Airborne bacteria: names and numbers, please…

Airborne bacteria: names and numbers, please…

Airborne bacteria:
n a m e s and numbers,
please…
Runar Thyrhaug
Dep. of Biology
University of Bergen
Norway
[email protected]
My background
 Microbial ecology
 Marine microbiology group- Bergen, Norway
 Focus: algae, bacteria, protozooa, virus
My background




Microbial ecology
Marine microbiology group- Bergen, Norway
Focus: algae, bacteria, protozooa, virus
Methods





Nutrient uptake/turnover
Production rates
Genetic diversity/biodiversity
Viral activity
Modelling (vs experiments)
W h y airborne bacteria?




Curiosity…new field
Biodiversity and transport rates
Microbial production
Improved/new methods…possibilities?
Bacteria in t h e air
… from w h e r e?
 Soil (terrestrial)
 Sea /fresh water
Bacteria
Virus
~109 g-1
~106 mL-1
??
~107 mL-1
Bacteria in t h e air
… from w h e r e?
 Soil (terrestrial)
 Sea /fresh water




Bacteria
Virus
~109 g-1
~106 mL-1
??
~107 mL-1
We know:
Most marine bacteria are assumed to be alive
Nutrient limited N , P or C
<1% growing on agar plates (sea water and soil samples!)
Counting airborne
bacteria
 Liquid samples!
 Rain or air sampled in liquids
 Methods:
 Flow cytometry (FCM)
 DNA/RNA staining - fluor /scatter signals from single particles
 Quantitative PCR (Q-PCR)
 DNA staining – amount of bacterial DNA in a bulk sample
 Colony forming units (CFU)
 Bacteria willing to grow on agar plates
 Epifluorescense microscopy (EFM)
 DNA/RNA staining - fluor signals from single particles
FCM plot / EFM picture
s e a water examples:
FCM plot / EFM picture
s e a water examples:
Marine bacteria
FCM plot / EFM picture
s e a water examples:
Marine bacteria
Marine viruses
FCM plot / EFM picture
s e a water examples:
Emiliania huxleyi virus
isolate (EhV)
Marine bacteria
Marine viruses
EhV
Sampling airborne
bacteria
 Xmx-cv (Dycor)
 0,7 m3 min-1
 5 mL PBS
 Suggested max
sampling time: 5 min
 Our max: 10 min
F l o w cytometry o f air
samples
Fluorescence
T0=Blank
Scatter signal
F l o w cytometry o f air
samples
Fluorescence
T0=Blank
Scatter signal
Fluorescence
T5~3,5m3 in 5 mL
Scatter signal
F l o w cytometry o f air
samples
Fluorescence
T0=Blank
Scatter signal
~103 pr m3
Fluorescence
T5~3,5m3 in 5 mL
Scatter signal
F l o w cytometry v s QPCR
8
 Q-PCR:FCM ~10
 Calibration going on!
 Air: yellow
 Rain: red
 Sea water:blue
)3
6
4
FCM (Log bact pr m
2
0
0
2
4
6
Q-PCR (Log bact pr m3)
8
F l o w cytometry v s QPCR
8
 Q-PCR:FCM ~10
 Calibration going on!
 Air: yellow
 Rain: red
 Sea water:blue
)3
6
4
CFU: around 1-2%
compared to FCM
FCM (Log bact pr m
Epifluorescence microscopy:
difficult to discriminate
signal/noise
2
0
0
2
4
6
Q-PCR (Log bact pr m3)
8
S n o w and r a i n samples
snow ~500-104 pr mL
Rain ~104 pr mL
S n o w and r a i n samples
snow ~500-104 pr mL
Bacterial production:
Addition of H3 leucine, 24 h incubation
Rain ~104 pr mL
S n o w and r a i n samples
snow ~500-104 pr mL
Rain ~104 pr mL
Bacterial production:
Addition of H3 leucine, 24 h incubation
Result: activity pr bacteria ~ as in sea water
Estimated generation time~ 1day
Comparison o f 3
bioaerosol samplers
 Time series
 FCM and CFU counts
 Liquid loss rates
Sampler 1
 Xmx-cv (Dycor)
 0,7 m3 min-1
 5 mL
 Suggested max
sampling time: 5 min
 Our sampling time 0-10 min
 Liquid: PBS
 ~10% PBS loss after 10 min
Sampler 2
 SKC 225-9595 (SKC)




0,0125 m3 min-1
Sampling time 0-60 min
Liquid: MilliQ
Refill~25% milliQ at 10 min
Sampler 3
 Kärcher DS 5550
Sampler 3
 Kärcher DS 5550





3,3 m3 min-1
1500 mL
Max sampling time: 120 min
Liquid: PBS
~10% pbs loss after 120 min
FCM c o u n t s - time series
Black: direct counts FCM ”bacteria pr mL”
Grey: estimated bacterial concentration pr m3 air
White: estimated bacterial conc pr m3 air after substracting blank (T0 sample)
XMX - FCM
3000
SKC - FCM
600
) -1
2500
500
2000
400
1500
300
1000
200
500
100
Karcher - FCM
1000
800
600
400
2000
1000
0
2
4
6
Sampling time (min)
8
10
Estimated bact conc (m
0
0
40000
Bacterial
concentration (mL-1)
Estimated bact conc (m
) -3
no correction
T0 substracted
Bacterial
) -3concentration (mL
Estimated bact conc (m
Bacterial
concentration (mL-1)
) -3
0
8000
6000
4000
200
no correction
T0 substracted
20000
12000
10000
8000
6000
4000
2000
0
0
20
40
Sampling time (min)
60
80
0
60000
40000
20000
no correction
T0 substracted
4000
3000
2000
1000
0
0
20
40
60
80
100
Sampling time (min)
120
140
CFU c o u n t s - time
series
Black: direct counts CFM ”bacteria pr mL”
Grey: estimated bacterial concentration pr m3 air
White: estimated bacterial conc pr m3 air after substracting blank (T0 sample)
XMX - CFU
40
SKC - CFU
50
) -1
) -1
) -1
50
Karcher - CFU
35
30
40
30
30
20
20
10
10
25
20
15
10
60
40
20
0
0
2
4
6
8
Sampling time (min)
10
0
3000
no correction
T0 substracted
2000
1000
200
180
160
140
120
100
80
60
40
20
0
0
20
40
Sampling time (min)
60
80
Bacterial
concentration (mL
Bact concentration (m
) -3
no correction
T0 substracted
Bacterial
Bact concentration (m
) -3 concentration (mL
Bacterial
) -3 concentration (mLBact concentration (m
0
80
5
0
no correction
T0 substracted
200
180
160
140
120
100
80
60
40
20
0
0
20
40
60
80
100
Sampling time (min)
120
140
CFU as % o f FCM
sampling data
CFU pr m3
FCM m3
CFU as % of FCM
n
FCM±std.
dev4
raw data
FCM±std.dev4
T0 substr
FCM raw data
FCM
data T0
subst.
18±13
4
1800±330
1600±330
1.0
1.1
0.261
33±35
4
2500±690
1400±690
1.3
2.4
60
0.0381
67±77
4
8800±2200
4200±2200
0.8
1.6
slit sampler
60
1.82
37
1
Mas-100
20
2.02
40±14
2
Sampler
time
(min)
volume
(m3)
CFU±err
XMX-CV
10
1.41
Karcher
120
SKC
3
Table xx. cfu and fcm bacterial counts in samples with the highest theoretical concentration factor in this experiment.
1. estimated air volume sampled pr ml sampling liquid.
2. Air volume sampled.
3. std dev except slitsamplers,
4. std dev of 3 dilutions
CFU as % o f FCM
sampling data
CFU pr m3
FCM m3
CFU as % of FCM
n
FCM±std.
dev4
raw data
FCM±std.dev4
T0 substr
FCM raw data
FCM
data T0
subst.
18±13
4
1800±330
1600±330
1.0
1.1
0.261
33±35
4
2500±690
1400±690
1.3
2.4
60
0.0381
67±77
4
8800±2200
4200±2200
0.8
1.6
slit sampler
60
1.82
37
1
Mas-100
20
2.02
40±14
2
Sampler
time
(min)
volume
(m3)
CFU±err
XMX-CV
10
1.41
Karcher
120
SKC
3
Table xx. cfu and fcm bacterial counts in samples with the highest theoretical concentration factor in this experiment.
1. estimated air volume sampled pr ml sampling liquid.
2. Air volume sampled.
3. std dev except slitsamplers,
4. std dev of 3 dilutions
CFU as % o f FCM
sampling data
CFU pr m3
FCM m3
CFU as % of FCM
n
FCM±std.
dev4
raw data
FCM±std.dev4
T0 substr
FCM raw data
FCM
data T0
subst.
18±13
4
1800±330
1600±330
1.0
1.1
0.261
33±35
4
2500±690
1400±690
1.3
2.4
60
0.0381
67±77
4
8800±2200
4200±2200
0.8
1.6
slit sampler
60
1.82
37
1
Mas-100
20
2.02
40±14
2
Sampler
time
(min)
volume
(m3)
CFU±err
XMX-CV
10
1.41
Karcher
120
SKC
3
Table xx. cfu and fcm bacterial counts in samples with the highest theoretical concentration factor in this experiment.
1. estimated air volume sampled pr ml sampling liquid.
2. Air volume sampled.
3. std dev except slitsamplers,
4. std dev of 3 dilutions
T h e d i c e is thrown…
Evaluation
Background noise
Liquid volume
Sampling volume
Sampling time
Sampling efficience – concentration factors
Reliability
T h e d i c e is thrown…
XMX-CV
+ Robust
+ Low backgroud
+ Fast sampling
- Low liquid volume
T h e d i c e is thrown…
XMX-CV
SKC
+ Robust
+ ok background
+ Low backgroud
-Very low efficiency
+ Fast sampling
-Highest liquid loss
rate
- Low liquid volume
T h e d i c e is thrown…
XMX-CV
SKC
Kärcher
+ Robust
+ ok background
+ High volume rates
+ Low backgroud
-Very low efficiency
+ High volumes
+ Fast sampling
-Highest liquid loss
rate
- High volumes
- Low liquid volume
- Highest backgroud
In t h e end
 Bacteria in air pr m3:
 CFU
10-50
 FCM
~103
In t h e end
 Bacteria in air pr m3:
 CFU
10-50
 FCM
~103

Bacteria in snow/rain pr mL:
 CFU
10-200
 FCM
~104
 Bacterial activity in snow/rain ~ SW bacteria
In t h e end
 Bacteria in air pr m3:
 CFU
10-50
 FCM
~103

Sampler test:
1. XMX-CV
2. Kärcher
3. SKC
Bacteria in snow/rain pr mL:
 CFU
10-200
 FCM
~104
 Bacterial activity in snow/rain ~ SW bacteria
Sampler test:
1. XMX-CV
2. Kärcher
3. SKC
In t h e end
 Bacteria in air pr m3:
 CFU
10-50
 FCM
~103

Bacteria in snow/rain pr mL:
 CFU
10-200
 FCM
~104
Thanks to:

University of Bergen, Dep Biology
 Jørn Einen
 Ruth-Anne Sandaa
 Gunnar Bratbak
 Mikal Heldal

Norwegian Defence Research Establishment
 Janet Martha Blatny
 Gunnar Skogan
 Else-Marie Fykse
 Tone Aarskaug
 Bacterial activity in snow/rain ~ SW bacteria