Microbial Air Sampler: Active vs. Passive Methods, Principle, Types, and How to Read the Results

In a hospital operating theatre, a pharmaceutical filling line, or a vaccine cleanroom, the air itself is a contamination route. A single settling bacterium can spoil a sterile product or seed a surgical-site infection. So these environments do not just filter the air, they count what is in it. That count is only meaningful if you measure the right thing. Leave an open agar plate on the bench and you learn how fast microbes fall onto a surface. Pull a known volume of air through a sampler and you learn how many microbes are actually suspended in that air. Those are two different numbers answering two different questions, and confusing them is the most common mistake in environmental monitoring.

A microbial air sampler is a device used to detect and count viable microorganisms suspended in air (bioaerosols). Unlike a particle counter, which counts all particles regardless of whether they are alive, a microbial air sampler collects organisms onto or into a growth medium so that only viable, culturable microbes are counted after incubation. The result is usually expressed as colony forming units per cubic meter of air (CFU/m³).

Air can harvest many different kinds of microorganisms. So, monitoring air quality is an essential criterion in quality control in laboratories. Air quality monitoring can be done either by collecting air samples or passively by settled plate methods. The microbial air sampler is a way to collect air samples for active air quality monitoring.

Microbial air samplers are laboratory equipment that helps in collecting a fixed volume of air through it from the testing area. The microbial air sampler can be of various types based on the size and medium of the air collected. It is widely applicable in quality control laboratories, pharmaceuticals, and food industries. It is a crucial requirement for laboratories that require the utmost sterile environment.

Passive Vs. Active Sampling

Air sampling methods fall into two families, and the difference is not a technical detail. It changes what the number means.

Passive sampling (settle plates, the sedimentation method): an open agar plate is exposed to the air for a set time. Microbes carried on dust and droplets settle onto the agar under gravity and grow into colonies. This is simple and cheap, and it tells you the rate at which viable particles are depositing onto a surface. It does not tell you how many microbes are in a given volume of air, because you never measure a volume of air. A common reporting convention is the Index of Microbial Air contamination (IMA), based on colonies settling on a 9 cm plate exposed for a defined time. Passive sampling favors larger, faster-settling particles and undersamples small ones that stay airborne.

Active sampling (volumetric samplers): a pump draws a known volume of air through or against a collection medium, so the result can be expressed per unit volume, CFU/m³. This is what regulated environments (pharmaceutical cleanrooms, operating theatres) require, because a concentration in air is what actually relates to contamination risk. Active samplers are more expensive and need calibration, but they answer the quantitative question a settle plate cannot.

The one-line rule: a settle plate measures deposition onto a surface; an active sampler measures concentration in a volume. Use passive sampling for a cheap, relative sense of "how dirty is the air here"; use active sampling when you need a defensible number in CFU/m³.

Active sampler types by capture mechanism

Active samplers are grouped by the physical mechanism they use to pull microbes out of the air.

Impaction samplers. Air is accelerated through a jet or slit and directed at a collection surface. Particles have too much inertia to follow the airstream as it bends away, so they impact and stick to the surface. Two common designs:

• Sieve (cascade) impactors, such as the Andersen sampler: air passes through a plate perforated with many holes onto an agar plate beneath. Multi-stage versions stack several such plates with progressively smaller holes, so each stage collects a narrower, smaller particle-size range. This sizes the bioaerosol as well as counting it, which matters because particle size determines how deep into the respiratory tract an organism can travel.
• Slit samplers: air is drawn through a narrow slit onto a slowly rotating agar plate, so the time of sampling maps to position on the plate. This lets you see how airborne counts change over time, useful during an event like a surgical procedure.

Impingement samplers. Air is drawn through a jet into a liquid collection medium. Microbes are trapped in the liquid rather than on a surface. The liquid can then be diluted, plated, or tested by non-culture methods, and it is gentler on fragile organisms and viruses than dry impaction. The classic design is the all-glass impinger (AGI-30).

Filtration samplers. A known volume of air is pulled through a membrane filter (or a gelatin filter) that retains microbes on its surface. The filter is then placed on or dissolved into a medium and incubated. Filtration is simple and good for spores and fungi, but drying stress during sampling can kill delicate vegetative bacteria, lowering the count.

Centrifugal (cyclone) samplers. Air is spun so that centrifugal force throws particles outward onto an agar strip lining the sampler. These are portable and popular for quick spot checks.

Reading the result: plate count to CFU/m³

An active sampler gives you colonies on a plate and a sampled air volume. The concentration is:

CFU/m³ = (number of colonies ÷ volume of air sampled in liters) × 1000

Two corrections matter in practice. First, sieve impactors can undercount at high colony numbers because two microbes may pass through the same hole and grow as one colony; a positive-hole (Feller) correction is applied to convert the raw count to a statistically likely true count. Second, results depend on the medium and incubation, since only organisms that grow under those conditions are counted. A single sampler run is a snapshot, not a full picture of the air.

Types of Microbial Air Samplers

Figure: Types of microbial air sampler based on collecting medium

Based on the state of the collecting medium

Based on the collecting medium, samplers are solid-medium (impactor, agar) or liquid-medium (impinger), both described under Active sampler types by capture mechanism above.

Based on size

Figure: Types of microbial air sampler based on size

1. Portable microbial air sampler: Sometimes, air quality monitoring might extend beyond indoor or laboratory conditions. In such cases, a portable battery-operated microbial air sampler is the best choice. It is easy to carry and, when properly calibrated, gives results comparable to a benchtop sampler.
2. Benchtop microbial air sampler. This type of microbial air sampler is larger than the portable one. It must be fixed to a specific portion of the bench in the laboratory and is best for research and laboratory air quality monitoring.

Principle of Impactor Microbial Air Sampler

The microbial air sampler is based on Andersen's principle of the sieve impactor method. Here for the collection cycle, the air is aspirated through a perforated plate. The stream of air is now directed onto the agar surface in a standard Petri dish. After the collection cycle, the agar plate is incubated at normal incubation temperature for one to two days. After incubation, the colonies are counted and expressed as colony-forming units per cubic meter of air (CFU/m³). This method is also known as the impactor method.

Two types of impactor are used: six-stage and two-stage. The six-stage impactor stacks six agar plates in cascade, each fed by progressively smaller orifices, so it both counts viable particles and separates them by size: the top stage collects the largest particles (roughly above 7 microns) and each lower stage collects a smaller range, down to submicron particles. This size distribution matters because particle size determines how deep into the respiratory tract an inhaled organism can travel. The two-stage impactor collapses this into two fractions, respirable and non-respirable, and is used when total count or a simple respirable split is needed rather than a full size distribution.

Accessories of Impactor Microbial Air Sampler

Figure: Parts of impactor microbial air sampler

The parts of the impactor microbial air sampler are as follows:

1. Sampling head cover: It covers the Petri plate and is present in the topmost part of the equipment.

2. Porous sampling head: It is the area at the top of the microbial air sampler which helps collect air samples.

3. Pump: It helps in pulling the air through the impactor.

4. Sampling pump cover: It separates the pump from the Petri plate.

5. Handle: It helps to carry the equipment from one place to another.

6. Operation panel: It has an ON/OFF switch for turning on the equipment. It also has start and stop buttons. Likewise, the mode switch present helps in changing volume and duration. The up and down arrow button allows navigation. Some may have a + or – control in them for navigation.

7. Petri Plate with media: It is required for the impactor method microbial air sampler. It is the part that collects the sample and provides growth conditions to the viable microorganisms.

8. Anemometer: A device used to calibrate the impactor's airflow.

   

Figure: Anemometer

Steps of Operating Impactor Microbial Air Sampler

The following are the steps for operating an impactor microbial air sampler:

1. Place the Petri plate of standard size (90 mm ✕15 mm) inside the microbial air sampler.
2. Turn the sampler on using the ON/OFF switch in the operation control panel.
3. Adjust the sample volume to the desired amount using the operation panel’s + or – sign or arrow sign.
4. Press the Start button to begin sampling. The red LED light symbolizes the equipment is running.
5. Press the Stop button to stop the sampling process. Some may have the option of presetting the duration for sampling.
6. Remove the Petri plate and incubate it at the appropriate temperature and air condition for 18-24 hours to observe the growth of viable organisms. Fungal growth may require a longer incubation period.

Calibration of Impactor microbial air sampler

Impactor samplers are calibrated for airflow, typically using an anemometer to verify the sampling flow rate, since an accurate volume is what makes the CFU/m³ result valid. Some units include automatic calibration software.

Principle of Impinger Microbial Air Sampler

Another active method of collecting air samples for microbial analysis is the impinger method. The suction pump pulls air from the surrounding into a small flask with a liquid medium. Once the air hits the surface of the liquid, it changes direction swiftly, and suspended particles impinge into the liquid medium. Then, the fluid is cultured to enumerate the viable microorganisms. It is also a quantitative method for calculating air volume using the flow rate and sampling time.

Accessories Required for Impinger Microbial Air Sampler

The parts of the impinger microbial air sampler are as follows:

1. Inlet: It directs air into the vessel.
2. Outlet: It directs air outside of the vessel.
3. Collection vessel: The air is collected in a container with a liquid medium.
4. Tangential nozzles: It is the part that comes in contact with the liquid medium, and their presence help in impinging air to the surface of the liquid.
5. Pump: It helps in providing pressure and direction for the air.

Figure: glass impinger with pump and holder

Extra accessories required

• Vial: It is required to transfer the liquid medium after sampling to the incubator.

• Rotameter: A device used to calibrate the impinger's airflow. It measures the volumetric flow rate of the air being drawn through the sampler, which is needed to convert the count to CFU/m³.

  

Figure: Rotameter

Operating an Impinger Microbial Air Sampler

The following are the steps of operating an impinger microbial air sampler:

1. Charge the pump for at least 48 hours before use.
2. Install the impinger holder inline to attach the impinger.
3. Separate the two parts of the impinger and place the bottom part in the impinger into the holder.
4. Pour the proper amount of sampling medium. Then place the top part of the impinger into the bottom part.
5. Attach one end of the tube to the pump and the other to the impinger’s side port or outlet port.
6. Turn on the pump and record the duration from the turning on of the pump.
7. Once sampling is completed, turn off the pump, and transfer the liquid medium into a vial.
8. Label and tighten the vial and then incubate the vessel at appropriate conditions to determine the number of viable microorganisms.

Calibrating Impinger Microbial Air Sampler

In the case of the impinger microbial air sampler, only the airflow needs to be calibrated from time to time. The following are the steps for calibrating the impinger microbial air sampler.

1. Preheat the pump for at least 48 hours before calibration.
2. Secure the impinger holder in the pump and place the bottom part of the impinger in the tube holder.
3. Pour the desired amount of distilled water into the bottom and place the impinger’s top part on the bottom.
4. Then attach the tube just like the sampling method.
5. Attach a rotameter in the top part or inlet part of the impinger.
6. Note the value in the rotameter; if the rate needs to be adjusted, turn the screw on the pump.
7. Correlate the value with the calibration standard provided in the rotameter.

Things to consider

1. From time to time, sterilization and cleaning are necessary for the equipment.
2. Calibrating the microbial air sampler before and after use is suggested.
3. Selecting the correct type of cultural media is essential.
4. Temperature and humidity may bring change in the final result of viable microorganisms.

Uses of Microbial Air Sampler

The microbial air sampler helps collect air samples. It has a variety of purposes in different laboratories, which are as follows:

1. Indoor air quality monitoring: It helps collect active air quality monitoring samples. Indoor air quality monitoring is essential, especially in hospital settings, microbiological laboratories, hospice care facilities, pharmaceutical industries, and food industries.
2. Research purposes: Since air carries harmful substances, air sampling can be helpful in various research, like research related to airborne illness.
3. Environmental monitoring in aseptic manufacturing: air sampling is a routine part of the environmental monitoring that supports sterile production in the pharmaceutical and food industries, alongside surface and personnel monitoring.

How to Remember

Passive falls, active pulls. A settle plate waits for microbes to fall onto it (deposition). An active sampler pulls air through a pump (volume). If the method involves a pump and a volume, you can report CFU/m³. If it's just an open plate, you cannot.

Impaction, impingement, filtration: hit, splash, sieve. Impaction, particles hit a solid surface. Impingement, particles splash into a liquid. Filtration, particles are sieved onto a membrane. Three verbs, three mechanisms.

Andersen sizes as it counts. The multi-stage Andersen sampler stacks plates with smaller and smaller holes, so higher stages catch bigger particles and lower stages catch the small ones that reach deep lung. It doesn't just count the bioaerosol, it sorts it by size.

Positive-hole correction: same hole, one colony. When two microbes go through one hole, you see one colony but there were two. The positive-hole correction adds back the ones you couldn't see. Remember it as "crowded holes hide colonies."

Key exam facts in one table

Where Students Get Confused

"A settle plate tells you how many microbes are in the air." It does not. It measures how fast microbes deposit onto a surface over the exposure time. Without a measured air volume, you cannot report a concentration. Only active, volumetric samplers give CFU/m³.

CFU/m³ versus particle count. A particle counter counts everything, living or not, and gives a result instantly. A microbial air sampler counts only viable microbes, and only after incubation. High particle counts do not directly equal high microbial counts.

Why the Andersen sampler has multiple stages. The stages are not redundant. Each collects a different particle-size range, because particle size determines how far into the airway an inhaled organism travels. The sampler characterizes the size distribution of the bioaerosol.

The positive-hole correction is not optional at high counts. When many colonies form, the raw count underestimates the true number because multiple microbes share holes. Skipping the correction under-reports contamination.

Sampler choice changes the number. Filtration can kill fragile bacteria by drying; impingement preserves them in liquid. The same air can give different counts on different samplers, so the method must be stated with the result.

References

• Cappuccino JG, Sherman N. Microbiology: A Laboratory Manual. 11th ed. Pearson Education; 2017.
• Gilmore BF, Denyer SP, editors. Hugo and Russell's Pharmaceutical Microbiology. 9th ed. Wiley; 2023.
• European Commission. EudraLex Volume 4, Annex 1: Manufacture of Sterile Medicinal Products. 2022 (in operation from 25 August 2023). Available at: https://health.ec.europa.eu/latest-updates/revision-manufacture-sterile-medicinal-products-2022-08-25_en