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ISSN: 1948-5948
Journal of Microbial & Biochemical Technology
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Isolation and Identifi cation of Bacterial Strains and Study of their Resistance to Heavy Metals and Antibiotics

Korrapati Narasimhulu1*, P. Sreenivasa Rao2 and A.Venu Vinod3

1Department of Biotechnology, National Institute of Technology, Warangal, India.506021

2Department of Biotechnology, National Institute of Technology, Warangal, India.506021

3Department of Chemical Engineering, National Institute of Technology, Warangal, India.506021

*Corresponding Author:
Dr. Korrapati Narasimhulu,
Department of Biotechnology,
National Institute of Technology,
Warangal, India.506021,
Tel: 919985470286,
E-mail: [email protected]

Received date: May 16, 2010; Accepted date: June 19, 2010; Published date: June 19, 2010

Citation: Narasimhulu K, Rao PS, Vinod AV (2010) Isolation and Identification of Bacterial Strains and Study of their Resistance to Heavy Metals and Antibiotics. J Microbial Biochem Technol 2:074-076. doi:10.4172/1948-5948.1000027

Copyright: © 2010 Narasimhulu K, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Heavy metal resistant bacteria; Antibiotic resistance; NITW waste water treatment plant


Sewage treatment is the process of removing contaminants from wastewater and household sewage, both runoff (effluents) and domestic. It includes physical, chemical, and biological processes to remove physical, chemical and biological contaminants. Its objective is to produce a treated effluent and a solid waste or sludge suitable for discharge or reuse back into the environment. This material is often inadvertently contaminated with many toxic organic and inorganic compounds. The main object of treatment units is to reduce the sewage contents (solids) from the sewage and remove all the nuisance causing elements and change the character of the sewage in such a way that it can be safely discharged in natural water course applied on the land. Screening is the very first operation carried out at a sewage treatment plant and consists of passing the raw sewage through different types of screens so as to trap and remove the floating matter such as tree leaves, paper, gravel, timber pieces, rags, fibre, tampons, cans, and kitchen refuse etc. Microorganisms present in sewage can tolerate metal concentrations toxic to human beings, such as cobalt, lead, nickel, chromium etc., and also antibiotics. Various experiments are to characterize and identify the microorganisms present in the domestic sewage water collected from the sewage treatment plant at NIT Warangal. This characterization aids in the further use of these strains for bioremediation of waste water.

Characterization of experiments

Four staining techniques were used to characterize the bacteria isolated.

1. Gram Staining

2. Morphology

3. Metal resistance

4. Antibiotic Resistance

Gram staining: It is a differential staining technique used to characterize bacteria as Gram positive and Gram negative. The fixed bacterial smear is subjected to Crystal Violet, Iodine Solution, Alcohol (decolorizing agent) and Safranin respectively. Gram-positive bacteria retain crystal violet and hence appear deep violet in color, while Gram-negative bacteria lose the crystal violet and are counterstained by the Safranin. Hence they appear red in color.

Morphology: Bacteria are generally spherical (cocci), rod-shaped (bacilli), comma shaped (vibrio) or spiral-shaped (spirilla). After Gram staining bacteria are observed under a Light Microscope.

Metal resistance: Bacterial strains present in sewage are resistant to heavy metals such as cobalt, nickel, chromium, lead etc. Here six metal salts are used in order to identify bacteria corresponding to them. Six nutrient agar plates were prepared, each consisting of the respective salts of a particular metal.

Antibiotic resistance: Incomplete metabolism in humans has resulted in release of large amounts of pharmaceutical drugs into municipal wastewater treatment plants. The presence of antibiotics in sewage water can lead to the development of antibiotic resistant strains. Hence tests were performed to detect the presence and tolerance for various antibiotics.

Materials and Methods

Preparation of culture media and stock solutions

The nutrient agar (NA) medium is prepared as follows: (see Table 1).

The following components are weighed (except for agar) and dissolved in 600 ml of DDW.

Sl. No. Component Percentage Amount for 1 litre (in grams)
1 Beef Extract 0.3 3
2 Peptone 0.5 5
3 Sodium Chloride 0.8 8
4 Agar 1.5 15

Table 1: NA medium composition.

Metal stock solutions were prepared as follows:

1. Metal salts were used to prepare stock solutions of concentration 10 mg/ml.

2. Weight of the salt required to make stock solution is calculated in the following Table 2.

Sl. No. Metal Salt used mol wt Atomic weight Weight % weight for 100 ml stock solution (in g)
1 Cr K2Cr2O7 294.2 51.99 17.67 5.65
2 Cd CdCl2 183.32 112.41 61.32 1.63
3 Pb (CH3COO)2Pb 325.29 207.2 63.7 1.57
4 Cu CuSO4 159.61 63.55 39.82 2.51
5 Co CoCl2 129.84 58.93 45.39 2.20
6 Ni NiCl2 129.6 58.69 45.28 2.21

Table 2: Stock solutions of metals.

Isolation of bacteria to get pure culture

NA medium was used to isolate the bacterial strains from the samples. The cell counting technique in agar plates was followed to determine the population density in colony forming units per ml (cfu/ml) for each sample. A series of dilutions were made to reduce the cells in the samples. One ml of diluted sample was spread onto the surface of NA medium in the petri dishes and incubated at 37ºC and allowed to grow for 24 h. Single developed colony was picked on the NA plates and subcultured in nutrient broth and plated again on NA medium after dilution to obtain pure cultures. Pure bacterial strains were obtained after successive transfer of individual colony in NA plates and incubated for 24 h at 37ºC temperature. Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of antimicrobial agents or metal ion concentration that will inhibit the visible growth of a micro-organism after overnight incubation.

MIC for heavy metals commonly found in waste water

Waste water from domestic sources has various heavy metal ions in varying concentration. In order to isolate bacteria tolerant of heavy metal ions, we checked for their respective MICs using various metal ions like chromium(Cr), cadmium(Cd), lead(Pb), copper(Cu), cobalt(Co) and nickel(Ni). The pure cultures isolated above were plated on NA medium with metal concentrations ranging between 50 µg/ml and 500µg/ml with an increment of 50µg/ml. Then the procedure was repeated with an increment of 10µg/ml in the range where visible growth was absent to determine the MIC for each metal (Shakoori et al., 1998). NA medium was prepared. 20 ml NA was poured into each Petri plate and the volume of metal stock solutions were calculated by the formula: C1xV1=C2xV2, where C1 is the metal concentration in stock solution, V1 is the volume of stock solution used, C2 is the concentration of metal in agar and V2 is the volume of agar.

The isolated strains were then streaked onto the NA medium containing metal salts using sterile loops. The plates were sealed and incubated for 7 days at 37ºC. The plates were checked for bacterial growth. The concentration of metal ion at which there was no growth observed is the MIC for that salt for that strain.

MIC for various antibiotics

The isolated strains were also checked for antibiotic resistance with Kanamycin, Streptomycin and Amoxicillin with concentrations ranging between 10 and 250µg/ml the results have been tabulated (Table 5). NA medium is prepared. 20 ml NA is poured into each petri plate and the volume of antibiotic stock solutions were calculated. The isolated strains were then streaked onto the NA medium using sterile loops. The plates were sealed and incubated for 7 days at 37ºC. The plates were checked for bacterial growth. The concentration of antibiotic at which there was no growth observed is the MIC for that salt for that strain. Antibiotic sensitivity and resistance of the isolated heavy metal resistant isolates were assayed according to the Kirby- Bauer disc diffusion method given by Bauer et al. (1996) (Mackey and McCartney, 1996).

Physical characterization

Physical characteristics like shape and staining properties play an important role in identification of strains. Isolated strains were observed under microscope and shapes were noted. They were stained using Gram stain by the following procedure: Smears were prepared and heat-fixed. The slides were stained as follows: The crystal violet stain was flooded for one minute. Excess dye was poured off and gently washed in tap water and the slide drained against a paper towel. The smears were exposed to Gram’s iodine for one minute by washing with iodine, then added more iodine and left it on the smear until the minute was over. The slide was washed with tap water and carefully drained. The slide was washed with 95% alcohol for 30 seconds. The slide was washed with tap water at the end of the 30 seconds to stop the decolonization and the water was drained. The bacteria were counterstained with 0.25% safranin for 30 seconds. It was then washed, drained, blotted.

Results and Discussion

Physical characterization

A total of 9 strains could be isolated from the sample collected from the oxidation pond stage of waste water treatment. The density of microorganisms was found to be 9x103 cfu/ml. After pure cultures were obtained they were checked for the Gram staining reactions and the morphology noted and the results are tabulated (Table 3).

S.No. Strain / Species Gram staining Morphology
1 Strain 1 positive cocci
2 Strain 2 negative rod-shaped
3 Strain 3 negative rod-shaped
4 Strain 4 negative cocco-bacillary
5 Strain 5 negative rod-shaped
6 Strain 6 negative rod-shaped
7 Strain 7 positive rod-shaped
8 Strain 8 negative rod-shaped
9 Strain 9 negative rod-shaped

Table 3: Physical Characterization Results.

Metal tolerance

Maximum resistance was shown for chromium and minimum for nickel. The strains were found to be resistant to various heavy metal ions in varying concentrations as shown as follows (Table 4).

Sl. No. Strain / Species MIC for various metals in µg/ml
Cu Co Ni Cr Pb Cd
1 Strain 1 90 140 160 490 160 140
2 Strain 2 Nil 130 110 200 160 100
3 Strain 3 Nil 170 90 140 Nil Nil
4 Strain 4 210 180 140 200 140 170
5 Strain 5 220 200 120 180 190 310
6 Strain 6 Nil 100 80 130 100 210
7 Strain 7 140 120 190 220 140 170
8 Strain 8 320 140 90 250 160 120
9 Strain 9 140 260 120 150 110 100

Table 4: MIC values for various heavy metals.

Antibiotic tolerance

Maximum resistance was shown for amoxilcillin and minimum for kanamycin. The strains showed antibiotic resistance to the above antibiotics and their respective MICs are tabulated as follows (Table 5).

Sl. No. Strain / Species MIC for various antibiotics in µg/ml
Kanamycin Streptomycin Amoxicillin
1 Strain 1 90 80 180
2 Strain 2 85 75 125
3 Strain 3 20 35 055
4 Strain 4 40 90 105
5 Strain 5 80 65 95
6 Strain 6 85 60 115
7 Strain 7 70 85 220
8 Strain 8 65 250 330
9 Strain 9 15 35 65

Table 5: Results for Antibiotic Resistance.


The effluent in NIT Warangal waste water treatment plant includes waste water from a variety of sources like hostels, mess and staff quarters. In addition to that there are various laboratories situated in the campus like chemistry, biochemistry, microbiology and molecular biology laboratories which contribute a large number of chemicals including heavy metals to the effluent. The waste water treatment plant was setup in NIT Warangal campus so as to recycle the water and use it for irrigation of lawns. Sample collected from the NIT Warangal waste water treatment has a lot of bacterial species which help in the breakdown of organic compounds. Apart from that these bacterial species have been found to reduce the levels of toxic metals present in sewage and industrial waste. The metal resistant strains isolated from the waste water sample can be used for bioremediation process by construction of bioreactors where the strains can be immobilized for treating waste water effluents from industrial or domestic sources. Further selection of strains can result in isolation of strains with higher resistance which could serve as an effective means of treating waste water.


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