In the Gram discoloration trial, 5 isolates are gram positive and 4 isolates are gram negative. Gram positive bacteriums will give blue or purple color consequence while gm negative bacteriums will give tap color consequence. Both gram positive and gram negative bacteriums are stained with the primary discoloration crystal violet to give a deep purple coloring material. Then, I is added as a mordant that will function to repair the primary dye. Iodine will adhere with crystal violet to make an indissoluble composite within the thick peptidoglycan bed of Gram-positive cells. Then decolourizer 95 % ethyl alcohol will fade out the lipoids that are found in the outer membrane of Gram negative bacteriums, this will do the crystal violet-iodine complex flights. So Gram negative bacteriums will go colourless and Gram positive bacteriums will still stay dark violet coloring material. The 2nd counterstain saffranine will dye the Gram negative with pink coloring material. Gram positive bacteriums will besides dye pink as good but the darker coloring material of crystal violet masks the lighter coloring material of the saffranine ( Barry Chess, 2009 ) .

5.3 Antibiotic Sensitivity Testing

Kirby-Bauer Method has been used in this testing. In this method, antibiotics paper discs are seeded on the surface of Mueller-Hinton agar. The medium can be used with complete assurance because it is rich in foods, able to turn fastidious beings. The usage of a medium with suited growing features is indispensable to prove the susceptibleness of micro-organisms to antibiotics. It is besides recommended for proving most normally encountered aerophilic and facultative anaerobic bacteriums ( Julia A. Kiehlbauch, et al. , 2000 ) . Mueller-Hinton Media contains beef extract and casamino acids, and amylum. Starch acts as a colloid that protects against toxic stuff in the medium. Beef extract and casamino acids are provided as a beginning of energy and foods. Agar is added when a hardening agent is needed. The degrees of Achromycin and sulfonamide inhibitors, thymidine, T, Mg and Ca ions are controlled so as non to interfere with susceptibleness proving and to give good growing ( Farmer, 1999 ) . Many factors are involved in sensitiveness disc proving. These include size of the inoculants, distribution of the inoculants, incubation period, deepness of the agar, diffusion rate of the antibiotic, concentration of antibiotics in the disc and the growing rate of bacterium ( John P.Harley, 2011 ) .

From Table 4.3 we can cognize the susceptibleness of the 9 bacterial isolates. Isolate 3S has the most figure of antibiotic oppositions which is 10. 3S is a Gram negative bacteria. The others Gram negative bacteriums which are 2LP, 2SP and 3S besides have higher figure of antibiotics opposition compared with Gram positive bacteriums. It might state us those antibiotics that being used in this testing are more effectual toward Gram positive bacteriums. So it shows higher figure of antibiotics opposition for Gram negative bacteriums compared with Gram positive bacteriums. From Table 4.3 we can besides cognize that imioenem is the most effectual antibiotic to suppress the growing of the 9 bacterial isolates. All bacterial isolates show susceptible against this antibiotic. Aztreonam and clindamycin show least consequence to against those 9 bacterial isolates. 7 isolates are opposition to these 2 antibiotics.

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5.4 Tolerance of Bacterial Isolates against Different Heavy Metallic elements

Heavy metals in low concentrations are non harmful to microorganisms, elements like Ni or Zn are even indispensable because of their incorporation in enzymes or cofactors ( Watt & A ; Ludden, 1999 ) . Nevertheless, high concentrations of heavy metals in the environment lead to an increasing intracellular concentration with the effect of suppression of enzymes or DNA harm by the production of reactive O species or irreversible binding to the active centres of enzymes

( Lopez-Maury et al. , 2002 ) . There are many illustrations of immune micro-organisms have been reported. These metal resistant isolates have developed really efficient and different mechanisms for digesting normal toxic degrees and have no consequence on cell growing ( Kaur & A ; Rosen, 1992 ; Collard, Taghavi & A ; Mergeay, 1993 ) . Heavy metals affect the microbial cell in assorted ways. On the macroscopic and microscopic degree general alterations in morphology, the break of the life rhythm, and production of pigments ( Figure 5.1 ) . It has been shown that the impact of metals on the metamorphosis depends on the growing signifier.

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( a ) ( B )

Figure 5.1: ( a ) Isolate 5P grows on lead LB agar ( 800AAµg/ml ) produces brown color pigment. ( B ) Isolate 3S grows on lead LB agar ( 1200AAµg/ml ) produces dark brown color pigment.

From Table 4.4.1, we can cognize that isolate 3S has the highest tolerance against assorted types of heavy metals. Isolate 3S has highest minimal repressive concentration ( MIC ) for 7 heavy metals. We can besides cognize that the ranking of toxicity for heavy metals against the 9 bacterial isolates. The most toxic heavy metal is quicksilver. Least toxic heavy metal is lead. The order is mercury, Ag, Cd, Co, Ni, Zn, Cu, manganese, ferrum, Cr and lead.

It is a fact of affair that current bioprocess research on metal remotion from treatable beginnings to place species of micro-organisms that are capable of efficient consumption of environmentally and economically of import metals ( Unz and Shuttleworth, 1996 ) . As a consequence of metal toxicity, populating cells may be inactivated ; hence most living-cell systems exploited to day of the month hold been used to decontaminate wastewaters incorporating metals at subtoxic concentrations ( Gadd and White, 1993 ) . However, the surveies introduced in the undermentioned focal point on metal immune micro-organism to look into the possible development of both the strictly physical sorption capacity and the active metal consumption with a subsequent intracellular segregation. Both mechanisms could back up schemes applicable to bioremediation when wastewaters contain toxic concentrations of metals.