Bio Lab Practical Review

These pipets have marked graduations on the side and are used to transfer liquids that are less than 1 ml. They are less accurate than using a pipetman

1. Transfer Pipet

P-20 for dispensing 2-20 µl P-200 for 20-200µl P-1000 for 100-1000µl

2. Pipetman P-20, P-200, P-1000

1. left click design tab in chart tools menu 2. find chart layouts and left click "add chart element" 3. left Clift axis titles and primary horizontal (e.g. concentration) 4. repeat for y axis (primary vertical) (e.g. absorbance)

Adding axis labels

1. go to design tab 2. click add chart element 3. under chart title select above chart (e.g. standard curve of a serial dilution)

Adding title

1. Design 2. Chart layouts 3. add chart element 4. find the trend line tab 5. click trend line and then linear 6. Right click on trend line and select format trend line 7. at bottom click checkboxes next to display equation and r-squared value

Adding trend line and R values

1. Collect 3 water samples using BOD sample bottles- bottles are black to BLOCK OUT LIGHT 2. Plug Dissolved Oxygen Probe into Channel 1 of the LabQuest 3. Unscrew clear protective plastic case 4. Rinse the rip of the probe with water and dry with Kimwipe 5. Set switch on DO probe to mg/L 6. Submerge probe to a depth of 4-6cm 7. Hold for 40 seconds 8. Collect data for atleast 10 seconds 9. Stop collection 10. Go to analyze then statistice and record average mg/L in the BOD table 11. flip the switch on the DO probe to percent and measure for 10 seconds, 12. Record the average percent saturation in the DO table 13. repeat for the remaining two bottles

Biochemical Oxygen Demand (BOD)

Reaction rate=|∆A|/∆T= |Absorbance final-Abs. initial|/(time final-time initial)

Calculating enzyme rate

=Average(B3:G3) =Median =STDEV.S

Calculations of Descriptive Statistics

1. click on formulas menu 2. in functions library section click pull down next to the more functions 3. go to statistical option and click on T.Test 4. click in the "Array1" box. Highlight cells B3 through G3 5. click in the "Array2" box. Highlight cells B4 through G4 6. Click in the "Tails" box and type "2" 7. Click in the "Type" box and type "3" 8. Click ok 9. decide whether or not significant

Calculations of inferential statistics (t-test)

1. Insert 2. Find charts and then scatter 3. Right click on the blank box and Select Data 4. Under the legend Entries Series box hit "Add" 5. Go to "Series X values" and click icon to right 6. select all of numbers from the concentration column and hit enter 7. Enter Y values similarly and hit OK

Creating a graph on excel

solve for x for concentration (independent variable)

Determining Unknown concentrations from trend line

C₁V₁=C₂V₂ C₁=concentration of stock solution V₁=volume of stock solution needed to make dilute sol. C₂=final conc. of dilute soln V₂=final volume of dilute soln Cs/Cu=As/Au IV→x DV→y

Determining concentrations of unknowns

-can be measured directly at the site from water samples -initial DO readings from BOD bottles were taken and recorded for DO value -units is mg/L or percent saturation

Dissolved Oxygen (DO)

P-1000; thousands (in red), hundreds, tens P-200; hundreds, tens, ones, P-20; tens, ones, tenths (in red)

How to read the volume settings for the three pipetman used in lab

starting buffer 1X in 2X step dilution starting volume 1ml C₁=starting conc. V₁=starting volume C₂=step dilution factor V₂=transfer volume (1X)(1ml)=(2X)(V₂) V₂=0.5 Step Dilution→2X Total Dilution→ 2, 4, 8, 16 Water= V₁-V₂=0.5 Starting cuvette has 1ml of stock buffer and rest have 0.5 of water pipette 0.5 stock from first into second and continue to last remove and discard 0.5 from last cuvette

Serial dilution example

1. with one hand squeeze the air valve (marked A) and then with the other hand squeeze the large bulb to expel the air. Release the A valve and the pipet bulb should stay collapsed 2. Place the end of the pipet with the cotton plug snuggly in the bottom of the siphon and then place the pointed tip into the liquid keeping the pipet vertical at all times. Slowly draw up the liquid by squeezing the suction valve (marked S) between the large bulb and the pipet 3. Place the pipet in the tube that you want to add the liquid and expel the liquid by pinching together the Empty valve (marked E) on the side arm of the pipet bulb

Serological pipet

1. go to "Sensors" in the menubar 2. Select "Calibrate" from drop down menu 3. Place cuvette labeled "B" (blank) in the cuvette holder 4. Press finish calibration, then ok 5. Go to "Sensors" and select "Data Collection" 6. Change the Mode from "Full Spectrum" to "Time Based" 7. Tap on the large red box on screen and select "Change Wavelength" 8. Change Wavelength from the drop down menu 9. Enter value (e.g 550) 10. Remove cuvette B and put cuvette 0 in the cuvette holder 11. Press the green play button and collect for at least 10 seconds 12. When sampling run is complete, stop data collection by pressing red square 13. Hit "Analyze" in the menubar at the top of the screen and select "Statistics" record average (mean) absorbance 14. Remove cuvette 0 and put cuvette 1 in holds 15. Hit "Discard" and start data collection for cuvette 1 16. Same for next cuvettes.

Spectrovis Labquest shiz for Serial Dilution to obtain Standard Curve

-use a temperature probe -measurements made in the field -calculate temperature change between two measurement locations and record value in data sheet

Temperature

1. Use a balance to measure the mass of each beaker before adding any water to it 2. Swirl sample water to keep it uniform 3. Place beakers into oven and allow water to evaporate at temp of 100-105 degrees for one week 4. After a week, measure the mass of the beaker and solids, allow to cool 5. Use a balance to measure the mass of each beaker with the solids now left behind. 6. Get mass by subtracting mass of empty beaker from mass of beaker with solids (atleast 0.025g)

Total Solids Test

1. Set up 3 double strength lactose broth; 6 durham tubes of single strength lactose broth 2. Shake the sample water bottle to resuspend any settled bacteria 3. with a 10 ml serological pipet, transfer 10 ml of sample water to each of the DSLB tubes 4. With the P-1000, transfer 1 ml of sample water to each of the middle set of 3 SSLB tubes 5. With the P-200, transfer 100 µl of sample water to each of the last three SSLB tubes 6. Incubate the tubes at 44.5 C for 24 hours. After 24 hours, your tubes will be refrigerated until Lab 5

WQI-Fecal Coliform

The Nitrate Ion-Selective Electrode (ISE) must be soaked in the Nitrate High Standard solution for 30 min Important: make sure the ISE is not resting on the bottom of the container and that the small white reference contacts are immersed. Make sure no air bubbles are trapped below ISE

WQI-Nitrates

-use a LabQuest pH probe -measure the pH for atleast 10 seconds -Analyze in menubar and select Statistics -record average value in data sheet

pH