refractometer use and scale 35 ppt STATION 4 MICROBIAL INDICATORS Enterococcus

Refractometer use and scale 35 ppt station 4

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refractometer use and scale = 35 ppt
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STATION #4: MICROBIAL INDICATORS ( Enterococcus ) **PLEASE REVIEW, BUT WILL BE DEMONSTRATED** Enterococcus Human and animal waste are loaded with microbes (x 10 10 cells g -1 !!!) including bacteria which constitute up to 60% of the dry mass of feces. A minority of these microbes are harmful to human and environmental health. Enterococcus are a genus of bacteria common to the mammalian gut. As such, they prefer to grow at high temperature (~37ºC). Moreover, Enterococcus survives well in water, including seawater, making it an ideal bioindicator of wastewater pollution. NOTE: Refer to STATION#3 instructions for proper use of the filter rig. NOTE: In this station be mindful of cross-contamination . Forceps should be sterilized by dipping in ethanol after each use. The filter rig should be sprayed with ethanol between each group’s series of dilutions (1 rinse after 3x filtering each unknown: 1, 5, and 100mL). 1) sterilize your filter rig by spraying with 70% ethanol in the spray bottle 2) rinse briefly with deionized water 3) unwrap and position a sterile membrane filter with the grid facing up on the filter rig base, reassemble the filter rig. 4) filter 100mL of deionized water through the membrane filter (this is your NEGATIVE CONTROL) 5) remove the filter using sterile forceps, place gently on the m-Enterococcus agar within a prepared plate. 6) repeat these steps with dilutions of your assigned water sample. for you will prepare 3 plates TOTAL from each of 3 volumes of filtered water. 1 x 1mL, 1 x 5mL and 1 x 100mL. You DO NOT need to sterilize the rig between volumes from the same depth. 7) label the bottom of each plate with your group number, depth, dilution volume (or control). 8) place in a 37ºC incubator for 24 hours. 9) remove the plates from the incubator and count the number of colonies. You may remove the lid to count and use a dissecting microscope if necessary. At all times you should wear gloves to prevent contaminating yourself! 10) calculate Enterococcus concentration by normalizing to 100mL. Thus, 100mL filtered requires no correction, 5mL counts require multiplying by 20, and 1mL requires multiplying by 100. = CFU 100mL -1 Enterococcus colonies growing on a gridded membrane placed over selective media. 7 of 8
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8 of 8 SPECIFIC ASSIGNMENTS : GROUP A: AMMONIUM, 50% dilution** (all 4 stations) ENTEROCOCCUS (station 1 only) TSS (station 1 only) pH & Salinity (all 4 stations) GROUP G: PHOSPHATE, 50% dilution** (all 4 stations) ENTEROCOCCUS (station 4 only) TSS (station 4 only) pH & Salinity (all stations) GROUP B: AMMONIUM, 50% dilution** (all 4 stations) ENTEROCOCCUS (station 1 only) TSS (station 1 only) pH & Salinity (all 4 stations) GROUP H: PHOSPHATE, 50% dilution** (all 4 stations) ENTEROCOCCUS (station 2 only) TSS (station 4 only) pH & Salinity (all stations) GROUP C: AMMONIUM, 50% dilution** (all 4 stations) ENTEROCOCCUS (station 2 only) TSS (station 2 only) ** to create pH & Salinity (all stations) of deionized a 50% dilution, mix 1mL of sample with 1mL water. Subsequently, you will multiply your final concentration by 2.
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  • Fall '16
  • Professor
  • Water supply, DI, Enterococcus

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