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Version A:
Version B:
Version C:
Version D:
A. Movie Taker
As the ring of void pattern was repetitively obtained, we get more and more concerned about the detailed formation process of this novel pattern. Several questions were arisen in our experiment: 1) How soon will this ring pattern be formed? 2) Was the ring formed once the bacteria get there or formed later on when cells have grow evenly on the plate? 3) What is the difference of the process when our programmed cells are compared to the wild type?
Therefore we tried to take pictures periodically after dropping the cell on the plates. It is a nontrivial process since we have to strictly control the quality of the photos as well as the time points on which the photo has been taken. These data are all crucial for a precise mathematical modeling. We have gone all the way through five versions of the picture capturing apparatuses. One thing worth noting is that, this procedure itself is exploration and creation in an unknown area for us. We have solved two major problems we encountered during this development, 1) How to capture high-quality pictures with appropriate light source and a clear back ground. 2) How to do the automated capturing without human interfering, since it usually take 15 to 20 hours for the pattern to be developed.
4 versions of devices
Version A:
As we want to have the plate photos with the same standards in order to compare patterns from different plates and to observe single plate cell growth, the way we take plate photos must be fixed, and that is the original motivation for designing such a camera holder. As shown in the picture, a tripod is fixed on an empty cuvette box with tapes, and a black sheet is attached at the bottom of the whole device(not shown). The foam directly below the tripod is removed from the box so the plate can be fixed there. In this way we are able to take plate photos under identical conditions each time (same distance between the camera lens and the plates, and same position of the plates each time we take photos).
Advantage: after all, a standard for taking plate photos is developed, and it is better than nothing.
Disadvantage: the device is very simple and crude. Since the tapes doesn’t always make the tripod firmly fixed on the foam base, slight differences are observed each time we take photos.
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Version B:
Having recognized that in the previous version, the camera is not fixed firmly on the holder, we took an umbrella holder with two sets of parallel tapes. In this way we eliminate the infirmity between the tripod and the cuvette box, and both plate size and plate position has not changed after this version of holder had been developed.
Advantage: the camera is fixed firmly on the holder, so the differences between plate positions and sizes from different photos are minimized.
Disadvantage: environmental light change is the main factor that has great influence on the brightness of the plates. Sometimes it is difficult for us to compare the colors and densities of the pattern from the photo source.
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Version C:
Because light change has become the main enemy for taking plate photos, we made another holder from a big solid box. A hole is drilled on the top of the box for digital camera connected to a computer, and a single constant light source is provided from a fixed direction so the effects of light change are minimized.
Advantage: very slight effects from the light source change are observed. Also, with the help of computer, we are able to take cell growth movie for a single plate automatically.
Disadvantage: only one plate can be placed in the box for movie taking, so the efficiency is very low as we have a comparatively great amount of plates movies to take in a relatively short time.
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Version D:
Multiple holes have been drilled on the top of the box, and multiple cameras are fixed firmly in the holes. Plate movies as many as four can be taken simultaneously, with minimum light source changes, minimum position changes and highly automation. This device answers for our needs perfectly, and as you can see, the best films and photos are taken in it.
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B. Reflection spectrophotometer
This part of data analysis is trying to analyze the relationship between plate brightness, cell concentration, and OD. Furthermore, we want to try out if plate brightness can be directly related to growth curve measurement.
The traditional OD test is the measurement of the amount of light absorbed by a suspension of bacterial cells or a solution of an organic molecule with the use of a colorimeter or spectrophotometer. The higher the optical density, the lower the transmittance. In microbiology, the values can be used to measure turbidity, which in turn is used to estimate the number of bacteria. The formula of OD (Optical Density):
O = the opacity per-unit
T = the transmittance per-unit
I0 = the intensity of the incident light beam
I = the intensity of the transmitted light beam
(Reference: http://en.wikipedia.org/wiki/Optical_density)
Our design is hoped to be an alternation of the traditional OD test in a more easy and convenient way. The method is that by imposing a unified, consistent light source to plates with cells migrating (swim or swarm), cells with different concentration will emit different light intensity which will be recorded and measured. We’re hoping to come up with a relation between the light intensity with cell concentration, and furthermore, relate light intensity directly to OD value. A formula representing the relationship is deducted.
Measuring bacteria growth curve has been a frustrating experiment for our lab work, especially for those cells with large doubling time (and we believe thousands of people have experiencing the same problem). During our experiment of measuring migration speed of cells, we come up with the idea that the total cell number may have some relationship with total brightness of the plate with static light source. By impose the unified, consistent light source to a single plate at different time point and record the light intensity of whole plate, the growth curve can be plot. As a result, we are hoping to implement our “automatic OD machine” to accomplish the task of growth curve measurement.
The design of plate picture taking is being progressively improved and optimized. During the design and setup phase, we have invented up to 5 version of “black room”, (Need to provide photos of each version)
Along with the version evolved, the problems solved/unsolved are as following:
Background light noise (solved): use black curtain as plate background. Also, black box is used to block other light source in order to keep consistent and unified light source.
Static light intensity (solved): firm light source intensity and distance to the plate each time.
Reflection, Refraction problem (need more work): The problem is that the brightness level we measured is from the light collected now is the reflection from light source not vertical to the plate. This method may relate more to the reflection of the surface cell, but less to the brightness of the cells inside Agar gel. We are planning to change the light source go in vertically from the other side of the plate, and the diascoptic light is more accurately related to the total cell number on the plate.
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