Detection of Micro-organisms in Compressed Gases

Transcription

1 Detection of Micro-organisms in Compressed Gases H. Zingre 1, R. Meier 2, Abstract MBV AG, Stäfa, Switzerland The International Standard ISO /2 1 demands the monitoring of bio-contamination in compressed gas systems which are used in pharmaceutical production processes. The norm describes the detection of biocontamination by sampling and enumerating viable colony forming units with appropriate methods. Two main questions were investigated: 1. Do micro-organisms survive in compressed gas systems? 2. If they survive, what appropriate method has to be applied in order to collect and enumerate them? 13 different medical relevant species of micro-organisms where grown and put under pressure at 10 and 170 bar. Their and behaviour under pressure and the effect of decompression was analysed. Based on the (Microbiological Air Sampler for Compressed Gas), this presentation shows that micro-organisms can be collected and grown from compressed gases and elucidates the possible pitfalls which have to be avoided in the count of colony forming units from gases under pressure. Key words: Microorganisms under pressure, detection of micro-organisms in compressed gases. 1. Do micro-organisms survive in compressed gas systems? 1.1 Introduction Compressed gases are commonly used in the pharmaceutical and the food industry for various purposes. In the food industry some gases are used to prevent products from being oxidised after the production process and to increase their shelf live. In the pharmaceutical production, compressed gases, such as compressed air, nitrogen and carbon dioxide, are used during manufacturing and filling of non-sterile and sterile active pharmaceutical ingredients and products. To answer the first questions about the survival of micro-organisms in pressurised systems, 13 relevant micro-organisms were chosen and cultivated under different pressures. 1.2 Method to cultivate and test microorganisms under pressure The test organisms were cultivated on Tryptone Soy Agar (bacteria) or Sabouraud Dextrose agar (fungi) respectively. The micro-organisms were suspended in NaCl solution and a serial dilution was prepared. 20 µl of the suspensions where inoculated into 1.5 ml of TSB (Tryptic Soy Broth) in sterile ampoules. The same quantity of sample was used for positive and negative s under normal atmospheric conditions. The ampoules where transferred into a stainless steel cylinder and pressurised with compressed air to 10 bar and 170 bar respectively. The incubation of the steel cylinders under pressure took place for 4 days at C. To avoid any damage of the organisms during the decompression phase of the gas cylinder, the time of decompression was chosen to be very slow (2 h in the case of 170 bar).

2 1.2.1 Table of tested organisms Tests at 10 bar No. Test organisms Class Microbial count Positive Negative at 10 bar at 1 bar A Bacillus atrophaeus spores ATTCC 9327 (1) n.t.* B Aspergillus niger spores ATCC C Micrococcus luteus BKL 564 (2) n.t. D Escherichia coli ATCC n.t. E Enterobacter cloacae BKL n.t. F Streptococcus faecalis ATCC n.t. G Klebsiella pneumoniae BKL n.t. H Salmonella newport BKL n.t. I Yersinia enterocolitica BKL n.t. K Proteus morganii BKL n.t. L Staphylococcus aureus BKL n.t. M Serratia marcescens ATCC n.t. N Saccharomyces cerevisiae DSM 426 (3) *n.t. test not necessary because of existing at 10 bar Tests at 170 bar No. Test organisms Class Microbial count Positive Negative at 170 bar A Bacillus atrophaeus spores ATTCC 9327 (1) B Aspergillus niger spores ATCC C Micrococcus luteus BKL 564 (2) D Escherichia coli ATCC E Enterobacter cloacae BKL F Streptococcus faecalis ATCC G Klebsiella pneumoniae BKL H Salmonella newport BKL I Yersinia enterocolitica BKL K Proteus morganii BKL L Staphylococcus aureus BKL M Serratia marcescens ATCC N Saccharomyces cerevisiae DSM 426 (3) (1) American Type culture Collection (2) Biological Control-Lab, Novartis Pharma AG Test performed by R. Meier, Microbiological Consultant (3) German Collection of Microorganisms at 1 bar

3 1.3 Interpretation of results The tests at 10 bar showed that 11 out of 13 microorganisms grew under 10 bar pressure and that the Aspergillus niger spores as well as Saccharomyces cerevisiae (yeast) grew again after incubation at atmospheric pressure. Observations from previous tests have provided evidence of this theory. The tests at 170 bar showed that all microorganisms could neither grow under this high pressure nor after recovery at atmospheric pressure. 1.4 Conclusions Most micro-organisms do survive and multiply in pressurised systems up to 10 bar and some are at least able to recover after being pressurised. 2. If micro-organisms survive in pressurised systems, what appropriate method has to be applied in order to collect and enumerate them? 2.1 Introduction Several methods are known to collect microorganisms from pressurised gas distribution systems. The filtration method combined with a rota meter and a stop watch to measure the collected volume is one of them. The impinger is another method where the gas is expanded and the gas flow directed into a bottle (impinger) which is filled with a broth. Other systems consists of a kind of an inverted funnel (see picture Fig1) where the gas is expanded into the funnel and aspirated to a microbiological air sampler. Fig1: Diffusor funnel (expansion takes place before collection, risk to damage micro-organisms) The is a fully automatic system which passes the compressed gas under pressure onto an agar Petri dish (impaction principle) and expands after the collection process (see picture Fig 2). The release of pressurised gas into atmospheric conditions leads to an explosive expansion of the compressed gas within a fraction of a second. (Example: 10 Liter at 10 bars will result in 100 liters at 1 bar; this signifies that the volume will increase by a factor of 10). Due to the fact that the micro-organisms are transported under pressure and then suddenly released into atmospheric conditions, they may be damaged by the immediate expansion of the gas and the resulting shearing forces. Fig2: Sampling head (expansion takes place after collection. The sampling head is used for a pressure of up to 10 bar.

4 2.2 Test methods Microbial behaviour during decompression 20 µl of the suspensions used in the previous tests were inoculated onto 2 TSA and 2 SDA agar Petridishes and spread by means of a spatula. One TSA and one SDA Petri-dish was used for the positive whereas the other plate was put under a pressure of 10 bar for one hour, then decompressed in 5 seconds and incubated at 30 C for 3 days. (The decompression time corresponded to the default decompression cycle in the ). The average microbial yield of the decompressed samples amounted to 94% of the yield of the plates (paired t-test= 1.41, 2p 0.05= 2.05) These tests showed that the decompression cycle was not influencing the viability of the microorganisms tested Collection of Micro-organisms with the Fig3: The is an instrument based on the impaction principle and was built with the experience of the MAS-100 Microbiological Air Sampler. It is designed to collect micro-organisms in pressurised gases. The major advantage of this instrument is that the sample is taken under pressure and the automatic decompression cycle after the sampling will avoid damages of the collected microorganisms. The system is pre-programmed for most gases used in the pharmaceutical and food industries: compressed air, nitrogen, carbon dioxide, argon, and oxygen. It may be used in explosion proof areas Comparison between and filtration Fig4: Test set up: 1: nebulizer, 2 Agar petri dish, 3 Sampling head, The system was set up as shown in figure 4. It was tested according to ISO with a suspension of Bacillus atrophaeus (ATCC 9372 Raven Biological Laboratory Inc.) in 80% ethanol. The nebulizer (1) was filled with 100 ml of test spore suspension (10 6 ) and a test pressure of 2.6 bar was applied. 250 liters of the spore containing aerosol was then automatically aspirated with the sampler onto a TSA Agar Petri Dish and incubated for 2 days at C. The same procedure was accomplished with a 0.45 µm membrane filter in an appropriate filter holder. Test series of 3 times 10 samples were performed with both the impaction and the filtration method. As required by the ISO the microbial yield of the impaction method was calculated in per cent of the yield of the membrane filtration. In addition a statistical evaluation, using paired t-test, was performed to evaluate the significance of the variations of the two methods.

5 Test Series of 3 x 10 samples with the impaction principle and filtration (under pressure) Test 1 Test 2 Test 3 MF MF MF r Pr r Pr r Pr m= 20.8 m= 18.3 m= 20.5 m= 19.6 m= 23.6 m= 21.4 t= 1.05 t= 0.32 t= 1.06 MF= Membrane Filter r= CFU Pr= corrected CFU (according Feller 2 ) t= paired t-test Summary of the collection Efficiency (Performance) of the No. Test 1 Test 2 Test 3 Membrane Filtraion (MF) m= 20.8 m=20.5 m= 23.6 m= 18.3 m=19.6 m=21.4 Performance 88% 96% 91% 2.3 Interpretation of results and conclusions Compared to the membrane filtration method, the has an average efficiency of 92%. The result of the paired t-test shows no significant variation between the two methods. The is an automatic instrument which is very easy to use and which shows a high performance. The collection of micro-organisms under pressure and the decompression after the collection cycle with this method show a high and reliable recovery of the micro-organisms. The author recommends to use air monitoring systems for pressurised gases which work under pressure during the collection cycle. Systems collecting micro-organisms after decompression should not be used due to probable damage of the organisms during expansion. It is very important to know the impaction speed of the micro-organisms (speed of the micro- organisms hitting the agar surface) during the sampling cycle. The has a constant impaction speed of 19.6 m/sec. It is of utmost importance to keep this speed constant to have an uniform and reliable sampling performance over the whole sampling period. To overcome difficulties as a consequence of big pressure variations in gas distribution systems, the has an integrated mass flow meter which maintains and guarantees a constant flow and as a result of this a constant impaction speed. 1 ISO ISO Cleanrooms and associated led environment Biocontamination -Part 1: General principles and methods (first edition ) 2 W.Feller, An introduction to the probability theory and its applications, Vol.1, p.175. John Wiley & Sons, Inc., New York,1950