Int. J. Biosci.

2016

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print), 2222-5234 (Online)
http://www.innspub.net
Vol. 8, No. 2, p. 16-24, 2016

RESEARCH PAPER OPEN ACCESS

Isolation and Identification of Salmonella sp. from different

food

Nasima Aktar*, Rabeya Bilkis, Mohammad Ilias

Department of Microbiology, University of Dhaka, Dhaka, Bangladesh

Key words: Salmonella sp., Food, Contamination, Biochemical tests.

http://dx.doi.org/10.12692/ijb/8.2.16-24 Article published on February 20, 2016

Abstract
The consumption of meat as well as dry fish and many different types of processed foods are dramatically
increasing in recent years. Although the productions are increasing, the quality is not maintained up to standard.
The microbial safety status of raw meat, dry fish and pudding was investigated in this study to determine the
incidence of Salmonella sp. contamination. Isolation and identification of Salmonella sp. by conventional tests
(microscopy and culture in differential media combined with several biochemical tests) was performed. Presence
of Salmonella sp. in the foods indicates fecal contamination.
* Corresponding Author: Nasima Aktar  [email protected]

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Introduction survival. Although it is an enteric organism, recent

The causative agent of Salmonellosis is Salmonella research indicates its survival in the environment. S.
sp. (Cross et al., 1989; Tauxe et al., 1991; Smith et al., typhi and the paratyphoid bacteria are normally
1994; Baumler et al., 2000), a gram-negative, non- caused septicemic and produce typhoid or typhoid-
spore forming rod and facultative anaerobe (Grimont like fever in humans. Foods other than eggs have also
et al., 2000). Most species are motile (except S. caused outbreaks of S. enteritidis disease. S. typhi
gallinarum and S. pullorum) by peritrichous flagella. and S. paratyphi A, B, and C produce typhoid and
Although optimum temperature for Salmonella sp. is typhoid-like fever in humans. In Bangladesh, meat,
32-37°C, it is also capable of growing at 6-46°C. dry fish, and processed foods are all very popular.
Generally, detection methods are based on Meat, major protein source, may contaminate by
physiological and biochemical markers of the cross contamination with carcass, contaminated
organism (Williams, 1981). Cultural methods are water used in cleaning, processing and
based on nutrient acquisition, biochemical transportation, handlers, container (Aseel et al.,
characteristics, and metabolic products unique to 2013). Presence of Salmonella sp. in dry fish and
Salmonella sp. (Ricke et al., 1998). processed food indicates cross contamination and
unhygienic food handling. Therefore, it is important
The natural habitat of Salmonella sp. is the intestinal to check microbiological quality and take necessary
tract of human and other animals (domestic and wild measures to the quality as per international standard
birds, reptiles, amphibians, and insects). They are and remove any existing flaws.
regular pathogen of human and other animals. They
possess a complex antigenic structure and produce Materials and methods
variety of toxins and other virulence factors. These Sample Collection
bacteria can enter via oral route usually with Dry fish (Chittagong), meat (Dhaka) and processed
contaminated food or drink and colonize in the foods (Dhaka) were collected in a clean box and
intestine within 8-48 hours to 7-20 days. The mean transported to laboratory promptly.
infective dose for Salmonella sp. to produce clinical
or sub-clinical infection in humans is 106-108 Pre-enrichment and Enrichment
CFU/ml, although as few as 103 CFU/ml can produce 25g of each sample was placed into 175 ml lactose
disease to infants and immune compromised broth and 10g of sample was dissolved in 90ml of
individuals. Host factors (gastric acidity, normal Ringer’s solution without homogenization to prevent
intestinal flora, and local intestinal immunity, age of injury to bacterial cells (Birol Özkalp et al.,).
person) contribute to resistance to Salmonella sp.
infection. People previously exposed to the organism Lactose broth (LB) was used for the pre-enrichment
have a high resistance to the organism. According to and homogenization of food samples were performed
World Health Organization (WHO) about 80% of all as this could damage bacterial cell and incubated at
diseases and sickness in the developing world are 37°C for 24 hour. Selenite broth (SB) (AOAC
caused by inadequate sanitation, contaminated water international, 2000; June et al., 1995; Hammack et
or unavailability of safe water (CDC training, 2012). al., 1999) was used for the selective enrichment media
In Asia and Pacific region, fecal pollution is one of the for Salmonella sp. for 24 hour at 37°C.
most serious problems affecting both surface water
and ground water bodies and leading to a tenacious Total Microbial Count
persistence of waterborne diseases. When Salmonella Pour Count Agar (PCA) plate was used for the
sp. is released from human or animal host to the counting of heterotrophic bacteria after incubation at
environment, it encounters nutritional and 37°C for 24 hours. Potato Dextrose Agar (PDA) was
physicochemical shocks causing limited time of used for the total fungal count after incubation at

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room temperature for at 72 hours without any incubated for 24 hour at 37°C. A positive result of
movement. As most fungal species are spore former motility was indicated by the spread of the stab line.
and movement will cause the spore to spread and that As only the motile bacteria can spread through the
will give false count. semi solid SIM media with their flagella and pilli. And
non-motile bacteria do not spread as they lack
Colony Morphology organelle required for motility.
Bismuth Sulfite Agar (BSA) was used as selective
media for the growth of Salmonella sp. A flow Indole Production Test
diagram of the total work is given in Fig. 1. This test was performed to determine the production
of indole from the amino acid tryptone. The
Morphological characteristics (size, shape, surface production of indole is indicated by the formation of
texture, edge, elevation, color, opacity etc.) developed cherry red color layer at the top after the addition of
after 24 hour of incubation, were carefully studied Kovac’s reagent. And the negative reaction is
and recorded. indicated by dark brown color.

Microscopic Study Hydrogen Sulfide Production Test

Microscopic characteristics (size, shape, arrangement, This test was performed on KIA (Kliggler’s Iron Agar).
color, Gram reaction) were observed according to the The butt was inoculated with the 24 hour old bacterial
methods described in Manual of Methods for General culture and the slant was inoculated by streaking. It
Bacteriology by American Society of Microbiology was incubated at 37°C for 24 hour. A positive reaction
(ASM, 1981). Gram staining was performed to for the fermentation of glucose and lactose was
observe size, shape, arrangement, and Gram reaction. indicated by the yellow coloration of both the butt and
slant. Presence of cracks indicates the production of
Biochemical Tests acids. Blackening of the medium indicates hydrogen
Several biochemical tests (CDC training, 2012) were sulfide production. Pink-red slant but yellow but
performed to detect the physiological activities of the indicates glucose fermentation but not lactose.
selected isolated strain.
MR-Test (Methyl red)
Oxidase Test Each broth media tube was inoculated with
A positive reaction forms a deep–purple hue within 5- organisms from sample and incubated at 37°C for 24
10 seconds. A delay reaction forms color within 10-60 hour.
seconds. A negative reaction do not form color or
form color after 60 seconds. Voges-Proskauer Test (VP)
This test is used to determine the capability of some
Catalase Test organisms to produce non-acidic or neutral
Few drops of hydrogen peroxide was placed onto a compound like acetylmethylcarbonyl from organic
clean glass slide and then a small amount of 24 hour acids derived from glucose metabolism. Most
old culture was picked and mixed with hydrogen Salmonella sp. is VP-negative (CDC and prevention
peroxide and immediate bubble formation indicates training, 2012). Each tube was inoculated by loop
positive reaction. using sterile technique and incubated over night at
37°C. Barritt’s reagent was added and waited for 15
Motility Test minutes.
The SIM (sulfide, indole, motility) medium
containing McCartney bottle was inoculated with the Citrate Utilization
test organism by a single stab with a straight wire and In the absence of fermentable sugar some microbes

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are able to use citrate as their energy source if they Results

possess citrase enzyme. Citrate agar were stabbed and Sample Collection
streaked with a needle by maintaining sterile Dry fish, meat and processed food were collected
technique and incubated at 37°C for 24 hours. from different places (Dhaka, Chittagong).

Carbohydrate Fermentation Pre-enrichment and Enrichment

Each tube was inoculated with a loop and no shaking Lactose broth (LB) was used for the pre-enrichment
was done as it may force bubble of air in the inverted and no homogenization of food samples were
glass vial which may give false positive result and performed as this could damage bacterial cell and
incubated at 37°C for 24 hour. turbidity after 24 hour incubation at 37°C (Fig. 2a).

Table 1. Colony characteristics on BSA.

Colony characteristics BSA agar
Size Moderate to small
Shape Round
Elevation Convex
Margin Entire
Surface Smooth
Color Black colony with silver metallic sheen at the edge
Opacity Opaque
Texture Gummy

Table 2. Microscopic observation.

Parameters Observation
Size Small
Shape Rod
Arrangement Single
Gram reaction Negative (Pink)

Salmonella sp. growth in selenite broth is indicated of the organisms on PCA agar (24 hour) and PDA (72
by changing of broth color after 24 hour incubation hour) agar was observed. And the bacterial and fungal
(Fig. 2b). count was performed (Fig. 3a & 3b). From bar
diagram it is clear that total bacterial count was
Total Microbial count highest in pudding and least in dry fish (Fig. 4).
After overnight incubation the cultural characteristics

Table 3. Biochemical test results.

Test / Sample Dry fish Meat Pudding
Oxidase - - -
Catalase + + +
Motility + + +
Indole production - + -
KIA A/A A/A A/K
Gas production + + +
H2S production - - -
Glucose fermentation + + +
Lactose fermentation - - -
Sucrose fermentation - - -
Starch hydrolysis + + +
Gelatin hydrolysis + + +
Citrate utilization + + +
MR test + + +
VP test - - -

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However highest fungal spore was present in raw Colony characteristics (Table. 1) of the isolates were
meat and least in pudding. Moderate amount of observed and Shiny black color colony on BSA plate
microbes were present in dry fish (Fig. 5). after incubation at 37°C for 48 hour confirms that the
isolates are potential Salmonella sp. (Fig. 6b).
Growth on BSA agar Plate

Fig. 1. Flow Diagram of conventional culture and biochemical tests to identify Salmonella sp. from different
sample.

Fig. 2. (a) Pre-enrichment and (b) Enrichment of bacteria.

Microscopic Observation revealed that the isolates are motile facultative

Gram reaction and microscopic observation (Table. 2) anaerobes possessing catalase but not oxidase
revealed that the isolates are small gram negative rod enzyme. They are Methyl Red positive but VP
shaped bacteria that are singularly arranged (Fig.6a). negative. They can utilize citrate as carbon source.
They showed variation in Kliglers Iron Agar test result
Biochemical Tests that is normally used for differentiating certain
Biochemical tests results presented in Table. 3 members of the Enterobacteriaceae by demonstrating

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hydrogen sulfide production. The isolates did not monosaccharide such as glucose and produces acidic
produce H2S but produced gas. There was also by products that turn the red media yellow by
variation in indole production tests (Fig. 7). decreasing the pH of the media but they are unable to
ferment disaccharide such as sucrose and lactose and
Carbohydrate Fermentation hence the media color remains unchanged (Fig. 8).
This bacterium is capable of breaking

Fig. 3. (a) Total bacterial count (b) Total fungal count.

Fig. 4. Bar diagram showing total bacterial count of different samples.

Discussion Molecular methods, immunological, and

Most Salmonella sp. is pathogen to human and other bacteriophage detection systems are used in many
animals. They are not normal inhabitants of diagnostic food microbiology labs. Novel technologies
environment as they are normally reside in human such as the application of biosensors, microarrays,
and animal feces so their presence in food indicates and nanotechnology might be used in near future.
fecal contamination. Different types of methods for Present study was designed to investigate the
sample preparation, isolation and detection of occurrence, distribution, and persistence of
Salmonella in foods and food ingredients are used. Salmonella sp. in different food samples by using cost

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effective microscopic, cultural and biochemical tests contained lower number of microbes indicating lower
results. Although total bacterial count was much survivality of bacteria due to lower water activity (aw),
higher in processed food (pudding) than raw meat moderate number of fungal spore was present
and dry fish, fungal count was much lower. Raw meat indicates their lower withstanding capacity at lower
contained highest number fungal spore compared to a w.
dry fish and pudding. On the other hand dry fish

Fig. 5. Bar diagram showing total fungal count of different samples.

Fig. 6. (a) Microscopic view of the organism (b) Colony characteristics on Bismuth Sulfite Agar.

The isolates producing black shiny colony on selective confirmed the isolated isolates are Salmonella sp as
media (BSA) were selected as potential Salmonella these species can only ferment monosaccharide but
sp. showed characteristics, indicating that the isolates unable to ferment disaccharides.
were Salmonella sp.
Most of the strains showed similar, but some showed
Microscopic characteristics and gram reaction results different biochemical characteristics indicating strain
as well as biochemical tests and glucose fermentation variation.

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Fig. 7. Biochemical tests. (a) Oxidase test (b) Catalase test (c) Urease test (d) MR test (e) Indole test (f) H 2S
production test (g) Starch hydrolysis test (h) VP test (i) Citrate utilization test (j) Motility test.

Fig. 8. Sugar fermentation test (a) Glucose (b) Sucrose (c) Lactose.

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