Day 3 - Wednesday - 31/10/2007
Whatsssuppp Peepers, woops I mean Peeps!!
As usual, we had bioprocess today and Immanuel and I are in charge of the Experiment 3…Which is the usual scientific inoculation, fermentation and Monitoring...Wait a min, no class monitor how to do monitoring?? LOL.Anyway, what happened today was pretty simple and procedural (Since when science is not procedural?) Basically, the laboratory technician or lab tech for short (why not VTEC?) transferred the seed culture (the culture dun have seed, but got the glowing
E.coli cells) into the fermenter using the peristaltic pump. By doing so, what this VTEC woops I mean lab tech is doing is to allow the
E.coli cells to grow at a larger scale (1 L is like large scale...-_______- "" ). After that, the lab tech then help us to collect the culture at T=0 as it is to be used as a control (or blank)...See, scientist always like to use symbols like T=1, [S] = 20,etc to make people feel that it is so deep, actually is much simpler. Okay, here comes the boring part, wait is boring or bo ring coz bo ring mean no ring in hokkien…
Er..Right. Basically what we have to do next is to assign our team members (2B), not 3A,3B or 6A,6B, this one not multi-storey car park! After assigning the team members, a pair of peeps will then collect the culture at every 1 hour interval for OD reading…So we are all good students such that the lab tech helped us to collect the culture at the last collection time which is 6pm. Actually right, no I mean left, the lab tech was very, ultra, super, ultimately nice to do it for us volunteeringly.
Procedure, Procedures, Procedures
1. Loosen the clamp on the input tube followed by pulling the plunger (backside) of the syringe to draw out the culture from the fermenter. The culture will then flow to the sampling test tube located on the side of the fermenter. Juz freaking push the plunger until the end.
2. After that, remember to tighten the irritating clamp back on the input.
3. Continuing on with the so boring procedure, loosen the clamp on the output tube. Next, pull out the syringe from the exhaust (How big is the size of the exhaust? Got 3" or 4" piping or not?) and air filter and pull the backside of the syringe.
4. Next, cap the syringe back to the exhaust air filter (Change Air Filter for more Horse Power!!!!) and push the backside.
5. The medium will flow to the collecting test tube and tighten the sickening clamp once and for all.
*Note that steps 3. and 5. is crucial. Failure to do so will result in eXpLoSiOn! Juz kidding…This is because if no tightening is done, the medium in the sampling will flow back to the fermenter, which might result in contamination.
*Step 2. is also required to ensure that the air is pushed back into the fermenter. Contamination will not occur in this case as there is an air filter (make sure that air filter got performance!) while pushing air back into the fermenter.
The sooo INTERESTING answers to questions are next!
1. For pH (Not referring to Philippines), the pH of the medium is detected by pH probe in the fermenter. Maintaining optimum (So Optimus Prime will come out), pH is controlled by the base and acid pumps on the automated machine. So if the pH is too acidic or too low, the base pump is activated by the machine and NaOH (Sodium Hydroxide) is added until the desired pH is reached. If the pH is too basic or too high, the acid pumps is activated and H
2SO
4 (Sulphuric acid) is added until desired pH is reached.
2. For temperature, in the batch fermentation process, the cooling jet (how bout jet plane? Not funny right?) will be activated if the temperature of the fermenter is too high.
3. For dissolved oxygen (how about unsolved ones? Hahahaa), the dissolved oxygen probe will measure the amount of dissolved oxygen in the medium to check whether it is sufficient or not. If it is insufficient, air will be released into the medium via sparger.
After all of that, Imauto and I, woops I mean Immanuel and I went to do the OD readings of what we had collected. Below is the table of the readings! Too bad got table but no chair, cannot sit down... haha...
| Sample | Hours | OD600 | log (x/x0) |
| Control | 0.0 | 0 | - |
| 1 | 1.0 | 0.152 | 0 |
| 2 | 2.0 | 0.189 | 0.095 |
| 3 | 3.0 | 0.789 | 0.715 |
| 4 | 4.0 | 1.152 | 0.880 |
| 5 | 5.0 | 1.570 | 1.014 |
| 6 | 6.0 | 1.800 | 1.073 |
| 7 | 7.0 | 1.940 | 1.106 |
| 8 | 8.0 | 1.980 | 1.115 |
| 9 | 9.0 | 2.030 | 1.126 |
| 10 | 10.0 | 2.040 | 1.128 |
From the different samples we took, we determined the optical density (OD) of the sample using a machine called a spectrophotometer. This machine uses light absorbance to determine concentration of a solution using the formula using the equation A=Elc. Because terms E and l are constant, the equation can be phrased as A=c. The higher an OD reading is, the more light is being absorbed and thus more concentrated. 600 is the wavelength in nanometers used for this cell density. Why? Cos this amount of light is optimally absorbed by cells and not other contents such as waste and nutrients. Cool huh?
The readings were then log-ified to another term used for cell growth. A graph was then plotted to show how the bacteria has grown over the 9 hours. As you guys can see, the cells are growing in the log phase. The protein GFP is a primary metabolite (produced during growth). Ideally we should have harvested the protein before the cells starts dying (stationary phase!). But... we are not those kinds of people who stays in school. So we will harvest the protein tomorrow when the cells begin to die (death phase).
Immanuel and Ice Man (Shang-Feng ) Signing out... Till then, Ja ~ ne~
