Mycobacterium tuberculosis (M.tuberculosis) vs Lactobacillus Casei (L.Casei)

M.tuberculosis is a BSL-3 (Figure 1) airborne pathogen as there is a high risk of being infected, potentially resulting in serious repercussions. In fact, the chances of being infected by M.tuberculosis are 'three to nine times higher' compared to handling other clinical specimens[1].

Figure 1; Definitions of various Biosafety Levels[2]

Therefore, we used Lactobacillus casei (BSL-1) instead of M. tuberculosis in the laboratory, as it is safe to use and has similar features to the M. tuberculosis bacterium such as the gram-positive cell wall.[3] L. casei is a suitable substitute for M. tuberculosis as it is gram-positive, much safer to use and will require the same conditions for thermal lysis.

Using L. casei

Our intended use for L. casei is to conduct tests to show and record the successful lysis of its cell wall using heat, and to use microscopy to confirm the successful lysis of the cell.

How?

Equipment

  • Bunsen burner

  • 12 jars of MRS Broth

  • Sterile test tubes

  • L. casei

  • Incubator

Protocol

  1. Flame the neck of the jar containing MRS broth and transfer to sterile test tubes

  2. Prepare inoculating loops for transfer of bacteria to broth jar

  3. Transfer L. casei culture from jar for isolation in MRS Broth

  4. Replace caps, but do not screw on tightly

  5. Incubate at 25°C for 3 days.

Prolonged heating of L.Casei at 65 C promotes lysis of bacteria

The baseline image at room temperature, taken before heating, shows the presence of only single cells. After heating the sample for 20 minutes, chains of L.Casei can be seen to have formed. This effect of increased chain formation and growth has been previously reported at high temperatures4. However, by 80 mins of incubation, change in morphology with disruption of the chain formation and increased debris indicates increased bacterial lysis.

Methodology

  1. Heat water bath to 65 C

  2. Remove L. casei jars from incubator

  3. Gram-stain sample from jar to create a negative control slide (see gram-staining methodology below)

  4. Transfer vials of L. casei into water bath

  5. Let vials sit for 20 mins, then remove and take a swab to procure a slide for gram-staining

  6. Use swab to transfer bacteria in vial to slide

  7. Repeat each time for periods at 40 mins, 60 mins and 80 mins

  8. Inspect slides for changes

Gram-staining methodology

  1. Dip swab into bacterial culture then smear thoroughly onto microscope slide

  2. Heat dry the slide by passing over Bunsen flame using forceps-this will also heat-fix the bacterial smear to the slide

  3. Wait for slide to cool down

  4. Switch off Bunsen flame

  5. Suspend slide over sink using slide racks then flood slide with crystal violet solution

  6. Wait for 1 min

  7. Pour off excess stain, then wash with bottle of deionised water

  8. Flood slide with Gram’s iodine solution and wait for 1 min

  9. Tilt slide using forceps, then wash down with a few drops of ethanol until run-off is clear

  10. Wash with deionised water

  11. Pour off water, then flood slide with Safranin counterstain, leave for 15 seconds

  12. Wash away with deionised water and wait for slide to dry

  13. Place slide under microscope for inspection

RPA and Lateral Flow

RPA

Part of our initial plan was to test RPA alongside lysis to prove that our selectively designed primers are correctly optimised, however, due to the renovations in some of our labs and delay in fundraising, we were unable to carry out the RPA in time for the Wiki freeze. Our successful fundraising has permitted us to carry out the testing of this component of our project, so we have opted to go ahead with the wet lab to obtain results before the Jamboree in Paris.

LFA Output

Due to the aforementioned issues encountered by our team, we were unable to access the laboratories to test the final stage of our system. The results below have been provided by similar research conducted by the Wyss Institute. They too, used the Cas12 enzyme in conjunction with lateral flow assay technology - detecting RPA amplicons using gRNA/Cas complexes, resulting in the collateral cleavage of FAM-Biotin probes.5

Using gRNAs modelled by Benchling, we are confident in our ability to procure successful results.

The figures below show the sensitivity of the LFA sensor.

References

[1] Philippe Herman, M. F.-D. B. B. V. V. K. P. C. D. D. T. M. S. M. W. R. S. &. W. M., 2006. Biosafety Recommendations for the Contained Use of Mycobacterium tuberculosis Complex Isolates in Industrialized Countries.. [Online]

Available at: https://www.biosafety.be/sites/default/files/mtub_final_dl.pdf

[Accessed 23 July 2022]

[2] GenoFAB, n.d. AN OVERVIEW OF BIOSAFETY LEVELS. [Online]

Available at: https://blog.genofab.com/overview-of-biosafety-levels

[Accessed 23 July 2022]

[3]  Hill, D., Sugrue, I., Tobin, C., Hill, C., Stanton, C. and Ross, R., 2018. The Lactobacillus casei Group: History and Health Related Applications. Frontiers in Microbiology, [online]

Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160870/#__ffn_sectitle

[Accessed 20 September 2022].

[4] Novitsky, T., Chan, M., Himes, R. and Akagi, J., 1974. Effect of Temperature on the Growth and Cell Wall Chemistry of a Facultative Thermophilic Bacillus. Journal of Bacteriology, 117(2), pp.858-865.

[5] Nguyen, P., Soenksen, L., Donghia, N., Angenent-Mari, N., de Puig, H., Huang, A., Lee, R., Slomovic, S., Galbersanini, T., Lansberry, G., Sallum, H., Zhao, E., Niemi, J. and Collins, J., 2021. Wearable materials with embedded synthetic biology sensors for biomolecule detection. Nature Biotechnology, 39(11), pp.1366-1374.