Bioaerosols

Contained Use of GMOs and pathogens
Bioaerosols and/or droplets
(Author: P. Herman) (Last revised: December 19, 2007 )

What are aerosols? Aerosols are defined as colloidal suspensions of liquid or solid particles dispersed in a gas (usually air).

What is a Bioaerosol ? A collection of airborne biological particles. Generally a bioaresol is generated as polydispersed droplets of particles of different sizes ranging from 0.5 to 30 micrometers in diameter.

Laboratory studies (air sampling determinations) of potential sources of infection have focused on hazards associated with bioaerosols produced from microbiological techniques (routine experiments, research and development, large scale production). Aerosols present two means of potential personnel exposure: through breathable infectious airborne particles and by the disposition of heavy droplets onto surfaces, equipment and personnel. Fig. 1 shows an example of aerosol formation through pipetting.

There exist different sized aerosols and as a consequence different routes of possible infections (fig. 2,

). Inhalation, ingestion and dermal contact are route of human exposure for airborne micro-organisms. Inhalation is the main route giving adverse health effects. The average human inhales approximately 10 cubic meters of air/day. Large airborne particles are lodged in the upper respiratory tract. Generally, particles < 6 micrometers in diameter are transported to the lung, but the greatest retention in the alveoli is of 1 to 2 micrometer particles. After penetration, infection also depends on the nature and concentration of the infectious agents present in aerosols.

Once bioaerosols are generated, settling velocity can vary greatly depending on the particle type. The greater the settling velocity is, the less the operator is exposed to a risk. Table 1 shows the size classification of aerosols.

Particle type	size range (micrometer)	settling velocity (fpm*)
Droplet	100 - 400	59 - 498
Dust	10- 100	0.59 - 59
Droplet nuclei	1 -10 0.0 - 0.1	0.007 - 0.59 0.00016 - 0.007

The larger droplets, greater than 100 micrometers in diameter settle quickly and contaminate the surfaces on wich they come to rest. The smaller droplets did not settle but evaporated very rapidly. For example : droplets with a diameter of 100 micrometers evaporated in 1.7 s and those with a diameter of 50 micrometer in 0.4 s. The bacteria (or any other biological agent) in droplets remain in a dried state as "droplet nuclei", also referred to as infected airborne particles. Such particles are moved around rooms and buildings by air currents generated by ventilation and the movement of people. The smaller they are the greater their potential for travelling long distances.

Biohazardous materials should not be manipulated unless suitable containment measures are applied.

The Table2 describes some sources of infection with pathogen organisms (viruses and bacteria) affecting human such as Hepatitis B, C or D virus as well as M. tuberculosis and others. The main aerosol sources of infection for these infectious agents are: blood, body fluids and/or various tissues. Some laboratory-acquired infections were reported for these pathogenic micro-organisms.

Table 2 : Some biological agents with known aerosol route of infection and recommended containment level to adopt

Biological agent	Containment Level: Laboratory facilities, equipment and work practices
Epstein-Barr Virus	L2
Neisseria meningitidis	L2 L3 if large quantities and high concentrations
Hepatitis B, C and D virus, HIV-1 and HIV-2	L2 L3 if large quantities and high concentrations
Mycobacterium tuberculosis	L3
Brucella spp.	L2 L3 for tissue culture of infected cells

A description of Containment (Biosafety) Level 2 and Containment Level 3 facilities, equipment and work practices may be found respectively at the following pages : containment level L2, containment level L3. All containment measures should be adequate for work with pathogenic micro-organisms with emphasis on potential spread by aerosols, (micro)droplets and/or contaminated surfaces and objects.

Common laboratory techniques that produce aerosols: Many common laboratory microbiological techniques produce aerosols consisting of various sizes of particles.

Opening containers;
Pipetting (no visible spill);
Mixing Test tubes;
Opening lyophilised cultures;
Centrifugation;
Cell sorting (cell analysis in a lesser extent) with flow cytometers*

*Cell-sorters are equipped with a nozzle to form a jet of microdroplets. Instrument failures such as clogged sort nozzle or air in the fluidic system can drastically increase aerosol formation. A droplet containment module should be installed to reduce the risk of exposure to generated droplets and aerosols.

Laboratory activities releasing particles smaller than 5 micrometers

Careful pouring;
Fixed-volume automatic pipettors;
Pipette mixing of fluid culture;
Harvesteing/dropping of infected eggs;
High-speed blenders;
Shaking machine;
Dropping tubes or flasks of cultures;
Pipette spills;

Use of centrifuges equiped with biosafety cups;
Do not use a syringe for mixing infectious fluids and check that only the tip of the needle is immersed below the level of fluid in the container avoiding the necessity of excessive force;
Prepare bacterial or infected cell plates in a class II biosafety cabinet;
Wherever possible aerosol-producing operation should be performed in a biosafety cabinet.

The larger particles of an aerosol drop onto the floor (or onto the bench), within seconds where they form an agregate of dust that is unlikely to be dispersed into the air. Droplets larger than 140 micrometers tend to fall onto the ground before they evaporate and those smaller than 140 micrometers are more likely to evaporate before contacting the floor or any solid surface. Table 3 summarizes the evaporation times and falling distance of droplets based on size.

(adapted from Fleming D.O et al., Laboratory Safety, principle and practices, Second edition. 1995)

The particles coming from evaporated droplets are able to remain in the air for very long period. These tiny particles are called "droplet nuclei". If droplet nuclei contain an infectious agent (e.g M. tuberculosis) airborne transmission to human or animal is possible. If the micro-organisms (pathogenic and/or genetically modified) are contained in proteinaceaous fluids (sputum, mucus, serum) instead of aqueous fluids, evaporation will be much slower as these material tend to retain water. The droplets will settle more rapidly; fewer will remain suspended in air and fewer infected airborne particles, available for wider dispersion, will be produced.

Lyophilised cultures;
Dried bacterial colonies;
Dried materials on stoppers and caps of culture tubes and bottles;
Dried exudates fungal and actinomycetes spores;
Dusts from deliberately - or not - infected animal cage and beddings.

It should be remembered that even the larger particles and droplets, which do not evaporate rapidly, may be a source of infection by contaminating surfaces (direct or indirect contacts). Hence, non glove-protected fingers may be contaminated by pathogenic micro-organisms and then transfered to mouth, nose and eyes.

Harding LA & Brandt Byers K. Epidemiology of Laboratory-Associated Infections, Chapter 4, pp. 35-54, in Biological Safety, Principles and practices, Third Edition. Edited by Fleming DO and Hunt DL. ASM Press, American Society for Microbiology, Washington DC, 2000.