use of bacteria as model non spherical aerosol particles

8/4/2019 Use of Bacteria as Model Non Spherical Aerosol Particles

http://slidepdf.com/reader/full/use-of-bacteria-as-model-non-spherical-aerosol-particles 1/8

J . Aeros ol Sc i . , V o l 1 6 . N o . 3 , p p 1 9 3 2 0 0 . 1 9 8 5 0 0 2 1 8 5 0 2 i 8 5 $ 3 . 0 0 + 0 0 0

P n n t e d i n G r e a t B r i t a i n q 1 9 85 P e r g a m o n P r e ss L t d

U S E O F BA C T E RI A A S M O D E L N O N S P H E R I C A L A E R O S O L

P A R T I C L E S

A. J . ADAMS, D. E. WENNERSTROM* a nd M . K . M A ZU M D ER

University of Arkansas, Graduate Institute o f Techno logy, P. O. Box 3017, Little Rock, AR 72203, U.S.A. and• Department o f Microbiology and Immun ology, University of A rkansas lot Medical Sciences,

Little R ock, AR 72205, U.S.A.

(F i r . s t r ece i r ed 27 A u y u s t 1984: a n d i n r e r i s e d J o r m I I D e c e m b e r 19841

Abstract--Nonviable rod-shaped bacteria Baci l lu s . s uh t i l i s an d E . w h e r i c h i a c o i l and the sphericalbacterium S t a p h y l o c o c c u s e p i d e r m i d i s w ere aerosolized us ing the conventional technique o1"pneumatic atomization. Single-particle aerodynamic relaxation tim e (SPA RT) analysis was carriedout in order to determine the orientation-averaged aerodynamic size distribution for the aerosol.Results indicate that the com pressed -air nebulizer used in this study is effective n gene rating bacterialaerosols of both un iform nonspherical and mono disperse spherical particles. The aerodynamicdiameters as measured by the SPAR T technique agree with published data for the cell dimensions andoptical microscopic analysis. Fu rthe r, the S PA RT me tho d distinguished the two rod-shapedorganisms based on their aerodynamic diameter. Th is study demon strates that nonviable bacterialcells can serve as a model system in studying aerosols of nonspherical particles and illustrates thepotential of the SP ART technique in the rapid detection and characterization of airborne biologicalparticles.

I N T R O D U C T I O N

O n e a s p e c t o f a e r o s o l s c i e n ce c u r r e n t l y r e c e i v i n g c o n s i d e r a b l e a t t e n t i o n i s t h e s t u d y o f

n o n s p h e r i c a l p a r t i cl e s . N u m e r o u s s t u d i e s a i m e d a t c h a r a c t e r i z i n g t h e p h y s i c a l p r o p e r t i e s a n d

d y n a m i c b e h a v i o r o f n o n s p h e r i c a l a i r b o r n e p a r t i c u l a t e s h a v e b e en r e p o r t e d ( C o l e tt i , 19 84 ;

D a h n e k e , 1 9 8 2 ; G a l l i l y e t a l . , 1 98 3; H e y d e r a n d S c h e u c h , 1 9 8 3; K a s p e r a n d S h a w , 1 98 3; L i u e t

a l . , 1 9 8 3 ; M a z u m d e r e t a l . , 1 98 2). T h e s e e f f o r t s h a v e b e e n p r o m p t e d b y t h e f in d i n g t h a t m a n y

n a t u r a l a n d m a n - m a d e a e r o s o l s c o n t a i n n o n s p h e r i c a l p a r t i cl e s , s o m e o f w h i ch a r e r e g u l a r l y

s h a p e d ( fi b er s, se a s a l t- c r y st a l s , a n d c y l i n d ri c a l m i c r o o r g a n i s m s ) w h i l e o t h e r s a r e i r r e g u l a r i n

g e o m e t r y ( d u st s , c o m b u s t i o n a n d i n d u s t r i a l a e ro s o ls ~ . T h e d e v i a t i o n i n t h e d y n a m i c b e h a v i o r

o f a n o n s p h e r i c a l p a r t ic l e f r o m a n e q u i v a l e n t s p h e r i c al o n e i s m o d e l e d b y a s c r i b i n g t o t h e

p a r t ic l e a d y n a m i c s h a p e f a c t o r 7 ,, a d i m e n s i o n l e s s p a r a m e t e r w h i c h r e l a te s t h e t r u e d r a g f o r c e

t o a n e q u i v a l e n t v o l u m e d i a m e t e r ( se e H i n d s , 1 98 2, p . 4 7). I t i s p o s s i b l e t o d e r i v e a n a l y t i c a l

e x p r e s s i o n s f o r Z i n t e r m s o f t h e p a r ti c l e d i m e n s i o n s o n l y f o r s o m e s i m p l e r e g u l a r g e o m e t r i c

s h a p e s , s u c h a s e ll i p so i d s o f r e v o l u t i o n ( F u c h s , 1 96 4). F o r m o s t o f th e p a r t i c l e s h a p e s o f

i n t e re s t i t is n e c e s s a r y to m a k e e m p i r i c a l m e a s u r e m e n t s o f ~ a n d t h is h a s b e e n d o n e f o r

v a r i o u s r e g u l a r a n d i r r e g u l a r n o n s p h e r i c a l p a r t i c l e s ( D a v i e s , 1 97 9; N i i d a e t a l . , 1983) . One

o b j e c t i v e in p r o b i n g t h e p h y s i c a l p r o p e r t i e s o f n o n s p h e r i c a l p a r t ic l e s h a s b e e n t h e

d e v e l o p m e n t o f i n s t r u m e n t a t i o n c a p a b l e o f d e t e c t i n g a n d c h a r a c t e ri z i n g a s p h e r i c a l p a r t i cl e s

( D e t e n b e c k , 1 9 8 0 ) .

O n e e x p e r i m e n t a l d i ff i cu l t y w h i c h h a s i m p e d e d t h e s y s t e m a t i c c h a r a c t e r i z a t i o n o f

n o n s p h e r i c a l p a r t i c u l a t e a e r o s o l s i s t h e l a c k o f s u it a b l e m o d e l s y s t e m s . H i n d s ( 19 8 2) h a s

e m p h a s i z e d t h e i m p o r t a n c e o f m o n o d i s p e r s e a e r o s o l s o f k n o w n s iz e, s h a p e, a n d d e n s it y i n

a e r o s o l s c i e nc e a n d t e c h n o l o g y . T h e r e c e n t p r o l i f e r a t i o n o f d a t a d e s c r i b i n g a e r o s o l s o f

s p h e r i c al p a r t ic l e s is i n la r g e p a r t d u e t o t h e d e v e l o p m e n t o f m o n o d i s p e r s e l a te x p o l y m e r s ( f o r

d i s c u s s i o n s e e M e r c e r , 1 9 7 31 . A l t h o u g h s e v e r a l r e c e n t s t u d i e s h a v e e m p l o y e d l i n e a r c h a i n s o f

s p h e r i c a l p a r t i c l e s a s a m o d e l s y s t e m ( D a h n e k e , 1 98 2; K a s p e r a n d S h a w , 1 9 8 3; K a s p e r , 1 9 84 ),

t h e r e i s a t p r e s e n t n o r e a d i ly a v a i l a b l e c o m m e r c i a l s o u r c e o f u n i f o r m n o n s p h e r i c a l p a r t ic l e sf o r u s e a s t e s t a e r o s o l s .

I n t h is r e p o r t w e d e s c r ib e t h e g e n e r a t i o n a n d c h a r a c t e r i z a t i o n o f u n i f o r m a e r o s o l s

c o n t a i n i n g c y l in d r i ca l p a r ti c le s . W e h a v e f o u n d t h a t n o n v i a b l e r o d - s h a p e d b a c t e r i a in t h e s iz e

r a n g e a p p r o x . 1 ~4 ~ m c a n s e r v e a s s u i t a b l e r e g u l a r l y s h a p e d n o n - s p h e r i c a l p a r t ic l e s i n m o d e l

193

A S 1 6 / ~ - A



a e r o s o l s y s t e m s . A l t h o u g h t h e r e g u l a r i t y a n d u n i f o r m i t y o f b a c te r i a l p a r ti c le s a s , ~= ii a~

t h e i r a b i l it y t o r e m a i n i n t a c t a n d i n s o m e c as e s v ia b l e w h e n a e r o s o l i z e d h a v e i o ~ : i~ cc ~

r e c o g n i z e d , c y l in d r i c a l ly s h a p e d b a c t e r i a a p p a r e n t l y h a ~ e n o t b e e n x ~ d e l v e x p l o i t e d il~

a e r o s o l s t u d ie s . W e n o t e t h a t w i t h i n t h e f i el d o f a p p l i e d a n d e n v i r o n m e n t a l b i o l o g y ~ i ~c re ~

c u r re n t i n t e re s t i n b i o l og i c a l a e roso l s , spe c i f i c a l l y t he ge ne ra t i on , c ha ra c t e r i z a t i ons , a nd

b e h a v i o r o f a i r b o r n e b a c t e r i a l a n d v i r al p a r t i c le s t S o r d e l l i et al.. 1984: M oh r , 't al .. 19~4i . H ere

w e d e m o n s t r a t e t h a t b a c t e r ia o f b o t h s p h e r ic a l a n d r o d - s h a p e d m o r p h o l o g i e s ~ at~ b e

a e r o s o l i z e d b y t h e c o n v e n t i o n a l t e c h n i q u e o f p n e u m a t i c a t o m i z a t i o n , a s e~ i d e n c e d b y ~ i ng le -

p a r t ic l e a e r o d y n a m i c r e l a x a ti o n t i m e ( S P A R T ) a n al y si s. *

E X P E R I M E N T A L P R O C E D U R E

B i o l o g i c a l a e r o s o l s t u d i e s w e r e c a r r i e d o u t w i t h t h r e e b a c t e ri a . Baci l lus subt i l i s i l o n g r o d t .

E sc he r i c h ia c o l i ( s h o r t r o d ) , a n d S taphy loc oc c us e p ide rm id i s { s p he r e) w e r e o b t a i n e d f r o m t h e

s t o ck c u l t u r e co l le c ti o n o f t h e D e p a r t m e n t o f M i c r o b i o lo g y a n d I m m u n o l o g y , U n i v e rs i ty o f

A r k a n s a s f o r M e d i c a l Sc ie n ce s. E a c h o r g a n i s m w a s m a i n t a i n e d a t 4 C o n s l a nt s o f T o d d

H e w i t t b r o t h ( D i f c o ) s o li d i f ie d w i t h 1 .5 o ,, a g a r . F o r e x p e r i m e n t s , c el ls w e r e t r a n s f e r r e d b y

s t er il e l o o p t o 2 0 0 c m 3 o f T o d d H e w i t t b r o t h a n d i n c u b a t e d a t 3 7 c C i n a n o s c i ll a ti n g w a t e rb a t h w i t h s h a k i n g a t t l 0 c y c l e s / m i n . A f t e r i n c u b a t i o n f o r 1 8 h r , c e ll s w e r e h a r v e s t e d b y

c e n t r i f u g a t i o n a t 1 9 ,0 0 0 0 f o r 1 0 r a i n a t 5 ~C , w a s h e d o n c e i n w a t e r a n d i n a c t i v a t e d b y t h e

a d d i t i o n o f f o r m a l d e h y d e a t a f in a l c o n c e n t r a t i o n o f 0.7 ? o a n d i n c u b a t i o n f o r 1 h r a t r o o m

t e m p e r a t u r e . T h e c e l l s w e r e t h e n w a s h e d o n e t i m e b y c e n t r i f u g a t i o n a s a b o v e a n d

r e s u s p e n d e d i n w a t e r t h a t h a d b e e n f i lt e re d b y p a s s ag e t h r o u g h a c e ll u lo s e a c e t a te m e m b r a n e

w i t h a n a v e r a g e p o r e d i a m e t e r o f 0 . 4 5 /a m ( M i l li p o re ) . G r a m - s t a i n e d s m e a r s w e r e e x a m i n e d a t

1 00 0 X u s i n g a n A m e r i c a n O p t i c a l M i c r o s c o p e e q u i p p e d w i t h an o i l i m m e r s i o n o b j e c ti v e .

C o m p r e s s e d - a i r n e b u l i z a t i o n w a s u s e d t o g e n e r a t e a e r o s o l s o f b a c t e r i a l p a r t ic l e s . T h e

g e n e r a t o r , a U - M i d ® s in g l e- u s e j e t n e b u l iz e r ( B a r d - P a r k e r , R u t h e r f o r d , N e w J e r se y , U .S .A .

o r B e c t o n D i c k i n s o n , L i n c o l n P a r k . N e w J e r s e y , U . S . A . ) h a s a l i q u i d u s a b l e c a p a c i t y o f

5 0 0 m l a n d p r o d u c e s a e r o s o l p a r t i c l e s e ff i c ie n t ly i n t h e 0 . 3 ~ , / a m d i a m e t e r r a n g e . T h i s t y p e o fg e n e r a t o r i s e q u i p p e d w i t h a d i al f o r s e l e c ti n g a r e q u i r e d o x y g e n d i l u t i o n . T h i s w a s s et t o

100° /o in o u r s t ud i e s . F i l t e re d d ry a i r a t a t yp i c a l p re s su re o f 1 .03 x 105 P a (15 ps i g ) wa s

a p p l i e d t o t h e n e b u l i z e r , r e s u l ti n g i n a n a i r f l o w o f a p p r o x i m a t e l y 7 l / m i n. P r e v i o u s s t u d i e s

u s i n g p o l y l a t e x s p h e r e s ( P L S ) i n t h e n e b u l i z e r h a v e s h o w n t h a t i n t h e 1 # m d i a m e t e r p a r t i c l e

s iz e r a n g e a n i n it i al l iq u i d s u s p e n s i o n o f 10 9 p a r t i c l e s /c m 3 r e s u l t s in a n a e r o s o l c o n c e n t r a t i o n

o f 1 0 4 / cm 3. C o m p a r a b l e c o n c e n t r a t i o n s w e r e n o t e d w i t h b a c t e r ia l s a m p le s .

T h e p r i n c i p a l m e t h o d f o r a n a l y z i n g t h e b a c t e r ia l a e r o s o ls w a s s in g le p a r t ic l e a e r o d y n a m i c

r e l a x a t i o n t i m e ( S P A R T ) a n a l y s i s ( M a z u m d e r et al., 1 97 9) . T h i s t e c h n i q u e e m p l o y s a d u a l -

b e a m l a se r D o p p l e r v e l o c i m e t e r t o m o n i t o r s in g le p a r t ic l e d y n a m i c s i n a n o s c i l l at o r y a co u s t i c

f ie ld o f 2 4 k H z . T h e a e r o d y n a m i c d i a m e t e r o f i n d i v i d u al p a r t ic l es i s d e t e r m i n e d b y m e a s u r i n g

t h e p h a s e l a g o f t h e p a r t i c le m o t i o n r e l a ti v e t o t h e d r i v i n g a c o u s t i c e x c i t a t io n . T h e t i m em e a s u r e m e n t u s e d i n t h e p h a s e d e t e r m i n a t i o n i s m a d e i n s u c h a w a y t h a t 0 - r t/ 2 p h a s e l a g is

m e a s u r e d t o 1 p a r t i n 1 2 8 . A m i c r o c o m p u t e r is u s ed t o r e c o r d t h e c o u n t s i n e a c h o f th e 1 28

c h a n n e l s , t h e t o t a l n u m b e r o f c o u n t s N , a n d t h e t o t a l s a m p l i n g t i m e in s e c o n d s . T h e s iz e

d i s t r i b u t io n is d i s p l ay e d i m m e d i a t e ly b y a n o n - b o a r d p r i n te r . A p l o t t i n g r o u t i n e g e n e r al ly

p r e s e n t s t h e d a t a a s d N / d ( l o g d a ) a s i s , o r i n a n o r m a l i z e d f o r m .

T h e p h a s e l a g , a d i r e c t m e a s u r e o f t h e p a r t i c l e a e r o d y n a m i c r e l a x a t i o n t i m e r p , v a r i e s

s i g n i fi c a n t ly w i t h a e r o d y n a m i c d i a m e t e r o n l y i n th e f r e q u e n c y r a n g e c e n t e r e d a t c o zp = 1,

w h e r e o9 i s t h e a n g u l a r f r e q u e n c y o f th e a c o u s t i c e x c i t a t i o n w a v e . F o r a n a c o u s t i c f r e q u e n c y o f

2 4 k H z t h e S P A R T i n s t r u m e n t c a n e f f e c ti v e l y s iz e p a r t i cl e s w i t h h i g h r e s o l u t i o n i n t h e

0 . 3 - 6 / a m a e r o d y n a m i c d i a m e t e r r a n g e . R e c e n t ly t h e o p e r a t i n g r a n g e o f t h e s y st e m w a s

* The results reported here were presented n part at the 1984Annu al BiophysicalSocietyMeeting,San Antonio,Texas, U.S.A., 19-23 Febru ary 1984.



Bacteria as aerosols 195

e x t e n d e d t o i n c lu d e p a r t ic l e s i n t he 3 - 2 0 p m r a n g e b y o p e r a t i n g a t a n a c o u s t i c f r e q u e n c y o f

1 k H z ( A d a m s et al., 1984).

P r i o r t o e a c h s t u d y t h e S P A R T a n a l y z e r w a s c a l i b r a t e d u s i n g u n i f o r m l a t e x p a r t i c l e s .

S e p a r a t e s u s p e n s i o n s o f 0. 6 a n d 1 .0 9 p m d i a m e t e r P L S w e r e su b j e c te d t o a e r o s o l i z a t i o n b y

p n e u m a t i c a t o m i z a t i o n , y i e l d i n g m o n o d i s p e r s e a e r o s o l s w i t h c o n c e n t r a t i o n s o f 1 0 4 - 1 0 5

p a r t i c l e s / c m 3. T h e s e a e r o s o l s c o n t a i n i n g P L S p a r t i c l e s o f k n o w n s iz e s w e r e u s e d t o v e r i f y t h e

S P A R T c a li b ra t io n .

W a s h e d b a c t e r i a w e r e s u s p e n d e d i n d i s t il l e d w a t e r t o y i e ld c o n c e n t r a t i o n s o f a p -

p r o x i m a t e l y 1 0 9 / c m 3. T h e r e s u l t i n g c e l l s u s p e n s i o n ( 5 0 - 1 0 0 m l ) w a s t h e n a d d e d t o t h e

r e s e r v o i r o f t h e n e b u l iz e r a n d c o m p r e s s e d a i r a p p l ie d . T w o s a m p l i n g s c h e m e s w e r e e m p l o y e d :

b o t h r e s u l t e d i n si m i l a r r e c o r d e d s i ze d i s t r i b u t i o n s . I n o n e c a se a e r o s o l p a r t i c l es w e r e

g e n e r a t e d d i r e c t l y i n t o a 4 -1 . f l as k a n d t h e s a m p l e w a s d r a w n d i r e c t l y f r o m t h e f la s k w i t h o u t

d r y i n g a n d c h a n n e l e d t h r o u g h t h e S P A R T r e l a x a t i o n c h a m b e r w i t h in m i n u t e s o f g e n e r a t io n .

I n t h e s e c o n d c a s e a e r o s o l p a r t i c l e s w e r e f i r s t c h a n n e l e d t h r o u g h a c o n v e n t i o n a l s i l i c a g e l

d r y i n g c o l u m n ( o u t e r d i a m e t e r 5 .0 c m , i n n e r s c r e e n d i a m e t e r 2 c m , l e n g th 6 0 c m t a n d t h e n

p a s s e d t h r o u g h a 1 -m p o l y v i n y l c h i o r i d e t u b e ( 5 c m i n d i a m e t e r ) t o t h e S P A R T a n a l y z er . T h e

s a m p l e f l o w r a te t h r o u g h t h e i n s t r u m e n t is t y p ic a l ly 5 0 c m 3 / m i n a n d a t y p i ca l c o u n t r a t e f o r

t h e S P A R T s y s t e m i s 1 0 0 0 - 2 0 0 0 p e r m i n .

I n o r d e r t o o b t a i n i n i ti a l d a t a c o n c e r n i n g t h e p h y s i c a l s t a t e o f t h e a e r o s o l i z e d b a c t e r i a l

p a r t i cl e s , a s t u d y o f c e ll v ia b i l it y a n d p a r t i c l e c o u n t r a t e a s a f u n c t i o n o f t i m e f o l l o w i n g

a e r o s o l i z a t i o n i n a f l a s k w a s c a r r i e d o u t i n s e p a r a t e r u n s u s i n g B. subt i l i s a n d E . c o i l In t h i s

s t u d y t h e t r e a tm e n t w i t h f o r m a l d e h y d e w a s o m i t t e d i n th e p r e p a r a t i o n p r o c e d u r e . A t c e rt a i n

s e t t i m e i n t e r v a l s t h e b a c t e r i a l a e r o s o l w a s s a m p l e d b y d r a w i n g 1 0 m l i n t o a p l a s t i c s y r i n g e

c o n t a in i n g a p p r o x i m a t e l y 1 m l o f w a r m g r o w t h m e d i u m . T h e s y ri n g e w a s ca p p e d a n d s h a k en

t o e n t r a p t h e a e r o s o l i z e d b a c t e r i a a n d t h e s a m p l e w a s i m m e d i a t e l y p l a t e d b y a d d i n g t h e

m e d i u m t o m o l t e n a g a r. A t t h e s am e t i m e , th e a e r o s o l w a s s a m p l e d b y t h e S P A R T a n a l y z e r

a n d t h e n u m b e r o f p ar t ic l e s p e r u n i t t i m e w a s d e t e r m i n e d .

S e v e ra l a d d i t i o n a l e x p e r i m e n t s w e r e p e r f o r m e d i n o r d e r t o h e l p f a ci l it a te d a t a i n t e r p r e t -

a t i o n . T h e e f fe c t o f f o r m a l d e h y d e - t r e a t m e n t o n t h e ce ll si ze d i s tr i b u t i o n o f E. co i l w as

e x p l o r e d b y t h e m e a s u r e m e n t o f ' li v e ' c e ll s w h i c h h a d o n l y b e e n w a s h e d i n d i s t il l e d w a t e r . I n

a n o t h e r e x p e r i m e n t a s u s p e n s i o n o f E. co l i w a s s o n i c a t e d a t 2 0 0 W f o r 5 m i n a n d t h e n

a e r o s o l i z e d a n d s a m p l e d . T o v e r i f y t h a t t h e S P A R T i n s t r u m e n t c o u l d d i s t in g u i s h t h e s h o r t

r o d E. co l i f r o m t h e lo n g r o d B. subt i l i s , 1 c m 3 a l i q u o t s o f E . coli , s t a n d a r d i z e d t o a t u r b i d i t y o f

4 0 0 K l e t t u n it s ( K l e t t - S u m m e r s o n p h o t o m e t e r w i t h b l u e f il te r) w e r e a d d e d s e q u e n t ia l l y to

7 5 c m 3 o f B. subt i l i s d i l u t e d 1 : 4 f r o m a n o r i g i n a l s u s p e n s i o n o f 4 0 0 K l e t t u n i t s . T h e s i z e

d i s t r i b u t i o n o f t h e p a r ti c le s w a s d e t e r m i n e d f o l l o w i n g a e r o s o l i z a ti o n a f t e r e a ch a d d i t i o n . I n a

s e p a r a t e e x p e r i m e n t , t h e a e r o s o l o f B. subt i l i s p a r t i c l e s w a s c o l l e c te d o n a 0 .2 p m N u c l e p o r e

p o l y c a r b o n a t e f i lt er , t h e p a r t i c u la t e s r e s u s p e n d e d i n w a t e r b y v i g o r o u s s h a k i n g , a n d t h e

s e d i m e n t a f t e r c e n t r i f u g a t i o n p l a c e d o n a o p t i c a l m i c r o s c o p e s l id e f o r o b s e r v a t i o n . A n

a d d i t i o n a l c o n t r o l e x p e r i m e n t i n w h i c h b a c t e r i a l s u s p e n s i o n s w e r e f i l te r e d t h r o u g h 0 . 2 # mN u c l e p o r e b y v a c u u m f i l t r a ti o n a n d t h e f i lt r a t e u s e d t o g e n e r a t e a e r o s o l w a s a l so c a r r i e d o u t .

R E S U L T S A N D D I S C U S S I O N

T h e m o r p h o l o g i e s o f B. subt i l i s , E . co l i , a n d S. ep idermidis a r e w e ll k n o w n . B e r g e y ' s M a n u a l

o f De te rmin a t i ve Ba c te r io lo g y ( B u c h a n a n a n d G i b b o n s , 1 9 7 4 ) l i s t s B. subt i l i s a s a r o d ,

0 . 7 - 0 . 8 / ~ m x 2 - 3 pro, E. coli a s a rod 0 .4 -0 .7 ~ tm × 1 -3 / ~ m , a nd S. ep idermidis a s a s p h e r e w i t h

d i a m e t e r 0 . 5 - 1 .5 p m . O p t i c a l m i c r o s c o p i c a n a l y s is o f d r i e d s t a in e d s m e a r s o f o u r p r e p a r -

a t i o n s c o n f i r m e d t h e s e s h a p e s a n d d i m e n s i o n s . T o c a l c u l a t e p r e d i c t e d m e a n a e r o d y n a m i c

d i a m e t e r s t h e f o l l o w i n g d i m e n s i o n s a r e a s s u m e d : 0 . 7 5 x 3 .0 # m f o r B. subt i l i s , 0.5 x 1.5 pmf o r E. coli, a n d 1 .0 0 g m f o r S. ep idermidis . N o t e t h a t t h e s e f i g u r es a r e c o n s i s t e n t w i t h B e rg ey ~s

d a t a . T h e a e r o d y n a m i c d i a m e t e r d a o f a n o n s p h e r i c a l p a r t i cl e c an b e r e l a t e d t o a n e q u i v a l e n t

v o l u m e d i a m e t e r d e b y :

cl~ = ppd~ , poZ, t l )



wh e re pp i s t he pa r t i c l e ma ss de n s i t y , P o i s un i t de ns i t y , a nd Z t s t he dyn a m i c sha p e I a c l o r lz~

o r d e r t o a p p l y e q u a t i o n ( l i f o r c a lc u l a ti n g p r e d i c t e d a e r o d y n a m i c d i am e t e r s , ~ al ue s t o t m a s s

d e n s i t y a n d s h a p e f a c t o r m u s t b e p r e s u m e d . S p e c i fi c d e n s i t y v a lu e s f r o m 1 0 7 t o i.25 g c m ?

h a v e b e e n r e p o r t e d f o r b ac t e r ia ( L a m a n n a e t al.. 1 9 7 3 L N o a t t e m p t t o i n d e p e n d e n t l ~

de t e rm i ne t he ma ss de n s i t y o f t he a e roso l i z e d b a c t e r i a l c el ls wa s ma de . B e c a use t~t t he

u n c e r t a i n t y i n p p w e c a lc u l a t e d p r e d i c t e d d i a m e t e r s f o r s p e c if i c d e n s i t 5 v a l u e s o i ! a nd .

1 .1 6 g / c m 3 {m e a n o f r a n g e c i t e d a b o v e ) . A s s u m i n g c y l i n d r i c al s h a p e f o r t h e r o d - s h a p e d

b a c t e r i a , p u b l i s h e d d a t a f o r t h e s h a p e f a c t o r s c h a r a c t e r i s t i c o f d is t i n ct L D v a l u es c a n b e

u s e d . I n o u r s a m p l i n g p r o c e d u r e t h e R e y n o l d s n u m b e r i s l es s t h a n 0 .1 a n d t h u s n o o r i e n t a t i o n

i s p r e s u m e d . A n o r i e n t a t i o n a v e r a g e d e t e r m i n e d b y

X., = 2Xh,3 + X~/3, (2 )

w h e r e Xh i s t he sha pe fa c t o r fo r c y l i nd r i c a l a x i s ho r i z on t a l i n a ve r t i c a l f l ow f i e l d a nd X, t he

v a l u e f o r l o n g i t u d i n a l o r i e n t a t i o n , i s u s e d t o c a l c u l a t e p r e d i c t e d v a l u e s f o r d ~. V a lu e s t b r Zh

a n d X~, we re t a ke n fo l l ow i ng F uc h s 11964 , p . 41) .

F i g u r e 1 p r e s e n t s a ty p i c a l n o r m a l i z e d s i z e d i s t r i b u t i o n f o r E. coil a s r e c o r d e d b y t h e

S P A R T a n a ly z e r . A s n o t e d i n T a b l e 1, w h i c h c o m p i l e s th e r e s u lt s o f c o m p a r i n g t h e m e a s u r e d

S P A R T v a lu e s w i th t h o s e c a l c u la t e d b y ( l t a n d (2 ), a n a v e r a g e a e r o d y n a m i c d i a m e t e r o f0 . 8 9 / J m w a s m e a s u r e d . T h e S P A R T v a l u e c o m p a r e s f a v o r a b l y w i t h t h e c a l cu l a t ed v a l u e o f

0 . 8 3 / ~ m ( a s s u m i n g a d e n s i t y o f 1 .1 6) . I n c l u d e d i n F i g . 1 is t h e d i s t r i b u t i o n f r o m t h e s o n i c a t e d

s u s p e n s i o n , i n d i c a t i n g t h a t ( 1) i n t h e i n i ti a l c a s e t h e p e a k i n t h e d i s t r i b u t i o n is d u e t o w h o l e

c e ll s a n d ( 2) u n d e r t h e s p e c i f ic s o n i c a t i o n c o n d i t i o n s s o m e i n t a c t c el ls r em a i n . W e n o t e h e r e

t h a t t h e r e w a s n o s i g n i f ic a n t d i f f e r e n c e in t h e s i ze d i s t r i b u t i o n b e t w e e n q iv e" c e ll s a n d t h o s e

p r e t r e a t e d w i t h f o r m a l d e h y d e ,

I n F i g . 2 a r e p r e s e n t a t i v e n o r m a l i z e d d i s t r i b u t i o n f o r B. subtilis i s g i ve n . An a ve r a ge va l ue

f o r fi v e p r e p a r a t i o n s o f 1 .2 9 ~ m w a s o b t a i n e d w i t h t h e S P A R T a n a l y z e r , w h i l e c a l c u l a t e d

. . . d

O' 1

Z

i . - . ,

1. O0

O. 900

O . 8 0 0 .

O.70 0

0.600

O.SO 0

O. 400

O. 300

0.:~00,

0 . 1 0 0 .

O.0 . 1 0

/

. /0.20 0.50 1.0 2.0 S.O

RERODYr4~IlC DIRI1ETER (IIICROMETERS]

F i g . 1. N o r m a l i z e d a e r o d y n a m i c s i z e d i s t r i b u t i o n [ ( 1 / N ) d N / d l l o g d =) v s d = ] f o r a e r o s o l i z e d E . ¢ o lt a,~

m e a s u r e d b y t h e S P A R T a n a l y z e r b e l o r e ( - . - o - - } a n d a f t e r [ - - O - - ) s o n i c a t i o n o f th e s u s p e n si o n .

T h e c o u n t m e d i a n a e r o d y n a m i c d i a m e t e r , f o r a p p r o x i m a t e l y 1 00 0 p a r t ic l e s s a m p l e d p e r m i n u t e , w a s

0 .8 91 ~ m b e f o r e s o n i c a t i o n a n d 0 . 6 1 4 # m a f t e r s o n i c a t i o n . T h e g e o m e t r i c s t a n d a r d d e v i a t i o n s w e r e1~15 and 1 .29 , r e spec t iv e ly .

1 0 .



B a c t e r i a a s a e r o s o l s

T a b l e 1. P r e d i c t ed a n d m e a s u r e d v a l u e s o f o r i e n t a t io n - a v e r a g e d a e r o d y n a m i c d i a m e t e r

19 7

M e a s u r e d

o r i e n t a t i o n

N u m b e r o f G e o m e t r i c a l A s s u m e d P r e d ic t e d a v e r a g e d D i f f er e n c e

S a m p l e p r e p a r a t i o n s s i ze (/ am ) d e n s i t y ( g / a m 3 ) d,, (urn) d o in (/lm ) ( ' :,,~

B. s ub t i l i s 5 0 .75 x 3 .0 1 .0 1 .19 1 .29 +- 0 .06 7 .71.16 1.29 0.0

E. co l i 5 0 .5 x 1 .5 1 .0 0 .76 0 .89+_ 0 .05 14 .6

1.16 0 .83 6 .7

S . e p i d e r m i d i s 2 1.0 1.00 1.00 0.9 2 +_ 0.0 8 8.(1

1.16 1.08 15.0

values of 1.19/~m and 1.29 were predicted based on the assumptions previously cited. We note

the presence o f a secondary peak at approximately 0.85 #m. The microscopic analysis of

aerosol particles collected by filtrat ion revealed that particles of this smaller size were present

to a minor extent in the aerosol. Rather than cell fragments these particles appeared to be

coalesced cytoplasmic material. We did find that when either Todd- Hewitt broth or istonicsaline was present in the cell suspension a significant amount of particles in the 0.65-0.85/~m

diameter size range was recorded by the SPART instrument. The photomicroscopy of

collected aerosol particles also revealed the minor presence of cell coats apparently emptied

of intracellular material. These 'shells' may also contribute to the secondary peak at 0.85/~m.

A typical SPART distribution for S. ep idermidis is given in Fig. 3.

The results of generating an aerosol from a mixture ofE . col i and B. subt i l i s are presented in

Fig. 4. The SPART technique is capable of distinguishing the two particles based on

aerodynamic behavior. The two curves represent successive additions of E. co l i suspension to

a suspension ofB. subtil is . We note here that in our control experiment, in which filtrate after

passage through a 0.2 #m filter was used to generate aerosol, the particle count rate dropped

from 1000 per min to 35 per min, with the bulk of these counts occurring at 0.5 #m.

.. J

L n

5

2

Ol f

1.00

O. 900

O. 800

O. 700

0.600

O. 500

O. 400

O. 300

O. 200

0.100

O.

0. I0

t

• , , , , , •

0.20 0.50 1.0 2.0 S.O 10.R£RODYHRMIC DIflrlETER (MICROMETERS]

F i g . 2 . R e p r e s e n t a t i v e n o r m a l i z e d a e r o d y n a m i c s iz e d i s t r i b u t i o n f o r B. s uh t i l i s a s m e a s u r e d b y t h e

S P A R T t e c h n iq u e , c o r r e s p o n d i n g t o a c o u n t m e d i a n a e r o d y n a m i c d i a m e t e r o f 1 . 2 9 ~ m a n d a

g e o m e t r i c s t a n d a r d d e v i a t i o n o f 1 . 2 9 .



Z

,...,

1 . 0 0

O . 9 0 0

O. 8 0 0

O . ? 0 0 •

O. 6 0 0

0 . 5 0 0

0 . 4 0 0 .

0 . 3 0 0 .

O. 2 0 0

0 . I 0 0

O.

0 . 1 0 0 . ' 2 0 0 . ' S 0 1 . 0 2 ~ 0 S . 0 1 0 ,

R ER OD YI~ R r l I c D IP ,M ETER C r l I cR o r IETER S)

F i g . 3 . N o r m a l i z e d a e r o d y n a m i c s i z e d i s t r i b u t i o n f o r S. epidermidis a s m e a s u r e d b y t h e S P A R T

analyzer . The peak in the s ize d is t r ibu t ion occurs a t 0 .88 .um.

_J

e,,t

Z

2,

1 . 0 0 ,

O . 900

O . 8 0 0 \

O. 7 0 0

0 . 6 0 0

o . S O 0

O. 4 0 0

O. 2 0 o

o . 2 0 0

0 . I 0 0

o .

0 . 1 0 0 . 2 0 0 . 5 0 1 . 0 2 . 0 S .OR ER OD YN R r l IC OIR r lETER ( t l IC R O r ET ER $ )

F i g . 4 . N o r m a l i z e d s iz e d i s t r i b u t i o n s f o r a e r o s o ls f r o m a m i x t u r e o f E. co / i a n d B. sub t i l i s . T h e

m i x t u r e s w e r e p r e p a r e d b y a d d i n g a t o t a l o f I c m 3 ( o ) o r 2 c m 3 g l ) o f E . c o l i s u s p e n s i o n t o 7 5 c m 3

o f B. subti l is .

1 0 .



Bacteria as aerosols

Table 2. T ime variation o f count rate and cell viability

199

Time following aerosol Relative count RelativeSamp le generation (mint rate viability

B. suh tilis < 1 1.130 1.005 0.47 0.17

12 0.32 0.0320 0.22 0.004

E. coil < 1 1.00 1.00

5 1.00 0.1810 0.83 0.1715 0.58 0.0525 0.38 0.02

T h e o u t c o m e o f t h e s t u d y o f t h e v a r i a t io n o f c o u n t r a te a n d c ell v ia b il it y is p r e s e n t e d i n

T a b l e 2 . G e n e r a l l y b a c t e r i a l a e r o s o l s w e r e g e n e r a t e d f o r a p p r o x i m a t e l y 1 m i n i n a 4 l it e r

f l as k a n d t h e s a m p l i n g t i m e s r e p o r t e d i n T a b l e 2 r e fe r to t h e d e la y s f o l l o w i n g th i s p r o c e d u r e .

R e l a t i v e c o u n t r a t e a n d r e l a ti v e v i a b i li t y r e fe r t o t h e i n it ia l v a lu e . F o r a e r o s o l g e n e r a t i o n u s i n gt he U - M i d ® n e b u li z er t h e S P A R T c o u n t r a t e fo r B . s u b t i l i s d e c r e a s e s m o d e r a t e l y f a s t ( t o

4 7 °//o i n 5 m i n ) a n d t h e r e l a t i v e v i a b i l i t y d r o p s t o 1 7 ~ o. A s c a n b e s e e n , m u c h o f t h e d r o p i n

v i ab l e c o u n t s i s d u e t o t h e r e d u c e d p a r t ic l e c o u n t . T h e c o u n t r a t e f o r E . c o i l d o e s n o t a p p e a r t o

d e c r e a s e i n th e f i r s t 5 r a i n w h i l e t h e v i a b l e c o u n t f a ll s to 1 8 ~ o. T h u s , t h e d a t a r e f l ec t a t r u e l o s s

i n v ia b i li ty . T h e g e n e r a l r e s u l t o f t h e g r e a t e r l o s s in p a r t i c le c o u n t f o r t h e l a r g e r p a r t i c l e s ( B.

s u b t i l i s ) , a l t h o u g h p r o b a b l y i n f lu e n c e d b y th e m e t h o d o f a e r o s o l i z a t i o n a n d p a r ti c le s h a p e , is

c o n s i s t e n t w i t h o u r e m p i r i c a l o b s e r v a t i o n s w i t h p o l y s t y r e n e l at e x a e r o s o l p r e p a r a t i o n s

( u n p u b l i s h e d ) .

C O N C L U S I O N S

W e h a v e d e m o n s t r a t e d t h a t E . c o l i , B . s u b t i l i s , a n d S . e p i d e r m i d i s c a n b e a e r o s o l i z e d b y

p n e u m a t i c a t o m i z a t i o n , t h a t t h e a i r b o r n e r o d - s h a p e d b a c t er i al p a r t ic l es c a n s e r v e a s te s t

a e r o s o l s o f n o n s p h e r i c a l p a r ti c le s , t h a t t h e S P A R T a n a l y z e r c a n a e r o d y n a m i c a l l y s i ze t h e

c e l ls o n a s i n g l e p a r t i c l e b a s i s t o w i t h i n 1 0 ~ o o f t h e e x p e c t e d v a l u e , a n d t h a t B . s u b t i l i s a n d E .

c o l i c a n b e c l e a r l y d i s t i n g u i s h e d b a s e d o n a e r o d y n a m i c s i z e . I n a d d i t i o n , v i a b i l i t y o f E . c o l i

a n d B , s u b t i l i s d e c r e a s e s ig n i fi c a n tl y af t e r 5 m i n . T h e s e r e s u l ts s h o w t h a t t h e S P A R T s y s t e m

r a p i d l y d e t e ct s a n d c h a r a c t e r i z e s a i r b o r n e m i c r o b e s . T h e r e f o r e , i t h a s p o t e n t i a l i n th e s t u d y o f

a e r o s o l i z a t i o n t e c h n i q u e s, c e ll v ia b i li ty , a n d t h e m o r p h o l o g i c a l r e s p o n s e o f b i o a e r o s o l

p a r ti c le s t o v a r y i n g e n v i r o n m e n t a l c o n d i t i o n s .

Ac k n o wle d g e me n t - - We gladly acknowledge helpful discussions w ith J. D. W ilson, technical help from R. Evans and

P. Archer, and secretarial assistance from D . Belk. The authors also wish to thank the reviewers of the m anuscript forconstructive suggestions.

R E F E R E N C E S

Adam s, A. J. , Tennal, K. B., Wilson, J. D . and Mazum der, M. K. (1984) Abstract, 15th Annual Meeting o f the FineParticle Society, 30 July -I August 1984. Orlando, FL, U .S,A.

Coletti, A. (1984) Aerosol Sci. Tech. 3, 39.Dahneke, B. (1982) Aerosol Sci. Tech. 1, 179.Davies, C. N . (1979) J. Aerosol Sci. 10. 477.Detenb eck, R. W. (1980)Technical Report o n Feasibility Study for an Asbestos Aerosol Monitor, R eport N o. EPA -

6002-80-200, U .S. Environmental Protection A gency.Fuch s, N . A . (19641 The Mechanics of Aerosols . Pergamon Press, New York.Gallily. I., Krushkal, E. M. and Garze, E. (1983) Aerosol Sci. Tech. 2, 209.

Heyder. J. and Scheuch, G. (1983) Aerosol Sci. Tech. 2, 41 .Hinds, W. C. (1982) Aerosol Technology. Wiley, New York.Kasper, G. and Shaw. D. T. (19831 Aerosol Sci. Tech. 2, 369.Kasper, G (1984~ Abstract, 15th Annual Meeting o f the Fine Particle Society, 30 July-1 August 1984. Orlando.Lamanna. C., Mailleta. M. F. and Zimmerman. L. N. (1973) Basic Bacteriology, p. 74. Williams and Wilkins,

Baltimore.



L i u , B . Y . H . , P u l D . Y . H . , W a n g , X . Q . a n d L e f t s . ( ' ~ W . I 1 9 8 3 1 A e r o s o / S e t . T ec h . 2 , 4 m ~

M a z u m d e r . M . K . . W a r e , R . E .. W i l s o n . J . D . , R e n n i n g e r , R . G . , H i l le r , F ( 2 , M c L e o d . P ( R a i b l c ~ ~ ,~ . a ~ d

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use of bacteria as model non spherical aerosol particles

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