airborne bacteria: names and numbers, please…vali/perugia07/talks/thyrhaug.pdf · airborne...
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AirborneAirborne bacteriabacteria::
namesnames and and numbersnumbers,,
pleaseplease……Runar Runar ThyrhaugThyrhaugDep. Dep. ofof BiologyBiologyUniversityUniversity ofof Bergen BergenNorwayNorway
[email protected]@bio.uib.no
My My backgroundbackground
MicrobialMicrobial ecologyecology Marine Marine microbiologymicrobiology group-group- Bergen, Bergen, NorwayNorway FocusFocus: : algaealgae, , bacteriabacteria, , protozooaprotozooa, virus, virus
My My backgroundbackground
MicrobialMicrobial ecologyecology Marine Marine microbiologymicrobiology group-group- Bergen, Bergen, NorwayNorway FocusFocus: : algaealgae, , bacteriabacteria, , protozooaprotozooa, virus, virus MethodsMethods
NutrientNutrient uptakeuptake/turnover/turnover ProductionProduction rates rates GeneticGenetic diversitydiversity//biodiversitybiodiversity ViralViral activityactivity ModellingModelling ( (vsvs experimentsexperiments))
WhyWhy airborneairborne bacteriabacteria??
CuriosityCuriosity……newnew fieldfield BiodiversityBiodiversity and transport rates and transport rates MicrobialMicrobial productionproduction ImprovedImproved//newnew methodsmethods……possibilitiespossibilities??
BacteriaBacteria in in thethe air air
……from from wherewhere??BacteriaBacteria VirusVirus
SoilSoil ( (terrestrialterrestrial)) ~10~1099 g g-1-1 ????
SeaSea / /freshfresh water water ~10~1066 mL mL-1-1 ~10~1077 mL mL-1-1
BacteriaBacteria in in thethe air air
……from from wherewhere??BacteriaBacteria VirusVirus
SoilSoil ( (terrestrialterrestrial)) ~10~1099 g g-1-1 ????
SeaSea / /freshfresh water water ~10~1066 mL mL-1-1 ~10~1077 mL mL-1-1
WeWe know: know: Most marine Most marine bacteriabacteria areare assumedassumed to be to be alivealive NutrientNutrient limited N , P or C limited N , P or C <1% <1% growinggrowing onon agar plates ( agar plates (seasea water and water and soilsoil samples!) samples!)
CountingCounting airborneairborne
bacteriabacteria LiquidLiquid samples! samples!
RainRain or air or air sampledsampled in in liquidsliquids MethodsMethods::
FlowFlow cytometrycytometry (FCM) (FCM) DNA/RNA DNA/RNA stainingstaining - fluor / - fluor /scatterscatter signals from single signals from single particlesparticles
QuantitativeQuantitative PCR (Q-PCR) PCR (Q-PCR) DNA DNA stainingstaining –– amountamount ofof bacterialbacterial DNA in a bulk sample DNA in a bulk sample
ColonyColony forming forming unitsunits (CFU) (CFU) BacteriaBacteria willingwilling to to growgrow onon agar plates agar plates
EpifluorescenseEpifluorescense microscopymicroscopy (EFM) (EFM) DNA/RNA DNA/RNA stainingstaining - fluor signals from single - fluor signals from single particlesparticles
FCM plot / EFM FCM plot / EFM picturepicture
seasea water water examplesexamples::
FCM plot / EFM FCM plot / EFM picturepicture
seasea water water examplesexamples::
Marine bacteria
FCM plot / EFM FCM plot / EFM picturepicture
seasea water water examplesexamples::
Marine viruses
Marine bacteria
FCM plot / EFM FCM plot / EFM picturepicture
seasea water water examplesexamples::
Marine viruses
Marine bacteria
Emiliania huxleyi virusisolate (EhV)
EhV
Sampling Sampling airborneairborne
bacteriabacteria
Xmx-cvXmx-cv ( (DycorDycor)) 0,7 m0,7 m33 min min-1-1
5 5 mLmL PBS PBS Suggested Suggested maxmaxsampling time: 5 minsampling time: 5 min OurOur maxmax: 10 min: 10 min
FlowFlow cytometrycytometry o fo f air air
samplessamplesT0=BlankT0=Blank
Fluo
res c
e nce
Scatter signal
FlowFlow cytometrycytometry o fo f air air
samplessamplesT0=BlankT0=Blank
T5~3,5m3 in 5 T5~3,5m3 in 5 mLmL
Fluo
res c
e nce
Scatter signal
Fluo
resc
ence
Scatter signal
FlowFlow cytometrycytometry o fo f air air
samplessamplesT0=BlankT0=Blank
T5~3,5m3 in 5 T5~3,5m3 in 5 mLmL
~103 pr m3
Fluo
res c
e nce
Scatter signal
Fluo
resc
ence
Scatter signal
FlowFlow cytometrycytometry vsvs Q- Q-
PCRPCR Q-PCR:FCM ~10Q-PCR:FCM ~10 CalibrationCalibration goinggoing onon!!
Q-PCR (Log bact pr m3)
0 2 4 6 8
FCM
(Log bact pr m3 )
0
2
4
6
8 Air: Air: yellowyellow RainRain: red: red SeaSea water:bluewater:blue
FlowFlow cytometrycytometry vsvs Q- Q-
PCRPCR Q-PCR:FCM ~10Q-PCR:FCM ~10 CalibrationCalibration goinggoing onon!!
Q-PCR (Log bact pr m3)
0 2 4 6 8
FCM
(Log bact pr m3 )
0
2
4
6
8
CFU: CFU: aroundaround 1-2% 1-2%comparedcompared to FCM to FCM
EpifluorescenceEpifluorescence microscopymicroscopy::difficultdifficult to to discriminatediscriminatesignal/signal/noisenoise
Air: Air: yellowyellow RainRain: red: red SeaSea water:bluewater:blue
SnowSnow and and rainrain samples samples
snowsnow ~500-10 ~500-1044 pr pr mLmL RainRain ~10 ~1044 pr pr mLmL
SnowSnow and and rainrain samples samples
snowsnow ~500-10 ~500-1044 pr pr mLmL RainRain ~10 ~1044 pr pr mLmL
Bacterial production:
Addition of H3 leucine, 24 h incubation
SnowSnow and and rainrain samples samples
snowsnow ~500-10 ~500-1044 pr pr mLmL RainRain ~10 ~1044 pr pr mLmL
Bacterial production:
Addition of H3 leucine, 24 h incubation
Result: activity pr bacteria ~ as in sea water
Estimated generation time~ 1day
ComparisonComparison o fo f 3 3
bioaerosolbioaerosol samplers samplers Time seriesTime series FCM and CFU FCM and CFU countscounts LiquidLiquid loss rates loss rates
Sampler 1Sampler 1
Xmx-cvXmx-cv ( (DycorDycor)) 0,7 m0,7 m33 min min-1-1
5 5 mLmL Suggested Suggested maxmaxsampling time: 5 minsampling time: 5 min OurOur sampling time 0-10 min sampling time 0-10 min LiquidLiquid: PBS: PBS ~10% PBS loss ~10% PBS loss afterafter 10 min 10 min
Sampler 2Sampler 2
SKC 225-9595 (SKC)SKC 225-9595 (SKC) 0,0125 m0,0125 m33 min min-1-1
Sampling time 0-60 minSampling time 0-60 min LiquidLiquid: : MilliQMilliQ Refill~25% Refill~25% milliQmilliQ at 10 min at 10 min
Sampler 3Sampler 3
KKäärcherrcher DS 5550 DS 5550
Sampler 3Sampler 3
KKäärcherrcher DS 5550 DS 5550 3,3 m3,3 m33 min min-1-1
1500 1500 mLmL Max sampling time: 120 minMax sampling time: 120 min LiquidLiquid: PBS: PBS ~10% ~10% pbspbs loss loss afterafter 120 min 120 min
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)
FCM FCM countscounts - time series - time series
Bacterial concentration (m
L-1)
0
200
400
600
800
1000
Sampling time (min)0 20 40 60 80 100 120 140
Estimated bact conc (m
-3)
0
1000
2000
3000
4000
200004000060000
no correctionT0 substracted
Karcher - FCM
Bacterial concentration (m
L-1)
0
500
1000
1500
2000
2500
3000
Sampling time (min)0 2 4 6 8 10
Estimated bact conc (m
-3)
0
1000
2000
400060008000
no correctionT0 substracted
XMX - FCM
Bacterial concentration (m
L-1
)
0
100
200
300
400
500
600
Sampling time (min)0 20 40 60 80
Estimated bact conc (m
-3)
0
2000
4000
6000
8000
10000
1200020000
40000no correctionT0 substracted
SKC - FCM
CFU CFU countscounts - time - time
seriesseries
Bacterial concentration (m
L-1
)
0
10
20
30
40
50
Sampling time (min)0 20 40 60 80
Bact concentration (m
-3)
020406080
100120140160180200
1000
2000
3000no correctionT0 substracted
SKC - CFU
Bacterial concentration (m
L-1
)
0
10
20
30
40
50
Sampling time (min)0 2 4 6 8 10
Bact concentration (m
-3)
0
20
40
60
80
no correctionT0 substracted
XMX - CFU
Bacterial concentration (m
L-1
)
0
5
10
15
20
25
30
35
Sampling time (min)0 20 40 60 80 100 120 140
Bact concentration (m
-3)
020406080
100120140160180200
no correctionT0 substracted
Karcher - CFU
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)
CFU as % o f FCM
240±142.0220Mas-100
1371.8260slit sampler
1.60.84200±22008800±2200467±770.038160SKC
2.41.31400±6902500±690433±350.261120Karcher
1.11.01600±3301800±330418±131.4110XMX-CV
FCMdata T0subst.
FCM raw dataFCM±std.dev4
T0 substr
FCM±std.dev4
raw datanCFU±err3volume
(m3)time(min)
CFU as % of FCMFCM m3CFU pr m3sampling data
Sampler
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
240±142.0220Mas-100
1371.8260slit sampler
1.60.84200±22008800±2200467±770.038160SKC
2.41.31400±6902500±690433±350.261120Karcher
1.11.01600±3301800±330418±131.4110XMX-CV
FCMdata T0subst.
FCM raw dataFCM±std.dev4
T0 substr
FCM±std.dev4
raw datanCFU±err3volume
(m3)time(min)
CFU as % of FCMFCM m3CFU pr m3sampling data
Sampler
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
240±142.0220Mas-100
1371.8260slit sampler
1.60.84200±22008800±2200467±770.038160SKC
2.41.31400±6902500±690433±350.261120Karcher
1.11.01600±3301800±330418±131.4110XMX-CV
FCMdata T0subst.
FCM raw dataFCM±std.dev4
T0 substr
FCM±std.dev4
raw datanCFU±err3volume
(m3)time(min)
CFU as % of FCMFCM m3CFU pr m3sampling data
Sampler
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
TheThe dicedice is is thrownthrown……
Evaluation
Background noise
Liquid volume
Sampling volume
Sampling time
Sampling efficience – concentration factors
Reliability
TheThe dicedice is is thrownthrown……
XMX-CV
+ Robust
+ Low backgroud
+ Fast sampling
- Low liquid volume
TheThe dicedice is is thrownthrown……
XMX-CV
+ Robust
+ Low backgroud
+ Fast sampling
- Low liquid volume
+ ok background
-Very low efficiency
-Highest liquid lossrate
SKC
TheThe dicedice is is thrownthrown……
XMX-CV
+ Robust
+ Low backgroud
+ Fast sampling
- Low liquid volume
+ ok background
-Very low efficiency
-Highest liquid lossrate
SKC KKäärcherrcher
+ High volume rates
+ High volumes
- High volumes
- Highest backgroud
In In thethe end end
BacteriaBacteria in air pr m in air pr m33:: CFUCFU 10-5010-50 FCMFCM ~10~1033
In In thethe end end
BacteriaBacteria in air pr m in air pr m33:: CFUCFU 10-5010-50 FCMFCM ~10~1033
BacteriaBacteria in in snowsnow//rainrain pr pr mLmL:: CFUCFU 10-20010-200 FCMFCM ~10~1044
BacterialBacterial activityactivity in in snowsnow//rainrain ~ SW ~ SW bacteriabacteria
In In thethe end end
BacteriaBacteria in air pr m in air pr m33:: CFUCFU 10-5010-50 FCMFCM ~10~1033
BacteriaBacteria in in snowsnow//rainrain pr pr mLmL:: CFUCFU 10-20010-200 FCMFCM ~10~1044
BacterialBacterial activityactivity in in snowsnow//rainrain ~ SW ~ SW bacteriabacteria
Sampler test:Sampler test:1.1. XMX-CVXMX-CV2.2. KKäärcherrcher3.3. SKCSKC
In In thethe end end
BacteriaBacteria in air pr m in air pr m33:: CFUCFU 10-5010-50 FCMFCM ~10~1033
BacteriaBacteria in in snowsnow//rainrain pr pr mLmL:: CFUCFU 10-20010-200 FCMFCM ~10~1044
BacterialBacterial activityactivity in in snowsnow//rainrain ~ SW ~ SW bacteriabacteria
Sampler test:Sampler test:1.1. XMX-CVXMX-CV2.2. KKäärcherrcher3.3. SKCSKC
UniversityUniversity ofof Bergen, Dep Bergen, Dep BiologyBiology JJøørn rn EinenEinen Ruth-Anne Ruth-Anne SandaaSandaa Gunnar Gunnar BratbakBratbak Mikal HeldalMikal Heldal
NorwegianNorwegian DefenceDefence ResearchResearch Establishment Establishment JanetJanet Martha Martha BlatnyBlatny Gunnar SkoganGunnar Skogan Else-Marie Else-Marie FykseFykse Tone Tone AarskaugAarskaug
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