positive from gram
Material sterilization negative organisms.19 A drop of water was placed on a clean slide and
a speck of bacterial growth was taken from the culture. The speck
Different laboratory apparatus was used for the research; the was emulsified on the slide to make a thin smear. The smear was then
apparatus was washed with detergent, rinsed with clean water and being passed over a flame to heat fix. It was then flooded with crystal
then dried. This was followed with a proper sterilization of the violet for 60seconds, washed with distilled water and iodine was
apparatus in a hot air oven at high temperature of about 1600C for added for 30seconds, washed with distilled water and decolonized

Citation: Orpin JB,Yusuf Z, Mzungu I, et al. Investigation of microorganisms associated with the spoilage of onions around dutsinma metropolis. MOJ Biol Med.
2017;2(4):280‒285. DOI: 10.15406/mojbm.2017.02.00057
 Copyright:
Investigation of microorganisms associated with the spoilage of onions around dutsinma metropolis ©2017 Orpin et al. 282

with alcohol. The smear was finally flooded with safranin to counter (30µg), chloramphenicol (30µg), ciproflaxin (10µg), ampiclox
stain for 60seconds. It will then be washed with distilled water and (30µg), erythromycin (10µg) and ampicilin (30µg). Each inoculums
allowed to air dry. Oil immersion was used and viewed at X100 of bacterial isolates was grown in separate plates at 37˚c in Mueller
objective lens. Purple coloration donated gram positive while red Hilton broth (agar plates) for 18hours, with shaking and subsequently
donated gram negative. For fungi: Little portion of the fungal growth diluted to an optical density of 0.1(0.5Mc Farland standard) and stored
was placed on a clean glass slide, a drop of the Lactophenol Cotton at 4˚c.The paper disk was gently but firmly placed on the inoculated
Blue (LPCB) was added, the speck was emulsified on the slide and plates using sterile forceps. The plates were incubated at 37˚c for
viewed at X10 and X40 objective lens. 24hours after which zones of inhibition was measured and interpreted.
Results obtained were classified as resistant or sensitive.
Characterization of bacteria (Biochemical Tests)
Statistical analysis
i. Motility test: Motility test is aimed at identifying motile
bacteria, it was carried out as described by,19,20 motility can The simple percentage was used as a statistical tool to determine
sometimes be referred to as the way an organism grow on solid the occurrence frequency of bacteria and fungi isolated from spoilt
media, and it determine the presence or absence of flagella. onions around Dutsinma metropolis.
Tubes containing the motility medium were inoculated by
making a fine stab with a loopful of the culture to a depth of Results
1-2cm and were then incubated at 370C for 48hrs. Motility is Data from (Table 1) show the total colony count in Colony
observed by spreading of the organism outwards from the stab Forming Unit (cfu/g) for twenty (20) collected from Wednesday
area. and Hayin Gada Markets respectively of various bacteria and
ii. Catalase test: This was used to differentiate those bacteria fungi obtained from the spoilt onion, the result shows the highest
that produce enzyme Catalase. Catalase test was carried out bacteria colony observed in Abe (Bacteria Isolated from Hayin Gada
as described by.19 A little portion of the bacterial growth was Market) was 5.92X105cfu/ml and the lowest was 1.04X105cfu/
transferred with a sterilized wire loop to a drop of hydrogen ml, the highest colony observed in Afe (Fungi Isolated from Hayin
peroxide on a clean glass slide. The presence of catalyses Gada Market) was 1.8X105cfu/ml and the lowest was 1.1X105cfu/
observed by bubbling indicated a positive test result while ml. While in Bbe (Bacteria Isolated from Wednesday Market) the
absence of bubble indicated negative test result of bacteria. highest colony observed was 5.4X105cfu/ml and the lowest colony
was 1.32X105cfu/ml, the highest colony observed in Bfe (Fungi
iii. Indole test: This test was carried out for Indole production by Isolated from Wednesday Market) was 5.8X105cfu/ml and the lowest
test organism which is important in identifying enterobacteria. colony observed was 1.0X105cfu/ml. Data from (Table 2) show
The test was carried out as described by.19 A little portion of the frequency of occurrence of bacteria and fungi isolated from the
each isolate was inoculated onto 5ml of sterile peptone-water onion samples, from the two sites and the percentages of occurrence
enriched with 1% tryptophan and incubated at 370C for 24 of each bacterium and fungus isolated, identified and characterized.
hours. To the culture, 0.5ml of Kovac’s reagent was added and The percentage of occurrence of bacteria and fungi isolated from the
gently stirred. A red colour indicated positive result while a samples was highest in E.coli with 25.00% which probably arose from
yellow colour indicated negative test result of bacteria. the use of water during harvesting, the presence of Staphylococcus sp
iv. Citrate utilization test: This test is based on the ability of an and Aspergillus sp (18.75%) due to contamination from spores carried
organism to use citrate as it is source of carbon.A little portion in the air. Bacillus sp showed 12.50% which may be due to post harvest
of each isolate was inoculated into Koser’s citrate medium handling. Pseudomonas sp, Yeast and Mucor showed, 9.38%, 9.38%,
and incubated at 370C for 72hours. A positive citrate test was and 6.25% respectively, probably due to environmental conditions,
confirmed by formation of bright blue color while the initial state of handling, processing, and storage of the onions. Data from
green color of the medium indicates a negative test.19 (Table 3) show the results of Biochemical tests from the spoilt onion
samples collected from Wednesday and Hayin gada Markets including
v. Methyl Red (MR): MR test was carried out to identify for Esherichia coli, Pseudomonas sp, Staphylococcus sp, and Bacillus
Enterobacteria based on the ability to produce and maintain sp. E.coli is a Gram negative rod, it is motile, positive in Catalase,
stable end product from glucose fermentation. It was carried out methyl red, Indole and negative in Citrate, and Coagulase. Bacillus
as described by (Ochei and Kolhatkar, 2000). A little portion of sp Gram positive rod, and Staphylococcus sp Gram positive cocci
each isolate was inoculated into the glucose phosphate peptone and were all Catalase and Coagulase positive, while Pseudomonas sp
water medium and was incubated at 370C for 48 hours. Few Gram negative rod, positive in Catalase, and Citrate it is Motile and
drops of methyl red were added to the culture. MR positive was negative in methyl red, Indole, and Coagulase.
indicated by red color.
Table 4 show the fungi identified and characterized, from the
Antibacterial sensitivity testing spoilt onion samples including Aspergillus sp, Yeast, and Mucor.
Mucor appeared whitish on Sabourand Dextrose Agar plate and
The antibacterial sensitivity test was used to determine the thick non septate hyphae on microscope, Yeast appeared as small,
susceptibility of various bacterial species to various antibiotics and creamy or whitish colonies that were somewhat more raised than
synthetic agents. A standardized disk diffusion method was used; bacteria colonies. Aspergillus sp appeared black colonies with
antibiotic susceptibility testing and the zone size interpretation whitish edges. Data from (Table 5) show the Antibiotics sensitivity
chart were used for the determination of the bacterial sensitive to of bacteria isolates, Pseudomonas sp recorded the highest sensitivity
the antibiotics selected. The commercially prepared paper disk to all the antibiotics, followed by Esherichia coli which also had
was impregnated with the various antibiotics that will be assessed no resistance to any of the antibiotics. Bacillus sp was sensitive to
against the isolates; gentamycin (10µg), streptomycin (10µg), septrin all the antibiotics except Chloramphenicol, Tarivid, Augmentin and

Citation: Orpin JB,Yusuf Z, Mzungu I, et al. Investigation of microorganisms associated with the spoilage of onions around dutsinma metropolis. MOJ Biol Med.
2017;2(4):280‒285. DOI: 10.15406/mojbm.2017.02.00057
 Copyright:
Investigation of microorganisms associated with the spoilage of onions around dutsinma metropolis ©2017 Orpin et al. 283

Sparfloxacine. While Staphylococcus sp had the highest resistance to Table 3 Biochemical test for bacteria isolated and identified
all the antibiotics used because there was no growth in the immediate
area around the disc used. Gram
Organisms CAT MOT MR IND CIT COA
RXN
Table 1 Total colony count from samples collected from hayin gada and
wednesday market Bacillus sp +SR + - - - - +
Staphylococcus
+C + - - - - +
S/N Sites Colony count sp

1 Abe 5.2X105 Pseudomonas

-LR + + - - + -
sp
2 Abe 5.0X10 5
Esherichia coli -R + + + + - -
3 Abe 1.2X105 +C, gram positive cocci; -, negative; +, positive; -R, gram negative rod; +R,
4 Abe 5.92X105 gram positive rod; CAT, catalase; MO, motility; IND, indole; CIT, citrate; COA,
coagulase; Gram RXN, gram reaction; MR, methyl red.
5 Abe 1.04X10
Table 4 Identification and characterization of fungi isolated
6 Afe 1.3X10 5

Organisms Microscopic Macroscopic

7 Afe 1 .4X105
Thick non-septate hyphae White, heavy, woolyfluffy
Mucor
8 Afe 1.72X105 with dark sporongiospore growth covering entire plate

9 Afe 1.1X105 Thick septate hyphae with

Black colonies with white
Aspergillus sp conidiaborn in chains from
10 Afe 1.8X10 5 edges
the sterigmata
11 Bbe 4.88X105 Oval in shape with very Small Creamy or Whitish
Yeast
short Multilateral budding colonies
12 Bbe 1.84X10
Table 5 Antibiotics sensitivity pattern of bacterial isolates obtained from the
13 Bbe 5.4X105 spoilt onions antibiotics used on bacterial isolates

14 Bbe 1.32X105
CH CN AM PEF OFX S AU SXT SP CPX
15 Bbe 2.4X105 11 22 24 25 10 24 10 20 6 34
16 Bfe 1.6X10 5
5 6 3 4 7 2 1 10 5 4

17 Bfe 2.64X105 23 30 31 31 32 32 32 25 32 34

18 Bfe 1.0X105 25 23 28 26 38 27 25 8 30 31

Greater than 19mm, susceptible; Less than 13mm, resistance; CH,

19 Bfe 2.1X10
chloramphenicol; SXT, septrin;Am, amoxicillin; CN, gentamicin; S, streptomycin;
20 Bfe 5.8X105 OFX, tarivid; Au, augmentin; AM, amoxicillin; PEF, pefloxacin; SP, sparfloxacine.

Abe, bacteria isolated from hayin gada market; Afe, fungi isolated from hayin Discussion
gada market; Bbe, bacteria isolated from wednesday market; Bfe, fungi isolated
from wednesday market; e, third test tube of serial dilution. In the present study, the microorganisms isolated were
Staphylococcus sp, Bacillus sp, Pseudomonas sp, Esherichia coli,
Table 2 Frequency of occurence of bacteria and fungi isolates from the spoilt Mucor, Yeast and Aspergillus sp. Growth on mould was identified on
onions around dutsin-ma
sabourand agar and most of the isolated bacteria grew on Nutrient
Number of Percentage (%)
agar. This was because sabourand agar contains antibiotics, which
Organisms makes it bacteriostatic, thereby inhibiting the growth of bacteria.
occurence of occurrence
On the other hand nutrient agar contains antimycotic agent, which
Bacillus sp 4 12.5 inhibit the growth of fungi. The fungal organisms isolated were
Pseudomonas sp 3 9.38 Aspergillus sp, Mucor and Yeast. This research agree with that of
Baiyewu et al.21 who isolated these fungi from garlic, pawpaw fruits
Staphylococcus sp 6 18.75 in Nigeria, Krogh (1992) who also isolated seven different fungal
genera including the one isolated from this research from different
Esherichia coli 8 25
fruits including sweet orange and when these isolates were aseptically
Mucor 2 6.25 inoculated into healthy susceptible fruits, the characteristic symptoms
originally observed were also noticed. All the three organisms isolated
Yeast 3 9.38
successfully take part in decay and are thus confirmed as the causal
Aspergillus sp 6 18.75 organisms of fruit decay.20 This research also agrees with Shehu et
al.22 who reported a high frequency of occurrence for Aspergillus sp
Total 32 100 in the onion bulbs they studied. The presence of these fungi in the

Citation: Orpin JB,Yusuf Z, Mzungu I, et al. Investigation of microorganisms associated with the spoilage of onions around dutsinma metropolis. MOJ Biol Med.
2017;2(4):280‒285. DOI: 10.15406/mojbm.2017.02.00057
 Copyright:
Investigation of microorganisms associated with the spoilage of onions around dutsinma metropolis ©2017 Orpin et al. 284

onion bulbs is attributable to the environmental conditions, handling 2. Yin MC, Cheng WS. Antioxidant activity of several allium members.
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of both fungal and bacterial microorganisms which have been known 13. Tyson JL, Fullerton RA. Effect of soil borne-inoculum on incidence of
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Septrin, Amoxicillin, Gentamicin, Streptomycin, Tarivid, Augmentin, 14. Muhammed S, Shehu K, Amusa NA. Survey of the market diseases
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Conflict of interest papaya L.) in South Western Nigeria. African Journal of Agricultural
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The author declares no conflict of interest.
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Citation: Orpin JB,Yusuf Z, Mzungu I, et al. Investigation of microorganisms associated with the spoilage of onions around dutsinma metropolis. MOJ Biol Med.
2017;2(4):280‒285. DOI: 10.15406/mojbm.2017.02.00057
 Copyright:
Investigation of microorganisms associated with the spoilage of onions around dutsinma metropolis ©2017 Orpin et al. 285

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Citation: Orpin JB,Yusuf Z, Mzungu I, et al. Investigation of microorganisms associated with the spoilage of onions around dutsinma metropolis. MOJ Biol Med.
2017;2(4):280‒285. DOI: 10.15406/mojbm.2017.02.00057