Morphological and Biochemical Characterization of Bacteria

PKISSN 0006 – 3096 (Print)
ISSN 2313 – 206X (On-line)
BIOLOGIA (PAKISTAN)
December, 2015, 61 (2), 271-277
Morphological and Biochemical Characterization of Bacteria isolated from Milk
Products
*
AMNA ALI, NAUREEN AKHTAR, UZMA BASHIR, RAHILA HAFEEZ, & MUHAMMAD SALEEM
HAIDER
Institute of Agricultural Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan
ABSTRACT
Microorganisms have been handed down from one generation to the next for use in fermented dairy
products. In addition some harmful bacteria are also present in dairy products which have detrimental effect
on human nature. The diversity of bacterial communities in dairy milk, dry milk, butter and yogurt samples
collected from different markets of Lahore Pakistan was evaluated in present study. Lactobacillus sp. was
isolated from dairy milk and yogurt while other isolates were identified as Citrobacter freundii, Ralstonia
pickettii, Salmonella typhii, Klebsiella pneumoniae, Salmonella gallinarum, Pasteurella sp. (hemolytical type
T and A), Shewanella putrefaciens, Acinetobacter jansani and Pantoea sp. and these are possible
pathogens of humans that cause infections. All strains were identified on the basis of morphological features
and metabolic processes. Maximum numbers of bacterial species were isolated from dry milk (brand A). The
results demonstrate that traditional fermented dairy products have diverse range of bacterial species which
are harmful for humans.
Key words: Identification, dairy products, gram negative, rods
INTRODUCTION
Milk is the basic dairy product and people
use it as basic supplement in food stuff. Liquid fresh
milk can be utilized in drinking purpose while after
processing cheese, cream, butter, yogurt, buttermilk
and other products are also used. It is truly an
amazing food when it is non-contaminated with any
other microorganisms. Conversely, it is a main
source for dispersal of food-borne pathogens
especially gram-negative bacteria in human life and
environment. Moreover, most of the dairy products
are unhygienic due to improper processing
conditions. Bacteria are ubiquitous in nature and
have the ability to colonize a wide variety of
substrates (Cleenwerck & Paul, 2008). Identification
of bacterial strains is not only valuable for
microbiologists but also for people associated with
medicines, as control and treatment of the
pathogenic bacteria is possible for properly
identified strains (Hasibe & Dilek, 2011). In the
absence of molecular data, morphological features
combined with certain biochemical studies provide
sufficient data required to identify the bacterial
unknowns (Ali & Naseem, 2012). The goal of
present work is to isolate and identify bacteria from
different products of dairy sold in Lahore markets
and to show the level of prevalence of pathogenic
bacteria in dairy products.
MATERIALS AND METHODS
Samples Collection
A range of dairy products: dairy milk
*Corresponding author: [email protected]
(pasteurized specifically in tetra packs or a milk
processing by plant), liquid milk (non- pasteurized
obtained from milk men), dry milk - brand A
(manufactured by local company), dry milk - brand B
(product of international company), loose packed
butter and yogurt were purchased randomly from
selected markets of Lahore. Properly labeled
samples, with sample type, collection site, date of
collection etc., were transported to the laboratory.
Isolation of bacterial species
Isolations were carried out using the freshly
collected samples by pour-plate technique on Luria
Bertani Agar (LBA) and Nutrient Agar (NA) media
(Ali & Naseem, 2011). The dry samples (butter,
yogurt, dry milk) 0.5 gm and liquid sample (liquid
milk) 0.5 ml were inoculated on prepared media
plates and incubated at 37°C for 3 days under
aerobic conditions. Purification was done by streak
plate technique, transferred the emerging colonies
on fresh media Petriplates under aseptic conditions
(Beishir, 1991). Pure bacterial cultures were stored
in 20% sterile glycerol at -20°C until further analysis.
Identification of bacterial species
First morphological and cultural features of
the bacterial colony/cells were recorded then certain
biochemical tests were performed as the routine
steps of bacterial identification.
Morphological Features: Morphological parameters
recorded for identification were cell shape, Gram
type, capsule stain, motility and pigmentation.
Growth on osmotic medium i.e., containing 2% NaCl
272
A. ALI ET AL
was also observed. Finally the ability of bacteria to
o
o
grow at 25 C and 40 C was also studied (Konem et
al., 1997).
Biochemical Analysis
Using the commercially available bacteria
identification
kit,
Microgen-TM
GnA+B-ID
Identification System (Microgen Bioproducts Ltd,
Surrey, UK), pure colonies was differentiated by
biochemical test. Initially preference of carbon
source of isolated bacteria was analyzed by
providing a wide range of carbohydrate sources that
include glucose, lactose, sucrose, inositol, sorbitol,
mannitol and xylose while sterile water was used as
control. Other biochemical analysis included study
of enzymatically catalyzed metabolic reactions such
as citrate, Indole, Methyl red, nitrate reductase,
oxidase, catalase, urease, malonate and gelatinase,
hydrogen sulphide, arginine and lysine (Holt et al.,
2000; Benson, 1996). Bacteria were identified by
providing the results of all above mentioned
biochemical tests to Microgen Identification System
software.
RESULTS AND DISCUSSION
In nutrition balancing science, dairy
products can play a definite and important role.
Therefore dairy products should be raw and
preferably certified. This cross-sectional study of
Gram-negative and Gram-positive staining bacterial
contamination of milk meant for human consumption
was carried out in some areas of Lahore, Pakistan.
Milk sampling points included smallholder’s milk
producers, dairy co-operatives, a milk processing
plant, and supermarkets. The hygienic procedures
applied
during
milking,
milk
collection,
transportation,
pasteurization,
and
postpasteurization storage conditions in these specified
dairy
products
were
evaluated.
Standard
bacteriological cultivation and biochemical assays
were used to isolate and identify bacterial
pathogens in the milk samples. During present
study, a total of thirteen different bacterial species
were isolated from various sources of dairy products
(Table I). Percentage occurrence of each species
was calculated. However maximum percentage of
bacterial species was obtained, six from dry milk
(brand A), two species from dry milk (brand B) and
butter whereas one species was isolated from dairy
milk, liquid milk and yogurt each (Figure 1). Thirteen
bacterial were isolated; 2 Gram-positive bacteria
BIOLOGIA (PAKISTAN)
and 11 Gram negative. Each isolate was given a
reference number (DP1 - DP13) that was used
throughout this study to represent the results of that
particular bacterium.
Table I: Detail of the substrates used to isolate
the bacterial strains.
Sample No.
Substrate
S1
Dairy milk
S2
Dry milk (Brand A)
S3
Dry milk (Brand B)
S4
Liquid milk
S5
Butter
S6
Yogurt
All the strains studied for this present work
were rod-shaped except DP1 and DP13 Gram
negative bacteria, likewise all strains were unable to
produce pigment except DP12. Furthermore only
DP12 managed to grow at 25°C although all strains
showed similar pattern of growth at 37°C. Only DP6
and DP7 were capsulated. Data recorded while
studying the morphology of bacteria is presented in
Table II. Naturally different bacterial strains have
different metabolic path ways (Sathishkumar et al.,
2008). Result demonstrated that all isolates gave
positive results with the carbohydrates (glucose)
except DP3 and DP7. In addition, DP1, DP2 and
DP13 had the ability to ferment lactose also while
others did not. Isolate DP2 exhibited positive result
with inositol among all species. Bacteria are referred
to as individuals or groups based on their patterns
of growth under various chemical (nutritional) or
physical conditions. Like all other living organisms,
different groups of bacteria utilize different sources
of energy to generate ATP, required for their
maintenance and reproduction. Most of the bacteria
use monosaccharides, for example glucose, as
energy source while few prefer disaccharides or
polysaccharides (Richard et al., 2011). Furthermore,
Fish (2002) reported that bacteria frequently secrete
chemicals into their environment in order to modify it
favorably and these secretions are often proteins
and may act as enzymes that digest some form of
food in the environment. Therefore, each species of
pathogen has a characteristic spectrum of
interactions with its human hosts. Capacity of
different bacterial strains to use various carbon
compounds as energy source is summarized in
Table III.
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BACTERIA FROM MILK PRODUCTS
273
Table II: Morphological and cultural features of bacteria studied.
Strain
No.
DP1
DP2
DP3
DP4
DP5
DP6
DP7
DP8
DP9
DP10
DP11
DP12
DP13
Gram
type
+
+
Capsule
stain
+
+
-
Motility
Pigment
+
+
+
+
+
+
+
+
-
Growth on
2% NaCl
+
+
+
+
-
Growth
o
at 25 C
+
-
Growth
o
at 40 C
+
+
+
+
+
+
+
+
+
+
+
Table III: Carbohydrate source preference analysis of bacterial isolates.
Strain No.
DP1
DP2
DP3
DP4
DP5
DP6
DP7
DP8
DP9
DP10
DP11
DP12
DP13
Glucose
+
+
+
+
+
+
+
+
+
+
+
Lactose
+
+
+
Sucrose Inositol
+
+
+
+
+
+
+
-
Biochemical tests actually exhibit the ability of an
enzyme to utilize different substrates. Such ability
can be assessed by the presence of products in a
biochemical reaction. Results of these tests help in
Sorbitol
+
+
+
-
Mannitol
+
+
+
+
-
Xylose
+
+
+
+
+
+
+
identification of bacteria (Harley, 2008). Enzymatic
activities of bacterial isolates are tabulated in Table
IV.
Table IV: Enzymatic activities of bacterial isolates.
Biochemical
test
Citrate
Indole
Methyl red
Nitrate
reductase
Oxidase
Catalase
Urease
Malonate
Gelatinase
H2S
Lysine
Arginine
DP1
+
-
DP2
+
+
DP3
-
DP4
+
DP5
+
+
+
-
+
-
+
-
+
+
-
+
+
+
-
Reference Strain No.
DP6 DP7 DP8
DP9
+
+
+
+
+
+
+
+
-
+
+
-
+
+
+
+
-
+
+
-
DP10
+
DP11
+
DP12
+
-
DP13
+
-
+
+
-
+
-
+
+
-
-
274
A. ALI ET AL
BIOLOGIA (PAKISTAN)
% of Bacterial species
50
40
30
20
10
0
Dairy milk
Dry milk
(Brand A)
Dry milk
(Brand B),
Butter
Yogurt
Liquid milk
Dairy Products
Fig., 1: Percentage of Bacterial Strains Isolated from each Sample of Dairy products
The identification of bacteria is essential in
microbiology by various aspects (Nitesh et al.,
2011). Identification was made by using the
Microgen Identification System software. Cultural
and biochemical data recorded for the isolates
(Table I-IV) was entered in the software to key out
the unknown bacteria. Lactobacillus sp. (n=2),
Citrobacter freundii (n=1), Ralstonia pickettii (n=1),
Salmonella typhii (n=1), Klebsiella pneumonia (n=1),
Salmonella gallinarum (n=1), Pasteurella sp.
(hemolytical type T) (n=2), Pasteurella sp.
(hemolytical type A ) (n=1), Shewanella putrefaciens
(n=1), Acinetobacter jansani (n=1) and Pantoea sp.
(n=1) were the identified species. Identified bacterial
species were deposited in First Fungal Culture Bank
of Pakistan (FCBP). All species versus their
reference no as well as their FCBP accession
numbers are given in Table V. Occurrence of each
bacterial strain in each sample is also shown in
Figure 2.
Table V: Bacterial species identified from dairy products
Sr. No.
S1
S2
Substrate
Dairy milk
Dry milk
(Brand A)
Ref. No. of Strain
DP1
DP2
C. freundii
DP3
DP5
DP6
DP8
R. pickettii
S. typhii
K. pneumoniae
S. gallinarum
Pasteurella sp.
(hemolytical type T)
Pasteurella sp.
(hemolytical type T)
Pasteurella sp.
(hemolytical type A)
S. putrefaciens
A. jansani
Pantoea sp.
Lactobacillus sp.
DP9
S3
Dry milk
(Brand B)
DP4
DP10
S4
S5
Liquid milk
Butter
S6
Yogurt
Species Identified
Lactobacillus sp.
DP7
DP11
DP12
DP13
FCBP accession No.
FCBP004
FCBP063
FCBP065
FCBP067
FCBP068
FCBP073
FCBP078
FCBP066
FCBP079
FCBP071
FCBP090
FCBP100
FCBP139
VOL. 61 (2)
BACTERIA FROM MILK PRODUCTS
Pantoea sp.
275
Lactobacillus sp.
A. jansani
Pasteurella sp.
(type A)
C. freundii
S.gallinarum
R. pickettii
S. putrefaciens
Pasteurella sp.
(type T)
K. pneumoniae
S. typhii
Fig., 2: Occurrence of Bacterial Strains in various Sample of Dairy products
Ranadheera et al. (2012) revealed that
some beneficial bacteria were used in multiple
sectors i.e., industry (Lactobacillus sp. in food, plant
and
dairy
fermentation)
and
agriculture
(Lactobacillus spp. as probiotic microorganism,
Azotobacter sp. as nitrifying agent, Pseudomonas
sp. as phosphate solubilizing agent). Results of
Wang et al. (2008) and Duskoval et al. (2012)
studies were similar to recent findings of bacterial
isolation from dairy product. Furthermore, it shows
that it is possible for dairy products to be
contaminated with pathogenic bacteria. This implies
that attention should be given to sanitary behavior of
food handlers. These pathogenic organisms release
toxins, which are the agents responsible for
illnesses such as diarrhea, dysentery, nausea and
vomiting, caused by these organisms upon
consumption of the contaminated foods (Okolie et
al., 2012). Unfortunately, even quite small numbers
of microbes can grow quickly into dangerous hordes
when products are not properly stored. The findings
of this study have confirmed that pathogenic
bacteria can exist in dairy products even though
they may physically appear to be quite wholesome;
thus, proper steps should be taken to ensure that
the occurrence of such organisms in products is
kept within limits (Okolie et al., 2012).
In addition present results also highlighted
that most of the sampled dairy milk was collected
under unclean environmental conditions and poor
preparation. Therefore, early study of Connor &
Charles (Connor & Charles, 1995) was also focused
on contamination in raw milk from soil, manure and
soiled bedding as well as direct contact with fecal
material during milking. Milk contains important
nutritional components for bacterial growth, and,
therefore, it is also an ideal medium for the growth
of many different bacteria. Temperature plays an
important role in bacterial growth. Many bacteria
prefer to grow at high temperature. As we know dry
milk is usually stored at room temperature while
liquid milk at cold temperature that’s why highest
percentage of bacteria was isolated from dry milk
(brand A) as compared to liquid milk. Further
findings of Champagne et al. (1994) and Jayaroo &
Wang (1999) about untreated water used in
equipment cleaning process, hands of milk producer
or improper udder preparation before milking were
main source of bacterial contamination in milk and
that might hold true in this study. Moreover, it is
necessary that the equipment be washed using
detergents then with cold water to remove as much
previous milk and dirt as possible followed by
washing with warm water to remove fatty deposits.
Afterwards, the equipment has to be washed again
with warm water and stored in a clean, dry and dust
free area (Robinson, 2002). Different bacterial
species isolated from the Dry milk (brand A) were
coliform bacteria including, K. pneumoniae, S.
276
A. ALI ET AL
gallinarum and S. typhii with frequency of 7.69%. It
has previously reported that dominant Gramnegative staining bacteria isolated from raw milk of
bulk tank milk were Escherichia coli and
Pseudomonas aeruginosa species (Khan et al.,
2008). Therefore these findings are similar to our
results. Additionally frequencies of bacterial isolation
observed in present investigation are also in
agreement with the studies performed to assess
bacteriological quality of raw milk in Ethiopia
(Tassew & Seifu, 2011). On the contrary, the
additional
bacterial
species
isolated
as Acinetobacter sp., Pantoea sp. and Citrobacter
sp. which were not isolated previously might be
attributed to higher environmental contamination
during transportation and/or contamination during
waiting along the roadside. Such kind of results
indicates that there are weaknesses in milk
transportation and processing. Ashenafi & Beyene
(1994) indicated that all microorganisms are not
killed by pasteurization especially when their
number is very high in raw milk. Therefore, it is
suggested that pasteurized milk should not be kept
at room temperature for days.
Recommendations
It is hereby recommended that routine
microbial analysis of dairy products sold at public
places be carried out to prevent outbreak of human
diseases. Also appropriate handling and hygienic
practices should be ensured by product vendors in
markets. Furthermore, product handlers should be
educated on appropriate storage temperature for
processed products. This kind of study should also
be conducted in other areas of Pakistan so as to
provide a comprehensive data for the local
government public health section.
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