Sampling microbes in compressed gases

In a specially developed nebulising chamber, a spore-containing aerosol was generated under pressure. The bacterial spore count of the aerosol was determined by the membrane filtration method and by means of a microbiological air sampler for pressure gases (the MAS-100 CG developed by MBV, of Switzerland). Based on three different test series, it could be proven that both test methods yielded statistically significant, reproducible results. By applying the impaction method a collection efficiency of 92% could be achieved with the MAS-100 CG compared with membrane filtration.

Identical conditions

Taking ISO standard 14698-11 into account, the test method using a MAS-100 CG and the membrane-filter method were compared in a given pressure range. Both methods were applied under identical test conditions. Compressed air was used as a pressure gas. Since the aforementioned ISO standard1 Annex B (Guidance on validating air samplers) contains no instructions regarding the generation of aerosols in the pressure range concerned, this methodology had to be specially developed (the spinning-top or spinning-disc aerosol generator cannot be used in this pressure range).

By using an ultrasonic nebulising chamber, the spore suspension of Bacillus atrophaeus given in the ISO standard1 could be nebulised and then the microbial count determined by membrane filtration or by the MAS-100 CG.

The MAS-100 CG Ex is an air-sampling system, which is based on the known impaction principle of the MAS-100.3 The micro-organisms are directly impacted on a 90mm standard Petri dish filled with 16-18mL of culture medium. This air sampler was specially developed for the microbiological monitoring of pressure gases. The micro-organisms are collected under working pressure without risking a sublethal damage by the subsequent decompression. The suction volume of 100 litres/min is electronically controlled over a pressure range of 1.6 to 10 bar absolute pressure.

Defined pressure range

Contrary to other systems, sampling is always performed in the defined pressure range. The instrument is delivered ex-works as an “ex-proof” version and thus is suitable for use in explosion-proof zones. The MAS-100 CG is calibrated for air, nitrogen, argon and carbon dioxide, but it can also be calibrated for other gases.

The equipment used included: a MAS-100 CG Ex with sieve plate, 300 x 0.6mm holes, made of aluminum (CG); membrane filter of mixed cellulose esters, 0.45µm pore size, sterilised (MF); air compressor Atlas-Copco, prefilter (type DD32) and afterfilter (type PD32), for separating oil, condensate and soil particles; ultrasonic nebulising chamber, capacity approx. 500mL, made of clear polycarbonate; Tryptic soy agar – Petri dishes, BioMérieux (TSA); aqueous, concentrated suspension of Bacillus atrophaeus ATCC 9372 (former Bac. subtilis v. niger), from Raven Biological Laboratories; and ethanol (80%).

The bacterial suspension was prepared according to ISO standard 14698-1;1 Annex B; B 2.2.1 ff., so as to obtain a concentration of 1.0–1.5 x 104 cfu/mL in 80% ethanol. To obtain this concentration, the required volume of bacterial stock suspension was diluted with 80 % ethanol.

The two methods are illustrated in Figures 1 and 2. Figure 1 shows the experimental set-up of the MAS-100 CG and Figure 2 the experimental set-up for the membrane filtration.

Nebulising chamber

Once set up, 100mL of the bacterial suspension (106 spores) were transferred into the nebulising chamber and a (absolute) pressure of 2.6 bar was applied to the whole system. For a period of 30 seconds a spore-containing aerosol was generated by means of ultrasonics. Subsequently this – visible – mist was either aspirated through a membrane filter, using a (dynamic) pressure of 1.6 bar, and placed on a Petri dish filled with TSA, or directly impacted onto a TSA Petri dish. (For technical reasons, the two test procedures could not be performed in parallel). The agar plates were then incubated at 30°C for two days.

With the filtration method, the aerosol was sucked off during one minute with an air flow rate of 10 litres/min, which corresponds to the 20-fold volume of the nebulising chamber. In order to attain a similarly long rinsing period of the system, a test-gas volume of 250 litres was selected, which, under a dynamic pressure of 1.6 bar, corresponds to a collection period of approximately 1.5 mins.

Efficiency comparison

With both procedures a series of 10 individual samples was tested on each of three different days. The microbial recovery attained with the MAS-CG was expressed according to the ISO standard as a percentage of the membrane-filtration recovery. Additionally, the differences between the two procedures were checked statistically using the paired test.

The results of the colony count/plate from 10 individual samples are shown in table 1.

Interpreted according to ISO standard 14698-1[1]; Annex B.3 “Interpretation of results”, the results in table 2 show the collection efficiency of the MAS-100 CG for particles of approximately 1µm size (mean value from 10 samples).

In comparison with the membrane-filter method, the MAS-100 CG has an average efficiency of 92%. According to the test there is no significant difference between the microbial counts determined by the two procedures.

The tests have shown that by using ultrasonic nebulisation a spore-containing aerosol could be generated under pressure and the microbial count of this aerosol could be determined using the MAS-100 CG. Membrane filtration served as the method of reference. On three different days a series of 10 individual samples was examined using each of the two methods and the results were statistically evaluated. All three test series yielded reproducible, corresponding results that could be statistically proven by the t test. The average efficiency of the MAS-100 CG for approx. 1µm particles is 92%. Since the cut-off value calculated4 amounts to 1.12 µm, this means that practically all particles of larger diameter are separated by the MAS-100 CG.

As regards the determination of the separated particles, the ISO standard specifies the use of five different concentrations of potassium iodide solution, which should yield particle sizes of 0.8-15 µm. As the separation rate for Bacillus atrophaeus spores – with a particle size of approx. 1µm – attained with the MAS-100 CG was about the same as with the membrane-filter method, a detection of the larger particles is not necessary.

Based on the present, reproducible results, the MAS-100 CG can enable the quantitative detection of microbe-carrying particles (approx. 1µm in size) under pressure conditions.