Paper_Continuos_Monitoring_2012 - Microbiological Air Sampler

A new Instrument for the Continuous Measure of Air Born 

Microorganisms in Isolators and RABS Systems 

Beat Glauser 1 , Hans Zingre 1 , Michael Hochstrasser 1 , Deborah Haefeli 2 and Lars Fieseler 2 

b.glauser@mbv.ch, h.zingre@mbv.ch, m.hochstrasser@mbv.ch , 

lars.fieseler@zhaw.ch, haee@zhaw.ch 

1 MBV AG, Stäfa, Switzerland 

2 Zurich University of Applied Sciences, Institute of Food and Beverage Innovation, Wädenswil, Switzerland 

Abstract 

A combination of active sampling head instruments and passive settling plates is routinely used to monitor 

microorganisms in the air of isolators and RABS systems. While the collection efficiency of an active head 

is superior to a settling plate the drawback is the limited sampling time. Applying a newly designed 

sampling head the exposure of agar plates was extended from 10 min to up to 4 h during air sampling 

without changing the collection efficiency. Both, the water activity and the microbial growth, were analysed 

on exposed agar surfaces to prove that the new sample collection head allows efficient detection of air borne 

microorganisms. 

Key words: Continuous measurement, air sampling, active sampling, impaction principle, biological 

contamination, isolator, RABS, settle plate, SQS 

1. Introduction 

Determination of air quality in an isolator or RABS 

system (restricted access barrier system) is typically 

performed applying a combination of active 

sampling points and settling plates. The collection 

efficiency of the active sampling points is usually 

high. But due to the short sampling time this 

method is less suited for continuous analyses. 

Hence settling plates are generally used, because 

these can be exposed to the environment for several 

hours. The drawback is the low collection efficiency 

as well as the lower sensitivity for smaller particles. 

A longer active sampling period can be achieved by 

dividing the total volume into several smaller 

samples taken in regular intervals (Fig. 1). While 

this sequential sampling (SQS) is not fully 

continuous the interval can be extended to hours 

instead of minutes. As long as the agar remains 

covered behind the perforated lid no negative 

impact from drying could be detected over several 

hours (Bijlenga and Obrist, 2006). 

500 

400 

300 

200 

100 Time 

Figure 1. Sampling volume divided into five 

fractions. 

In this study we introduce a novel method for 

continuous active sampling for up to 4 h without 

changing the collection efficiency compared to 

standard active sampling heads. 

2. The Impaction principle 

The basis of active air sampling by the impaction 

principle was introduced by Anderson (Anderson, 

1958). It is based on the inertia of air borne 

particles: to pass through a restriction the air 

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A new Method for Continuous Measurement; B.Glauser, MBV AG page 1/7 

1. sampling 

pause 

2. sampling 

pause 

3. sampling 

pause 

4. sampling 

pause 

5. sampling

(including the air borne particles) speeds up. After 

the restriction the air path direction abruptly 

changes. While the air molecules can change their 

direction, heavier particles keep going straight and 

are captured on the agar surface (Fig. 2). 

Figure 2. Air sampling by the impaction principle. 

A measurement based on the impaction principle is 

defined by the flow rate and the measurement time. 

Hence the total volume sampled can be described 

as: 

volume � flowrate * time 

(1) 

The smaller the area of the restriction, the more the 

air is accelerated to pass through: 

flowrate 

airspeedrestricted � � vimpaction 

(2) 

area 

restricted 

The impaction speed defines the collection 

efficiency. The higher the impaction speed, the less 

mass is needed to have enough inertia to get 

captured leading to a wider range of sampled 

particle sizes. 

3. A new measurement setup 

For the new setup described here the sampling time 

should increase without changing the collection 

efficiency, e.g. the impaction speed, so that the 

results are comparable to previous burst 

measurements and the same pass criteria can be 

applied. 

3.1. Reduced flow rate 

According to (2) the impaction speed remains the 

same when the flow rate and the restricted area are 

reduced by the same factor. In the case of the 

proposed setup of the MAS-100 Iso NT ® 

Continuous the reduction factor is 12 or 24 while 

maintaining the impaction speed of the 6 th stage of 

an Anderson air sampler. The smaller the flow rate 

the longer is the sampling time (1). 

3.2. Rotating agar 

With a reduced number of aspiration holes the agar 

surface in direct contact with the air jets would be 

less than 1% of the total agar surface. This would 

lead to an excess water loss underneath the air jets 

reducing the survival rate of microorganisms at the 

area where they are actually collected. Therefore, a 

rotating agar dish similar to known slit samplers is 

used here. To identify the starting point of the 

measurement the rotation stops just before the 12 

o’clock position (Fig. 3). 

Figure 3. Surface coverage by the air jets. 

As a side effect of the rotation each collection is 

now time resolved; a microorganism growing at the 

“quarter past” position was captured after a quarter 

of the total measurement time had elapsed. 

3.3. Increased total sample volume 

The total measurement time can be extended by 

sampling more air. As this further reduces the water 

content of the agar, this parameter should be 

carefully examined (chapter 5). 

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A new Method for Continuous Measurement; B.Glauser, MBV AG page 2/7

4. The new instrument 

4.1. Sampling head 

A new developed active sampling head allows the 

rotation of the agar plates applied. It is designed for 

easy cleaning (Fig. 4) and optimized for a small 

form factor (Fig. 5). 

Figure 4. Easy cleanable surface of the sampling 

head. 

Figure 5. Compact head with rotating agar plate. 

4.2. D50 cut off value 

According to Nevalainen et al. (1993) the physical 

sampling efficiency for air samplers can be 

described by the size of particles they are able to 

collect. The d50 value represents the smallest 

particle size where half of the particles are still 

captured (3). 

d 

50 

9�Dh 

Stk50 

� (3) 

�UC 

Where � is the dynamic viscosity, Dh is the 

hydraulic diameter of the holes in the aspiration 

sieve, Stk50 is the Stokes number of the opening, � 

is the particle density, U is the impact velocity, and 

C is the Cunningham correction factor. 

For the round openings of the aspiration sieve the 

physical diameter is used for Dh and ¼ for Stk50. 

The particle density is assumed 1 g/cm 3 and the 

Cunningham correction factor was set to 1 (for 

particles > 1µm). For an environment with 1013hPa 

and 20°C the d50 value translates to 1.1µm (equal to 

the MAS-100 NT ® ). To keep this value constant the 

number of holes in the aspiration sieve were 

changed for different air flows (4, 8, 16 l/min) while 

the diameter of the holes remained the same. 

4.3. Pump unit 

The pump unit is similar to the proven design of the 

MAS-100 Iso NT ® . It handles the pump control and 

controls the agar rotation. 

5. Testing 

5.1. Comparison to the MAS-100 NT ® 

instrument 

The new method was compared to a standard 

instrument (MAS-100 NT ® ) sampling a burst of 

1000 l of ambient air at 100 l/min (10min). A 

second instrument (also MAS-100 NT ® ) sampled in 

SQS mode 1000 l in 10 fractions over 1, 2 and 4 h 

(also at 100 l/min). The continuous measurement 

sampled 1000 l at 16, 8 and 4 l/min with matching 

sampling heads so that for all measurements the 

impaction speed was 19.6 m/s and the d50 value 

1.1µm (same as the MAS-100 NT ® ). All 

instruments run at the same time in close proximity. 

Fig. 6 depicts that microbial growth is not affected 

by the new sampling head compared to established 

standards and the SQS measurement. 

The variations are mainly due to an uneven 

distribution of microorganisms in the ambient air so 

that the measurement time and the measurement 

duration influence the results. 

The shorter the measurement time becomes the 

smaller the averaging effect is and therefore a 

higher variation in the results can be observed. This 

is especially true for the standard measurement time 

of 10 min. 

________________________________________________________________________________________________________________________ 


Figure 6. Comparison of microbial growth applying 

different sampling instruments. 

5.2. Water content of agar plates 

As discussed above the walk-away time can be 

expanded by sampling more air but this further 

reduces the water activity of the agar plates. 

Most Gram-negative bacteria require a water 

activity aw = 0.97-0.96. However, many other 

bacteria can still grow at aw = 0.95-0.91 while some 

yeasts can tolerate aw = 0.65-0.60. Below this 

threshold the survival rate of any vegetative 

microbial cell is dramatically affected. 

Using the new sampling head the water activity was 

determined before and after the collection of 

different air volumes, respectively (Fig. 7). 

Figure 7. Sampling points for the determination of 

the water activity. A: sample point before air 

collection; B-D: sample points after air collection. 

Each area of an agar plate used was analysed in 

triplicates. Because the agar samples had to be 

removed from every plate for the analysis, 

measurements of the base line (A) were performed 

using independent plates from the same batch so 

that the airflow was not disturbed from the missing 

piece of agar. The analyses revealed moderate 

reduction of the water activity for runs applying 

1000 l and 2000 l. When 3000 l were sampled the 

water activity of the agar was aw = 0.96. Sampling 

4000 l resulted in aw < 0.6. 

Figure 8. Water activity (aw) after sampling 

different volumes of air (average of point b, c and d 

(Fig 7)). 

There was no significant difference in the water 

activity between sampling areas b, c and d. 

Therefore, it can be concluded that the main factor 

for drying is the accelerated air jet underneath the 

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aspiration hole. Slow flowing air did not 

significantly contribute to the drying effect for the 

first 4 h. This finding is also supported in a study 

about the SQS measurement mode (Bijlenga and 

Obrist, 2006). 

Due to the asymmetric placement of the exhaust all 

air exits toward the outer rim of the plate. To 

examine a possible build up effect a second analysis 

with more sampling areas was conducted (Fig. 9). 

Figure 9. Additional sampling points for the 

determination of the water activity. 

Rather than excess water loss at the outer regions of 

the agar plate (b, d, f) slightly lower water contents 

were monitored at the center of the agar plate (c, g, 

e) (Tab. 1). This is probably due to a higher air 

volume to surface ratio at the center. 

Table 1. Water activity at the sampling points a-g. 

The equidistant spacing of the aspiration holes was 

changed for later tests. The space between two holes 

now increased towards the center to achieve a more 

homogenous distribution. 

5.3. Microorganisms used 

To monitor microbial growth experiments were 

conducted applying Gram-positive (Staphylococcus 

aureus, Micrococcus luteus, and Bacillus subtilis) 

and Gram-negative bacteria (Pseudomonas 

aeruginosa and Acinetobacter lwoffii). For all tests 

20 ml TSA agar plates were used (Oxoid, Art. Nr: 

PO5012A). 

5.4. Test setup 

Maintaining a constant level of evenly distributed 

and viable microorganisms suspended in air is 

rather difficult. Therefore bacteria were placed onto 

the sampling plates using a 45 pin replicator tool 

and donor plates (Fig. 10). 

Figure 10. Pin replicator tool for bacterial transfer 

onto the sampling plates. 

To simulate a capture at the beginning of the 

sampling period the test agar was inoculated and 

placed into the sampling head which was operated 

in a biosafety flow cabinet. To simulate a capture at 

the end of the measurement the process was 

reversed. First the defined amount of clean air was 

sampled in the biosafety flow cabinet and then the 

agar was inoculated using the pin replicator tool. 

The agar plates were incubated at 30°-37°C and 

bacterial counts were enumerated after 24 h and 

48 h of incubation, respectively. Acinetobacter 

lwoffii, Pseudomonas aeruginosa and Micrococcus 

luteus were incubated at 30°C, Staphylococcus 

aureus and Bacillus subtilis at 37°C. In Fig. 11 an 

inoculated agar plate is shown after incubation. 

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Figure 11. Test agar plate after 48 h of incubation 

and grown deposited colonies. 

5.5. Results 

The pin replicator consists of 45 pins in total. The 

survivalrate was calculated as the percentage of 

grown colonies (4). 

colonies 

pins 

counted 

survivalrate� (4) 

total 

5.6. Maximum volume 

A first question was if the maximum acceptable 

volume of air, which did not dramatically reduce the 

water activity also supported growth of selected 

microorganisms. 

Figure 12. Survival rate after 48 h; inoculation 

before air sampling. 

Fig. 12 illustrates that bacteria responded differently 

to the total air volume applied. In general no 

adverse effect was evident for an air volume of 

2000 l. Application of 3000 l revealed that growth 

of Acinetobacter lwoffii was slightly affected, while 

growth of the other bacterial species used was not 

altered. After 4000 l all three bacteria species 

showed a reduction in growth. Taken together 

2000 l of sampling air volume can be safely applied 

to monitor bacterial contamination in air. 

5.7. Inoculation of test agar plates at the 

beginning vs. the end of a sampling period 

A second question was if microbial growth depends 

on whether a bacterial cell was present at the 

beginning or if it was collected just before the end 

of the sampling period. If the bacteria were 

transferred before the air flow started colonies 

appeared slightly slower at 2000 l air flows after 

24 h (Fig. 13). However, after 48 h of incubation 

colonies remained equal in size (Fig 14). 

Figure 13. Survival rate after 24 h of incubation; 

inoculation before air sampling. 


inoculation before air sampling. 

If the bacteria were transferred after the collection 

of air the effect was still present but less 

pronounced (Fig. 15, 16). 

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inoculation after air sampling. 


inoculation after air sampling. 

These results demonstrate that the survival rate 

remains equal from the beginning to the end of the 

sampling period. 

5.8. Influence of total sampling time 

Applying a 4 l/min head the measurement time 

becomes a considerable factor: 4 h are needed to 

sample 1000 l; 8 h for 2000 l and 12 h for 3000 l. In 

contrast to the 8 l/min and 16 l/min measurement 

the water activity was significantly reduced if an air 

volume of 2000 l or more was applied (Fig. 17). 

Figure 17. Water activity of test agar plates at a 

flow rate of 4 l/min. 

Hence Micrococcus luteus and Acinetobacter lwoffii 

exhibited a clear decline in the survival rate at 

2000 l air flow (8 h sampling time) and 

Pseudomonas aeruginosa exhibited limited growth 

at 3000 l air flow (12 h sampling time) (Fig. 18). 

Figure 18. Survival rate after 48 h of incubation at 

an air flow rate of 4l/min.; inoculation before air 

sampling. 

6. Conclusions 

Continuous measurements of up to 4 hours and an 

air volume of up to 2000 l can be achieved with the 

same collection efficacy as todays burst 

measurements. This allows the construction of air 

samplers with the same walk away time as settling 

plates combined with all the benefits of an active 

measurement. 

References 

Anderson, V. (1958): New sampler for the 

collection, sizing and enumeration of viable 

airborne particles, U.S. Army Chemical Corps 

Proving Ground, Dugway, Utah, U.S.A. 

Bijlenga, R., Obrist D. (2006): A comparative 

study of two different Microbial Air Monitoring 

Methods: Conventional and Sequential Sampling, 

Laboratory of Applied Microbiology / University of 

Applied Sciences of Geneva (EIG), Switzerland 

Nevalainen A, Willeke K, Liebhaber F, Pastuszka J, 

Burge H, Henningson E. Bioaerosol sampling. In: 

Willeke K, Baron PA, eds. Aerosol Measurement: 

Principles, Techniques and Applications. New 

York: Van Nostrand Reinhold, 1993: 471-492. 

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Paper_Continuos_Monitoring_2012 - Microbiological Air Sampler

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