This sampling potocol allows the isolation and enumerate Aspergillus spp spores in air.
Sampling Procedure (general)
The air sample is aspirated through the instrument at a nominal rate of 180 liters per second for a preselected period of between 20 seconds and 6 minutes giving a volume range between 60 litres and 1080 litres. The airflow is directed towards the agar surface of a 50 mm diameter contact plate which contains 12.5 ml. of agar so that particles whose aerodynamic diameter causes them to leave the airstream are deposited on the agar surface. The plate is then removed for incubation.
Sampling Location
Sampling location is dependent on circumstances but should accord broadly with the breathing zone of potentially affected personnel. The choice of a sampling height of 1.2 meters for room hygiene. With other samples taken for exploratory purposes near suspected or potential sources of contamination. Multiple samples are preferable to single samples as they will highlight temporal and spatial variation in spore levels within any environment.
Selection of Sampling Time
Selection of an appropriate sampling period is vital to the success of the sampling operation. If the sampling time selected is long in a heavily contaminated environment then the colonies, once cultivated, can only be expressed as exceeding a particular number. Where confluent growth occurs the colonies may even be uncountable. A chart is supplied with the instrument to assist in selection of the sampling time which, on the basis of an assumption regarding the level of contamination in the environment permits the number of sampling units (periods of 20 ) to be estimated. In practice, whilst some knowledge of the levels of contamination may build up over time, certainly initially the selection of the sampling period must largely be done by trial and error.
Sampling Steps
1. Unscrew the top cover plate avoiding contact with the inner
or outer surfaces of the
drilled area. The cover plate should be cleaned after each
use.
2. Insert a contact plate with Czapek Dox agar with the lid still
in place, remove the contact plate lid and replace the instrument
cover plate.
3. Set the digital selector on the instrument to zero units.
4. Switch on the battery pack.
5. Turn the timer to the desired setting.
6. Press the instrument start button.
7. Following completion of the sampling the instrument will switch
off. The cover plate can then be removed and the exposed contact
plate agar surface immediately covered by replacing the contact
plate lid.
8. The contact plate should then be removed for incubation.
Laboratory Procedure
1. On receipt of the contact plates, these are placed in a
pre-heated incubator ot 28oC for 48 hrs to permit
germination and colony formation.
2. The plates are then microscopically examined at x100
magnification to enumerate colonies growing on the plate.
3. Identification of fungal colonies is based on colony
characteristics and micromorphological characteristics ascertained
through microscopic examination ot x400 magnification.
4. Specimens for examination should be prepared using a wet needle
mount using Lactophenol with Cotton Blue Stain (0.75%).
5. A colour key is available for the specific identification of
different Aspergillus species grown on Czapek Dox agar or
broth.
N.B. Czapek Dox (Oxoid) is recognised as a suitable medium for isolation and culturing of Aspergillus species whilst permitting growth of the majority of airborne fungal species. This permits general levels of fungal contamination to be simultaneously appraised as an indicator of building hygiene.
Enumerating the Colony Forming Units
There is a possibility that any colony which grows on the contact plate derives from more than one colony forming unit passing through a single hole in the cover plate. This possibility increases with the number of colonies on the plate and for higher counts a correction factor is applied according to the following formula:-
Pr = n(1/n+ 1/(n-1) + 1/(n-2) +.......................1/(n-r+1))
Where
Pr is the probable statistical total
N is the number of holes in the sampling head
r is the number of colonies counted
Tables have been prepared by the manufacturer which can be read off to give a value for P once r has been established.
The number of colony forming units calculated from the above formula is normally expressed as CFUs/m3. This is calculated using the formula:-
| X = | Adjusted colony count on plate x 1000 |
| Volume of air drawn into sampler (litres) |
[accepted thresholds here for different types of hospital enviroments]
In published outbreaks of invasive aspergillosis, local fungal contamination of ducts, grids, and filters may release spores intermittently as may nearby demolition and building works. Although in some outbreaks of aspergillosis the patient's isolates may have been different from those in the environment (refs), some degree of control is more important than measuring air counts, because these are taken only at one points in time within the ward or clinic, and if an outbreak of infection has occurred, it reflects an episode of air contamination that happened some days or weeks before. "Normal" air counts are therefore not a reason for complacency, and a search for a specific source of air contamination is required, together with continued vigilance against the introduction of large numbers of spores into the patient's room.