Team:CCU Taiwan/Test paper

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We want our Carindex to be an easy-to-use product for the customers. So we apply our functional bacteria onto the test paper. By doing it, our test paper along with the bacteria can be preserved for a very long time.
Finally, these freeze-dried test paper will be placed between two pieces of glass slides and become the consumptive material of Carindex.


When customer want to use Carindex, the revived liquid (LB broth) will be dipped into the test paper between two glass slides to revive the functional bacteria. Once the bacteria have awake, the CSP and lactate level detecting functions will be activated. Hence, the bacteria will detect the concentrations of CSP and lactate in the sample.


Experiment and result of test paper

Purpose

We designed experiments to test out whether the bacteria on the test paper will survive or not after freeze-drying procedure.

Method

First of all, we cultured the bacteria in LB broth (Vtotal=5 ml) for 16 hours and add fixed proportion of glycerol (4.5%) and glucose (7.5%) into the it. Then, we dipped 50 µl of liquid culture onto the test paper and took it to the freeze dryer. After freeze-drying, we do the revived experiment to test our the bacteria survival.

Protocol (before freeze-drying)

  • 1. Culture E. coli DH5α Interlab Positive Strain in 5 mL LB broth with antibiotics for 16 hours at 37oC and 170 rpm.
  • 2. Measure the OD600 value of the overnight culture.
  • 3. Add glycerol (4.5 %) and glucose (7.5 %) into the overnight culture.
  • 4. Dilute the liquid culture with LB broth to different OD600 value.
  • 5. Apply 50 μL liquid culture onto the test paper (Whatman #42, size: 15 mm*15 mm).
  • 6. Put the test paper into the microcentrifuge tube.
  • 7. Some microcentrifuge tubes are added 1 ml LB broth, some tubes are not.
  • 8. Freeze rapidly by liquid nitrogen.

Protocol (after freeze-drying)

  • 1. After freeze-drying, dip 50 µl of LB broth onto the agar plate.
  • 2. Apply the test papers on it.
  • 3. Incubate the agar plate at 37oC for 16 hours or place at room temperature for 16 hours.

Result

  • 1. Incubate the plate at 37oC for 16 hours with different OD600 value.
  • (OD600=5.3, 1.3, 0.6, 0.1)


  • 2. Place the plate at room temperature for 16 hours with different OD600 value.
  • (OD600=5.3, 1.3, 0.6, 0.1)


  • 3. Incubate the plate at 37oC for 16 hours with different OD600 value.
  • No adding glycerol and glucose before freeze-drying.
  • (OD600=5.3, 1.3, 0.6, 0.1)


  • 4. Place the plate at room temperature for 16 hours with different OD600 value.
  • (OD600=5.3, 1.3, 0.6, 0.1)


Discussion

After carrying out a series of experiments, we prove that bacteria will survive on the test paper after freeze-drying process. It would grow better if it is incubated after freezed drying. And one more thing to notice is that without adding glucose and glycerol, the survival of bacteria decreases drastically.

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