"Experiment 3" - Beakers and Growth Rate

Goal: See what effect glassware size has on Haemophilus influenzae growth (i.e. change in OD600 over time). Bonus: also get to see if cfu/mL correctly corresponds with OD600.

Method:

1. Get different sized beakers
2. Get H. influenzae from common source
3. Give each beaker 1.5 cm worth of solution and give duplicate beakers 1.0 cm worth of solution
4. Incubate, periodically measure OD600 over time
5. Plate cells every few OD measurements

 Here's a table to clarify:

Beaker #	Radius	Height of Liquid	Total Volume	Volume of Cell Solution
1	2.5 cm	1.5 cm	20 mL	400 µL
2	2.5 cm	1.0 cm	13.3 mL	267 µL
3	3.5 cm	1.5 cm	39.8 mL	770 µL
4	3.5 cm	1.0 cm	16.7 mL	333 µL
5	4.0 cm	1.5 cm	50 mL	1000 µL

Results:

  

Figure 1: OD600 over time

• The most obvious thing impacting the slope appears to be the height of the liquid in the beaker. There is a clear distinction between the beakers with 1.0 cm of liquid (2 and 4) and the others with 1.5 cm of liquid. This could actually be the effect of total volume rather than hight (since they are related) however it is interesting to note that beaker 1 and 4 had comparable volumes and were still distinct. 
• Most likely, the decreasing amount of liquid over time (each measurement took 1 mL of solution) had a larger impact on available resources in the beakers with a shorter liquid height than the beakers with a larger height of solution. 
• Surprisingly, there seems to be no obvious effect of the size of the beaker (at least not one that can be noticed simply by looking at the graph)  

Figure 2: cfu/mL at different OD600 

•  The measurement for cfu/mL was calculated based on the number of colonies growing on plates the following day
•  No strong trends seem to appear between the beakers but beaker 4 appears to be the least viable and beaker 3 appears the most viable  (though it's impossible to say anything for certain without repeating the experiment since two measurements from the same beaker are not independent)
•  From what little I know about Haemophilus influenzae growth, 10^8 and 10^9 are reasonable values at higher OD600's

Figure 3: Pretty much the same as the previous graph but with colony counts and a fitted curve

• I couldn't add a line for each beaker in the previous graph because there are not enough measurements per beaker to fit the model I was trying to fit (exponential)
• Also note that I had to leave out 5 data points (around 0.3) because when I measured the OD, I had not noticed that the pipette I was using was used by someone else who had changed the volume (I didn't notice until after 2 sets of readings)

What I learned:

• I learned how to code in R over the weekend, these graphs where made entirely from typing code into the R console
• I learned to check the pipette volume allocation every time you put it down and pick it up later
• I learned how much liquid should be added to a plate when plating bacteria (I got a lot less contamination compared to last time, however I still had a few alien colonies growing on some plates)
• Marcelo has informed me that if I want to measure accurate cfu/mL that I plate colonies at multiple dilutions. This sounds reasonable and will be something I keep in mind for future experiments (this will prevent 0 colony plates from affecting the cfu measurement)