APPLIED MICROBIOLOGY

January, 1964
Vol. 12, No. 1, p. 27-31
Copyright 1964 by the American Society for Microbiology
Printed in U.S.A.

Penetration by Gases to Sterilize Interior Surfaces of Confined Spaces

JOHN B. OPFELL, YUI-LOONG WANG,' ALLAN L. LOUDERBACK, AND CURTIS E. MIILLER
Dynamic Science Corporation, South Pasadena, California

Received for publication 29 August 1963

ABSTRACT

OPFELL, JOHN B. (Dynamic Science Corp., South Pasadena,

Calif.), YUI-LoONG WANG, ALLAN L. LOUDERBACK, AND CURTIS
E. MILLER. Penetration by gases to sterilize interior surfaces of
confined spaces. Appi. Microbiol. 12:27-31. 1964.-The rate
of penetration of gaseous sterilizing agents into confined spaces
can be predicted from physical and chemical considerations.
The exposure times required to obtain sterilizing concentrations
of ethylene oxide in several configurations of confined space
were predicted by computation and illustrated by experiment.
The results of the computations are presented graphically.
Exposure to ethylene oxide vapor was proposed as a
method for the terminal sterilization of space vehicles
(Davies and Communtzis, 1960; Phillips and Hoffman,
1960) because it is highly effective and highly penetrating
(Phillips, 1961). The penetration of ethylene oxide or other
sterilizing chemicals proceeds by the mechanisms of
diffusion and convection. Although the empirical diffusion
coefficients are not easy to establish with precision (Reamer
et al., 1956), the mechanism of diffusion has been studied
extensively (Bird, Stewart, and Lightfoot, 1960; Chapman
and Cowling, 1939; Opfell and Sage, 1955; Sherwood and
Pigford, 1952). In the gas phase, the estimation of the
rate of diffusion for substances such as ethylene oxide
requires relatively little empirical information. On the
other hand, quantitative analysis of the processes of
absorption and diffusion of ethylene oxide in plastic and
in elastomers (Myers et al., 1961) requires information
about the equilibrium ethylene oxide content of these
materials as a function of temperature and pressure, as
well as information about the diffusivity of ethylene oxide
in these materials.
When ethylene oxide can be transported to the vicinity
of the microorganisms to be sterilized by methods other
than molecular diffusion (e.g., by flow into an evacuated
space or by forced convection), then the rate and extent
of penetration can be increased substantially. The discussion which follows will pertain specifically to the penetration of ethylene oxide into confined spaces filled with air at
atmospheric pressure and devoid of convection currents.
The ethylene oxide will be assumed not to react with the
air or the walls confining the space. The analysis will apply
to other sterilants in the gas phase under these same
assumptions. For the purposes of the analysis, the ethylene
oxide in the environment exterior to the confined space will
1 Present address: Burroughs Corp., Pasadena, Calif.

ANALYSIS

The diffusion of a sterilizing gas through a small orifice

into a relatively large confined space has not been analyzed
previously, though the importance of this process in the
design of sterile space vehicles has been emphasized
(Phillips and Hoffman, 1960). For purposes of mathematical convenience, the particular configuration shown
in Fig. 1 was chosen for analysis in detail. The configurations shown in Fig. 2 and 3 can be analyzed in a similar
manner. The results of such an analysis were presented
elsewhere (Carslaw and Jaeger, 1947) and are presented
again in Fig. 2 and 3.
In Fig. 1, the circle of radius a belongs to a small spheie
of the same radius, whose (mathematical) surface is at a
steady uniform concentration of gas sterilant, Ca. Except
for the orifice which includes the small sphere, the surface
of the hollow hemisphere of radius b is not penetrable by
gases. All gas transport between outside and inside the
hemisphere passes through the small sphere. The concentration of sterilizing gas, C (lb/ft3), is determined at all
points inside and outside the hemisphere (except inside
the small sphere) by Fick's second law (Bird et al., 1960):
-c = D i2C
At

27

V87.32

2 aCf
r

r.

(1)

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be assumed to be dispersed in air at a known and steady

concentration.
Three types of configuration of confined space will be
considered (Fig. 1, 2, and 3). In each of these configurations, the ethylene oxide is diffusing into a space filled
with still air. The molar diffusion rate of the air will be
equal and opposite to the molar diffusion rate of the ethylene oxide at every point in the confined space.
Table 1 presents some values for the diffusion coefficient
for several gases, including several sterilizing gases, diffusing through air at normal conditions of temperature and
pressure. The calculated values were computed by use of
the semiempirical equation of Sherwood and Pigford
(1952). The diffusion coefficient for,-propiolactone is,
interestingly, almost as large as that for ethylene oxide.
Its observed low penetrating ability is due to its low saturation pressure in the vapor state. The same property
limits the penetration of formaldehyde vapor at temperatures below the decomposition temperature of paraformaldehyde.

APPL. MICROBIOL.

OPFELL ET AL.

28

with boundary conditions inside the hemisphere:

ac

at r = b

(2)

C=CCa at r=a
C = 0 at t = 0

(3)

-= 0

the diffusion coefficient in square feet per hour. The symbol

a represents partial differentiation.
By use of the transformations:
(5)

C= u/r

(4)
where t is time in hours, r is the radial distance in feet
from the center of the small sphere of radius a, and D, is
TABLE 1. Diffusion coefficients, Dv, for gaseous diffusion in air at
one atmosphere
Substance

Temp

ft2l/hr

2.37
0.853
0. 587*
0.482*
0.440*
0.337*
0.196

32
32
80
80
80
80
32

(6)

Ob2/ID

(7)

r = bR

in which u is a concentration parameter (lb/ft2), 0 is a time

parameter (dimensionless), and R is a distance parameter
(dimensionless), equations 1 through 4 may be written in
the more convenient forms:
Ou
M0

O'u
dR2

(8)

ou

at R = 1

-= u

OR

(9)

u = aCa at R = b/a = a

(10)

u =0

(11)

at 0= 0

where a is the orifice parameter (dimensionless).

Application of the Laplace transformation (Carslaw
*
Calculated value obtained by the method of Sherwood and
and
Jaeger, 1947) to equation 8 with respect to 0 yields a
Pigford (1952).
second-order differential equation for the Laplace transform of the function u, viz., ui, whose general solution is:
TIME IN HOURS
u = Ae Rp + BeRV/

(12)

The parameter p is a complex variable related to the Laplace transform of 1; A and B are parameters of integration. Application of the boundary condition expressed in
equation 9 yields the following relationship among A, B,
and p:
B = A _V/P + 1 e-2-,/p
(13)

\/p -

hence
= A txe-R/\p +

+\; + e-2V/+RV}

(14)

From the boundary condition expressed in equation 10

and the transformation:

id

-\/-p

(15)

N%.

the following equations result:

C

Ca

(q

1)e-Rq + (q + 1)e-2q+Rq

Rp (q

1)e-aq + (q + 1)e-2q+aq

(16)

or

10

20

Dvt/g

30

40

X$DIMENSIONLESS

FIG. 1. Concentration of a gas sterilant at the remote wall in a

confined space. To permit the use of the information in the figure to
deduce penetration rates for gases other than ethylene oxide in various
sizes of confined space, the abscissa scale at the bottom of the figure is
expressed in terms of a dimensionless parameter. The upper abscissa
scale applies only to ethylene oxide in the baby-food jars discussed in
the test. The three curves correspond to the following values of a, the
orifice diameter parameter: (1) 0.100, (2) 0.010, and (3) 0.001.

C
Ca

=q cosh (1- R)q -

sinh (1- R)q

q cosh (1- a)q - sinh (1 - a)q

(17)

The function C/Ca has poles at q = 0 and at the roots of

the equation:
tanh(1 - a) q

(18)
Because these roots lie along the imaginary axis (Jahnke
and Emde, 1945), they are at the points i X, . Thus:
q =iA at p = -X,,2
(19)
which means that in the plane of p the roots lie along the
= q

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Hydrogen ......................
Water vapor ...................
Formaldehyde .................
Ethylene oxide .................
Ethylene imine ................
,-Propiolactone ................
n-Octane ......................

Dv

t =

PENETRATION- BY GASEOUS STERILAN-TS

VlOL. 12, 1964

negative real axis. The expression for the concentration

C can be obtained as the inverse of the Laplace transform.
Thus:
C

Ca R

(0

Jif~
1f'~
27ri J_-it

R)q
cosh (1
q cosh (1 -a)q

sinh (1
sinh (1

R)q
a)q

(20)

PJ

value of the real part of p greater than the real

any of its singularities. The value of the integral can be obtained from the sum of the residues of the
integrand at its poles. At the pole at q = 0, the residue is
equal to R/a. At the poles (simple) at p = -X2, the
residue is:
with

-y

part of

as a
p

at

acsX)

cos

cos

(1

R)X, -

(1

a)X,

(1 -

sin

R)X5n

(1

a)XAn2 sin (1

e-XA20

a)XJ

and:

2a

C
Ca

1+-R

R?
(21)

information presented by Carslaw and Jaeger, and shows

the concentration of ethylene oxide at the center of the
cylinder expressed as a fraction of the concentration along
the circumference and as a function of time. F'or all values
of d, distance between the discs, greater than the minimum
for which the gas behaves like a continuous medium, the
rate of diffusion of the sterilizing gas into the cylindrically
confinied space is independent of d.
Carslaw and Jaeger also analyzed and presented numerical information about the heat transfer analogue of
diffusion down a long narrow cylindrical tube. The information presented in Fig. 3 was derived from that presented
by Carslaw and Jaeger. Again, it is interesting to note that,
for all diameters of tubing larger than the minimum for
continuous fluid behavior, the rate of diffusion of the
sterilizing gas into the tube is independent of the diameter.
EXPERIMENT AND RESULTS
To confirm the practical applicability of the conclusion
presented in Fig. 1, a relatively simple experiment was

An Cos (1 R) -sin (1
R)X,,
-n2l
n=1 ax"n cos (1 - as)Xs - (1 - a)Xn2 sin (1 - a)X?e
a! E

and at R

1:
Qe-XnDDt /b2

=1+

(1-

cC)X

a)X

sin (1

-a)Xt

(22)

The

curve

in

Fig.

was

calculated from

equation

22.

Carslaw and Jaeger (1947) analyzed the heat transfer

analogue of diffusion into a cylindrical space such as that
shown in Fig. 2. The curve in Fig. 2 was computed from

0.8

06

TIME

IN

3
HOURS

FIG. 3. Concentration of ethylene oxide in air at closed end

0.4

o~~~~~~~~~~~~~~Al

of a

long tube. The lengths of the tuibes, L, for the several curves have the
following values: (1) 0.78 ft, (2) 1.10 ft, (3) 1.55 ft, (4) 2.20 ft, and
(5) 3.10 ft.
TABLE 2. Effect of penetration of ethylene oxide through a small
orifice on the viability of spores of Bacillus subtilis
var. niger in a confined space
Viable cells (survivors)
Orifice diam

TIME

IN

HOURS

2. Concentration of ethylene oxide in air resulting fromn radial

diffusion between two discs. The curves pertain to the center of the
space between the discs. T'he radii of the discs, a, for the several cu rves
have the following values: (1 ) 0.78 ft, (2) 1.10 ft, (3) 1.55 ft, and (4)
2.20 ft.
FIG.

Specimen no. 1

Specimen no. 2

3,500,000
2,500,000
2,000,000
1,000,000

3,900,000
2,400,000
3,000,000
1,200,000
1,800

in.

Control
0.000
0.001
0.010
0.100

0.00000
0.00000
0.000267
0.00267
0.0267

360

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X
a,

29

30

30OPFELL ET AL.

DISCUSSION
Ernst and Shull (1962) recently presented information
about the sterilizing effectiveness of ethylene oxide under
conditions comparable in temperature and concentration
to those of the experiment discussed here. The sterilizing
effectiveness of ethylene oxide vapor is a complicated
function of length of exposure, temperature, relative
humidity, and concentrationi (Ernst and Shull, 1962;
Opfell, Hohmann, and Latham, 1959; Phillips, 1961). At a
particular length of exposure, temperature, and relative
humidity for a specified surface, the effectiveness increases
with concentration up to a certain critical concentration of
about 1,500 mg per liter. Further increases in concentra
tion produce no further increase in effectiveness.

Under the condition of temperature, concentration, and

relative humidity used in this experiment, the data of
Ernst and Shull indicate that for a population of about
5 X 105 spores of B. subtilis var. niger the thermochemical death time should be about 150 min. For concentrations lower than 500 mg per liter, increasing numbers of
spores would survive. Because the concentration of ethylene oxide in the vicinity of the spores was transient
throughout the period of exposure, because water vapor
and Freon were diffusing simultaneously with the ethylene
oxide, and because the geometry of the jar was not really
hemispherical, comparison of the experimental results
with thermochemical death time measurements cannot
be very precise. Because the concentration of ethylene
oxide was very low for a portion of the time, the total
exposure was not enough to cause us to expect sterilization of the inoculums. For the exposure period used,
however, the data of Ernst and Shull with the information
in Fig. 1 would indicate that the population exposed
through the 0.100-in. orifice should be nearly destroyed,
while those populations exposed through the holes of
smaller diameter should be relatively unaffected. The
experiment confirmed these indications.
The information in Fig. 1 can be used to predict the
minimal time required for the concentration of a sterilizing gas to reach a certain level at a remote surface in a
confined space of appropriate geometry. The actual time
required can be greater for a variety of reasons, such as
chemical reaction or polymerization in conjunction with
the diffusion. A gas sterilant cannot reasonably be expected to penetrate confined spaces much more rapidly
than is indicated in Fig. 1, 2, and 3. For rapid penetration,
convection in addition to diffusion is essential. Often
evacuation of the confined space to a very low pressure
before exposure to the gas sterilant will produce rapid
penetration through the mechanism of forced convection.
LITERATURE CITED
BIRD, R. B., W. E. STEWART, AND E. N. LIGHTFOOT. 1960. Transport phenomena. John Wiley & Sons, Inc., New York.
CARSLAW, H. S., AND J. C. JAEGER. 1947. Conduction of heat in
solids. Oxford University Press, London, England.
CHAPMAN, S., AND T. G. COWLING. 1939. Mathematical theory of
non-uniform gases. Cambridge UJniversity Press, New York.
DAkVIES, R. W., AND M. G. CoMMUNTZIS. 1960. The sterilization of
space vehicles to prevent extraterrestrial biological contamination. External publication 698, Jet Propulsion Laboratory,
California Institute of Technology, Pasadena.
ERNST, R. R., AND J. J. SHULL. 1962. Ethylene oxide gaseous sterilization. I. Concentration and temperature effects. Appl.
Microbiol. 10:337-341.
JAHNKE, E., AND F. EMDE. 1945. Tables of functions. Dover Publications, New York.
MYERS, A. W., J. A. MEYER, C. E. ROGERS, V. STANNETT, AND
M. SZWARC. 1961. The gas and vapor permeability of plastic
films and coated papers. VI. The permeation of water vapor.
Tappi 44:58-64.
OPFELL, J. B., J. P. HIOHMANN, AND A. B. LATHAM. 1959. Ethylene

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performed in the laboratory, Inoculums of 4 million spores

of Bacillus subtilis var. niger- were deposited from suspension in 0.01 ml of distilled water on the bottom inside surface of each of several clean, sterile, 8-oz, glass, baby-food
jars (3.75 in. deep). The inoculums were dried overnight
at 37 C and 20 % relative humidity. Each jar was covered
with aluminum foil (0.002 in. thick) which was sealed to
the jar with paraffin wax. Holes of various sizes were
punched in the center of the aluminum foil to permit
passage of ethylene oxide into the jar.
The jars were placed in a temperature-regulated bath of
ethylene oxide in Freon-12 at a concentration of 300 mg
per liter at 50'S relative humidity and at 23 C. The jars
remained in this environment for 180 min. During startup, the ambient ethylene oxide atmosphere had a transient
period as the jars adjusted temperature, and air from some
of the jars escaped relatively rapidly. This transient
period was short relative to the total period of exposure.
The volume of the temperature-regulated bath was 7.1
liters. The gas mixture was passed continuously through
the bath and helped to maintain the bath temperature at
23 i 0.5 C throughout the 180-min exposure of the jars.
Because the jars were in thermal equilibrium with the
temperature-regulated bath when the gas was first introduced, the jars were at all times less than 0.5 C different
in temperature from the gas.
At the completioni of the exposure period, 20 ml of
sterile distilled water were put into each of the jars. The
jars were then immersed in an ultrasonic scrubber to suspend the inoculum in the water. Serial dilutions of this
suspension of the inoculum were made. Samples of the
suspension and the dilutions were placed on Trypticase
Soy Agar and incubated at 37 C for 48 hr. From the numbers of colonies which developed on the agar, the numbers
of viable cells (survivors) shown in Table 2 were deduced.
The controls were treated in the same manner as were the
test inoculums, except that no hole was punched in the
cover and the jars were not exposed to ethylene oxide. The
entries for the controls show that the scrubbing technique
removed nearly all the inoculum from the bottom of the
jar and suspended it uniformly in water.

APPL. -MICROBIOL.

VOL. 12, 1964

PENETRATION BY GASEOUS STERILANTS

oxide sterilization of spores in hygroscopic environments. J.

Am. Pharm. Assoc. Sci. Ed. 48:617-619.
OPFELL, J. B., AND B. H. SAGE. 1955. Relations in material transport. Ind. Eng. Chem. 47:918-923.
PHILLIPS, C. R. 1961. Recent developments in the sterilization of
surgical materials. The Pharmaceutical Press, London,
England.

31

PHILLIPS, C. R., AND R. K. HOFFMAN. 1960. Sterilization of interplanetary vehicles. Science 132:991-995.
REAMER, H. H., J. B. OPFELL, B. H. SAGE, AND C. H. DUFFY. 1956.
Diffusion coefficients in hydrocarbon systems. Methanedecane-methane in liquid phase. Ind. Eng. Chem. 48:275-282.
SHERWOOD, T. K., AND R. L. PIGFORD. 1952. Absorption and extraction. McGraw-Hill Book Co., Inc., New York.

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