MICROBIOLOGICAL EXAMINATION OF WASTEWATER Water sampling (i) For aerobic samples: Samples were collected aseptically (100mL) in clean

n & sterile 500mL Schott bottles and kept on ice. The water temperature was determined this is to decide the incubation temperature for culturing microorganisms later on. For anaerobic samples: Samples were collected aseptically (100mL) in clean & sterile 100mL Schott bottles and stored in an anaerobic jar (with CO2 gaspak). The water temperature was also determined.

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Serial dilution & enumeration of microorganism (i) Serial dilution Fresh samples were serially diluted (1:10) in Nutrient Broth (Difco) up to 109. Serial dilutions were repeated until at least half of the samples were used up. Lawn 100-200L of each serial dilution onto Nutrient Agar, NA (Difco), and Plate Count Agar, PCA (Difco) for bacteria, and Potato Dextrose Agar , PDA (Difco) for fungi; and spread evenly using a sterile hockey stick. Allow the agar media to dry & incubate in the following temperature(s) 30C & 37C for 24hours. ** For anaerobic samples, incubate the samples in an anaerobic jar supplied with CO2 gaspak. Enumeration of microorganisms Agar media that was incubated with the samples for 24 hours was enumerated on colony counter. [!! Only plates showing 30-300 colonies were taken into consideration. Below or above this number, they are labelled as Too little to count, TL or Too numerous to count, TNTC] ** At this point, we still have a mixture of microorganisms that have not been identified.

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Isolation for purification of colonies Distinct colonies on NA, PCA & PDA were purified by streaking on fresh, sterile agar media and incubated at their respective incubation temperatures of 30C or 37C. This step is repeated for a few times until we obtained a pure culture.

When the cultures on the agar media seemed pure (e.g. one colour & one shape & one size, etc.), they are further determined by using Grams stain (for bacteria) & methylene blue (for fungi). Grams staining procedure: a. Pick a single colony from a solid media using a flamed-sterile inoculating loop and spread thinly onto a clean glass slide. b. Fix the cells by heating briefly and allow the slide to cool to room temperature. c. To stain, cover the heat-fixed cells with methylene blue. Allow to stand for one minute. d. Wash away the dye by replacing it with iodine and allow it to stand for another one minute. e. Rinse the iodine under slow running tap water. f. Add alcohol for 10 seconds to de-stain and immediately wash the alcohol away under slow running tap water. g. Counter stain using safranin by allowing it to stand on the slide for 20 seconds. h. Remove excess safranin by washing under slow running tap water. i. Allow the prepared slides to dry. j. Observe under microscope (from lower to higher magnification power)

Characterization and differentiation of microorganisms (i) Grams staining characterization Grams stained cells were characterized according to their colour, shape, and spore formation. From here, we can also determine its purity. Selection & differentiation on selective and differential media Isolated, pure colonies were streaked onto available selective media MacConkey Agar (MCA), Eosin Methylene Blue Agar (EMBA), Mannitol Salt Agar (MSA), Salmonella-Shigella (SS) Agar & - for quick screening of microorganisms. Biochemical characterization A series of biochemical tests was carried out according to Bergeys manual for preliminary identification of the microorganisms.

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Identification using commercially available kits & 16SrRNA sequencing (i) BIOLOG Microbial ID system Protocol is done according to the manufacturers recommendation. User guide is available online. http://wiki.bio.dtu.dk/teaching/images/8/82/MicroStation_System_Vers ion_4.2_User_Guide.pdf 16SrRNA sequencing Colony PCR Overnight cultures on Nutrient Agar was picked using a sterile toothpick and suspended in 250L sterile ddH2O. Cell lysis was induced by heating in water bath (95C) for 5 minutes. Aliquot 5L of cell lysis suspension into PCR tube containing: i. H2O add to 50 l ii. 10x Optimized DyNAzyme buffer 5 l 1x (1.5 mM MgCl2) iii. 10 mM dNTPs 1 l (200 M each) iv. Primer A x l 0.5 M* v. Primer B x l 0.5 M* vi. Template DNA (cell lysis solution) 5 l vii. DyNAzyme II DNA Polymerase 0.251 l** 0.01-0.04 U/ M (0.52 U/50 l) * The recommendation for final primer concentration is 0.5 M but it can be optimized between 0.21.0 M, if needed. ** Possible enzyme dilutions are recommended to be made in 1x reaction buffer or H2O immediately before use. Load PCR product in 1.0% agarose gel electrophoresis, 75V 90mins. Purify the PCR product and have them sent for sequencing. Upon receiving the sequence result, BLAST the sequence http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SP EC=WGS&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSear ch

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