In the Gram stain test, 5 isolates are gram positive and 4 isolates are gram negative. Gram positive bacteria will give blue or purple colour result while gram negative bacteria will give pink colour result. Both gram positive and gram negative bacteria are stained with the primary stain crystal violet to give a deep purple colour. Then, iodine is added as a mordant that will serve to fix the primary dye. Iodine will bind with crystal violet to create an insoluble complex within the thick peptidoglycan layer of Gram-positive cells. Then decolourizer 95% ethanol will dissolve the lipids that are found in the outer membrane of Gram negative bacteria, this will make the crystal violet-iodine complex escapes. So Gram negative bacteria will become colourless and Gram positive bacteria will still remain dark purple colour. The second counterstain safranin will dye the Gram negative with pink colour. Gram positive bacteria will also dye pink as well but the darker colour of crystal violet masks the lighter colour of the safranin (Barry Chess, 2009).
5.3 Antibiotic Sensitivity Testing
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Kirby-Bauer Method has been used in this testing. In this method, antibiotics paper disks are seeded on the surface of Mueller-Hinton agar. The medium can be used with complete confidence because it is rich in nutrients, able to grow fastidious organisms. The use of a medium with suitable growth characteristics is essential to test the susceptibility of microorganisms to antibiotics. It is also recommended for testing most commonly encountered aerobic and facultative anaerobic bacteria (Julia A. Kiehlbauch, et al., 2000). Mueller-Hinton Media contains beef infusion and casamino acids, and starch. Starch acts as a colloid that protects against toxic material in the medium. Beef infusion and casamino acids are provided as a source of energy and nutrients. Agar is added when a solidifying agent is needed. The levels of tetracycline and sulfonamide inhibitors, thymidine, thymine, magnesium and calcium ions are controlled so as not to interfere with susceptibility testing and to yield good growth (Farmer, 1999). Many factors are involved in sensitivity disk testing. These include size of the inoculums, distribution of the inoculums, incubation period, depth of the agar, diffusion rate of the antibiotic, concentration of antibiotics in the disk and the growth rate of bacteria (John P.Harley, 2011).
From Table 4.3 we can know the susceptibility of the 9 bacterial isolates. Isolate 3S has the most number of antibiotic resistances which is 10. 3S is a Gram negative bacterium. The others Gram negative bacteria which are 2LP, 2SP and 3S also have higher number of antibiotics resistance compared with Gram positive bacteria. It might tell us those antibiotics that being used in this testing are more effective toward Gram positive bacteria. So it shows higher number of antibiotics resistance for Gram negative bacteria compared with Gram positive bacteria. From Table 4.3 we can also know that imioenem is the most effective antibiotic to inhibit the growth of the 9 bacterial isolates. All bacterial isolates show susceptible against this antibiotic. Aztreonam and clindamycin show least effect to against those 9 bacterial isolates. 7 isolates are resistance to these 2 antibiotics.
5.4 Tolerance of Bacterial Isolates against Different Heavy Metals
Heavy metals in low concentrations are not harmful to microorganisms, elements like nickel or zinc are even essential because of their incorporation in enzymes or cofactors (Watt & Ludden, 1999). Nevertheless, high concentrations of heavy metals in the environment lead to an increasing intracellular concentration with the consequence of inhibition of enzymes or DNA damage by the production of reactive oxygen species or irreversible binding to the active centers of enzymes
(Lopez-Maury et al., 2002). There are many examples of resistant microorganisms have been reported. These metal resistant isolates have developed very efficient and different mechanisms for tolerating normal toxic levels and have no effect on cell growth (Kaur & Rosen, 1992; Collard, Taghavi & Mergeay, 1993). Heavy metals affect the microbial cell in various ways. On the macroscopic and microscopic level general changes in morphology, the disruption of the life cycle, and production of pigments (Figure 5.1). It has been shown that the impact of metals on the metabolism depends on the growth form.
C:\Documents and Settings\Kang\Desktop\DSC03371.JPGC:\Documents and Settings\Kang\Desktop\DSC03373.JPG
Figure 5.1: (a) Isolate 5P grows on lead LB agar (800ÂÂµg/ml) produces brown colour pigment. (b) Isolate 3S grows on lead LB agar (1200ÂÂµg/ml) produces dark brown colour pigment.
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From Table 4.4.1, we can know that isolate 3S has the highest tolerance against various types of heavy metals. Isolate 3S has highest minimum inhibitory concentration (MIC) for 7 heavy metals. We can also know that the ranking of toxicity for heavy metals against the 9 bacterial isolates. The most toxic heavy metal is mercury. Least toxic heavy metal is lead. The order is mercury, silver, cadmium, cobalt, nickel, zinc, copper, manganese, ferrum, chromium and lead.
It is a fact of matter that current bioprocess research on metal removal from treatable sources to identify species of microorganisms that are capable of efficient uptake of environmentally and economically important metals (Unz and Shuttleworth, 1996). As a result of metal toxicity, living cells may be inactivated; therefore most living-cell systems exploited to date have been used to decontaminate effluents containing metals at subtoxic concentrations (Gadd and White, 1993). However, the studies introduced in the following focus on metal resistant microorganisms to investigate the possible exploitation of both the purely physical sorption capacity and the active metal uptake with a subsequent intracellular sequestration. Both mechanisms could support strategies applicable to bioremediation when effluents contain toxic concentrations of metals.