Workshop Calculos Bioindicadores PDF

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 (r2) 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 (r2) 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 N0 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 N0 = 1.7 x 105.
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
THSK
D
Log10 N 0 0.2507
d d
THSK Tk x r
2 n
THSK = mean time to sterility

Log10 N0 = spore population
0.2507 = Euler’s constant
Tk = 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
f1= exposure time or dose where all units are positive Exposure time r
i (# units negative)
(at all shorter times or doses, all units are positive) 1
and 2
fk = exposure time or dose where all units are negative 3
(at all longer times or doses, all units are negative) 4
Fill in the appropriate data in the blanks below: 5
Time (Tk) for achieving results fk 6
Difference between adjacent times (d) 7
Sample size (n) 8
Sum of the negative replicates (r) from f1 to fk-1 9
(fk-1 is the time prior to fk) 10
Average spore count per carrier (No) 11
Log No= (round to 4 decimal places) 12
Calculate mean heating time (THSK) for achieving complete kill by the equation: 13
14
THSK = Tk- d/2 - (d/n * r) (r) from f1 to fk-1
(fk-1 is the time prior to fk)
THSK = (round to 4 decimal places)
Calculate D-value (D) by the equation:

D = (THSK)/ (Log No + 0.2507)
D= (round to 4 decimal places)
* values ≥0.0950 are rounded to 1 decimal place.

D-VALUE (rounded to 1 decimal place*)
* values ≤0.0949 are rounded to 2 decimal places.
Calculation by: Date:
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Sample Identification #: Exposure conditions: 121°C
(at all shorter times or doses, all units are positive) 1 8 0
and 2 9 2
fk = exposure time or dose where all units are negative 3 10 5
(at all longer times or doses, all units are negative) 4 11 11
Fill in the appropriate data in the blanks below: 5 12 18
Time (Tk) for achieving results fk 13 6 13 20
Difference between adjacent times (d) 1 7
Sample size (n) 20 8
Sum of the negative replicates (r) from f1 to fk-1 36 9
Average spore count per carrier (No) 1.7 x 105 11
Log No= 5.2304 (round to 4 decimal places) 12
14
THSK = Tk- d/2 - (d/n * r) (r) from f1 to fk-1 36
1  1 
 13    x36 
2  20 
 13  (0.5)  (1.8)  10.7
THSK = 10.7000 (round to 4 decimal places)

D = (THSK)/ (Log No + 0.2507)
10.7000 10.7000
   1.9522
5.2304  0.2507 5.4811
D= 1.9522 (round to 4 decimal places)

D-VALUE (rounded to 1 decimal place*) 2.0
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 BI’s and you do not have a data point with
all BI’s 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.
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
(D1 + D2 + Dn )
D=
n
U1
D1 =
log10 N 0 − log 10 N u1
n
Nui = ln
r
U = exposure time
N0 = starting spore population
Nu = most probable number of surviving spores
n = number units exposed
r = number units sterile (negative)
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CALCULATION OF D-VALUE USING

STUMBO-MURPHY-COCHRAN METHOD
Sample Identification #: ________________________ Exposure Conditions: ___________________________
In the table on the right, fill in the exposure times and # of units Exposure Number Calculated
i
killed starting with the shortest exposure. Round all numbers to 4 Time (U) Killed (r) D-value
decimal places*. 1
Sample size (n) = 2
Average spore count per carrier (No) = ___________________ 3
Log No =___________________ 4
5
Calculation of D1
6
NU1 = ln (n/r) = _______________
7
log NU1 = __________________ 8
D1 = U1/(log N0 – log NU1) = ___________________ 9
∑D-values = ____________
Calculation of D2 average D-value = ______________
NU2 = ln (n/r) = _______________ D-value = ______________
log NU2 = __________________ (round to 1 decimal place*)
D2 = U2/(log N0 – log NU2) = ___________________
Calculation of D6
Calculation of D3 NU6 = ln (n/r) = _______________
NU3 = ln (n/r) = _______________ log NU6 = __________________
log NU3 = __________________ D6 = U6/(log N0 – log NU6) = ___________________

D3 = U3/(log N0 – log NU3) = ___________________
Calculation of D7
NU4 = ln (n/r) = _______________ log NU7 = __________________
D4 = U4/(log N0 – log NU4) = ___________________
Calculation of D8
NU5 = ln (n/r) = _______________ log NU8 = __________________
D5 = U5/(log N0 – log NU5) = ___________________

Calculation by: _________________________________________________________________________ Date: __________________

Reviewed by: __________________________________________________________________________ Date: __________________
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Sample Identification #: ________________________ Exposure Conditions: 121°C
i
decimal places*. 1 8 0 NA
Sample size (n) = 20 2 9 2 1.8487
Average spore count per carrier (No) = 1.7 x 105 3 10 5 1.9652
Log No = 5.2304 4 11 11 2.0169
Calculation of D1
5 12 18 1.9331
6 13 20 NA
NU1 = ln (n/r) = 2.3026
7
log NU1 = 0.3622 8
9 9
D1 = U1/(log N0 – log NU1) = = 1.8487 ∑D-values = 7.7639
4.8682
average D-value = 1.9410
D-value = 1.9
Calculation of D2 (round to 1 decimal place*)
NU2 = ln (n/r) = 1.3863
log NU2 = 0.1419
Calculation of D5
10
D2 = U2/(log N0 – log NU2) = = 1.9652 NU5 = ln (n/r) = _______________
5.0885
log NU5 = __________________
D5 = U5/(log N0 – log NU5) = ___________________
Calculation of D3
NU3 = ln (n/r) = 0.5978 Calculation of D6
log NU3 = -0.2234 NU6 = ln (n/r) = _______________
11 log NU6 = __________________
D3 = U3/(log N0 – log NU3) = = 2.0169
5.4538 D6 = U6/(log N0 – log NU6) = ___________________
Calculation of D7
Calculation of D4
NU7 = ln (n/r) = _______________
NU4 = ln (n/r) = 0.1054
log NU7 = __________________
log NU4 = -0.9772
D7 = U7/(log N0 – log NU7) = ___________________
12
D4 = U4/(log N0 – log NU4) = = 1.9331
6.2076 Calculation of D8
NU8 = ln (n/r) = _______________
* values ≥0.0950 are rounded to 1 decimal place. log NU8 = __________________
* values ≤0.0949 are rounded to 2 decimal places. D8 = U8/(log N0 – log NU8) = ___________________

Reviewed by: __________________________________________________________________________ Date: __________________
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 N0 = 2.2 x 105
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 r2 for the data.
Data Collected
Exposure Time Population Recovered
0.0 minutes 2.2 x 105
1.8 minutes 3.0 x 104
3.6 minutes 2.6 x 103
5.4 minutes 1.0 x 103
7.2 minutes 2.1 x 101
The Survivor Curve D-value using the Log Linear Regression
Method
m = slope of the regression line
m=
[n∑ [x log y]] − [(∑ ( x))(∑ (log
10 10 y) ) ]
[n∑ ( x )] − [∑ ( x)]
2 2
n = number of data points

y = recovered spore population
x = exposure time
r2 = coefficient of determination which indicates how close the data points are to the
predicted line
an r2 = 1.000 indicates all data points are on the predicted line
2
  ∑ (log10 y ) 
[ ]
∑ [x(log10 y )] − ∑ ( x) n 
  
r2 =
 (∑ ( x) ) 
2
∑
∑ ( x ) −
2
[(
 ∑ (log10 y ) −
2
)] (log10 y ) 2
 n  n
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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
Exposure 2 2
Population = log10 y x x(log10 y) (log10 y)
Time = x
y
2 2
∑(x)= A ∑(log10 y)= B ∑(x )= C ∑[x(log10 y)]= G ∑(log10 y )]= E
Assigned Variable A= B= C= G= E=
Calculation of slope (m) and D-value Calculation of coefficient of determination (r2 )

2
   B  
Number of data points (n) = ________ (G ) − ( A)  
   n  
r2 =
  A 2    B 2 
 (C ) −  
 n   ( E ) −  
     n 
[(n) (G )] − [( A)( B )]
m=
[(n) (C )] − ( A 2 ) 2
    
( ) − ( )  
m=
[( )( )] − [( )( )] r2 =
    
[( )( )] − ( 2
)   2
   2

( ) −   ( ) −  
     
m=
[( )] − [( )]
[( )] − ( )
[( ) − [( )] ] 2
r =
2
[( )] [( )−( )][( )− ( )]
m=
[( )]
r =
2 [( ) ]2
m = __________ [( )][( )]
1
D value = −1  
m r2 =
( )
( )
 1 
D value = −1  
  r2 = __________
D value = −1 ( ) Calculations by: ____________________________ _____________

Date
D value = __________ ≈ _______________ Reviewed by: ______________________________ _____________
(rounded to one decimal)
Date
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Recovered
Exposure 2 2
Time = x
y
1.7 x 105 0.0 5.2304 0.0000 0.0000 27.3571
3.0 x 104 2.0 4.4771 4.0000 8.9542 20.0444
1.0 x 103 4.0 3.0000 16.0000 12.0000 9.0000
6.0 x 102 6.0 2.7782 36.0000 16.6692 7.7184
1.1 x 101 8.0 1.0414 64.0000 8.3312 1.0845
2 2
Assigned Variable A = 20 B = 16.5271 C = 120.0000 G = 45.9546 E = 65.2044
Calculation of coefficient of determination (r2 )

Calculation of slope (m) and D-value 2
   B  
Number of data points (n) = 5 (G ) − ( A)  
   n  
r2 =
  A 2    B 2 
m = slope of the regression line  (C ) −  
 n   ( E ) −  
     n 
[(n) (G )] − [( A)( B )]
m=
[(n) (C )] − ( A 2 ) 
2
  16.5271  
(45.9546 ) − (20 )  
[(5 )(45.9546 )] − [(20 )(16.5271)]    5  
m= r =
2
[(5 )(120 )] − (20 2 )   20 2  

(120 ) − 
 16.52712 
 (65.2044 ) −  
  5    5 
m=
[(229.7730 )] − [(330.5420)] [(45.9546) − [(66.1084)] ] 2
[(600)] − (400) r2 =
[(120) − (80)][(65.2044) − (54.6290)]
[(− 100.7690)] r2 =
[(− 20.1538) ] 2
m=
[(200)] [(40)][(10.5754)]
m = -0.5038 r2 =
(406.1757 )
1 (423.0160)
D value = −1  
m
r2 = 0.9602
 1 
D value = −1  
 − 0.5038 
D value = −1 (− 1.9849) Calculations by: ____________________________ _____________

Date
D value = 1.8911 ≈ 2.0 Reviewed by: ______________________________ _____________
Date
Example of Plotting the Survivor Curve on Semi Log Graph Paper
1.9 Min
Sterilizing Value, Minutes


Calculate the Z-value Using the Following Data:
D121 = 1.9522
D124 = 1.3127
D127 = 0.8650
ISO 11138 states three D-values between 110 and 130°C

Temperature Coefficient Z-value Using the Log Linear
Regression Method
m = the slope of the regression line
m=
[(n )(∑ [x log y])] − [(∑ (x ))(∑ (log y ))]
10 10
[(n )∑ (x )] − [∑ (x )]
2 2

y = D-value
x = exposure temperature
r2 = coefficient of determination
2
  ∑ (log10 y ) 
[ ]
∑ [x(log10 y )] − ∑ ( x) n 
r2 =   
 ( ( x)) 
∑ ( x 2 ) − ∑
2
∑ (log10 y ) 2
[(∑ 10
(log y 2
)]
) −
 n  n
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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.
Exposure
D value = Temperature = 2 2
log10 y x x(log10 y) (log10 y)
y x
2 2
Assigned
Variable A= B= C= G= E=

Calculation of slope (m) and Z-value 2
  B 
(G ) − ( A)  n 
Number of D values (n) = ________   
r2 =
  A  
2
 B 2 
 n   ( E ) −  
     n 
[(n) (G )] − [( A)( B )]
m=
[(n) (C )] − ( A 2 )     
2
( ) − ( )  
[( )( )] − [( )( )] r2 =     
m=
[( )( )] − ( )      
2 2
( ) −   ( ) − 
2

     
m=
[( )] − [( )]
[( )] − ( ) [( ) − [( )] ] 2
r =
2
[( )−( )][( )− ( )]
m=
[( )]
[( )] [( ) ]2
r =
2
m = __________
[( )][( )]
1
Z value = −1  
r2 =
( )
m
( )
 1 
Z value = −1   r2 = __________
 
Z value = −1 ( )
Calculations by: ____________________________ _____________
Date
Z value = _________ ≈ ______________
Reviewed by: ______________________________ _____________
Date
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Exposure
y x
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
2 2
Assigned
Variable A = 372 B = 0.3457 C = 46146 G = 41.8063 E = 0.1024

  B 
(G ) − ( A)  n 
Number of D values (n) = 3   
r2 =
  A  
2
 B 2 
 n   ( E ) −  
     n 
[(n) (G )] − [( A)( B )]
m=
[(n) (C )] − ( A 2 )    0.3457  
2
(41.8063) − (372 )  
   3  
m=
[(3)(41.8063)] − [(372)(0.3457 )] r =
2
  372 2   
[(3)(46146)] − (372 2 )  (46146 ) − 
 3   (0 . 1024 ) −
 0.3457 2
 
     3 
m=
[(125.4189 )] − [(128.6004)]
[(138438)] − (138384) r =
2 [(41.8063) − [(42.8668)] ]
2
[(46146) − (46128)][(0.1024) − (0.0398)]

m=
[(− 3.1815)] [(− 1.0605) ]
2
[(54)] r =
2
[(18)][(0.0626)]
m= -0.0589
r2 =
(1.1247 )
1
Z value = −1   (1.1268)
m
 1 
r2 = 0.9981
Z value = −1  
 − 0.0589 
Z value = −1 (− 16.9779 )
Calculations by: ____________________________ _____________
Date
Z value = 16.9779 ≈ 17.0
Reviewed by: ______________________________ _____________
Date
Example of Plotting the Z-value Curve on Semi Log Graph Paper
16.8°C
Plotting the Z-value Curve on Semi Log Graph Paper
Process Equivalent Time F
−  Tref −TA 
 
FTref , Z A = t A x10  Z 
Process Equivalent Lethality FTref , Z A = ∑ FT1 , Z A + FT2 , Z A + FT3 , Z A ....FTi , Z A
tA = process clock time interval, usually 1 minute

Tref = process reference temperature
ZA = actual Z-value for the spore challenge
Process Equivalent Time
A typical time interval is 1 minute. tA = 1 minute, Tref = 121°C, ZA = 8°C.
Measure average temperature during 1 minute interval to calculate the FTi , Z i

Time Temp. Formula Process
(Min.) Equivalent Time
T1 110°C 

− 

FT1 , Z = 1 min x10 −   = 1x10
T2 115°C 

− 

FT2 , Z = 1 min x10 −   = 1x10
T3 118°C  − 
 
FT3 , Z = 1min x10 −   = 1x10
T4 120°C  − 
 
FT4 , Z = 1 min x10−   = 1x10
T5 121°C  − 
 
FT5 , Z = 1 min x10−   = 1x10
T6 122°C  − 
 
FT6 , Z = 1 min x10−   = 1x10
T7 124°C  − 
 
FT7 , Z = 1 min x10−   = 1x10
T8 121°C  − 
 
FT8 , Z = 1min x10 −   = 1x10
T9 118°C  − 
 
FT9 , Z = 1min x10 −   = 1x10
T10 107°C  − 
 
FT10 , Z = 1 min x10 −   = 1x10
T11 105°C  − 
 
FT11 , Z = 1 min x10 −   = 1x10
∑F
T ,Z
Process FTref ,Z = ∑F Ti , Z

(Min.) Equivalent
Time
T1 110°C  121−110 
 
FT1 , Z = 1 min x10 −  8  = 1x10 −1.3750 0.0422
T2 115°C  121−115 
 
FT2 , Z = 1 min x10 −  8  = 1x10 −0.7500 0.1778
T3 118°C  121−118 
− 
FT3 , Z = 1 min x10  8  = 1x10 −0.3750 0.4217
T4 120°C  121−120 
 
FT4 , Z = 1 min x10 −  8  = 1x10 −0.1250 0.7499
T5 121°C  121−121 
 
FT5 ,Z = 1 min x10 −  8  = 1x10 0 1.0000
T6 122°C  121−122 
 
FT6 , Z = 1 min x10 −  8  = 1x10 0.1250 1.3335
T7 124°C  121−124 
 
FT7 , Z = 1 min x10 −  8  = 1x10 0.3750 2.3714
T8 121°C  121−121 
 
FT8 , Z = 1 min x10 −  8  = 1x10 0 1.0000
T9 118°C  121−118 
 
FT9 , Z = 1 min x10 −  8  = 1x10 − 0.3750 0.4217
T10 107°C  121−107 
 
FT10 , Z = 1 min x10 −  8  = 1x10 −1.7500 0.0178
T11 105°C  121−105 
− 
FT11 , Z = 1 min x10  8  = 1x10 − 2.0000 0.0100
∑F
T ,Z 7.5460
Calculate the Process Spore Log Reduction Value
 
 
Process Equivalent Time  F 
 T ,Z 
SLR =  ref A 
Process D - value
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.
 
 
 T ,Z 
SLR =  ref A 
Process D - value
SLR = =
Process Spore Log Reduction (SLR) Value
58.5 min
SLR = = 13
4.5 min
Your process delivers 13 spore log reductions.

Sterility Assurance Level
SAL = 10 (log10 N 0 −SLR )

Calculate the Sterility Assurance Level
Your process used a spore challenge of 1.5 x 106 therefore, N0 = 1.5 x 106.
Process equivalent lethality is equal to 13 spore log reductions (SLR).
Calculate the sterility assurance level.
Calculation
SAL = 10logNo – SLR
N0 =
logN0 =
Process equivalent lethality =
SAL =
SAL =
Calculation
N0 = 1.5 x 106
logN0 = 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.

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
THSK
D
Log10 N 0 0.2507
d d
THSK Tk x r
2 n
THSK = mean time to sterility

Log10 N0 = spore population
0.2507 = Euler’s constant
Tk = 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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Sample Identification #: Exposure conditions:
(at all shorter times or doses, all units are positive) 1
and 2
fk = exposure time or dose where all units are negative 3
(at all longer times or doses, all units are negative) 4
Fill in the appropriate data in the blanks below: 5
Time (Tk) for achieving results fk 6
Difference between adjacent times (d) 7
Sample size (n) 8
Sum of the negative replicates (r) from f1 to fk-1 9
Average spore count per carrier (No) 11
Log No= (round to 4 decimal places) 12
14
THSK = Tk- d/2 - (d/n * r) (r) from f1 to fk-1
THSK = (round to 4 decimal places)

D = (THSK)/ (Log No + 0.2507)
D= (round to 4 decimal places)

D-VALUE (rounded to 1 decimal place*)
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Sample Identification #: Exposure conditions: 121°C
(at all shorter times or doses, all units are positive) 1 8 0
and 2 9 2
fk = exposure time or dose where all units are negative 3 10 5
(at all longer times or doses, all units are negative) 4 11 11
Fill in the appropriate data in the blanks below: 5 12 18
Time (Tk) for achieving results fk 13 6 13 20
Difference between adjacent times (d) 1 7
Sample size (n) 20 8
Sum of the negative replicates (r) from f1 to fk-1 36 9
Average spore count per carrier (No) 1.7 x 105 11
Log No= 5.2304 (round to 4 decimal places) 12
14
THSK = Tk- d/2 - (d/n * r) (r) from f1 to fk-1 36
1  1 
 13    x36 
2  20 
 13  (0.5)  (1.8)  10.7
THSK = 10.7000 (round to 4 decimal places)

D = (THSK)/ (Log No + 0.2507)
10.7000 10.7000
   1.9522
5.2304  0.2507 5.4811
D= 1.9522 (round to 4 decimal places)

D-VALUE (rounded to 1 decimal place*) 2.0
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 BI’s and you do not have a data point with
all BI’s 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.
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
(D1 + D2 + Dn )
D=
n
U1
D1 =
log10 N 0 − log 10 N u1
n
Nui = ln
r
U = exposure time
N0 = starting spore population
Nu = most probable number of surviving spores
n = number units exposed
r = number units sterile (negative)
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Sample Identification #: ________________________ Exposure Conditions: ___________________________
i
decimal places*. 1
Sample size (n) = 2
Average spore count per carrier (No) = ___________________ 3
Log No =___________________ 4
5
Calculation of D1
6
NU1 = ln (n/r) = _______________
7
log NU1 = __________________ 8
D1 = U1/(log N0 – log NU1) = ___________________ 9
∑D-values = ____________
Calculation of D2 average D-value = ______________
NU2 = ln (n/r) = _______________ D-value = ______________
log NU2 = __________________ (round to 1 decimal place*)
D2 = U2/(log N0 – log NU2) = ___________________
Calculation of D6
NU3 = ln (n/r) = _______________ log NU6 = __________________

D3 = U3/(log N0 – log NU3) = ___________________
Calculation of D7
NU4 = ln (n/r) = _______________ log NU7 = __________________
D4 = U4/(log N0 – log NU4) = ___________________
Calculation of D8
NU5 = ln (n/r) = _______________ log NU8 = __________________
D5 = U5/(log N0 – log NU5) = ___________________


Reviewed by: __________________________________________________________________________ Date: __________________
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Sample Identification #: ________________________ Exposure Conditions: 121°C
i
decimal places*. 1 8 0 NA
Sample size (n) = 20 2 9 2 1.8487
Average spore count per carrier (No) = 1.7 x 105 3 10 5 1.9652
Log No = 5.2304 4 11 11 2.0169
Calculation of D1
5 12 18 1.9331
6 13 20 NA
NU1 = ln (n/r) = 2.3026
7
log NU1 = 0.3622 8
9 9
D1 = U1/(log N0 – log NU1) = = 1.8487 ∑D-values = 7.7639
4.8682
average D-value = 1.9410
D-value = 1.9
Calculation of D2 (round to 1 decimal place*)
NU2 = ln (n/r) = 1.3863
log NU2 = 0.1419
Calculation of D5
10
D2 = U2/(log N0 – log NU2) = = 1.9652 NU5 = ln (n/r) = _______________
5.0885
log NU5 = __________________
D5 = U5/(log N0 – log NU5) = ___________________
Calculation of D3
NU3 = ln (n/r) = 0.5978 Calculation of D6
log NU3 = -0.2234 NU6 = ln (n/r) = _______________
11 log NU6 = __________________
D3 = U3/(log N0 – log NU3) = = 2.0169
5.4538 D6 = U6/(log N0 – log NU6) = ___________________
Calculation of D7
Calculation of D4
NU7 = ln (n/r) = _______________
NU4 = ln (n/r) = 0.1054
log NU7 = __________________
log NU4 = -0.9772
D7 = U7/(log N0 – log NU7) = ___________________
12
D4 = U4/(log N0 – log NU4) = = 1.9331
6.2076 Calculation of D8
NU8 = ln (n/r) = _______________
* values ≥0.0950 are rounded to 1 decimal place. log NU8 = __________________
* values ≤0.0949 are rounded to 2 decimal places. D8 = U8/(log N0 – log NU8) = ___________________

Reviewed by: __________________________________________________________________________ Date: __________________
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 N0 = 2.2 x 105
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 r2 for the data.
Data Collected
Exposure Time Population Recovered
0.0 minutes 2.2 x 105
1.8 minutes 3.0 x 104
3.6 minutes 2.6 x 103
5.4 minutes 1.0 x 103
7.2 minutes 2.1 x 101
The Survivor Curve D-value using the Log Linear Regression
Method
m=
[n∑ [x log y]] − [(∑ ( x))(∑ (log
10 10 y) ) ]
[n∑ ( x )] − [∑ ( x)]
2 2

y = recovered spore population
x = exposure time
r2 = coefficient of determination which indicates how close the data points are to the
predicted line
an r2 = 1.000 indicates all data points are on the predicted line
2
  ∑ (log10 y ) 
[ ]
∑ [x(log10 y )] − ∑ ( x) n 
  
r2 =
 (∑ ( x) ) 
2
∑
∑ ( x ) −
2
[(
 ∑ (log10 y ) −
2
)] (log10 y ) 2
 n  n
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Recovered
Exposure 2 2
Time = x
y
2 2
Assigned Variable A= B= C= G= E=
Calculation of slope (m) and D-value Calculation of coefficient of determination (r2 )

2
   B  
Number of data points (n) = ________ (G ) − ( A)  
   n  
r2 =
  A 2    B 2 
 (C ) −  
 n   ( E ) −  
     n 
[(n) (G )] − [( A)( B )]
m=
[(n) (C )] − ( A 2 ) 2
    
( ) − ( )  
m=
[( )( )] − [( )( )] r2 =
    
[( )( )] − ( 2
)   2
   2

( ) −   ( ) −  
     
m=
[( )] − [( )]
[( )] − ( )
[( ) − [( )] ] 2
r =
2
[( )] [( )−( )][( )− ( )]
m=
[( )]
r =
2 [( ) ]2
m = __________ [( )][( )]
1
D value = −1  
m r2 =
( )
( )
 1 
D value = −1  
  r2 = __________
D value = −1 ( ) Calculations by: ____________________________ _____________

Date
D value = __________ ≈ _______________ Reviewed by: ______________________________ _____________
Date
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Recovered
Exposure 2 2
Time = x
y
1.7 x 105 0.0 5.2304 0.0000 0.0000 27.3571
3.0 x 104 2.0 4.4771 4.0000 8.9542 20.0444
1.0 x 103 4.0 3.0000 16.0000 12.0000 9.0000
6.0 x 102 6.0 2.7782 36.0000 16.6692 7.7184
1.1 x 101 8.0 1.0414 64.0000 8.3312 1.0845
2 2
Assigned Variable A = 20 B = 16.5271 C = 120.0000 G = 45.9546 E = 65.2044

Calculation of slope (m) and D-value 2
   B  
Number of data points (n) = 5 (G ) − ( A)  
   n  
r2 =
  A 2    B 2 
 n   ( E ) −  
     n 
[(n) (G )] − [( A)( B )]
m=
[(n) (C )] − ( A 2 ) 
2
  16.5271  
(45.9546 ) − (20 )  
[(5 )(45.9546 )] − [(20 )(16.5271)]    5  
m= r =
2
[(5 )(120 )] − (20 2 )   20 2  

(120 ) − 
 16.52712 
 (65.2044 ) −  
  5    5 
m=
[(229.7730 )] − [(330.5420)] [(45.9546) − [(66.1084)] ] 2
[(600)] − (400) r2 =
[(120) − (80)][(65.2044) − (54.6290)]
[(− 100.7690)] r2 =
[(− 20.1538) ] 2
m=
[(200)] [(40)][(10.5754)]
m = -0.5038 r2 =
(406.1757 )
1 (423.0160)
D value = −1  
m
r2 = 0.9602
 1 
D value = −1  
 − 0.5038 
D value = −1 (− 1.9849) Calculations by: ____________________________ _____________

Date
D value = 1.8911 ≈ 2.0 Reviewed by: ______________________________ _____________
Date
1.9 Min


Calculate the Z-value Using the Following Data:
D121 = 1.9522
D124 = 1.3127
D127 = 0.8650
ISO 11138 states three D-values between 110 and 130°C

Temperature Coefficient Z-value Using the Log Linear
Regression Method
m = the slope of the regression line
m=
[(n )(∑ [x log y])] − [(∑ (x ))(∑ (log y ))]
10 10
[(n )∑ (x )] − [∑ (x )]
2 2

y = D-value
x = exposure temperature
r2 = coefficient of determination
2
  ∑ (log10 y ) 
[ ]
∑ [x(log10 y )] − ∑ ( x) n 
r2 =   
 ( ( x)) 
∑ ( x 2 ) − ∑
2
∑ (log10 y ) 2
[(∑ 10
(log y 2
)]
) −
 n  n
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Exposure
y x
2 2
Assigned
Variable A= B= C= G= E=

  B 
(G ) − ( A)  n 
Number of D values (n) = ________   
r2 =
  A  
2
 B 2 
 n   ( E ) −  
     n 
[(n) (G )] − [( A)( B )]
m=
[(n) (C )] − ( A 2 )     
2
( ) − ( )  
[( )( )] − [( )( )] r2 =     
m=
[( )( )] − ( )      
2 2
( ) −   ( ) − 
2

     
m=
[( )] − [( )]
[( )] − ( ) [( ) − [( )] ] 2
r =
2
[( )−( )][( )− ( )]
m=
[( )]
[( )] [( ) ]2
r =
2
m = __________
[( )][( )]
1
Z value = −1  
r2 =
( )
m
( )
 1 
Z value = −1   r2 = __________
 
Z value = −1 ( )
Calculations by: ____________________________ _____________
Date
Z value = _________ ≈ ______________
Reviewed by: ______________________________ _____________
Date
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Exposure
y x
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
2 2
Assigned
Variable A = 372 B = 0.3457 C = 46146 G = 41.8063 E = 0.1024

  B 
(G ) − ( A)  n 
Number of D values (n) = 3   
r2 =
  A  
2
 B 2 
 n   ( E ) −  
     n 
[(n) (G )] − [( A)( B )]
m=
[(n) (C )] − ( A 2 )    0.3457  
2
(41.8063) − (372 )  
   3  
m=
[(3)(41.8063)] − [(372)(0.3457 )] r =
2
  372 2   
[(3)(46146)] − (372 2 )  (46146 ) − 
 3   (0 . 1024 ) −
 0.3457 2
 
     3 
m=
[(125.4189 )] − [(128.6004)]
[(138438)] − (138384) r =
2 [(41.8063) − [(42.8668)] ]
2
[(46146) − (46128)][(0.1024) − (0.0398)]

m=
[(− 3.1815)] [(− 1.0605) ]
2
[(54)] r =
2
[(18)][(0.0626)]
m= -0.0589
r2 =
(1.1247 )
1
Z value = −1   (1.1268)
m
 1 
r2 = 0.9981
Z value = −1  
 − 0.0589 
Z value = −1 (− 16.9779 )
Calculations by: ____________________________ _____________
Date
Z value = 16.9779 ≈ 17.0
Reviewed by: ______________________________ _____________
Date
Example of Plotting the Z-value Curve on Semi Log Graph Paper
16.8°C
Plotting the Z-value Curve on Semi Log Graph Paper
Process Equivalent Time F
−  Tref −TA 
 
FTref , Z A = t A x10  Z 
Process Equivalent Lethality FTref , Z A = ∑ FT1 , Z A + FT2 , Z A + FT3 , Z A ....FTi , Z A
tA = process clock time interval, usually 1 minute

Tref = process reference temperature
ZA = actual Z-value for the spore challenge

(Min.) Equivalent Time
T1 110°C 

− 

FT1 , Z = 1 min x10 −   = 1x10
T2 115°C 

− 

FT2 , Z = 1 min x10 −   = 1x10
T3 118°C  − 
 
FT3 , Z = 1min x10 −   = 1x10
T4 120°C  − 
 
FT4 , Z = 1 min x10−   = 1x10
T5 121°C  − 
 
FT5 , Z = 1 min x10−   = 1x10
T6 122°C  − 
 
FT6 , Z = 1 min x10−   = 1x10
T7 124°C  − 
 
FT7 , Z = 1 min x10−   = 1x10
T8 121°C  − 
 
FT8 , Z = 1min x10 −   = 1x10
T9 118°C  − 
 
FT9 , Z = 1min x10 −   = 1x10
T10 107°C  − 
 
FT10 , Z = 1 min x10 −   = 1x10
T11 105°C  − 
 
FT11 , Z = 1 min x10 −   = 1x10
∑F
T ,Z

(Min.) Equivalent
Time
T1 110°C  121−110 
 
FT1 , Z = 1 min x10 −  8  = 1x10 −1.3750 0.0422
T2 115°C  121−115 
 
FT2 , Z = 1 min x10 −  8  = 1x10 −0.7500 0.1778
T3 118°C  121−118 
− 
FT3 , Z = 1 min x10  8  = 1x10 −0.3750 0.4217
T4 120°C  121−120 
 
FT4 , Z = 1 min x10 −  8  = 1x10 −0.1250 0.7499
T5 121°C  121−121 
 
FT5 ,Z = 1 min x10 −  8  = 1x10 0 1.0000
T6 122°C  121−122 
 
FT6 , Z = 1 min x10 −  8  = 1x10 0.1250 1.3335
T7 124°C  121−124 
 
FT7 , Z = 1 min x10 −  8  = 1x10 0.3750 2.3714
T8 121°C  121−121 
 
FT8 , Z = 1 min x10 −  8  = 1x10 0 1.0000
T9 118°C  121−118 
 
FT9 , Z = 1 min x10 −  8  = 1x10 − 0.3750 0.4217
T10 107°C  121−107 
 
FT10 , Z = 1 min x10 −  8  = 1x10 −1.7500 0.0178
T11 105°C  121−105 
− 
FT11 , Z = 1 min x10  8  = 1x10 − 2.0000 0.0100
∑F
T ,Z 7.5460
Calculate the Process Spore Log Reduction Value
 
 
 T ,Z 
SLR =  ref A 
Process D - value
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.
 
 
 T ,Z 
SLR =  ref A 
Process D - value
SLR = =
Process Spore Log Reduction (SLR) Value
58.5 min
SLR = = 13
4.5 min
Your process delivers 13 spore log reductions.

Sterility Assurance Level
SAL = 10 (log10 N 0 −SLR )

Calculation
N0 =
logN0 =
Process equivalent lethality =
SAL =
SAL =
Calculation
N0 = 1.5 x 106
logN0 = 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 (r2) 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 (r2) 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 N0 minus the SLR value delivered by the
process.

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Biological Indicator

The process set at 121.0° C.

Calculate the D-value using the data sheet provided.

THSK = mean time to sterility

THSK = (round to 4 decimal places)

Calculate D-value (D) by the equation:

D= (round to 4 decimal places)

* values ≥0.0950 are rounded to 1 decimal place.

THSK = 10.7000 (round to 4 decimal places)

Calculate D-value (D) by the equation:

D= 1.9522 (round to 4 decimal places)

* values ≥0.0950 are rounded to 1 decimal place.

The initial population N0 = 1.7 x 105.

CALCULATION OF D-VALUE USING

log NU3 = __________________ D6 = U6/(log N0 – log NU6) = ___________________

* values ≥0.0950 are rounded to 1 decimal place.

Calculation by: _________________________________________________________________________ Date: __________________

CALCULATION OF D-VALUE USING

Calculation by: _________________________________________________________________________ Date: __________________

Calculate the coefficient of determination r2 for the data.

n = number of data points

Calculation of D-value by Log Linear Regression (Survivor Curve)

Calculation of slope (m) and D-value Calculation of coefficient of determination (r2 )

D value = −1 ( ) Calculations by: ____________________________ _____________

Calculation of D-value by Log Linear Regression (Survivor Curve)

Calculation of coefficient of determination (r2 )

[(5 )(120 )] − (20 2 )   20 2  

D value = −1 (− 1.9849) Calculations by: ____________________________ _____________

Sterilizing Value, Minutes

Sterilizing Value, Minutes

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

n = number of data points

Calculation of Z-value by Log Linear Regression

Calculation of coefficient of determination (r2 )

Calculation of Z-value by Log Linear Regression

Calculation of coefficient of determination (r2 )

[(46146) − (46128)][(0.1024) − (0.0398)]

Process Equivalent Lethality FTref , Z A = ∑ FT1 , Z A + FT2 , Z A + FT3 , Z A ....FTi , Z A

tA = process clock time interval, usually 1 minute

Measure average temperature during 1 minute interval to calculate the FTi , Z i

Measure average temperature during 1 minute interval to calculate the FTi , Z i

The process D-value is 4.5 minutes.

Calculate the spore log reduction.

Your process delivers 13 spore log reductions.

SAL = 10 (log10 N 0 −SLR )

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

Calculate the sterility assurance level.

Process equivalent lethality =

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

Calculate the sterility assurance level.

Process equivalent lethality = 13 SLR

SAL = 10(6.176 – 13)

The process set at 121.0° C.

Calculate the D-value using the data sheet provided.

THSK = mean time to sterility

THSK = (round to 4 decimal places)

Calculate D-value (D) by the equation:

D= (round to 4 decimal places)

* values ≥0.0950 are rounded to 1 decimal place.

THSK = 10.7000 (round to 4 decimal places)

Calculate D-value (D) by the equation:

D= 1.9522 (round to 4 decimal places)

* values ≥0.0950 are rounded to 1 decimal place.

log NU3 = D6 = U6/(log N0 – log NU6) = _

Calculation by: _______________________________________________________ Date:

Calculation by: _______________________________________________________ Date:

D value = −1 ( ) Calculations by: ________________ _

D value = −1 (− 1.9849) Calculations by: ________________ _

log NU3 = D6 = U6/(log N0 – log NU6) = _

Calculation by: _______________________________________________________ Date:

Calculation by: _______________________________________________________ Date:

D value = −1 ( ) Calculations by: ________________ _

D value = −1 (− 1.9849) Calculations by: ________________ _