Team:HZAU-China/InterLab
The HZAU-China team also participated in the InterLab project as we did in the past years. At first we read and analyzed the protocol provided by the headquarters carefully, then we checked out if we had all the essential material and equipment. After everything was well prepared, we formally carried out the experiment.
To obtain the OD600 reference point of a particular plate reader is rather necessary since we can only obtain ABS from a plate reader which is different from OD gained from a spectrophotometer. The ABS data obtained from a plate reader is volume-dependent while the sample of a spectrophotometer are volume-fixed.
As the protocol instructed, we added 100μL LUDOX solution into wells A1, B1, C1, D1 while added 100μL ddH2O into wells A2, B2, C2, D2. Then, we measured OD600 of the samples in the plate reader (Table 1).
Table 1. The result of OD600 reference point measuring.
| LUDOX CL-X | H2O | |
|---|---|---|
| Replicate 1 | 0.057 | 0.036 |
| Replicate 2 | 0.055 | 0.039 |
| Replicate 3 | 0.056 | 0.038 |
| Replicate 4 | 0.056 | 0.039 |
| Arith. Mean | 0.056 | 0.038 |
| Corrected Abs600 | 0.018 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 3.500 |
The data showed that the OD600 reference point of our plate reader is 3.500.
The particle standard curve experiment aims to obtain the relationship curve between the ABS600 of the solution and the concentration of bacteria. To achieve this goal, we need to find a kind of material which resembles bacteria very much. The headquarters sent us with Silica Beads, a kind of little particle which met our needs.
We made up a series of solution of different but continuous magnitude. To make the experiment more precise, we also set up another three parallel groups (Figure 1).
Figure 1. The result of Particle Standard Curve.
The fluorescence standard curve experiment targets to obtain the relationship curve between the strength of fluorescence and the concentration of fluorescein which has similar excitation and emission properties to GFP.
We prepared a series of fluorescein solution of different but continuous magnitude. As every experiment required, we also set up 4 parallel groups. Then we measured the strength of the fluorescence of each sample (Figure 2).
Figure 2. The result of Fluorescence Standard Curve.
Cell measurement was the most complicated experiment we've ever countered in the whole InterLab. It nearly took us a whole week to complete and diagnose the data. Each step of the experiment was so crucial that it could impact the result to a detectable extent.
However, we fixed up all the mistakes and completed the experiment satisfactorily (Table 2).
Table 2. Our raw data of cell measurement.
After repeated experiments we chose the best data and upload them to headquarters.
The CFU experiment is another challenging mission for its large amount of work and complicated operations.
We diluted the bacteria solution to OD600 = 0.1, then made up a sort of magnitude as the protocol required.
After reconducting the experiment for three times, we successfully achieved the data required by headquarters and finished the InterLab satisfactorily (Table 3).
Table 3. Our raw data of cell measurement.
| Replicate 1 | Replicate 2 | Replicate 3 | |
|---|---|---|---|
| Positive 1 | 0.084 | 0.0945 | 0.0875 |
| Positive 2 | 0.098 | 0.084 | 0.0875 |
| Negative 1 | 0.0945 | 0.084 | 0.0875 |
| Negative 2 | 0.1015 | 0.098 | 0.1015 |
| Replicate 1 | aTMTC | 11 | 1 |
| Replicate 2 | 206 | 18 | 6 |
| Replicate 3 | 174 | 17 | 2 |
| Replicate 1 | TMTC | 9 | 4 |
| Replicate 2 | 228 | 25 | 8 |
| Replicate 3 | 278 | 2 | 0 |
| Replicate 1 | TMTC | 23 | 1 |
| Replicate 2 | 370 | 28 | 10 |
| Replicate 3 | TMTC | 11 | 1 |
| Replicate 1 | TMTC | 89 | 1 |
| Replicate 2 | TMTC | 16 | 0 |
| Replicate 3 | TMTC | 48 | 4 |
aTMTC in our forms means the colony forming units in this plate is too much to count (over 300).
With this, we are delighted to announce that we completed the work on punctual with satisfaction from Measurement Committee.
The only suggestion we want to submit is that, in the Cell Measurement Experiment, we don’t think it’s correct to put the sample at 0 hour on ice. Because even if you put it on ice, the bacteria will also definitely grow, which results in high systematical error in the experiment.
