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Safe High Pressure Freezing of Infectious Microorganisms

Safe sample preparation with Leica EM PACT2

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Especially in core EM facilities one is confronted with material (microorganisms and cells) which are infectious. It is a must to follow the safety rules according to the National regulations. However even when safe laboratories are available it is very convenient to know that the applied high pressure freezing system is in itself safe.

The Leica EM PACT2

By applying 3 bar on a piston with diameter 8 cm one generates a force of about 150 kp. This force transduced to a piston with a diameter of 3 mm generates a pressure of about 2,000 bar.

Fig. 1: The principle of high pressure freezing

The platelets

The membrane platelet for the Leica EM PACT2 is the only available system which can be safely sealed, because the sample is sealed by defined surfaces once introduced into the pod. This is not the case for all other platelet systems of the available high pressure freezing systems.

Fig. 2: The membrane platelet for Leica EM PACT2 (courtesy of Studer D, University of Bern, Anatomy)

Control experiment

Test to check if the microorganisms are alive and healthy. In Figure 3 the corresponding growth curve is shown. Streptococcus pneumoniae was cultured and centrifuged. 0.5 µl of the pellet was aspired and

  • (positive control) pipetted into 10 ml medium
  • (negative control) 0.5 µl sterile medium was pipetted into 10 ml medium.

Result

The bacteria are healthy (Figure 3, red line) and the medium sterile (Figure 3, grey line).

Fig. 3: Positive (0.5 µl S. pneumoniae) and negative control (0.5 µl medium) in 10 ml medium.

Experiment 1

Filling the membrane platelet with a pellet of the sample. Bacteria (Streptococcus pneumoniae) were cultured and centrifuged. 0.5 µl of the pellet was aspired and filled into the membrane platelet (∼0.35 µl), subsequently the platelet was fixed in a pod.

Fig. 4: Correct filling of the sample

The platelet has to be correctly filled. Overfilling as well as putting not a sufficient amount of sample into the platelet leads to heavy defomation of the platelet during high pressure freezing (for more information see [1].

Fig. 5: Correct filling of the sample

Sterilization of the platelet fixed in the pot

Once the platelet is mounted in the pod it is disinfected in Gigasept and then rinsed in bidistilled water.

Fig. 6a: Gigasept, Sigma.
Fig. 6b: Bidistilled water

Testing if bacteria are surviving he encapsulation into the pod (Figure 7, turquoise line) and if the surface of the pod, the manipulator and the platelet are sterile (Figure 7, green line).

Fig. 7: Growth curve after loading: Platelet filled with S. pneumoniae fixed in pod and incubated 5 min. in GigaSept and thereafter washed with bidistilled water. Dark green: Content of platelet in 10 ml medium; light green: Pod (incl. platelet) after washing in 10 ml medium.

Result

The turquoise curve proves that the conent of the platelet is unaffected. The microorganisms are alive in the pod. The green curve shows that there are no living bacteria at the pod surface after disinfection.

Experiment 2

Checking influence of pressure to viability of bacteria (Figure 9, red line) and if the platelet sealed in the pod is leaky (Figure 9, orange line).

Same procedure as described in Figures 4–6, but before checking outside and content of the platelet in the pod the samples were pressurized without freezing.

Fig. 8: Pressurization of the pod after loading: The sample platelet filled with S. pneumoniae fixed in pod and incubated 5 min. in GigaSept and thereafter washed with bidistilled water. This sample is pressurized to 2,000 bar (red line), however, not cooled (blue line indicates room temperature).

Fig. 9: Growth curve after pressurization: Platelet filled with S. pneumoniae fixed in pod and incubated 5 min. in GigaSept and thereafter washed with bidistilled water and pressurised. Red: Content of platelet in 10 ml medium; orange: pod (incl. platelet) after washing in 10 ml medium.

Result

The orange line (Figure 9) shows that during pressurization the pod remains sealed (no growth of bacteria), however, the bacteria in the containment survive to be a quite high degree the pressurization (red line in Figure 9).

Experiment 3

Checking influence of high pressure freezing to viability of bacteria (Figure 11, dark blue line) and if the platelet sealed in the pod is leaky after pressurization and cooling (Figure 11, light blue line).

Same procedure as described in Figures 4–6, but before checking bacterial growth the sample was high pressure frozen.

Fig. 10: Typical pressure/temperature diagram of HPF machines.
Fig. 11: Growth curve after high pressure freezing: Platelet filled with S. pneumoniae fixed in pod and incubated 5 min. in GigaSept and thereafter washed with distilled water and high pressure frozen. Dark blue: Content of platelet in 10 ml medium; light blue: pod (incl. platelet) after washing in 10 ml medium.

Result

The light blue line shows that the sample is sealed. No bacteria are on the closed pod after a high pressure freezing cycle. However (dark blue line) surprisingly part of the content is still living. That means that bacteria can survive high pressure freezing and subsequent thawing!!!

Conclusion

Fig. 12: All growth curves put together: Conclusion of the diverse experiments described on the precedent pages.
  • Streptococcus pneumoniae survives high pressure freezing (dangerous when a sample holder leaks).
  • The content of a well prepared membrane platelet fixed in the pod of the Leica EM PACT2 does not leak when pressurised or high pressure frozen.
  • Careful preparation is a prerequisite for safe high pressure freezing.

Last but not least:

  • The authors cannot be held responsible in the event of sample leakage.

References

  1. Vanhecke D, Zuber B, Brugger SD and Studer D: Safe high-pressure freezing of infectious micro-organisms, Journal of Microscopy 246 (2): 124–128 (2012); doi: 10.1111/j.1365-2818.2011.03598.x.

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