Many studies on gas plasma sterilization and disinfection conducted so far by physical researchers contain serious mistakes to be corrected. These points were quite important to consider about sterilization.
First, it was not seriously considered whether biological indicator (BI) have clumps or not. Clumps are the phenomenon whereby spores or microorganisms overlap (Figure 1) [1
]. As can be seen in Figure 2, BI must be completely free from overlapping (clumps) to attain reproducible sterility assurance. Clumping may cause tailing in survival curves (Figure 3).
Tailing means survival curve does not present straight line from initial population to fraction negative portion of SLR (spore log reduction) of 5 to 10-2
]. Self-prepared BIs or commercially available Bls have clumps in most cases (SEM data are skipped). BI from Bacillus atrophaeus
ATCC 9372 was used for ethylene gas exposure sterilization and fry heating sterilization, while Geobacillus stearothermophilus
ATCC 7953 was used for autoclaving and formaldehyde sterilization (ISO 11138-1) and gas plasma sterilization, even though gas plasma is not defined as BI officially, but it considers the most tolerance towards gas plasma exposure.
These conventional sterilization procedures have sufficient depth of penetration, therefore tailing phenomena were rarely observed even if the BIs had clumps. However, gas plasma from several sorts of gases has a quite shallow depth of penetration. The depth of penetration of gas plasma was around 10-40 nm.
As one spore is around 1x3 µm (1000×3000 nm), the penetration depth by gas plasma was around one layer of spores. If multiple layers of spores on the carrier material exist as shown in Figure 1 [1
], the interior layers of the clumps will be protected by the inactivated outer layer [3
]. Thus, the apparent D (decimal reduction value) was, as a whole, greater than the exact D value. In addition, the tailing phenomenon in which the slope was not straight was observed (Figure 3A) [2
]. If tailing phenomenon can be observed, a sterility assurance level (SAL) of 10-6
required for health care products can be hard to achieve. The simultaneous achievement of SAL of 10-6
and material and functional compatibility was also overlooked in plasma sterilization papers published so far.
In order to avoid clumping in the BI, spores must be distributed evenly on the carrier material (Figure 2). If the hydrophobicity and hydrophilicity of spores differ (Table 1), so the characteristics of carrier materials must coincide with those of spores. Furthermore, as bioburden (bioburden means viable microorganisms on/in products) was around a few CFU (colony forming unit), so observations of clumps in bioburden could be quite rare in exact status. As an initial population of 106
CFU/carrier was so often required in validation study, but from the exact status the requirement of 106
CFU/carrier can be confirmed quite large number, so less than 106
CFU as an initial population was quite enough to evaluate gas plasma sterilization procedure. For another requirement of 106
CFU BI, an absolute bioburden method or combined BI/bioburden method can be presented in ISO 14161 and sterilization validation [4
]. As BI of gas plasma sterilization, G. stearothermophilus
ATCC 7953 must be utilized because this spore is the most tolerable to gas plasma sterilization. BI defined as the most tolerable spore towards a definite sterilization (ISO 11138-1). BI is inoculated onto a carrier material such as a polyhydroxymethylmethacrylate (PHMMA), partially modified PS (polystylene) or surface modified SUS to coincide Aw of surface and that of the spore in Table 1.
The hydrophobicity and/or hydrophilicity of PHMMA or partially modified PS or SUS are around 50/50, coinciding with hydrophobicity of G. stearothermophilus
in Table 1.
Another way to prepare a monolayer of Bl free from clumps resembles the preparation of DNA tips. This procedure atomizes the spores onto the carrier material instead of the drop procedure, conventionally used for BI preparation. DNA tips procedure is not easily applicable to BIs used for gas plasma sterilization for both economic and technical reasons. In case of using drop procedure, the correct carrier material selection and careful dropping procedures are required to avoid clumping. DNA tipping procedure is completely free from clumps, but quite expensive and time consuming.
ATCC 7953 does not show tailing in traditional sterilization procedures because they have no repair enzymes [5
]. In the case of Deinococcus radiodurans
(Figures 4,5), which tolerated the radiation procedure, a straight survivor curve was not attained due to the presence of repair enzymes [6
]. Significant shoulder can be observed (Figure 5) [6
Readers must keep in mind that only a limited number of bacterial spores present tailing phenomenon or shouldered survival curves [7
]. As far as the author is concerned, almost all papers published so far on gas plasma sterilization presented non-straight survival curves; this is quite exceptional in the real status.
The reason of the non-straight survivor curve is due to clumping. You can imagine how it is too much difficult task to inoculate 106
CFU in the circle of 2 mm diameter without clumping. As mentioned above, most of physical researchers cannot understand the importance avoiding clumps in BIs to attain straight survivor curve when conducting gas plasma sterilization, mostly due to quite shallow penetration depth (10 nm) of radicals and other sterilization factors from gas plasma. Up to date, no correct sterilization factors of gas plasma are defined.