!"#$%&'(#)%"Indoor air quality and mould inspections are increasingly requested by owner occupiers, tenants, landlords, property managers and insurers. There is a strong need for a rapid and reliable testing method giving accurate measurements, Specieslevel data and at a competitive price point.

Indoor air quality (IAQ) investigations rely on different methods to quantify levels of the airborne biota (bacteria, yeasts or fungi) in different rooms/areas. It is important that each method can be compared against other methods and that there is consistent interpretation.

Inhalation of mould spores, hyphae and cell wall fragments is the dominant route of infection with dermal contact being reported far less frequently. Therefore, air sampling that addresses the airspace is preferred over streak/swab testing. Different air sampling methods are either (i) active or (ii) passive. Perhaps the best known active method is the Anderson-type sampler that takes a known volume of air across a stage and through a set of holes where air plus viable cells are deposited onto the agar surface of Petri plates. The other active testing method also uses a pump to take a known volume of air and deposit it onto a sticky microscope slide and then spores are counted and classified. Spore traps are a non-viable method.

Both methods rely on a numerical value that indexes air quality. Disadvantages are that both methods require the use of expensive pumps and specialised equipment.

Figure 1. (a) Bio pump for air-o-cell cassettes. Pump cost $AU1,350 with each cassette approx. $AU8.50ea. (b) Bio pump for petri plates with calibration kit for airflow cost $AU3,750.

The alternative method which is the subject of this paper is called the settling plate method. This method does not rely on any equipment and uses open Petri plates for pre-determined time periods in target locations. Other advantages of this method are that Petri plates are readily available in different nutrient formulations, so IAQ testing can be made selective for different objectives.

*+#,$)+-. +"& /,#0%&.This paper reviews the literature regarding the use of the settling plate method for performing IAQ investigations. One criticism of viable settling plates is that the unit of measurement, the colony forming unit (CFU per plate) is not readily comparable to the CFU per m3 of air criterion used by spore traps. This review summarises multiple examples of passive settling plates being used in healthcare and industry to index air quality by exploiting the Omeliansky formula on the CFU to convert the values into CFU per m3 following: N=5a x 104 (bt)-1 Where: N=CFU per m3, a=CFU counted per Petri plate, b=Petri plate surface area and t=exposure time in minutes. An Excel spreadsheet was developed. This method is used as part of the mail order DIY mould testing kits available from: mould.net.au

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3,4),5Table 1 reviews the PubMed literature between 1990 and 2016 for the terms: settling plate, sedimentation technique, Omelianskyformula and passive sampling.

Table 1 reveals that passive sampling is routinely used to provide quantitative data regarding air quality in industry, public-places and healthcare.

6%"(-'.)%".The Australian Mould Guideline (2010) defines a viable fungal hygiene guide for indoor surfaces (p.11) that is based on the measurement of CFU per plate. However, the original table was designed for use with 55mm press plates. Notably though, this scale appears equally valid for air samples using 90mm plates since there is excellent congruence seen between the active sampling at 28.3L/min for 2 min using the conversion (Nastov et al. 2003) from plate count to CFU/m3 following: plate count * 35.3146667210648/sample time and the Omeliansky formula for 45minutes using settling plates. This means that using Table 2, it is now possible to quantify airborne room hygiene using either active air samplers or passive settle plates.

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7)#,$+#'$,2()#,&A.A. Abdel Hameed, A.M. Ayesh, M. Abdel Razik Mohamed, and H.F. Abdel Mawla. “Fungi and some mycotoxins producing species in the air of soybean and cotton mills: a case study”. Atmospheric Pollution Research, Vol. 3, pp. 126=131, 2012.

A. Abdel Hameed and H. A. Mawla. “Sedimentation with the Omeliansky Formula as an Accepted Technique for Quantifying Airborne Fungi”. Pol. J. Environ. Stud. Vol. 21, pp. 1539-1541, 2012.

A.A. Abdel Hameed and T. Habeeballah. “Air Microbial Contamination at the Holy Mosque, Makkah, Saudi Arabia”. Current World Environment., Vol. 8, pp. 179-187, 2013.

M.A. Afshari, M. Riazipour, R. Kachuei , M. Teimoori and B. Einollahi. “A Qualitative and Quantitative Study Monitoring Airborne Fungal Flora in the Kidney Transplant Unit”. Nephro-Urology Monthly. Vol. 5, pp. 736-740, 2013.

A. Agodi, F. Auxilia, M. Barchitta, M.L. Cristina, D.D’Alessandro, I. Mura, M. Nobile, C. Pasquarella on behalf of the Italian Study Group of Hospital Hygiene (GISIO). “Operating theatre ventilation systems and microbial air contamination in total joint replacement surgery: results of the GISIO-ISChIA study”. Journal of Hospital Infection, Vol. 90, pp. 213-219, 2015.

H. Aydogdu, A. Asan and M.T. Otkun. “Indoor and outdoor airborne bacteria in child day-care centres in Edirne City (Turkey), seasonal distribution and influence of meteorological factors”. Environmental Monitoring and Assessment, Vol. 164, pp. 53-56, 2010.

L. Bârlean, L. S. Iancu, M.L. Minea, I. Danila and D. Baciu. “Airborne microbial contamination in dental practices on Iasi, Romania.” Oral Health and Dental Management. Vol. 9, pp. 16-20. 2010.

E. Bogomolova and I. Kirtsideli. “Airborne fungi in four stations of the St. Petersburg Underground railway system”. International Biodeterioration & Biodegradation, Vol. 63, pp. 156-160, 2009.

D. Borda, R. Nastase-Bucur, C. Borda and I. Gorban. “The Assessment of the Airborne Microorganismes in Subterranean Environment - Preliminary Data”. Bulletin UASVM, Veterinary Medicine Vol. 66, pp. 236-242, 2009.

S. Borrego, P. Lavin, I. Perdomo, S.G. de Saravia, and Patricia Guiamet. “Determination of Indoor Air Quality in Archives and Biodeterioration of the Documentary Heritage”. International Scholarly Research Network, ISRN Microbiology, Vol. 2012, pp. 1-10, 2012.

F.L. Bowling, D.S. Stickings, V. Edwards-Jones, D.G. Armstrong and A.J.M. Boulton. “Hydrodebridement of wounds: effectiveness in reducing wound bacterial contamination and potential for air bacterial contamination”. Journal of Foot and Ankle Research, Vol. 2, pp. 1-8, 2009.

H.N. Burr, F.R. Wolf and N.S. Lipman.“Corynebacterium bovis: Epizootiologic Features and Environmental Contamination in an Enzootically Infected Rodent Room”.Journal of the American Association for Laboratory Animal Science, Vol. 51, pp. 189–198, 2012.

M. Chadeganipour, S. Shadzi, S. Nilpour and G. Ahmadi. “Airborne fungi in Isfahan and evaluation of allergenic responses of their extracts in animal model”. JundishapurJournal of Microbiology. Vol. 3, pp. 155-160, 2010.

M. Chadeganipour, R. Ojaghi, H. Rafiei, M. Afshar and S. T. Hashemi. “Bio-deterioration of library materials: study of fungi threatening printed materials of libraries in Isfahan University of Medical Sciences in 2011”. Jundishapur Journal of Microbiology. Vol. 6, pp. 127-131, 2013.

C.P. Cornea, M. Ciuc!, C. Voaides, V. Gagiu and A. Pop. “Incidence of fungal contamination in a Romanian bakery: a molecular approach”. Romanian Biotechnological Letters,Vol. 16, pp. 5863-5871, 2011.

D. Diaconu, M. Tatarciuc, D. Tatarciuc and A. Vitalariu. “Quantitative Analysis of Bacterial Contamination In Dental Laboratory Air”. Romanian Journal of Oral Rehabilitation, Vol. 4, pp. 27-29, 2012.

S. Fekadu and B. Getachewu. “Microbiological assessment of indoor air of teaching hospital wards: a case of JimmaUniversity specialized hospital”. Ethiopian Journal of Health Sciences. Vol. 25, pp. 117-122, 2015.

B. Friberg, S. Friberg and L.G. Burman. “Correlation between surface and air counts of particles carrying aerobic bacteria in operating rooms with turbulent ventilation: an experimental study. Journal of Hospital Infection. Vol. 42, pp. 61-69, 1999.

M.L. Grbic, M. Stupar, J. Vukojevic, I. Maricic and N. Bungur. “Molds in museum environments: Biodeterioration pf art photographs and wooden sculptures.” Archives of Biological Science Belgrade, Vol. 65, pp. 955-962, 2013.

S.Fekadu Hayleeyesus and A.M. Manaye. “Microbiological quality of indoor air in university libraries.” Asian Pacific Journal of Tropical Biomedicine. Vol. 4, pp. S312-S317, 2014.

Ignatova-Ivanova Ts., D.Bachvarova, A.Doychinov, S.Etem, K.Jordanova, M.Dimitrova, R.Ivanov and Dobromir Enchev. “Study of Biofouling in Books stored at the Archive of the Library of Shumen University”. European Journal of Biology and Medical Science Research, Vol.3, pp. 17-23, Aug. 2015.

A. Kazemi, E. Ahmadpour, B. Naghili, A.Z. Mahmoudabadi, A Jafari, and A.M. Ayatollahi. “Airborne Fungi in Tabriz, Comparing Airborne and Clinical Samples of A. fumigatus (2011), Survey and Literature Review”. Jundishapur J Microbiol., Vol. 6, 2013.

P. Kemp and H. Neumeister-Kemp. Australian Mould Guideline. The Enviro Trust. 2nd ed., 2010.

V.J. Khojasteh, V. Edwards-Jones, C. Childs and H.A. Foster. “Prevalance of toxin producting strains of Staphylococcus aureus in a pediatric burns unit”. Burns, Vol.33, pp. 334-340, 2007.

C. Misca, I. David, C. Jianu, G. Bujanca, C.B. Misca, O-M. Marginean and L.C. Misca. “Evaluation of the microbiological contamination of air in a company designed to obtain wooden packaging and sticks used in the food industry”. Journal of Horticulture, Forestry and Biotechnology, Vol. 18, pp. 122-125, 2014.

C. Napoli, V. Marcotrigiano and M.T. Montagna. “Air sampling procedures to evaluate microbial contamination: a comparison between active and passive methods in operating theatres”. BMC Public Health, 12(594), pp. 1-6, 2012.

J. Nastov, R. Tan and P. Dingle. The Use of Fibre technology to Control Surface Dust and Bacteria Contamination. Environmental Science Report Series. 2003-1. School of Environmental Science, Murdoch University, WA.

C. Pasquarella, O. Pitzurra and A. Savino. “The index of microbial air contamination”. Journal of Hospital Infection, Vol. 46, pp. 241–256, 2000.

A.F. Sahab, N.M. Sidkey., N.N. Abed and A. Mounir. “Studies on Indoor Air Quality in the Repositories of the National Library and Archives of Egypt”. International Journal of Science and Research (IJSR), Vol. 3, pp. 2122-2128, Nov. 2014.

G. Setlhare, N. Malebo, K. Shale and R. Lues. ( 2014). “Identification of airborne microbiota in selected areas in a health-care setting in South Africa”. BMC Microbiology,14(100), pp. 1-10, 2014.

S. Shadzi, M. H. Zahraee and M. ChadeganiPour. “Incidence of airborne fungi in Isfahan, Iran”. Mycoses, Vol. 36, pp. 69-73, 1993.

A.P. Verhoeff, J.H. van Wijnen, J.S.M. Boleij, B. Brunekreef, E.S. van Reenen-Hoekstra and R.A. Samson. “Enumeration and identification of airborne viable mould propagules in houses”. Allergy. Vol. 45, pp. 275-284, 1990.

:'$#0,$ )";%$/+#)%"© Copyright Cameron L. JonesEmail: info@biologicalhealthservices.com.auRelevant website connected with this poster: mould.net.auTelephone: 1300 13 23 50Head Office: Level 1, 459 Toorak Rd, Toorak, VIC, 3142Laboratory: 7/4 Weddell Court, Laverton North, VIC, 3026

Review Of The Settling Plate Method For Indoor Air Quality And Mould Inspections

biologicalhealth biologicalhealthservices drcameronjonesdrcameronjones

Table 1. Key literature that used settling plates for analysis of airborne contamination. The purpose of each study along with exposure times and Petri plate nutrient medium formulation is given. List of Petri Plate Media: BA, Blood agar; BEA, Bile esculine azide agar; BHI, Brain heart infusion agar; CAB, Columbia agar base + sheep blood; CAN Columbia colistin-nalidixic-acid + 5% sheep blood; CBA, Columbia blood agar; CDA, Czapek’s dox agar; Col, Brilliance E. coli/Coliform agar; DG18, Dichloran 18% glycerol agar; DRBC, Dichloran Rose Bengal chloramphenicol agar; GCA, Glucose chloramphenicol agar; GNB, Levine medium; HB, Horse blood agar; MEA, Malt Extract Agar; MEAC, Malt extract agar + chloramphenicol; MPA, Meat peptone agar; MSA, Manitol salt agar; NA, Nutrient agar; OGYA, Oxytetracycline glucose yeast agar; PCA, Plate count agar; PDA, Potato dextrose agar; SAB, Sabouroad agar; SabC, Sabouraud chloramphenicol dextrose agar; SCA, Starch casein agar; SC, Sabourauud dextrose agar and chloramphenicol; SPH, Chapmann medium; STP, Holmes medium; TAG, Beef-extract medium; TSA, trypticase soya agar; YGC, Yeast extract agar, glucose and chloramphenicol

There was a large diversity in exposure times and nutrient medium formulation used by the different authors. A commonly cited reference (Pasquarella et al. 2000) used a 1hr exposure time, 1m from the ground and 1m from a wall. The mean exposure time across this literature review was 37 min. However, we have found in practice that for ease of use for persons unskilled at taking environmental air samples, a time exposure time of 45 min, 1m from the ground and 1m from a wall gave reliable results. Any type of agar medium can be used and we have successfully used PDA or MEA for general fungal cultivation or NA, CHROM or HB for bacteria/yeasts from air samples. All authors used 90mm plates, where b = 3.14 *4.5cm2

Exposure Time/Conditions Purpose & Media Used Authors5 min, 2 m above ground Air sampling in a library

YGC, NAIgnatova-Ivanova et al. 2015

30, 60, 90 min, 1 m above the floor in centre of room

Air sampling in a teaching hospitalNA, SAB

Fekada and Getachewu. 2015

1 hr, 1 m off floor, 1 m away from walls Air sampling in an operating theatreTSA

Agodi et al. 2015

5 min Air sampling in wood processing and packing facilityPCA, Col, MSA, BEA, BA, SAB

Misca et al. 2014

30 min, 1 m from the ground Air sampling in library and archivesPDA, NA

Ahmed Sahab et al. 2014

30, 60, 90 min, 1 m above the floor in centre of room

Air sampling in University librariesNA, SAB

Hayleeyesus and Manaye. 2014

1 hr, 1 m off floor, 1 m away from walls Air sampling in a hospital inc. wards and kitchen

Setlhare, G. et al. 2014

15 min, 1.5 m off floor, middle of room Air sampling in a librarySC

Chadeganipour et al. 2013

30 min, 2 m above floor Air in museum depots and display roomsMEA + streptomycin

Grbic et al. 2013

20 min 1-1.5 m above ground Air sampling in a kidney transplant unitSDA

Afshari et al. 2013

15 min Air in Iran (Tabriz)SDA

Kazemi et al. 2013

10-20 min and 1-3 m above ground Air sampling at Holy MosqueTSA, CDA, SCA

Abdel Hameed and Habeeballah. 2013

1 hr, 1 m off floor, 1 m away from walls Air sampling in operating theatresTSA, SabC,

Napoli et al. 2012

15 min Air sampling in a dental labNot given

Diaconu et al. 2012

30 min, 2 m above ground Air sampling in archivesNA, YGC

Borrego et al. 2012

10 min, 1.5 m above ground Air sampling in an agricultural workplaceMEAC

Abdel Hameed et al. 2012, Abdel Hameed and Mawla. 2012

60 min or 8-24 hrs Corynebacterium bovis contamination of a rodent roomCNA

Burr et al. 2012

15 min Air sampling in bakeryGCA

Cornea et al. 2011

10 min indoors, 15 min outdoors Air in child care centresBHI

Aydogdu et al. 2010

15 min Air in Iran (Isfahan)SC, PDA

Chadeganipour et al. 2010

15 min Air sampling in dental practicesTSA, BA, SAB

Barlean et al. 2010

60 min, on floor Air sampling during hydrodebridement of woundsTSA

Bowling eta al. 2009

120 min Air sampling for Staphylococcus aureus contamination of a burns unitCBA

Javid Khojasteh et al. 2007

30 min Air sampling inside cavesTAG, GNB, SPH, SAB

Borda et al. 2009

30 min, 1 m above ground Air sampling in underground railway systemCDA, MEA, MPA

Bogomolova and Kirtsideli. 2009

1 hr, 1 m off floor, 1 m away from walls Air sampling in any environment at riskPCA, BA

Pasquarella et al. 2000

30 min Air sampling in operating theatreCAB

Fridberg et al., 1999

15 min Air in Iran (Isfahan)SAB

Shadzi et al. 1993

60 min Damp housesMEA, DG18, OGYA, DRBC

Verhoeff et al. 1990

Table 2. Statistical equivalence between active sampling and passive sampling for the assessment of room hygiene by air sampling for fungi.

<0= #0). ). )/>%$#+"#The measurement of indoor air quality has become increasingly important. On-site inspections are always preferred but this entails significant expense. Petri plates can be easily sent through the mail and they are relatively inexpensive. Many people just want to know if their indoor air (mould level) is normal or if one of more fungal Genera/Species are elevated or high. This method demonstrates how such information can be provided easily and reliably.

This poster was presented as part of: Australian Society for Microbiology Annual Scientific Meeting 2016, Perth, 3rd - 6th July