Biological Indicator

2010

Process Lethality
Calculation and
Workbook
The biological data derived from a sterilization process is qualitative information, such as
sterile or non-sterile, established by observing either growth or no growth of a biological
challenge. The process is challenged with calibrated bacterial spores with a defined
resistance to the sterilization process. The process is effective if the spore challenge is
killed (no growth). The process is not effective when the spore challenge survives.
When we expose replicate samples to replicate physical conditions we are able to expand
our knowledge of the lethality being delivered by the sterilization process. This is
usually expressed as a probabilistic value and is capable of predicting results with a high
level of certainty.

This workbook is intended to provide you with the ability to express biological
measurements in numbers using standard mathematical formulas. These biological
numbers provide the quantative assessment of the sterilization process. When used
properly, the bacterial spore provides the most accurate measure of the effectiveness of
the sterilization process.

The population of the spore challenge is established using standard microbiological plate
count procedures and is used in the following mathematical equations. The D-value is
the first assessment of the resistance of a biological challenge to a particular sterilization
process. The D-value is defined as the time in minutes that it takes at a specified set of
conditions to reduce the population of the biological challenge by one log or a factor of
ten. There are two basic approaches to establish the D-value. One approach is referred to
as the survivor curve method and the other is the fraction negative method. In the
survivor curve method, high levels of spores are exposed to successive short time periods
of sterilizing conditions. The data collected is the number of spores that survived the
sterilization conditions. The exposures are performed over increasing durations of clock
time. The surviving spores are recovered using standard microbiological plate count
techniques. The data is plotted on a semi log graph. The X axis is clock time and the
Y axis is the log scale of the number of spores recovered at each of the exposure times.
The slope of the curve is the D-value. The coefficient of determination (r
2
) is also
calculated. This coefficient indicates how close the data points are to the calculated
linear regression plot.

The D-value can also be calculated using fraction negative data from units exposed in the
quantal zone. There are two approaches to analyze this data. The first approach is
referred to as the Stumbo, Murphy, Cochran formula. This method calculates a D-value
from each fraction negative data point. When more than one data set is available, the
individual point D-values are summed and divided by the number of data points. This
method is quite useful for determining a process D-value when it may be difficult to
collect more than one fraction negative data set.

The second approach using fraction negative data sets is referred to as the Limited
Holcomb, Spearmen, Karber method. This method not only focuses on the quantal zone
data points, but it looks at the shortest time to all negative units and the longest time to all
positive units. It uses all the quantal zone values and the exposure interval to calculate
the mean time to sterility. This approach is a little more robust than the Stumbo,
Murphy, Cochran method.

Now that spore populations have been identified and D-values have been established at
specific sets of conditions, the effect of varying temperature conditions can be evaluated.
The temperature coefficient or Z-value is defined as the temperature change required to
alter the D-value by one log. This temperature coefficient can be applied to steam, dry
heat and ethylene oxide processes. The Z-value is best calculated using three D-values.
This can be performed graphically as well as calculated. The graphic plot is a semi log
plot with the X axis being the linear temperature scale and the Y axis the log plot of
the D-values. The slope of the curve is the Z-value. The formula to calculate the slope is
the same log linear regression plot used for the survivor curve. The coefficient of
determination (r
2
) is also calculated using the same formula as in the survivor curve
method.

The Z-value allows the integration of different lethal rates for different temperatures. A
reference temperature or process set point must be identified. As temperatures increase,
spores die faster. The Z-value provides an accurate assessment of lethality over the
normal process temperature variance as seen in come-up time, hold time and come-down
time process phases.

The temperature coefficient is now used to establish an equivalent process lethality (F-
value) at a defined reference temperature. The F-value integrates the varying process
conditions into an expression of equivalent process lethality. The equivalent process
lethality is usually described as equivalent process minutes at the reference temperature.
Complete and accurate temperature profiles are required for this calculation. This
establishes an accurate accumulated lethality value for a known biological challenge and
a dynamic process.

The F equivalent process lethality is now used to establish the equivalent spore log
reductions that are delivered by this equivalent process lethality value. This is
accomplished by dividing the F-value for the process by the process D-value. The result
is the spore log reductions (SLR) provided by the process.

The spore log reduction value is used to establish the sterility assurance level (SAL). The
sterility assurance level is used to assess the microbiological lethality of the process.
This value is the probability of a non-sterile unit (PNSU) occurring in the process.
Sterility assurance level is expressed as 10
-x
. X is the log of the microbial challenge
either spores or bioburden, which is labeled N
0
minus the SLR value delivered by the
process.








Calculate the D-value Using the Limited Holcomb, Spearman,
Karber Method

You have collected the following fraction negative data that you will apply to the Limited
Holcomb-Spearman-Karber equations.

The process set at 121.0 C.
The initial population N
0
= 1.7 x 10
5
.
Twenty (20) replicate BIs were used at each exposure.

Calculate the D-value using the data sheet provided.

Exposure Time Number of Units Exposed (n) Number of Units Sterile (r)
8 20 0
9 20 2
10 20 5
11 20 11
12 20 18
13 20 20











The Limited Holcomb, Spearman, Karber Method for
Fraction Negative Data

2507 . 0
0 10
N Log
T
D
HSK


r x
n
d d
T T
k HSK
2


T
HSK
= mean time to sterility
Log
10
N
0
= spore population
0.2507 = Eulers constant
T
k
= shortest time to all units sterile
d = time interval between data points
n = number of replicate units per test
r = number of units sterile








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CALCULATION OF D-VALUE USING LIMITED-HOLCOMB-
SPEARMAN-KARBER (USP)

Sample Identification #: Exposure conditions:
In the table on the right, fill in the exposure times and # of units killed where: Data
f
1
= exposure time or dose where all units are positive
i Exposure time
r
(# units negative)
(at all shorter times or doses, all units are positive)
1
and 2
f
k
= exposure time or dose where all units are negative
(at all longer times or doses, all units are negative)
3
4
Fill in the appropriate data in the blanks below: 5
Time (T
k
) for achieving results f
k

6
Difference between adjacent times (d)
7
Sample size (n)
8
Sum of the negative replicates (r) from f
1
to f
k-1

9
(f
k-1
is the time prior to f
k
)

10
Average spore count per carrier (N
o
)
11
Log N
o
=
(round to 4 decimal places) 12
Calculate mean heating time (THSK) for achieving complete kill by the equation: 13
THSK = T
k
- d/2 - (d/n * r)
14
(r) from f
1
to f
k-1

(f
k-1
is the time prior to f
k
)


THSK = (round to 4 decimal places)
Calculate D-value (D) by the equation:
D = (THSK)/ (Log N
o
+ 0.2507)

D =

(round to 4 decimal places)
D-VALUE (rounded to 1 decimal place*)
* values 0.0950 are rounded to 1 decimal place.
* values 0.0949 are rounded to 2 decimal places.
Calculation by: Date:


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CALCULATION OF D-VALUE USING LIMITED-HOLCOMB-
SPEARMAN-KARBER (USP)

Sample Identification #: Exposure conditions: 121C
In the table on the right, fill in the exposure times and # of units killed where: Data
f
1
= exposure time or dose where all units are positive
i Exposure time
r
(# units negative)
(at all shorter times or doses, all units are positive)
1 8 0
and 2 9 2
f
k
= exposure time or dose where all units are negative
(at all longer times or doses, all units are negative)
3 10 5
4 11 11
Fill in the appropriate data in the blanks below: 5 12 18
Time (T
k
) for achieving results f
k
13
6 13 20
Difference between adjacent times (d) 1
7
Sample size (n) 20
8
Sum of the negative replicates (r) from f
1
to f
k-1
36
9
(f
k-1
is the time prior to f
k
)

10
Average spore count per carrier (N
o
) 1.7 x 10
5

11
Log N
o
=
5.2304 (round to 4 decimal places) 12
Calculate mean heating time (THSK) for achieving complete kill by the equation: 13
THSK = T
k
- d/2 - (d/n * r)
14
(r) from f
1
to f
k-1

(f
k-1
is the time prior to f
k
)
36
|
.
|

\
|
= 36
20
1
2
1
13 x
7 . 10 ) 8 . 1 ( ) 5 . 0 ( 13 = =
THSK = 10.7000 (round to 4 decimal places)
Calculate D-value (D) by the equation:
D = (THSK)/ (Log N
o
+ 0.2507)
9522 . 1
4811 . 5
7000 . 10
2507 . 0 2304 . 5
7000 . 10
= =
+
=
D =
1.9522
(round to 4 decimal places)
D-VALUE (rounded to 1 decimal place*) 2.0
* values 0.0950 are rounded to 1 decimal place.
* values 0.0949 are rounded to 2 decimal places.
Calculation by: Date:








Using the Stumbo, Murphy, Cochran Method for Calculating the D-
Value

You have collected fraction negative data; your initial intent was to use the Limited Holcomb-
Spearman-Karber equation, but your data does not include two of the necessary data points. You
do not have a data point that indicated all surviving BIs and you do not have a data point with
all BIs killed. You must use the Stumbo, Murphy, Cochran method. Your process was set to
control at 121.0 C.

Calculate the D-value using the formula on the reverse side of this question.

The initial population N
0
= 1.7 x 10
5
.

Exposure Time Number of Exposed Units (n) Number of Units Sterile (r)
9 20 2
10 20 5
11 20 11
12 20 18







The Stumbo, Murphy, Cochran Fraction Negative Data

( )
n
D D D
D
n
+ +
=
2 1



1
10 0 10
1
1
log log
u
N N
U
D

=


Nu
i
=
r
n
ln



U = exposure time
N
0
= starting spore population
N
u
= most probable number of surviving spores
n = number units exposed
r = number units sterile (negative)







CALCULATION OF D-VALUE USING
STUMBO-MURPHY-COCHRAN METHOD

Sample Identification #: ________________________ Exposure Conditions: ___________________________









































* values 0.0950 are rounded to 1 decimal place.
* values 0.0949 are rounded to 2 decimal places.



Calculation by: _________________________________________________________________________ Date: __________________
Reviewed by: __________________________________________________________________________ Date: __________________

Sample size (n) =
Average spore count per carrier (N
o
) = ___________________
Log N
o
=___________________
In the table on the right, fill in the exposure times and # of units
killed starting with the shortest exposure. Round all numbers to 4
decimal places*.
i
Exposure
Time (U)
Number
Killed (r)
Calculated
D-value
1
2
3
4
5
6
7
8
9
D-values = ____________
average D-value = ______________

D-value = ______________
(round to 1 decimal place*)

Calculation of D
1

N
U1
= ln (n/r) = _______________
log N
U1
= __________________
D
1
= U
1
/(log N
0
log N
U1
) = ___________________
Calculation of D
2

N
U2
= ln (n/r) = _______________
log N
U2
= __________________
D
2
= U
2
/(log N
0
log N
U2
) = ___________________
Calculation of D
3

N
U3
= ln (n/r) = _______________
log N
U3
= __________________
D
3
= U
3
/(log N
0
log N
U3
) = ___________________
Calculation of D
7

N
U7
= ln (n/r) = _______________
log N
U7
= __________________
D
7
= U
7
/(log N
0
log N
U7
) = ___________________
Calculation of D
5

N
U5
= ln (n/r) = _______________
log N
U5
= __________________
D
5
= U
5
/(log N
0
log N
U5
) = ___________________
Calculation of D
8

N
U8
= ln (n/r) = _______________
log N
U8
= __________________
D
8
= U
8
/(log N
0
log N
U8
) = ___________________
Calculation of D
6

N
U6
= ln (n/r) = _______________
log N
U6
= __________________
D
6
= U
6
/(log N
0
log N
U6
) = ___________________
Calculation of D
4

N
U4
= ln (n/r) = _______________
log N
U4
= __________________
D
4
= U
4
/(log N
0
log N
U4
) = ___________________

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CALCULATION OF D-VALUE USING
STUMBO-MURPHY-COCHRAN METHOD

Sample Identification #: ________________________ Exposure Conditions: 121C











































* values 0.0950 are rounded to 1 decimal place.
* values 0.0949 are rounded to 2 decimal places.


Calculation by: _________________________________________________________________________ Date: __________________
Reviewed by: __________________________________________________________________________ Date: __________________
Sample size (n) = 20
Average spore count per carrier (N
o
) = 1.7 x 10
5

Log N
o
= 5.2304
In the table on the right, fill in the exposure times and # of units
killed starting with the shortest exposure. Round all numbers to 4
decimal places*.
i
Exposure
Time (U)
Number
Killed (r)
Calculated
D-value
1 8 0 NA
2 9 2 1.8487
3 10 5 1.9652
4 11 11 2.0169
5 12 18 1.9331
6 13 20 NA
7
8
9
D-values = 7.7639
average D-value = 1.9410

D-value = 1.9
(round to 1 decimal place*)
Calculation of D
1

N
U1
= ln (n/r) = 2.3026
log N
U1
= 0.3622
D
1
= U
1
/(log N
0
log N
U1
) = 8487 . 1
8682 . 4
9
=
Calculation of D
2

N
U2
= ln (n/r) = 1.3863
log N
U2
= 0.1419
D
2
= U
2
/(log N
0
log N
U2
) = 9652 . 1
0885 . 5
10
=
Calculation of D
3

N
U3
= ln (n/r) = 0.5978
log N
U3
= -0.2234
D
3
= U
3
/(log N
0
log N
U3
) = 0169 . 2
4538 . 5
11
=
Calculation of D
7

N
U7
= ln (n/r) = _______________
log N
U7
= __________________
D
7
= U
7
/(log N
0
log N
U7
) = ___________________
Calculation of D
5

N
U5
= ln (n/r) = _______________
log N
U5
= __________________
D
5
= U
5
/(log N
0
log N
U5
) = ___________________
Calculation of D
8

N
U8
= ln (n/r) = _______________
log N
U8
= __________________
D
8
= U
8
/(log N
0
log N
U8
) = ___________________
Calculation of D
6

N
U6
= ln (n/r) = _______________
log N
U6
= __________________
D
6
= U
6
/(log N
0
log N
U6
) = ___________________
Calculation of D
4

N
U4
= ln (n/r) = 0.1054
log N
U4
= -0.9772
D
4
= U
4
/(log N
0
log N
U4
) = 9331 . 1
2076 . 6
12
=

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Calculate the D-value Using the Survivor Curve Method

You are performing a D-value test using the survivor curve method. You collect the following
plate count data at the various exposure times. Your process is set to run at 121.0 C. The initial
population N
0
= 2.2 x 10
5


Calculate the survivor curve D-value using the log linear regression method on the worksheet on
the back of this question.

Calculate the coefficient of determination r
2
for the data.

Data Collected
Exposure Time Population Recovered
0.0 minutes 2.2 x 10
5

1.8 minutes 3.0 x 10
4

3.6 minutes 2.6 x 10
3

5.4 minutes 1.0 x 10
3

7.2 minutes 2.1 x 10
1









The Survivor Curve D-value using the Log Linear Regression
Method

m = slope of the regression line


[ ] [ ] ( )( ) [ ]
[ ] [ ]
2
2
10 10
) ( ) (
) (log ) ( log

=
x x n
y x y x n
m

n = number of data points
y = recovered spore population
x = exposure time
r
2
= coefficient of determination which indicates how close the data points are to the
predicted line
an r
2
= 1.000 indicates all data points are on the predicted line

[ ] [ ]
( )
( ) [ ]
n
y
y
n
x
x
n
y
x y x
r

(
(

(
(

|
|

\
|

=
2
10 2
10
2
2
2
10
10
) (log
) (log
) (
) (
) (log
) ( ) (log
2



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Calculation of D-value by Log Linear Regression (Survivor Curve)
Sample Identification # Round all values to 4 decimal places. Use rounded values in calculations.
Recovered
Population =
y
Exposure
Time = x
log
10
y x
2
x(log
10
y) (log
10
y)
2





(x)= A (log
10
y)= B (x
2
)= C [x(log
10
y)]= G (log
10
y
2
)]= E
Assigned Variable A = B = C = G = E =

Calculation of slope (m) and D-value

Number of data points (n) = ________

m = slope of the regression line

) ( )] ( ) [(
)] )( [( )] ( ) [(
2
A C n
B A G n
m

=

( )( ) [ ] ( )( ) [ ]
( )( ) [ ] ( )
2

= m

( ) [ ] ( ) [ ]
( ) [ ] ( )

= m

( ) [ ]
( ) [ ]
= m

m = __________
|

\
|
=
m
value D
1
1

|
|

\
|
=
1
1 value D

( ) 1 = value D

D value = __________ _______________
(rounded to one decimal)



Calculation of coefficient of determination (r
2
)
( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
n
B
E
n
A
C
n
B
A G
r
2 2
2
2


( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

(
(

|
|

\
|

=
2 2
2
2
r

( ) ( ) [ ] [ ]
( ) ( ) [ ] ( ) ( ) [ ]

=
2
2
r

( ) [ ]
( ) [ ] ( ) [ ]
2
2
= r

( )
( )
=
2
r

r
2
= __________
Calculations by: ____________________________ _____________
Date

Reviewed by: ______________________________ _____________
Date




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Calculation of D-value by Log Linear Regression (Survivor Curve)
Sample Identification # Round all values to 4 decimal places. Use rounded values in calculations.
Recovered
Population =
y
Exposure
Time = x
log
10
y x
2
x(log
10
y) (log
10
y)
2
1.7 x 10
5
0.0 5.2304 0.0000 0.0000 27.3571
3.0 x 10
4
2.0 4.4771 4.0000 8.9542 20.0444
1.0 x 10
3
4.0 3.0000 16.0000 12.0000 9.0000
6.0 x 10
2
6.0 2.7782 36.0000 16.6692 7.7184
1.1 x 10
1
8.0 1.0414 64.0000 8.3312 1.0845
(x)= A (log
10
y)= B (x
2
)= C [x(log
10
y)]= G (log
10
y
2
)]= E
Assigned Variable A = 20 B = 16.5271 C = 120.0000 G = 45.9546 E = 65.2044

Calculation of slope (m) and D-value

Number of data points (n) = 5

m = slope of the regression line

) ( )] ( ) [(
)] )( [( )] ( ) [(
2
A C n
B A G n
m

=

( )( ) [ ] ( )( ) [ ]
( )( ) [ ] ( )
2
20 120 5
5271 . 16 20 9546 . 45 5

= m

( ) [ ] ( ) [ ]
( ) [ ] ( ) 400 600
5420 . 330 7730 . 229

= m

( ) [ ]
( ) [ ] 200
7690 . 100
= m

m = -0.5038
|

\
|
=
m
value D
1
1

|

\
|

=
5038 . 0
1
1 value D

( ) 9849 . 1 1 = value D

D value = 1.8911 2.0
(rounded to one decimal)
Calculation of coefficient of determination (r
2
)
( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
n
B
E
n
A
C
n
B
A G
r
2 2
2
2


( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
5
5271 . 16
2044 . 65
5
20
120
5
5271 . 16
20 9546 . 45
2 2
2
2
r
( ) ( ) [ ] [ ]
( ) ( ) [ ] ( ) ( ) [ ] 6290 . 54 2044 . 65 80 120
1084 . 66 9546 . 45
2
2

= r
( ) [ ]
( ) [ ] ( ) [ ] 5754 . 10 40
1538 . 20
2
2

= r

( )
( ) 0160 . 423
1757 . 406
2
= r

r
2
= 0.9602
Calculations by: ____________________________ _____________
Date

Reviewed by: ______________________________ _____________
Date

Sterilizing Value, Minutes
Example of Plotting the Survivor Curve on Semi Log Graph Paper
1.9 Min

Example of Plotting the Survivor Curve on Semi Log Graph Paper
Sterilizing Value, Minutes







Calculate the Z-value Using the Following Data:

D
121
= 1.9522

D
124
= 1.3127

D
127
= 0.8650

ISO 11138 states three D-values between 110 and 130C







Temperature Coefficient Z-value Using the Log Linear
Regression Method

m = the slope of the regression line

( ) [ ] ( ) [ ] ( ) ( ) ( ) ( ) [ ]
( ) ( ) [ ] ( ) [ ]
2
2
10 10
log log

=
x x n
y x y x n
m

n = number of data points
y = D-value
x = exposure temperature
r
2
= coefficient of determination

[ ] [ ]
( )
( ) [ ]
n
y
y
n
x
x
n
y
x y x
r

(
(

(
(

|
|

\
|

=
2
10 2
10
2
2
2
10
10
) (log
) (log
) (
) (
) (log
) ( ) (log
2



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Calculation of Z-value by Log Linear Regression
Crop or Lot Number Round all values to 4 decimal places. Use rounded values in calculations.
D value =
y
Exposure
Temperature =
x
log
10
y x
2
x(log
10
y) (log
10
y)
2



(x)= A (log
10
y)= B (x
2
)= C [x(log
10
y)]= G (log
10
y
2
)]= E
Assigned
Variable
A = B = C = G = E =

Calculation of slope (m) and Z-value

Number of D values (n) = ________

m = slope of the regression line

) ( )] ( ) [(
)] )( [( )] ( ) [(
2
A C n
B A G n
m

=

( )( ) [ ] ( )( ) [ ]
( )( ) [ ] ( )
2

= m

( ) [ ] ( ) [ ]
( ) [ ] ( )

= m

( ) [ ]
( ) [ ]
= m

m = __________
|

\
|
=
m
value Z
1
1

|
|

\
|
=
1
1 value Z

( ) 1 = value Z

Z value = _________ ______________
(rounded to one decimal)



Calculation of coefficient of determination (r
2
)
( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
n
B
E
n
A
C
n
B
A G
r
2 2
2
2


( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

(
(

|
|

\
|

=
2 2
2
2
r


( ) ( ) [ ] [ ]
( ) ( ) [ ] ( ) ( ) [ ]

=
2
2
r

( ) [ ]
( ) [ ] ( ) [ ]
2
2
= r

( )
( )
=
2
r

r
2
= __________


Calculations by: ____________________________ _____________
Date


Reviewed by: ______________________________ _____________
Date




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Calculation of Z-value by Log Linear Regression
Crop or Lot Number Round all values to 4 decimal places. Use rounded values in calculations.
D value =
y
Exposure
Temperature =
x
log
10
y x
2
x(log
10
y) (log
10
y)
2
1.9522 121 0.2905 14641 35.1505 0.0844
1.3127 124 0.1182 15376 14.6568 0.0140
0.8650 127 -0.0630 16129 -8.0010 0.0040
(x)= A (log
10
y)= B (x
2
)= C [x(log
10
y)]= G (log
10
y
2
)]= E
Assigned
Variable
A = 372 B = 0.3457 C = 46146 G = 41.8063 E = 0.1024

Calculation of slope (m) and Z-value

Number of D values (n) = 3

m = slope of the regression line

) ( )] ( ) [(
)] )( [( )] ( ) [(
2
A C n
B A G n
m

=

( )( ) [ ] ( )( ) [ ]
( )( ) [ ] ( )
2
372 46146 3
3457 . 0 372 8063 . 41 3

= m

( ) [ ] ( ) [ ]
( ) [ ] ( ) 138384 138438
6004 . 128 4189 . 125

= m

( ) [ ]
( ) [ ] 54
1815 . 3
= m

m = -0.0589
|

\
|
=
m
value Z
1
1

|

\
|

=
0589 . 0
1
1 value Z

( ) 9779 . 16 1 = value Z

Z value = 16.9779 17.0
(rounded to one decimal)
Calculation of coefficient of determination (r
2
)
( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
n
B
E
n
A
C
n
B
A G
r
2 2
2
2


( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
3
3457 . 0
1024 . 0
3
372
46146
3
3457 . 0
372 8063 . 41
2 2
2
2
r


( ) ( ) [ ] [ ]
( ) ( ) [ ] ( ) ( ) [ ] 0398 . 0 1024 . 0 46128 46146
8668 . 42 8063 . 41
2
2

= r
( ) [ ]
( ) [ ] ( ) [ ] 0626 . 0 18
0605 . 1
2
2

= r

( )
( ) 1268 . 1
1247 . 1
2
= r

r
2
= 0.9981


Calculations by: ____________________________ _____________
Date


Reviewed by: ______________________________ _____________
Date

Example of Plotting the Z-value Curve on Semi Log Graph Paper
16.8C

Plotting the Z-value Curve on Semi Log Graph Paper












Process Equivalent Time F

=
Z
T T
A Z T
A ref
A ref
t F 10 x ,

Process Equivalent Lethality
+ + =
A i A A A A ref
Z T Z T Z T Z T Z T
F F F F F , .... , , , ,
3 2 1


t
A
= process clock time interval, usually 1 minute
T
ref
= process reference temperature
Z
A
= actual Z-value for the spore challenge

Process Equivalent Time

A typical time interval is 1 minute. t
A
= 1 minute, T
ref
= 121C, Z
A
= 8C.
Measure average temperature during 1 minute interval to calculate the
i Z T
i
F ,


Time
(Min.)
Temp. Formula Process
Equivalent Time
T
1
110C
10 x 1 10 x min 1
,
1
= =

Z T
F

T
2
115C
10 x 1 10 x min 1
,
2
= =

Z T
F

T
3
118C
10 x 1 10 x min 1
,
3
= =

Z T
F

T
4
120C
10 x 1 10 x min 1
,
4
= =

Z T
F

T
5
121C
10 x 1 10 x min 1
,
5
= =

Z T
F

T
6
122C
10 x 1 10 x min 1
,
6
= =

Z T
F

T
7
124C
10 x 1 10 x min 1
,
7
= =

Z T
F

T
8
121C
10 x 1 10 x min 1
,
8
= =

Z T
F

T
9
118C
10 x 1 10 x min 1
,
9
= =

Z T
F

T
10
107C
10 x 1 10 x min 1
,
10
= =

Z T
F

T
11
105C
10 x 1 10 x min 1
,
11
= =

Z T
F


Z T
F
,

=
Z T Z T
i ref
F F
, ,
Process

Process Equivalent Time

A typical time interval is 1 minute. t
A
= 1 minute, T
ref
= 121C, Z
A
= 8C.
Measure average temperature during 1 minute interval to calculate the
i Z T
i
F ,


Time
(Min.)
Temp. Formula Process
Equivalent
Time
T
1
110C
3750 . 1
8
110 121
,
10 x 1 10 x min 1
1

= =
Z T
F
0.0422
T
2
115C
7500 . 0
8
115 121
,
10 x 1 10 x min 1
2

= =
Z T
F
0.1778
T
3
118C
3750 . 0
8
118 121
,
10 x 1 10 x min 1
3

= =
Z T
F
0.4217
T
4
120C
1250 . 0
8
120 121
,
10 x 1 10 x min 1
4

= =
Z T
F
0.7499
T
5
121C
0
8
121 121
,
10 x 1 10 x min 1
5
= =

Z T
F
1.0000
T
6
122C
1250 . 0
8
122 121
,
10 x 1 10 x min 1
6
= =

Z T
F
1.3335
T
7
124C
3750 . 0
8
124 121
,
10 x 1 10 x min 1
7
= =

Z T
F
2.3714
T
8
121C
0
8
121 121
,
10 x 1 10 x min 1
8
= =

Z T
F
1.0000
T
9
118C
3750 . 0
8
118 121
,
10 x 1 10 x min 1
9

= =
Z T
F
0.4217
T
10
107C
7500 . 1
8
107 121
,
10 x 1 10 x min 1
10

= =
Z T
F
0.0178
T
11
105C
0000 . 2
8
105 121
,
10 x 1 10 x min 1
11

= =
Z T
F
0.0100

Z T
F
,
7.5460

=
Z T Z T
i ref
F F
, ,
Process











Calculate the Process Spore Log Reduction Value

SLR =
value - D Process
A
Z ,
ref
T
F Time Equivalent Process

Calculate the Process Spore Log Reduction (SLR) Value

The process equivalent time is 58.5 minutes.

The process D-value is 4.5 minutes.


Calculate the spore log reduction.

SLR =
value - D Process
A
Z ,
ref
T
F Time Equivalent Process

SLR = =














Process Spore Log Reduction (SLR) Value

SLR =
13
min 5 . 4
min 5 . 58
=


Your process delivers 13 spore log reductions.















Sterility Assurance Level

( ) SLR N log
0 10
10 SAL

=












Calculate the Sterility Assurance Level

Your process used a spore challenge of 1.5 x 10
6
therefore, N
0
= 1.5 x 10
6
.

Process equivalent lethality is equal to 13 spore log reductions (SLR).

Calculate the sterility assurance level.

Calculation

SAL = 10
logNo SLR


N
0
=

logN
0
=

Process equivalent lethality =

SAL =


SAL =







Calculate the Sterility Assurance Level

Your process used a spore challenge of 1.5 x 10
6
therefore, N
0
= 1.5 x 10
6
.

Process equivalent lethality is equal to 13 spore log reductions (SLR).

Calculate the sterility assurance level.

Calculation

SAL = 10
logNo SLR


N
0
= 1.5 x 10
6


logN
0
= 6.176

Process equivalent lethality = 13 SLR

SAL = 10
(6.176 13)

SAL = 10
-6.824


Plotting the Survivor Curve on Semi Log Graph Paper








Calculate the D-value Using the Limited Holcomb, Spearman,
Karber Method

You have collected the following fraction negative data that you will apply to the Limited
Holcomb-Spearman-Karber equations.

The process set at 121.0 C.
The initial population N
0
= 1.7 x 10
5
.
Twenty (20) replicate BIs were used at each exposure.

Calculate the D-value using the data sheet provided.

Exposure Time Number of Units Exposed (n) Number of Units Sterile (r)
8 20 0
9 20 2
10 20 5
11 20 11
12 20 18
13 20 20











The Limited Holcomb, Spearman, Karber Method for
Fraction Negative Data

2507 . 0
0 10
N Log
T
D
HSK


r x
n
d d
T T
k HSK
2


T
HSK
= mean time to sterility
Log
10
N
0
= spore population
0.2507 = Eulers constant
T
k
= shortest time to all units sterile
d = time interval between data points
n = number of replicate units per test
r = number of units sterile








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CALCULATION OF D-VALUE USING LIMITED-HOLCOMB-
SPEARMAN-KARBER (USP)

Sample Identification #: Exposure conditions:
In the table on the right, fill in the exposure times and # of units killed where: Data
f
1
= exposure time or dose where all units are positive
i Exposure time
r
(# units negative)
(at all shorter times or doses, all units are positive)
1
and 2
f
k
= exposure time or dose where all units are negative
(at all longer times or doses, all units are negative)
3
4
Fill in the appropriate data in the blanks below: 5
Time (T
k
) for achieving results f
k

6
Difference between adjacent times (d)
7
Sample size (n)
8
Sum of the negative replicates (r) from f
1
to f
k-1

9
(f
k-1
is the time prior to f
k
)

10
Average spore count per carrier (N
o
)
11
Log N
o
=
(round to 4 decimal places) 12
Calculate mean heating time (THSK) for achieving complete kill by the equation: 13
THSK = T
k
- d/2 - (d/n * r)
14
(r) from f
1
to f
k-1

(f
k-1
is the time prior to f
k
)


THSK = (round to 4 decimal places)
Calculate D-value (D) by the equation:
D = (THSK)/ (Log N
o
+ 0.2507)

D =

(round to 4 decimal places)
D-VALUE (rounded to 1 decimal place*)
* values 0.0950 are rounded to 1 decimal place.
* values 0.0949 are rounded to 2 decimal places.
Calculation by: Date:


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CALCULATION OF D-VALUE USING LIMITED-HOLCOMB-
SPEARMAN-KARBER (USP)

Sample Identification #: Exposure conditions: 121C
In the table on the right, fill in the exposure times and # of units killed where: Data
f
1
= exposure time or dose where all units are positive
i Exposure time
r
(# units negative)
(at all shorter times or doses, all units are positive)
1 8 0
and 2 9 2
f
k
= exposure time or dose where all units are negative
(at all longer times or doses, all units are negative)
3 10 5
4 11 11
Fill in the appropriate data in the blanks below: 5 12 18
Time (T
k
) for achieving results f
k
13
6 13 20
Difference between adjacent times (d) 1
7
Sample size (n) 20
8
Sum of the negative replicates (r) from f
1
to f
k-1
36
9
(f
k-1
is the time prior to f
k
)

10
Average spore count per carrier (N
o
) 1.7 x 10
5

11
Log N
o
=
5.2304 (round to 4 decimal places) 12
Calculate mean heating time (THSK) for achieving complete kill by the equation: 13
THSK = T
k
- d/2 - (d/n * r)
14
(r) from f
1
to f
k-1

(f
k-1
is the time prior to f
k
)
36
|
.
|

\
|
= 36
20
1
2
1
13 x
7 . 10 ) 8 . 1 ( ) 5 . 0 ( 13 = =
THSK = 10.7000 (round to 4 decimal places)
Calculate D-value (D) by the equation:
D = (THSK)/ (Log N
o
+ 0.2507)
9522 . 1
4811 . 5
7000 . 10
2507 . 0 2304 . 5
7000 . 10
= =
+
=
D =
1.9522
(round to 4 decimal places)
D-VALUE (rounded to 1 decimal place*) 2.0
* values 0.0950 are rounded to 1 decimal place.
* values 0.0949 are rounded to 2 decimal places.
Calculation by: Date:








Using the Stumbo, Murphy, Cochran Method for Calculating the D-
Value

You have collected fraction negative data; your initial intent was to use the Limited Holcomb-
Spearman-Karber equation, but your data does not include two of the necessary data points. You
do not have a data point that indicated all surviving BIs and you do not have a data point with
all BIs killed. You must use the Stumbo, Murphy, Cochran method. Your process was set to
control at 121.0 C.

Calculate the D-value using the formula on the reverse side of this question.

The initial population N
0
= 1.7 x 10
5
.

Exposure Time Number of Exposed Units (n) Number of Units Sterile (r)
9 20 2
10 20 5
11 20 11
12 20 18







The Stumbo, Murphy, Cochran Fraction Negative Data

( )
n
D D D
D
n
+ +
=
2 1



1
10 0 10
1
1
log log
u
N N
U
D

=


Nu
i
=
r
n
ln



U = exposure time
N
0
= starting spore population
N
u
= most probable number of surviving spores
n = number units exposed
r = number units sterile (negative)







CALCULATION OF D-VALUE USING
STUMBO-MURPHY-COCHRAN METHOD

Sample Identification #: ________________________ Exposure Conditions: ___________________________









































* values 0.0950 are rounded to 1 decimal place.
* values 0.0949 are rounded to 2 decimal places.



Calculation by: _________________________________________________________________________ Date: __________________
Reviewed by: __________________________________________________________________________ Date: __________________

Sample size (n) =
Average spore count per carrier (N
o
) = ___________________
Log N
o
=___________________
In the table on the right, fill in the exposure times and # of units
killed starting with the shortest exposure. Round all numbers to 4
decimal places*.
i
Exposure
Time (U)
Number
Killed (r)
Calculated
D-value
1
2
3
4
5
6
7
8
9
D-values = ____________
average D-value = ______________

D-value = ______________
(round to 1 decimal place*)

Calculation of D
1

N
U1
= ln (n/r) = _______________
log N
U1
= __________________
D
1
= U
1
/(log N
0
log N
U1
) = ___________________
Calculation of D
2

N
U2
= ln (n/r) = _______________
log N
U2
= __________________
D
2
= U
2
/(log N
0
log N
U2
) = ___________________
Calculation of D
3

N
U3
= ln (n/r) = _______________
log N
U3
= __________________
D
3
= U
3
/(log N
0
log N
U3
) = ___________________
Calculation of D
7

N
U7
= ln (n/r) = _______________
log N
U7
= __________________
D
7
= U
7
/(log N
0
log N
U7
) = ___________________
Calculation of D
5

N
U5
= ln (n/r) = _______________
log N
U5
= __________________
D
5
= U
5
/(log N
0
log N
U5
) = ___________________
Calculation of D
8

N
U8
= ln (n/r) = _______________
log N
U8
= __________________
D
8
= U
8
/(log N
0
log N
U8
) = ___________________
Calculation of D
6

N
U6
= ln (n/r) = _______________
log N
U6
= __________________
D
6
= U
6
/(log N
0
log N
U6
) = ___________________
Calculation of D
4

N
U4
= ln (n/r) = _______________
log N
U4
= __________________
D
4
= U
4
/(log N
0
log N
U4
) = ___________________

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CALCULATION OF D-VALUE USING
STUMBO-MURPHY-COCHRAN METHOD

Sample Identification #: ________________________ Exposure Conditions: 121C











































* values 0.0950 are rounded to 1 decimal place.
* values 0.0949 are rounded to 2 decimal places.


Calculation by: _________________________________________________________________________ Date: __________________
Reviewed by: __________________________________________________________________________ Date: __________________
Sample size (n) = 20
Average spore count per carrier (N
o
) = 1.7 x 10
5

Log N
o
= 5.2304
In the table on the right, fill in the exposure times and # of units
killed starting with the shortest exposure. Round all numbers to 4
decimal places*.
i
Exposure
Time (U)
Number
Killed (r)
Calculated
D-value
1 8 0 NA
2 9 2 1.8487
3 10 5 1.9652
4 11 11 2.0169
5 12 18 1.9331
6 13 20 NA
7
8
9
D-values = 7.7639
average D-value = 1.9410

D-value = 1.9
(round to 1 decimal place*)
Calculation of D
1

N
U1
= ln (n/r) = 2.3026
log N
U1
= 0.3622
D
1
= U
1
/(log N
0
log N
U1
) = 8487 . 1
8682 . 4
9
=
Calculation of D
2

N
U2
= ln (n/r) = 1.3863
log N
U2
= 0.1419
D
2
= U
2
/(log N
0
log N
U2
) = 9652 . 1
0885 . 5
10
=
Calculation of D
3

N
U3
= ln (n/r) = 0.5978
log N
U3
= -0.2234
D
3
= U
3
/(log N
0
log N
U3
) = 0169 . 2
4538 . 5
11
=
Calculation of D
7

N
U7
= ln (n/r) = _______________
log N
U7
= __________________
D
7
= U
7
/(log N
0
log N
U7
) = ___________________
Calculation of D
5

N
U5
= ln (n/r) = _______________
log N
U5
= __________________
D
5
= U
5
/(log N
0
log N
U5
) = ___________________
Calculation of D
8

N
U8
= ln (n/r) = _______________
log N
U8
= __________________
D
8
= U
8
/(log N
0
log N
U8
) = ___________________
Calculation of D
6

N
U6
= ln (n/r) = _______________
log N
U6
= __________________
D
6
= U
6
/(log N
0
log N
U6
) = ___________________
Calculation of D
4

N
U4
= ln (n/r) = 0.1054
log N
U4
= -0.9772
D
4
= U
4
/(log N
0
log N
U4
) = 9331 . 1
2076 . 6
12
=

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Calculate the D-value Using the Survivor Curve Method

You are performing a D-value test using the survivor curve method. You collect the following
plate count data at the various exposure times. Your process is set to run at 121.0 C. The initial
population N
0
= 2.2 x 10
5


Calculate the survivor curve D-value using the log linear regression method on the worksheet on
the back of this question.

Calculate the coefficient of determination r
2
for the data.

Data Collected
Exposure Time Population Recovered
0.0 minutes 2.2 x 10
5

1.8 minutes 3.0 x 10
4

3.6 minutes 2.6 x 10
3

5.4 minutes 1.0 x 10
3

7.2 minutes 2.1 x 10
1









The Survivor Curve D-value using the Log Linear Regression
Method

m = slope of the regression line


[ ] [ ] ( )( ) [ ]
[ ] [ ]
2
2
10 10
) ( ) (
) (log ) ( log

=
x x n
y x y x n
m

n = number of data points
y = recovered spore population
x = exposure time
r
2
= coefficient of determination which indicates how close the data points are to the
predicted line
an r
2
= 1.000 indicates all data points are on the predicted line

[ ] [ ]
( )
( ) [ ]
n
y
y
n
x
x
n
y
x y x
r

(
(

(
(

|
|

\
|

=
2
10 2
10
2
2
2
10
10
) (log
) (log
) (
) (
) (log
) ( ) (log
2



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Calculation of D-value by Log Linear Regression (Survivor Curve)
Sample Identification # Round all values to 4 decimal places. Use rounded values in calculations.
Recovered
Population =
y
Exposure
Time = x
log
10
y x
2
x(log
10
y) (log
10
y)
2





(x)= A (log
10
y)= B (x
2
)= C [x(log
10
y)]= G (log
10
y
2
)]= E
Assigned Variable A = B = C = G = E =

Calculation of slope (m) and D-value

Number of data points (n) = ________

m = slope of the regression line

) ( )] ( ) [(
)] )( [( )] ( ) [(
2
A C n
B A G n
m

=

( )( ) [ ] ( )( ) [ ]
( )( ) [ ] ( )
2

= m

( ) [ ] ( ) [ ]
( ) [ ] ( )

= m

( ) [ ]
( ) [ ]
= m

m = __________
|

\
|
=
m
value D
1
1

|
|

\
|
=
1
1 value D

( ) 1 = value D

D value = __________ _______________
(rounded to one decimal)



Calculation of coefficient of determination (r
2
)
( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
n
B
E
n
A
C
n
B
A G
r
2 2
2
2


( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

(
(

|
|

\
|

=
2 2
2
2
r

( ) ( ) [ ] [ ]
( ) ( ) [ ] ( ) ( ) [ ]

=
2
2
r

( ) [ ]
( ) [ ] ( ) [ ]
2
2
= r

( )
( )
=
2
r

r
2
= __________
Calculations by: ____________________________ _____________
Date

Reviewed by: ______________________________ _____________
Date




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Calculation of D-value by Log Linear Regression (Survivor Curve)
Sample Identification # Round all values to 4 decimal places. Use rounded values in calculations.
Recovered
Population =
y
Exposure
Time = x
log
10
y x
2
x(log
10
y) (log
10
y)
2
1.7 x 10
5
0.0 5.2304 0.0000 0.0000 27.3571
3.0 x 10
4
2.0 4.4771 4.0000 8.9542 20.0444
1.0 x 10
3
4.0 3.0000 16.0000 12.0000 9.0000
6.0 x 10
2
6.0 2.7782 36.0000 16.6692 7.7184
1.1 x 10
1
8.0 1.0414 64.0000 8.3312 1.0845
(x)= A (log
10
y)= B (x
2
)= C [x(log
10
y)]= G (log
10
y
2
)]= E
Assigned Variable A = 20 B = 16.5271 C = 120.0000 G = 45.9546 E = 65.2044

Calculation of slope (m) and D-value

Number of data points (n) = 5

m = slope of the regression line

) ( )] ( ) [(
)] )( [( )] ( ) [(
2
A C n
B A G n
m

=

( )( ) [ ] ( )( ) [ ]
( )( ) [ ] ( )
2
20 120 5
5271 . 16 20 9546 . 45 5

= m

( ) [ ] ( ) [ ]
( ) [ ] ( ) 400 600
5420 . 330 7730 . 229

= m

( ) [ ]
( ) [ ] 200
7690 . 100
= m

m = -0.5038
|

\
|
=
m
value D
1
1

|

\
|

=
5038 . 0
1
1 value D

( ) 9849 . 1 1 = value D

D value = 1.8911 2.0
(rounded to one decimal)
Calculation of coefficient of determination (r
2
)
( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
n
B
E
n
A
C
n
B
A G
r
2 2
2
2


( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
5
5271 . 16
2044 . 65
5
20
120
5
5271 . 16
20 9546 . 45
2 2
2
2
r
( ) ( ) [ ] [ ]
( ) ( ) [ ] ( ) ( ) [ ] 6290 . 54 2044 . 65 80 120
1084 . 66 9546 . 45
2
2

= r
( ) [ ]
( ) [ ] ( ) [ ] 5754 . 10 40
1538 . 20
2
2

= r

( )
( ) 0160 . 423
1757 . 406
2
= r

r
2
= 0.9602
Calculations by: ____________________________ _____________
Date

Reviewed by: ______________________________ _____________
Date

Sterilizing Value, Minutes
Example of Plotting the Survivor Curve on Semi Log Graph Paper
1.9 Min

Example of Plotting the Survivor Curve on Semi Log Graph Paper
Sterilizing Value, Minutes







Calculate the Z-value Using the Following Data:

D
121
= 1.9522

D
124
= 1.3127

D
127
= 0.8650

ISO 11138 states three D-values between 110 and 130C







Temperature Coefficient Z-value Using the Log Linear
Regression Method

m = the slope of the regression line

( ) [ ] ( ) [ ] ( ) ( ) ( ) ( ) [ ]
( ) ( ) [ ] ( ) [ ]
2
2
10 10
log log

=
x x n
y x y x n
m

n = number of data points
y = D-value
x = exposure temperature
r
2
= coefficient of determination

[ ] [ ]
( )
( ) [ ]
n
y
y
n
x
x
n
y
x y x
r

(
(

(
(

|
|

\
|

=
2
10 2
10
2
2
2
10
10
) (log
) (log
) (
) (
) (log
) ( ) (log
2



www.sgmbiotech.com
info@sgmbiotech.com

Calculation of Z-value by Log Linear Regression
Crop or Lot Number Round all values to 4 decimal places. Use rounded values in calculations.
D value =
y
Exposure
Temperature =
x
log
10
y x
2
x(log
10
y) (log
10
y)
2



(x)= A (log
10
y)= B (x
2
)= C [x(log
10
y)]= G (log
10
y
2
)]= E
Assigned
Variable
A = B = C = G = E =

Calculation of slope (m) and Z-value

Number of D values (n) = ________

m = slope of the regression line

) ( )] ( ) [(
)] )( [( )] ( ) [(
2
A C n
B A G n
m

=

( )( ) [ ] ( )( ) [ ]
( )( ) [ ] ( )
2

= m

( ) [ ] ( ) [ ]
( ) [ ] ( )

= m

( ) [ ]
( ) [ ]
= m

m = __________
|

\
|
=
m
value Z
1
1

|
|

\
|
=
1
1 value Z

( ) 1 = value Z

Z value = _________ ______________
(rounded to one decimal)



Calculation of coefficient of determination (r
2
)
( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
n
B
E
n
A
C
n
B
A G
r
2 2
2
2


( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

(
(

|
|

\
|

=
2 2
2
2
r


( ) ( ) [ ] [ ]
( ) ( ) [ ] ( ) ( ) [ ]

=
2
2
r

( ) [ ]
( ) [ ] ( ) [ ]
2
2
= r

( )
( )
=
2
r

r
2
= __________


Calculations by: ____________________________ _____________
Date


Reviewed by: ______________________________ _____________
Date




www.sgmbiotech.com
info@sgmbiotech.com

Calculation of Z-value by Log Linear Regression
Crop or Lot Number Round all values to 4 decimal places. Use rounded values in calculations.
D value =
y
Exposure
Temperature =
x
log
10
y x
2
x(log
10
y) (log
10
y)
2
1.9522 121 0.2905 14641 35.1505 0.0844
1.3127 124 0.1182 15376 14.6568 0.0140
0.8650 127 -0.0630 16129 -8.0010 0.0040
(x)= A (log
10
y)= B (x
2
)= C [x(log
10
y)]= G (log
10
y
2
)]= E
Assigned
Variable
A = 372 B = 0.3457 C = 46146 G = 41.8063 E = 0.1024

Calculation of slope (m) and Z-value

Number of D values (n) = 3

m = slope of the regression line

) ( )] ( ) [(
)] )( [( )] ( ) [(
2
A C n
B A G n
m

=

( )( ) [ ] ( )( ) [ ]
( )( ) [ ] ( )
2
372 46146 3
3457 . 0 372 8063 . 41 3

= m

( ) [ ] ( ) [ ]
( ) [ ] ( ) 138384 138438
6004 . 128 4189 . 125

= m

( ) [ ]
( ) [ ] 54
1815 . 3
= m

m = -0.0589
|

\
|
=
m
value Z
1
1

|

\
|

=
0589 . 0
1
1 value Z

( ) 9779 . 16 1 = value Z

Z value = 16.9779 17.0
(rounded to one decimal)
Calculation of coefficient of determination (r
2
)
( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
n
B
E
n
A
C
n
B
A G
r
2 2
2
2


( ) ( )
( ) ( )
(

|
|

\
|

|
|

\
|

\
|

=
3
3457 . 0
1024 . 0
3
372
46146
3
3457 . 0
372 8063 . 41
2 2
2
2
r


( ) ( ) [ ] [ ]
( ) ( ) [ ] ( ) ( ) [ ] 0398 . 0 1024 . 0 46128 46146
8668 . 42 8063 . 41
2
2

= r
( ) [ ]
( ) [ ] ( ) [ ] 0626 . 0 18
0605 . 1
2
2

= r

( )
( ) 1268 . 1
1247 . 1
2
= r

r
2
= 0.9981


Calculations by: ____________________________ _____________
Date


Reviewed by: ______________________________ _____________
Date

Example of Plotting the Z-value Curve on Semi Log Graph Paper
16.8C

Plotting the Z-value Curve on Semi Log Graph Paper












Process Equivalent Time F

=
Z
T T
A Z T
A ref
A ref
t F 10 x ,

Process Equivalent Lethality
+ + =
A i A A A A ref
Z T Z T Z T Z T Z T
F F F F F , .... , , , ,
3 2 1


t
A
= process clock time interval, usually 1 minute
T
ref
= process reference temperature
Z
A
= actual Z-value for the spore challenge

Process Equivalent Time

A typical time interval is 1 minute. t
A
= 1 minute, T
ref
= 121C, Z
A
= 8C.
Measure average temperature during 1 minute interval to calculate the
i Z T
i
F ,


Time
(Min.)
Temp. Formula Process
Equivalent Time
T
1
110C
10 x 1 10 x min 1
,
1
= =

Z T
F

T
2
115C
10 x 1 10 x min 1
,
2
= =

Z T
F

T
3
118C
10 x 1 10 x min 1
,
3
= =

Z T
F

T
4
120C
10 x 1 10 x min 1
,
4
= =

Z T
F

T
5
121C
10 x 1 10 x min 1
,
5
= =

Z T
F

T
6
122C
10 x 1 10 x min 1
,
6
= =

Z T
F

T
7
124C
10 x 1 10 x min 1
,
7
= =

Z T
F

T
8
121C
10 x 1 10 x min 1
,
8
= =

Z T
F

T
9
118C
10 x 1 10 x min 1
,
9
= =

Z T
F

T
10
107C
10 x 1 10 x min 1
,
10
= =

Z T
F

T
11
105C
10 x 1 10 x min 1
,
11
= =

Z T
F


Z T
F
,

=
Z T Z T
i ref
F F
, ,
Process

Process Equivalent Time

A typical time interval is 1 minute. t
A
= 1 minute, T
ref
= 121C, Z
A
= 8C.
Measure average temperature during 1 minute interval to calculate the
i Z T
i
F ,


Time
(Min.)
Temp. Formula Process
Equivalent
Time
T
1
110C
3750 . 1
8
110 121
,
10 x 1 10 x min 1
1

= =
Z T
F
0.0422
T
2
115C
7500 . 0
8
115 121
,
10 x 1 10 x min 1
2

= =
Z T
F
0.1778
T
3
118C
3750 . 0
8
118 121
,
10 x 1 10 x min 1
3

= =
Z T
F
0.4217
T
4
120C
1250 . 0
8
120 121
,
10 x 1 10 x min 1
4

= =
Z T
F
0.7499
T
5
121C
0
8
121 121
,
10 x 1 10 x min 1
5
= =

Z T
F
1.0000
T
6
122C
1250 . 0
8
122 121
,
10 x 1 10 x min 1
6
= =

Z T
F
1.3335
T
7
124C
3750 . 0
8
124 121
,
10 x 1 10 x min 1
7
= =

Z T
F
2.3714
T
8
121C
0
8
121 121
,
10 x 1 10 x min 1
8
= =

Z T
F
1.0000
T
9
118C
3750 . 0
8
118 121
,
10 x 1 10 x min 1
9

= =
Z T
F
0.4217
T
10
107C
7500 . 1
8
107 121
,
10 x 1 10 x min 1
10

= =
Z T
F
0.0178
T
11
105C
0000 . 2
8
105 121
,
10 x 1 10 x min 1
11

= =
Z T
F
0.0100

Z T
F
,
7.5460

=
Z T Z T
i ref
F F
, ,
Process











Calculate the Process Spore Log Reduction Value

SLR =
value - D Process
A
Z ,
ref
T
F Time Equivalent Process

Calculate the Process Spore Log Reduction (SLR) Value

The process equivalent time is 58.5 minutes.

The process D-value is 4.5 minutes.


Calculate the spore log reduction.

SLR =
value - D Process
A
Z ,
ref
T
F Time Equivalent Process

SLR = =














Process Spore Log Reduction (SLR) Value

SLR =
13
min 5 . 4
min 5 . 58
=


Your process delivers 13 spore log reductions.















Sterility Assurance Level

( ) SLR N log
0 10
10 SAL

=












Calculate the Sterility Assurance Level

Your process used a spore challenge of 1.5 x 10
6
therefore, N
0
= 1.5 x 10
6
.

Process equivalent lethality is equal to 13 spore log reductions (SLR).

Calculate the sterility assurance level.

Calculation

SAL = 10
logNo SLR


N
0
=

logN
0
=

Process equivalent lethality =

SAL =


SAL =







Calculate the Sterility Assurance Level

Your process used a spore challenge of 1.5 x 10
6
therefore, N
0
= 1.5 x 10
6
.

Process equivalent lethality is equal to 13 spore log reductions (SLR).

Calculate the sterility assurance level.

Calculation

SAL = 10
logNo SLR


N
0
= 1.5 x 10
6


logN
0
= 6.176

Process equivalent lethality = 13 SLR

SAL = 10
(6.176 13)

SAL = 10
-6.824


Plotting the Survivor Curve on Semi Log Graph Paper

Biological Indicator
2010

Process Lethality
Calculation and
Workbook
The biological data derived from a sterilization process is qualitative information, such as
sterile or non-sterile, established by observing either growth or no growth of a biological
challenge. The process is challenged with calibrated bacterial spores with a defined
resistance to the sterilization process. The process is effective if the spore challenge is
killed (no growth). The process is not effective when the spore challenge survives.
When we expose replicate samples to replicate physical conditions we are able to expand
our knowledge of the lethality being delivered by the sterilization process. This is
usually expressed as a probabilistic value and is capable of predicting results with a high
level of certainty.

This workbook is intended to provide you with the ability to express biological
measurements in numbers using standard mathematical formulas. These biological
numbers provide the quantative assessment of the sterilization process. When used
properly, the bacterial spore provides the most accurate measure of the effectiveness of
the sterilization process.

The population of the spore challenge is established using standard microbiological plate
count procedures and is used in the following mathematical equations. The D-value is
the first assessment of the resistance of a biological challenge to a particular sterilization
process. The D-value is defined as the time in minutes that it takes at a specified set of
conditions to reduce the population of the biological challenge by one log or a factor of
ten. There are two basic approaches to establish the D-value. One approach is referred to
as the survivor curve method and the other is the fraction negative method. In the
survivor curve method, high levels of spores are exposed to successive short time periods
of sterilizing conditions. The data collected is the number of spores that survived the
sterilization conditions. The exposures are performed over increasing durations of clock
time. The surviving spores are recovered using standard microbiological plate count
techniques. The data is plotted on a semi log graph. The X axis is clock time and the
Y axis is the log scale of the number of spores recovered at each of the exposure times.
The slope of the curve is the D-value. The coefficient of determination (r
2
) is also
calculated. This coefficient indicates how close the data points are to the calculated
linear regression plot.

The D-value can also be calculated using fraction negative data from units exposed in the
quantal zone. There are two approaches to analyze this data. The first approach is
referred to as the Stumbo, Murphy, Cochran formula. This method calculates a D-value
from each fraction negative data point. When more than one data set is available, the
individual point D-values are summed and divided by the number of data points. This
method is quite useful for determining a process D-value when it may be difficult to
collect more than one fraction negative data set.

The second approach using fraction negative data sets is referred to as the Limited
Holcomb, Spearmen, Karber method. This method not only focuses on the quantal zone
data points, but it looks at the shortest time to all negative units and the longest time to all
positive units. It uses all the quantal zone values and the exposure interval to calculate
the mean time to sterility. This approach is a little more robust than the Stumbo,
Murphy, Cochran method.

Now that spore populations have been identified and D-values have been established at
specific sets of conditions, the effect of varying temperature conditions can be evaluated.
The temperature coefficient or Z-value is defined as the temperature change required to
alter the D-value by one log. This temperature coefficient can be applied to steam, dry
heat and ethylene oxide processes. The Z-value is best calculated using three D-values.
This can be performed graphically as well as calculated. The graphic plot is a semi log
plot with the X axis being the linear temperature scale and the Y axis the log plot of
the D-values. The slope of the curve is the Z-value. The formula to calculate the slope is
the same log linear regression plot used for the survivor curve. The coefficient of
determination (r
2
) is also calculated using the same formula as in the survivor curve
method.

The Z-value allows the integration of different lethal rates for different temperatures. A
reference temperature or process set point must be identified. As temperatures increase,
spores die faster. The Z-value provides an accurate assessment of lethality over the
normal process temperature variance as seen in come-up time, hold time and come-down
time process phases.

The temperature coefficient is now used to establish an equivalent process lethality (F-
value) at a defined reference temperature. The F-value integrates the varying process
conditions into an expression of equivalent process lethality. The equivalent process
lethality is usually described as equivalent process minutes at the reference temperature.
Complete and accurate temperature profiles are required for this calculation. This
establishes an accurate accumulated lethality value for a known biological challenge and
a dynamic process.

The F equivalent process lethality is now used to establish the equivalent spore log
reductions that are delivered by this equivalent process lethality value. This is
accomplished by dividing the F-value for the process by the process D-value. The result
is the spore log reductions (SLR) provided by the process.

The spore log reduction value is used to establish the sterility assurance level (SAL). The
sterility assurance level is used to assess the microbiological lethality of the process.
This value is the probability of a non-sterile unit (PNSU) occurring in the process.
Sterility assurance level is expressed as 10
-x
. X is the log of the microbial challenge
either spores or bioburden, which is labeled N
0
minus the SLR value delivered by the
process.