1. INTRODUCTION:
High level of all metal concentrations in the sterilized jars. Soil samples were kept at4oCuntil
environment may have negative impacts because they examined. Soil samples were diluted, spread plate
can be stored in many foods and food chains [1-2, method and streak plate method were used for
27].Chromium is one of the most common heavy culturing, and isolated bacterial colonies were
metal in the environment [14]. The range of average observed on the bases of the colony morphology on
concentrations of chromium differs from 7 to 150 agar. The standards for colony morphology of
ppm and the type of the soil’s mother rock affects its isolated bacterial colonies were based on colony
content and distribution in the soil [23]. Chromium is form, edge, surface, elevation and colony color. For
one of the highest poisonous heavy metals settled identification of chromium resistant bacterial strains,
into the environment through many industrial a chromium stressed media was prepared [26]. The
practices has been reported as a severe health issue progress of the isolate under diverse chromium
[9]. Hexavalent chromium has highest oxidative state concentrations were measured after 24 hours of
with very stable oxidizing characteristics [6]. incubation at 37oC at incubator (Binder, Germany).
Hexavalent form is more toxic than trivalent. The Morphologically different colonies of isolates were
hexavalent chromium is considered as severe characterized by using standard bacteriological
pollutant whereas trivalent chromium does not techniques as gram staining and spore staining.
possess serious concerns [19]. The contamination of Biochemical tests as catalase, eosin methylene blue,
soil by heavy metals is often permanent and may kill oxidase test, citrate test, MacConkey agar test and
or reduce the microbial populations as well as the sulphate indole motility test were performed.
exposure to metals lead to the establishment of a
resistant microbial population [25]. 3. RESULTS:
Among processed samples, ten isolates were selected
In a variety of industrial applications, common use of and further purified on the basis of colony
hexavalent chromium to clean up contaminated area morphologies. These isolates were labeled from iso-
in bioremediation, which uses the metabolic potential one to iso-ten. These isolates were observed and
of microorganisms to eliminate poisonous metals [8]. analyzed for staining and biochemical analysis. Pure
Studies have been done to evaluate the isolates were selected on the basis of five colony
transformation of hexavalent chromium bacterial morphological features as mentioned in Cappuccino
species in to trivalent chromium bacterial species. and Sherman (2005) laboratory manual. These five
Many bacteria have ability to transformCr6+, into less morphological features were whole colony, edge of
mobile and less toxic formCr3+, [5, 9, 15]. colony, colony surface, elevation, color of colony in
Table 1.
2.MATERIALS AND METHODS:
The soil samples were collected (0-15cm depth) from
different locations of chromite mines of Muslim bagh
district Kila Saifullah using aseptic techniques in
Bacteria were classified by direct examination with Citrate utilization test was performed on all the
the light microscope (Leica). From the isolates under isolates and were observed for growth of bacterial
study, iso-one, two, three, five, six and ten were colony and also the color change, 10% showed
round (cocci) in shape shown in Fig.1a, b, c, d, e and positive. All isolates were observed to be negative for
f among which iso-six was in cluster form oxidase, 60% isolates showed negative result for
(staphylococcus) and remaining were in pair form MacConkey test in Table 5. 90% showed sulphur
(diplococci), while iso-four, seven, eight, nine were positive result while 10% were negative, 80%
(bacilli) rod shaped and among which iso-nine was isolates showed indole negative and 20% isolates
in chain form (Streptobacilli) shown in Fig. 2g, h, I showed positive as well as for motility test 70%
and j [Table 2].It was found that six isolates were isolates showed positive result. The observed
gram negative and four were gram positive. The gram resistance against chromium is as, iso-one salmonella
negative isolates were iso-one, two, three, five, six, enterica 10%, iso-two p.fluorescen 15%, iso-three
ten, while gram positive isolates were iso-four, seven, A.creatinolyticus 15%, iso-four B.pumilus 20%, iso-
eight and nine shown in Table 3. In spore staining it five E.arogenes 15%, iso-six S.aureus 10%, iso-
was found that all isolates were spore forming shown seven B.circulans 30%, iso-eight B.subtilis 100%,
in Table 4. Among samples, 90% showed catalase iso-nine B.sphaericus 0% and iso-ten micrococcus
positive.50% showed eosin methylene blue positive. luteus 5% in Fig.3
1a 1b 1c
1d 1e 1f
Fig. 1: a, b, c, d, e and f.
iso-1, iso-2, iso-3, iso-5, iso-6 and iso-10 respectively
2g 2h
2i 2j
Fig. 2: g, h, i and j
iso-4, iso-7, iso-8 and iso-9 respectively
Isolates Iso-1 Iso-2 Iso-3 Iso-4 Iso-5 Iso-6 Iso-7 Iso-8 Iso-9 Iso-10
Gram - + - + - + - - + -
Negative/Positive
Iso-1 + _ Central
Iso-2 + _ Sub-terminal
Iso-3 + _ Central
Iso-4 + _ Central
Iso-5 + _ Central
Iso-6 + _ Sub-terminal
Iso-7 + _ Central
Iso-8 + _ Sub-terminal
Iso-9 + _ Central
Iso-10 + _ Central
Catalase + + + + + - + + + +
EMB test + + + - + - + - - -
Citrate + - - - - - - - - -
utilization test
Oxidase test - - - - - - - - - -
MacConkey - + - - + + - - - +
test
Sulphur - - - - + - - - - -
Indole - - - - + - - - + -
Motility + + + + - + + - + -
4. DISCUSSION:
In this study iso-one identified as Salmonella enteric luteus has an unusual ability to tolerate and to use
with 75% biochemical similarity. These observations very toxic organic molecules as carbon sources, and
are compared with morphological characteristics of metals but it showed less tolerance against chromium
Salmonella enterica which is defined in Bergey’s though it can be used in the degradation of metals
Manual [13]. Salmonella enteric has been such as zinc, lead and nickel. It has been sequenced
documented as pathogen responsible for health risk because these features are important for potential
[3]. Iso-two placed close to Pseudomonas applications in bioremediation and biotechnology
fluorescens, four isolates as iso-four, seven, eight and [20]. All the bacterial isolates mentioned above were
nine identified which belonged to genus Bacillus, and examined against chromium (potassium dichromate)
iso-six places close to Staphylococcus aureus. at different levels. For that purpose five different
Effective chromium reduction in different Bacillus concentrations were selected from low to quite high
specie had been reported [4, 10, 22, 21, 24] while concentration. Then the bacterial
different researchers also reported chromium growth was evaluated to on that diverse
resistance in gram negative bacteria such as concentration, so iso-eight demonstrated the highest
pseudomonas species [17, 18, 21, 22] and Cr(VI) tolerant ability, with 64μg/ml Cr(VI) being
staphylococcus species [12,26]. Iso-three placed removed within 24 h [11]. However, iso-nine showed
close to Arthrobacter creatinolyticus. These the lowest tolerance on nutrient media plate (64μg/ml
observations are homogenous with morphological Cr (VI).
characteristics of Arthrobacter creatinolyticus
defined in Bergey’s Manual. Results obtained from 5. CONCLUSION:
biochemical analysis of isolates, iso-five identified as In this study it was concluded that selective bacteria
Entrobacterarogenes with 75% biochemical can grow successfully in chromium stressed medium.
similarity. These observations are homogenous with Microorganisms which reside in heavy metal polluted
morphological characteristics of soil, usually adopt resistance against those metals by
Entrobacterarogenes with defined in Bergey’s shifting their intrinsic structural, physiological and
Manual [13]. Entrobacteraerogenes occurs in soil, biochemical properties. Therefore, the survival of
sewage, water, dairy products, and the feces of microbes in polluted soil is prone to show higher
animals and humans. It produces infections and resistance to heavy metals. In the present study it was
bacteria in humans [16]. Results obtained from also concluded that biochemical test can efficiently
biochemical analysis of isolates, iso-ten identified as identify and characterize bacterial specie. Among
Micrococcus luteus with 60% biochemical similarity. those ten isolated bacterial species, iso-eight Bacillus
These observations showed similarity with subtilis showed highest resistance against potassium
morphological characteristics of Micrococcus luteus dichromate (source of chromium) and remaining
which is also defined in Bergey’s Manual [13]. These were showed lowest resistance while iso-nine showed
physical and biochemical features for isolates, iso-ten 0% resistance against chromium.
placed close to Micrococcus luteus. Micrococcus