Team:HZAU-China/InterLab
The HZAU-China team also participated into the InterLab project as we did in the past years.At first we read and analyzed the protocol provided by the HQ carefully, than 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. Data obtained are demonstrated in the sheet below:
| 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 HQ 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. Figures obtained are shown below:
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. The HQ explained why we didn’t use
e 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. Graphs obtained are shown below:
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. Any steps of the experiment were so crucial that they could impact the result to a detectable extent.
We made a lot of mistakes during the experiment, such as forgetting to mix up the solution, forgetting to measure ABS600 of the samples. We also unexpectedly came into a mutation happened on Device 4, which made the data of the two parallel groups vary significantly from each other.
owever, we fixed up all the mistakes and completed the experiment satisfactorily. Data achieved are shown below:
We have to point out that we directly measured the data at 0 hour without putting the sample on ice for 6 hours. We couldn’t understand why the protocol required us put the sample at 0 hour on the ice but not to measure it immediately. The protocol asked us to dilute the sample to ABS600 = 0.02. However, it also asked us to put the sample on ice for 6 hours. Even if you put it on ice, the bacteria would grow up a little definitely.
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. But we still came into some problems, including forgetting to cool the spreader and scalding the bacterial to death, forgetting to read the questionnaire on HQ’s website to check out all the data required before disposal the plates.
After reconducting the experiment for three times, we successfully achieved the data required by HQ and finished the InterLab satisfactorily. The CFU data are demonstrated below:
| 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 | TMTC | 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 |
We completed the work on punctual with satisfaction from Measurement Committee, which delighted us very much.
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.
