N.18 – Compressed air microbiological monitoring

TRIO

The present Application Note intends to be a simple Guide Line for the operators involved in the compressed air monitoring applying the ISO 8573-7.

The ISO 8573 consists of nine different parts:

1. Contaminants and purity classes. 2. Test methods for aerosol oil content. 3. Test methods for measurement of humidity. 4. Test methods for solid particle count. 5. Test methods for oil vapour and organic solvent content. 6. Test methods for gaseous contaminant content. 7. Test method for viable microbiological contaminant content. 8. Test methods for solid particle content by mass concentration. 9. Test methods for liquid water content.

The ISO 8573-7 has the following summary:

1. Scope. 2. Normative references. 3. Terms and definitions. 4. Method for verifying presence of viable micro-organisms by partial flow sampling. 5. Operating conditions. 6. Determination of viable, colony forming organisms. 7. Test report statement. Annex A ”Sample test report”, Annex B “Quantitative sampling method”, Annex C “Sampling endotoxins”. Annex D ”Preparation of Petri dish with culturable media. Bibliography.

Glossary The following terms are defined under the Chapter 3 Terms and definitions:

CFU, colony forming unit, culturable number, microbiological organisms, number of viable micro-organisms.

Scope

The ISO 8573-7 was prepared by the Technical Committee ISO/TC 118 Compressor, pneumatic tools and pneumatic machines, subcommittee SC4, Quality of compressed air and consists of the following parts:

Part 1: Contaminants and purity class

Part 2: Test methods for aerosol oil content

Part 3: Test methods for measurement of humidity

Part 4: Test methods for solid particle content

Part 5: Test methods for oil vapour and organic solvent content

Part 6: Test methods for gaseous contaminant content

Part 7: Test method for viable microbiological contaminant content

Part 8: Test methods for solid particle content by mass concentration

Part 9: Test methods for liquid water content. The ISO 8573-Part 7 specifies how to perform a method for evaluate viable, colony-forming units organisms which may be present in compressed air and provides a means of sampling, incubating and determining the number of microbiological particles.

Principle

The method for verifying presence of viable micro-organisms by partial flow sampling is to expose an agar nutrient to the compressed air sample. A type of impaction air tester shall be used operating with isokinetic sampling. The test should be performed with gas at atmospheric pressure conditions. Air from a compressed air installation is channelled through a specially designed connecting link and accelerated towards a moist agar surface. The micro-organisms, due to their weight, are flung into the agar surface, whereas the air molecules are deflected. Suitably incubated, they multiply into colonies, which are counted on the assumption that one micro-organism gives rise to one colony.

The measurements shall be taken within 4 hours if the test intends to discriminate non-microbiological from microbiological particles.

At the end of incubation time, the surface of the nutrient agar shall be visually examined to confirm the presence of viable, colony forming micro-organisms

Material

EQUIPMENT

– Microbiological air sampler

– Compressed air tester

– Incubator

DISPOSABLES

– Standard 90 mm disposable Petri dishes or Contact Plate  with PCA medium

– Standard 90 mm disposable Petri dishes or Contact Plate with SDA medium

– Sterile plastic bags

– Spray and liquid 70% ethanol disinfectant

Petri dish preparation

The described procedure should be applied for culturable media like Plate Count Agar (PCA) for total bacterial count and Sabouraud Dextrose Agar (SDA) for fungi.

(a) Weight out the culturable media in the quantity specified by the producer and dissolve it in water.

(b) Sterilize the media by autoclaving at 121°C for 15 minutes.

(c) After cooling a 50°C, measure the pH and, if necessary, according to the producer instructions, adjust the pH by HCl or NaOH.

(d) Transfer the medium in each Petri dish. The volume of the media should be suitable to guarantee a correct impact of the air on the agar surface (follow the manufacturer indications reported in the Instruction Manual of the air sampler).

(e) When the culturable medium is cool and stiff, pack each Petri dish in two sterile plastic bags. Close the first bag with a simple double fold-over seal. Seal the second bag positively with a welded edge.

(f) Label the dish with information about date, content and batch number.

Sampling Protocol

The sampling methodology involves the adoption of aseptic techniques. The use of a disinfecting agent such as 70% ethanol is recommended. When the air sampler is not in use, precaution should be taken to avoid the growth of micro-organisms in the equipment. All operation in which the test equipment is to be opened should be carried out with the minimum of delay in order to avoid possible ingress of contaminants from the environment. Precautions should also be taken against possible effects of draughts.

(1) The complete sampling unit is sterilized by a suitable disinfecting agent immediately before use.

(2) Allow a test sample to pass through the sampling equipment and associated tubes and hoses without the Petri dish and agar. This operation determines the evaporation of the disinfecting agent and adjusts the air sampler.

(3) Perform a blind test before and after the actual measurement inserting a Petri dish in the air sampler without starting it. The Petri dish should then be incubated together with the other Petri dishes. It should not subsequently show growth to demonstrate that it was sterile.

(4) Insert a Petri dish with the suitable media (PCA for bacteria; SDA for fungi) into the air sampler. The Petri dish shall have a label fixed to the bottom with traceability information (date, time of start, test site address, code, etc.). Remove the Petri dish lid and store it in a sterile plastic bag.

(5) Connect quickly the aspirating head to the sampler immediately after removing the Petri dish lid.

(6) Start the sampling. Note the starting time, sampling time, test location and the observation that could influence the measurement.

(7) At the end of sampling time (e.g.: 1000 litres of air = 1 cubic metre of air), unscrew the head of the sampler, apply the plastic lid to the Petri dish and remove the Petri dish from the sampler. Seal the Petri dish with tape and transfer it to the incubator.

(8) Incubate the Petri dish at a convenient temperature and time. In general, the most appropriate temperature during incubation is that near to the habitat in which the micro-organisms were present before sampling. Mesophilic bacteria or fungi should be cultivated at temperature between 20°C and 30°C. For specific thermo-tolerant bacteria other temperatures may be requested. Incubation period of up to fourteen days are normal for fungi, while those for mesophylic bacteria normally vary from 2 to 14 days. Other incubation temperatures may be considered. Selective media may be used for isolation of, for example, gram-negative enterobacteria and the counting shall take place within a given time period.

(9) At the end of the incubation time, count the CFU on the Petri dish. Non-selective media may be examined and the growth counted as early as 24 hours after the beginning of incubation and then recounted every 24 hours for 10-14 days. Regular observations shall be made during the incubation period to count and record colonies as they emerge and to prevent loss of counting accuracy by overgrowth of colonies.

Bibliography

ISO 8573-7:2003 Compressed air – Part 7: Test method for viable microbiological contaminant content

ISO7954-Microbiology – General guidance for enumeration of yeast and moulds – Colony counting technique at 25°C.