Validation of A Laboratory

Validation of a Laboratory
Incubator Using Wireless

and Cabled Datalogger
Part II
By Laura Valdes-Mora
Elite MicroSource Corporation
and Pamela Hardt-English
PhF Specialists, Inc.
he following article describes

the various steps regarding
the Installation Qualification
(IQ), Operational Qualification (OQ)
and Performance Qualification (PQ)
that were executed to complete the
validation of a pharmaceutical microbiology laboratory incubator. This
validation was designed and performed from the viewpoint of a
microbiologist with assistance from
validation experts. The protocol used
for this validation was previously
published in the Journal of Validation Technology.1
This validation
was designed
and performed
from the
viewpoint of a
microbiologist
with assistance
from validation
experts.
ber 31487. This is a gravity convection unit model number 6M with an

operating temperature range of 25C
to 70C. The incubator has the following electrical characteristics:
Volts- 120
Hertz- 50/60
Watts- 300
Description of the Incubator Unit
The overall dimensions of the

unit are 42 X 21 X 39 with chamber dimensions 36 X 18 X 27. The
incubator contains a dual hydraulic
thermostat, a mercury-in-glass thermometer, two shelves, two heaters, double inner
doors, and double outer doors.
The incubator is a conventional unit where fresh

air enters though an air intake at the bottom. The air
is heated at a plenum below the chamber and flows
into the chamber in a uniform flow pattern. Exhaust
air is vented through a port at the top of the incubator.
The unit is a Precision Thelco Incubator catalog num-
Installation Qualification (IQ)

During the IQ, personnel documented all items listed in the Incubator IQ Form. Refer to Appendix A of
validation protocol, previously published in the
Journal of Validation Technology.1 A second person
reviewed and approved all verifications.
162
Journal of Validation Technology
Laura Valdes-Mora & Pamela Hardt-English
Purpose
IQ was conducted to demonstrate that the piece
of equipment was inspected, and contains all the
required parts for its proper functioning. The presence/absence of external equipment aides (recorder
charts, etc.) was also documented.
Equipment and Materials
Incubator
Appendix A
IQ Observations
Incubator Description The unit is a Precision
Thelco Incubator, catalog number 31487. The
temperature range it is designed for is from
room temperature to 70C. The unit uses 120
volts and 5.2 amps. This incubator had not been
previously validated, and was 29 years and
seven months old at the time this validation was
conducted.
Incubator Model Number is 6M.
Incubator Serial Number 22-AG-8.
Incubator was purchased on July 14, 1971 from
Precision Scientific.
Vendor Manual is available.
Location of Vendor Manual The manual can be
found in the file cabinet of the microbiology laboratory.
Drawings for the incubator are available and
located in the vendor manual.
Utilities required Electricity is required for this
unit. The power source was found to be present
and functional. A member of the maintenance
department measured the Volts and the amperage reading of the circuit of the electrical outlet
where the incubator is plugged in. The data were
recorded as found.
Utility Data
Circuit Rating:
130 Volts
1 Phase
Equipment Rating:
125 Volts
1 Phase
Field Measurement:
122.9 Volts 1 Phase
60 Hertz
20 Amps
60 Hertz
5.2 Amps
60 Hertz
4.8 Amps
Cord and Plug NEMA Designation: 5-15 P

Wall receptacle NEMA Designation: 5-15R
Notes: The ground wire is connected. Non-load

cycle Amps = 0.5
An Uninterrupted Power Supply (UPS) is not
used on this incubator.
This unit is not hooked to a back-up generator.
List of Standard Operating Procedures (SOPs)
associated with this incubator; the SOPs were listed by number, title, and revision number. They
are not included in the article as SOPs, and are
confidential documents of the company where we
conducted this validation. SOPs were reviewed
and were found to be appropriate.
The incubator does not have a temperature display.
There is an external temperature recorder in use
for this unit, which is a Dickson Temptrace Recorder Chart. The recorder chart is identified as
number one and contains a replaceable seven-day
chart (graph).
Acceptance criteria were met.
Operational Qualification: (OQ)
In this section the performance of the equipment
and its operational procedure was verified. The OQ
of an incubator consists of an empty chamber temperature mapping.
Purpose
The OQ was performed to confirm that the empty
chamber works within the specified limits of temperature (32 to 37C) throughout the chamber.
Precision Thelco Incubator (32-37C)
Real Time Datalogger - Ellab Model TM9616
Real Time Datalogger
Serial Number 00270110141
Thermocouples Type T (Cu/Con)
Wireless Data Loggers
Ellab Model TSM9803
Serial Number 2056
Temperature Sensor
Serial Number
LC 1
20022 6422
LC 2
20753 5757
LC 3
20770 5747
LC 4
20764 5770
LC 5
20804 5766
LC 6
20717 5309
February 2002 Volume 8, Number 2
163
LC 7
20713 5219
LC 8
20703 4521
LC 9
11542 5679
LC 10
11537 4553
Windows-based PC or laptop computer to operate data loggers
External Temperature Device
Timer
In the diagrams seen for the top and lower shelf,

each number corresponds to the thermocouple and
the logger placed at the location. The two devices
were side-by-side for each one of the locations indicated. The box in the lower shelf with an R in the
center indicates the location of the chart recorder, as
shown in Figure 2.
Figure 2
Relative Position of the

Thermocouples and
Wireless Sensors
Equipment Picture
See Figure 1.
Figure 1
Empty Chamber Set-Up
Top Shelf
1
2
3
5
Front
Lower Shelf
6
8
9
Empty
Acceptance Criterion
All sites measured are within 32-37C.
OQ Observations
Calibration
The calibration of thermocouples was performed
using a Kaye LTR-140 serial number 5C339H, owned
by the pharmaceutical company where we conducted
this validation. A total of 12 Thermocouples (TCs)
were tested, and 10 of those were used for the temperature profiles. Calibrations were satisfactorily conducted according to the SOPs. The two extra calibrated thermocouples were kept in reserve in case of a
failure of one of the others.
Location of Thermocouples and Loggers- Diagrams: The purpose of the diagram is to show the
relative position of the thermocouples and wireless
sensors. The figure is not to scale.
164
10
Front
Tests for Temperature Setting

The incubator was set to 35-37C or 36 + 1C.
While TCs were being placed, the incubator was
opened for 20 minutes. It took 30 minutes after closing the doors of the incubator for the unit to reach
35C. Note that the chart recorder was reading 32.4C.
An additional reading of the recorder indicated the
temperature to be 35.5C.
Test of temperature setting number one (lowest
temperature setting on this incubator). In order to
expedite the decrease in temperature, we opened the
door of the incubator for a few minutes. The temperature decreased to a range between 25-29C. After
closing the door and allowing TCs to stabilize, the
lowest temperature recorded was 29C, as shown in
Figure 3.
Test of Temperature Setting #9 (highest safe
temperature setting on this incubator). Safety light
came on at 66C. Chart recorder read 49.9C, while
the TC next to the recorder chart read 59.2C as
Figure 3
Figure 4
Results of Low
Temperature Setting
Results of High
Temperature Setting
40.0
70.00
35.0
60.00
30.0
50.00
25.0
40.00
20.0
00.32
TC 1
TC 2
TC 3
TC 4
TC 5
TC 6
TC 7
00:37
Setting
Top Shelf Back Left

Top Shelf Back Right
Top Shelf Center
Top Shelf Front Left
Top Shelf Front Right
Bottom Shelf Back Left
Bottom Shelf Back Right
TC 8
TC 9
TC 10
TC 13
TC 14
TC 15
TC 16
00:42
Bottom Shelf Center
Bottom Shelf Left Front
Bottom Shelf Front Right
Description
Description
Description
Description
shown in Figure 4. It appears that the chart recorder

responds more slowly than the thermocouples to
temperature changes.
Due to the fact that the safety light came on during
this test, it was decided to record current temperatures, and set the knob back to its regular setting of
number five. The incubator manual was reviewed,
and it was found that the safety light is related to a
safety thermostat, which operates alternatively to the
control thermostat.
After increasing the temperature as per step number three, it took the incubator 75 minutes to come
down to the set temperature range of 35-37C.
Once all thermocouples were within the temperature range established by the protocol (32-37 C),
with the understanding that temperature readings
can range from 31.5 up to 37.4 C (per rounding
rules), the 24-hour mapping study was started.
24-Hour Temperature Mapping
The empty chamber test was conducted for over
24 hours. Temperature readings were recorded every
two minutes. From the time the run was started
(02:10:40 PM) to its end, the time was determined to
30.00
20.00
10.00
0.00
01:08
TC 1
TC 2
TC 3
TC 4
TC 5
TC 6
TC 7
01:13
Top Shelf Back Left

Top Shelf Center
01:18
Setting
TC 8
TC 9
TC 10
TC 13
TC 14
TC 15
TC 16
01:23
Bottom Shelf Center

Description
Description
Description
Description
be 26 hours, 44 minutes, and 39 seconds.

At 03:52:40 PM, all TCs were within temperature
range. There were still some hot pockets left from
the previous study. It took the chamber an additional 42 minutes to stabilize all locations.
The data obtained show that all thermocouples
were within the specified temperature range for 24
hours as shown in Figure 5.
Wireless Datalogger, Tracksense: The study started at 08:43:00. All wireless loggers (LCs) were at the
target temperature range at 10:07:00 as shown in
Figure 6. It took the sensors 1 hour and 24 minutes to
all be stabilized. This is almost double the amount of
time it took the TCs to get into temperature range.
165
Figure 5
Figure 6
Results of 24-Hour Empty

Chamber Mapping
Results of 24-Hour Empty

Chamber Mapping
Thermocouples
Wireless Tracksense
40.0
40.0
30.0
30.0
20.0
20.0
10.0
10.0
0.0
02:40
TC 1
TC 2
TC 3
TC 4
TC 5
TC 6
TC 7
07:25
12:10 16:55
Setting
Top Shelf Back Left

Top Shelf Center
TC 8
TC 9
TC 10
TC 13
TC 14
TC 15
TC 16
21:40
Bottom Shelf Center

Description
Description
Description
Description
In order for this to be a 24-hour run, the readings

needed to last until 10:07:00 on day number two. The
last reading on day number two was made at
08:41:00, which completes the 24-hour run, but does
not map the temperature within specified ranges for
24 hours. This is one hour and 20 minutes short of the
requirement. This is due to the fact of not being able
to see the data until the study is complete. Note that
the wireless system is being used to determine its
suitability for validating incubators by comparing it
to the real time system. This validation study is based
on the data obtained from traditional thermocouples.
Thermocouple Test: Started at 2:40:40 and ended
at 26:52:40.
All TCs reached the target temperature range at
2:40:40. During the first hour and eight minutes, there
were 10 temperature excursions: one on TC 5 of
0.1C and the other seven on TC 8. Of the excursions
on TC 8, six were of 0.1C, and one was of 0.2C.
This was not observed at any other point in time during the 24-hour study. This indicates that there were
still a few hot spots in the chamber after the highest
166
0.0
08:43-1
LC 1
LC 2
LC 3
LC 4
LC 5
13:28-1
Top Shelf Back Left

Top Shelf Center
18:13-1 22:58-1
Setting
LC 6
LC 7
LC 8
LC 9
LC 10
03:43-2

Bottom Shelf Center
temperature setting test was completed immediately

prior to the empty chamber mapping. Future mapping
studies should allow for more time between tests
either by adding two extra hours, or by running the
mapping study for 30 hours in order to compensate
for the adjustment period. There are companies that
change temperature ranges of their incubators on a
regular basis, due to the variety of tests conducted and
the lack of incubators. In this situation, the chamber
should be allowed several hours to equilibrate prior to
loading it. However, this is not the case of this particular piece of equipment. Therefore, this phenomenon
observed during the 24-hour empty chamber test
should not occur during day-to-day use.
Tracksense: All wireless sensors were within
specified temperature range at 9:43:00. During the
first 24 minutes, three sensors reached 37.5C. This
was not observed at any other point in time during
the 24-hour study.
During the review of TC temperatures, the excursions stated above were not found. The highest temperature found during this review using the computer screen was 37.1C
All temperature devices were checked, and all were

within the specified limit of + 2C from each other.
Note that the temperature loggers recorded different
actual clock times. The loggers were not synchronized
for this study, neither were they set to local time. Each
one was set for a different time zone. Areal time elapsed
adjustment was made to account for the difference.
Performance Qualification: (PQ)
In this section it was verified that the loaded incubator chamber was capable of reaching and maintaining the target temperature range.
Purpose
The PQ confirms that the loaded chamber works
within the specified limits of temperature throughout the chamber. The effect of opening doors for two
different lengths of time, and the effect of a power
failure on the incubators ability to get back into the
temperature profile were also evaluated.
Precision Thelco Incubator (32-37C)
Real Time Data Logger Ellab Model TM9616
Real Time Data Logger
Serial Number 00270110141
Thermocouples Type T (Cu/Con)
Wireless Data Loggers
Ellab Model TSM9803
Serial Number 2056
Temperature Sensor Serial Number
LC 1
20022 6422
LC 2
20753 5757
LC 3
20770 5747
LC 4
20764 5770
LC 5
20804 5766
LC 6
20717 5309
LC 7
20713 5219
LC 8
20703 4521
LC 9
11542 5679
LC 10
11537 4553
Windows-based PC or laptop computer to
operate data loggers.
External Temperature Device (chart recorder)
Timer
Agar plates and culture broths to create a maximum loaded chamber pattern.
Acceptance Criterion
All sites measured are within 32-37C
PQ Observations
A maximum load pattern was selected for the
PQ. In addition, the effect of opening doors for two
different lengths of time, and the effect of a power
failure on the temperature profile were evaluated in
this phase. Typically, these evaluations are conducted during the OQ, however, we chose the
loaded chamber for these tests as we consider this
scenario more realistic, therefore, more meaningful.
Loaded Chamber Description
Top Shelf:
4* 2000 mL screw cap bottles of Lactose
Broth
4
1000 mL screw cap bottles containing 500 mL each of Lactose Broth
1
Rack of 40 screw cap test tubes with
10 mL each of Nutrient Broth
2
Baskets each containing 11 Whirlpak
bags each with 100 mL TSB**
Lower Shelf:
1
Chart recorder
1
Water bottle (used by laboratory for
temperature determinations)
15
Stacks of plates- total of 72 plates- 42
Blood Agar and 30 TSA***
12
Whirlpak bags with 250 mL Lactose
Broth each (in a basket)
4
API strips
*
Two (2) 2000 mL bottles were removed from

the study. See observations for explanation.
** TSB= Tryptic Soy Broth
*** TSA= Tryptic Soy Agar
Loading Pattern Diagrams: The purpose of the

diagrams in Figure 7 is to show the relative position
of the various items in the load. The diagrams are
not to scale.
Numbers on each of the above rectangles denote
number of plates on each stack. If the number is followed by mL, it denotes the volume of medium on
each container.
167
Figure 7
Figure 8
Relative Position of the Various

Items in the Load
Picture of Fully Loaded Chamber
Top Shelf
T1
1
0
0
0
mL
2000
mL
1
0
0
0
mL
2000
mL
T4
1
0
0
0
mL
T3
Test Tube
Rack
T2
Basket
#1
1
0
0
0
mL
Basket
#2
Full
T5
Bottom Shelf
T6
10
A
3B
2B
3
A
10
B
4
B
4
B
T9
T8
3B
4
A
R= Chart recorder
A= TSA plates
C= Water Control
B= Blood Agar Plates
T7
5
A
6
B
8
A
2B
4
B
4
B
Basket
API
T10
T= Thermocouple number (a
wireless sensor is next to
each thermocouple)
Loaded Chamber Picture

An example of a loaded chamber can be seen in
Figure 8.
Maximum Load Pattern
After loading the chamber and waiting one hour
and 45 minutes, it was observed that only 2 TCs were
within the target temperature range. We analyzed the
content of the load, and laboratory personnel reviewed the loading pattern. It was concluded that the
incubator was loaded above the worst-case scenario.
Four 2-liter flasks are never placed into this incubator
by laboratory personnel. This was done only once
during the validation of a microbial limit test, but as it
is not a regular procedure, it was chosen to decrease
168
the loaded chamber by two 2-liter flasks, based on

previously performed work.
Following this action, it took the chamber two
hours and 45 minutes to reach the set temperature.
Five-Minute Opened Door Test
This incubator consists of two double doors.
During this test all doors were opened for five-minutes. The purpose of the test is to simulate a worse case
scenario when laboratory analysts are loading inoculated samples and their controls into the incubator.
During this test time, the temperature of the incubator decreased. Temperature ranged from 22.9 to
30.2C across the loaded chamber as shown in Figure 9. The chart recorder reading was 30.5C. It took
50.5 minutes for the loaded chamber to return to the
set temperature.
Three-Hour Power Outage Test
The purpose of the test was to simulate the effect
of a power outage on the temperature profile of a
fully loaded incubator chamber. The TCs were
checked to ensure all sites were at the set temperature
and proceeded to disconnect the incubator. After
three hours, the temperature across the chamber was
reviewed and the incubator was reconnected to its
power supply.
There was a decrease in temperature that took the
loaded chamber outside the set temperature and outside the target temperature. Temperatures ranged
from 27.7 to 29.4C as shown in Figure 10. The chart
recorder was reading 28.7C.
Figure 9
Figure 10
Results of Five-Minute
Opened Door Test
Results of Three-Hour
Power Outage Test
70.00
70.00
60.00
60.00
50.00
50.00
40.00
40.00
30.00
30.00
20.00
20.00
10.00
10.00
0.00
02:34
TA 1
TA 2
TA 3
TA 4
02:44
Top Back Left

Top Back Right
Top Center
Top Front Left
02:54
03:04 03:14 03:24 03:34

Setting
TA 5
TA 6
TA 7
TA 8
Top Front Right

Bottom Back Left
Bottom Back Right
Bottom Center
Following the power start-up, it took one hour

and 35.5 minutes for the temperature to reach the set
point of 35-37C. This unexpectedly long ramp time
signals that the load also dropped in temperature,
thus requiring a longer time to return to the set temperature.
One-Minute Opened Door Test:
The protocol was originally written for a fiveminute and a 15-minute opened door test. After performing the five-minute opened door test, it was
determined that most of the time the incubator doors
do not remain open longer than just a few minutes,
typically not even reaching the five-minute mark.
Based on this, it was decided to change the 15minute test to a one-minute test in order to represent
a true scenario. In addition, for this test, only one side
0.00
03:36
TC 1
TC 2
TC 3
TC 4
TC 5
04:31
Top Back Left

Top Back Right
Top Center
Top Front Left
Top Front Right
05:26
06:21
Setting
07:16
TC 6 Bottom Back Left

TC 7 Bottom Back Right
TC 8 Bottom Center
TC 9 Bottom Front Left
TC 10 Bottom Front Right
of the double doors was opened, not both, for the

same reason of mimicking actual use.
During the one-minute opened door test, the temperature decreased and was outside the set temperature in some areas of the chamber. The temperatures
ranged from 29.3 to 35.2C as shown in Figure 11.
The chart recorder was reading 33.6C. It took 15 minutes for the chamber to return to the set temperature
once the door was closed.
24-Hour Loaded Chamber Temperature Mapping
The loaded chamber test was conducted for 24
hours. Temperature readings were recorded every
two minutes. The study started at 08:32:00 and ended
at 32:16:00. This is 16 seconds short of precisely 24hours, however, due to rounding rules, this small
169
Figure 12
Figure 11
Results of 24-Hour Loaded

Chamber Temperature Mapping
Results of One-Minute
Open Door Test
Thermocouples
40.0
45.0
30.0
40.0
20.0
35.0
10.0
30.0
0.0
08:32
13:17
25.0
20.0
07:58
TC 1
TC 2
TC 3
TC 4
TC 5
08:08
Top Back Left

Top Back Right
Top Center
Top Front Left
Top Front Right
08:18
Setting
08:28
TC 6 Bottom Back Left

TC 7 Bottom Back Right
TC 8 Bottom Center
TC 9 Bottom Front Left
TC 10 Bottom Front Right
shortage is considered acceptable as this is a 24-hour

run, not a 24.0-hour run.
All thermocouples were within temperature range
at the start of the mapping, as shown in Figure 12.
No excursions were found at any point in time,
thus corroborating that the excursions seen during
the empty chamber test were due to hot pockets left
from the highest temperature setting test.
The loaded chamber maintained a tight temperature
range throughout the 24-hour mapping study on a per
TC basis. The lowest temperature recorded was
34.5C found on TC 8, while the highest was 36.9C
found on TC 9.
Tracksense: All loggers were within temperature
range at the start of the mapping as shown in Figure
13. The study started at 09:50:00, and ended at
33:52:00, for a total of 24 hours and two seconds.
There were no excursions found at any point in time.
170
TC 1
TC 2
TC 3
TC 4
TC 5
Top Back Left

Top Back Right
Top Center
Top Front Left
Top Front Right
18:02 22:47
Setting
TC 6
TC 7
TC 8
TC 9
TC 10
27:32
Bottom Back Left

Bottom Back Right
Bottom Center
Bottom Left Front
Bottom Front Right
The lowest temperature recorded was 34.3C found on

LC 8, while the highest was 37.0C found on LC 6.
All temperature devices were checked and all
were within the specified limit of + 2C from each
other.
Note that the temperature loggers recorded different actual clock times. The loggers were not synchronized for this study, neither were they set to local
time. Each one was set for a different time zone.
Comparisons Between Data Collected by

Thermocouples and by Wireless Loggers
The thermocouples used are type T, copper-constantan thermocouples. The quality of wire and construction can significantly affect the accuracy of the
thermocouples. The thermocouples used in these
tests are Ellabs sealed thermocouples with a stainless steel tip. The Ellab Tracksense logger sensor is
a thermister made of a Silicon chip. Each logger and
sensor has a separate ID number, and is factory calibrated at five points in the range of -40 to 140C.
The accuracy of the Ellab thermocouples and the
Ellab Tracksense sensors is +/-0.2C.
Figure 13
Results of 24-Hour Loaded

Chamber Temperature Mapping
Wireless Tracksense
40.0
30.0
20.0
10.0
0.0
09:50
LC 1
LC 2
LC 3
LC 4
LC 5
14:35
19:20 24:05
Setting
L # 20022 S # 6422
L # 20753 S # 5757
L # 20770 S # 5747
L # 20764 S # 5770
L # 20804 S # 5766
LC 6
LC 7
LC 8
LC 9
LC 10
28:50
L # 20717 S # 5309
L # 20713 S # 5219
L # 20703 S # 4521
L # 11542 S # 5679
L # 11537 S # 4553
Can these data collection systems be used interchangeably? Figure 14 shows a summary of 10 hours of
data from both the empty chamber 24-hour test and the
fully loaded incubator test for both the wireless and
thermocouple studies. The thermocouple data were collected on a real time system, EVAL, and the wireless
data were collected using Ellabs Tracksense system.

This summary demonstrates that both systems
generated comparable results. The differences in values are well within the accuracy of both the thermocouples and the thermisters, which is +/-0.2C.
When complementing thermocouples with wireless loggers or using wireless loggers in place of thermocouples, the issue of real time versus historical
data collection must be addressed. Thermocouples are
connected to real time data loggers, i.e., the data are
displayed as collected. The wireless system is historical, i.e., the data are downloaded and displayed at the
conclusion of the test. Since the data cannot be seen,
preliminary tests might need to be conducted to determine the start time on a test, such as the 24-hour fully
loaded incubator, because the temperature of the incubator might drop during loading.
With a wireless system, there are no wires to get
tangled and assembly can proceed quickly. The Ellab
wireless data loggers are more stable and sealed, and
need less frequent calibration than thermocouples.
Thermocouples are frequently not sealed, and therefore subject to corrosion and degradation that will
affect the calibration. It must be also kept in mind
that not all thermocouples and wireless data loggers
are as accurate as the sensors used in this study.
Summary of Deviations:
The following are the deviations that occurred during the execution of this validation. All deviations from
Figure 14
Data Collection Comparison Between Thermocouples and Wireless Datalogers

All Values are in Degrees Centigrade
Type of
Test
Data File
Name/System
Time
Interval
(10
Hours)
Minimum
for all
Minimum
Values
Maximum
for all
Maximum
Values
Average
of Minimum
Values
Average
Delta T Average
of Maximum (Range of all
Values
from
Values
Average)
Empty
Chamber
Eval1.dt
4/EVAL
6:00:0016:00:00
36.3
37.4
36.5
37.1
0.3
36.8
Track1.dt
4/Tracksense
6:00:0016:00:00
36.3
37.4
36.7
37.1
0.2
36.8
Eval 22.dt
4
12:00:00- 35.5
22:00:00
36.9
35.9
36.3
0.3
36.1
Track2.dt
4/Tracksense
12:00:00- 35.4
36.8
35.9
36.3
0.5
36.2
Loaded
Chamber
171
the original protocol were justified and approved.

There was no reference available for the Kaye
dry bath. Equipment was sent out to the manufacturer for routine calibration. The internal bath reference
was used for all calibrations of the dry bath performed for this validation.
The protocol indicated that the chamber should be
allowed to be at temperature range for at least 30
minutes prior to conducting the five-minute opened
door test. Based on OQ data, it was shown that the
incubator is capable of maintaining the temperature
within the range for extended periods; therefore waiting 30 minutes would not have made a difference.
A 15-minute Opened Door Test was described on
step 15 of the PQ protocol. This was changed to a oneminute Opened Door Test, as typically incubator doors
are opened for not more than a couple of minutes. The
new test length was considered to be reflective of a
real situation.
The final post calibration of the thermocouples had
to be repeated because there was not enough data collected to confirm the high temperature calibration at
40C. The EVAL Datalogger collects and displays
data every two seconds, but the program was set to
save data every two minutes, and the system was shut
down before the final data were stored. Therefore, the
calibration was repeated at Star Services, Hayward,
California, using a Kaye Liquid bath at two temperatures, 30 and 40C to confirm that the thermocouples
were within specifications.
Conclusions
IQ
It is concluded that the unit was installed properly. All necessary components were found to be present, and all the required SOPs were found to be in
place. The utilities (power) supplied to the unit, as
per testing, were found to meet the requirements.
OQ
All test results met the protocol acceptance criteria. The unit is capable of maintaining the target
temperature.
PQ
All test results met the protocol acceptance criteria. Based on data obtained from the opened door
tests and power failure, it can be concluded that nor172
mal laboratory practices are acceptable, and that

extending the incubation time for the same length of
time of the power failure is an acceptable solution.
Note that time should be calculated from the time
the incubator reaches the temperature range (see recommendations).
Conclusion
The incubator was tested and verified to operate
properly according to the manufacturer and protocol
requirements. This unit is therefore considered validated.
Recommendations
In order to have enough data collected, one should
consider running the temperature mappings with a
safety factor of six hours, that is, for a 24-hour run,
conduct a 30-hour mapping study, especially if wireless sensors will be used.
The validation presented here was completed in
three days of continuous work. Scheduling and executing the work in this manner is taxing and complicated. At times, one will be working very early or
very late hours. Careful pre-scheduling is highly recommended. Consider adding extra time to the schedule for unforeseen situations (example: chamber may
not get to temperature in the time you have predicted).
Older incubators that do not have a digital temperature display can benefit from having one installed.
Digital temperature displays are available; they have
a Real Time Datalogger (RTD) which is placed in the
incubator either through a port (if available) or
through the top vent.
Microbiologists should consider that after opening the incubator doors, the equipment loses heat,
causing a decrease in temperature, which typically
falls outside the tests temperature range. Based on
this, incubation times should be recorded from the
time the incubator is at temperature range, not from
the time samples are placed in the incubator.
Caution must be exercised when reading temperature from an external chart recorder. Data from this
validation clearly showed that chart recorders are
slow in capturing accurate temperature.
About the Authors

Laura Valdes-Mora serves as Technical Services Director, acting as Principal Consultant and National
Speaker on USP Microbiology Updates and related
topics at Elite MicroSource Corporation, a Resource
Center for Pharmaceutical and Biotechnology Microbiology. She received a B.S. degree in Biology from the
University of Puerto Rico in 1982, and an M.S. degree
in Microbiology from North Carolina State University in
1986.Ms. Valdes-Mora was a Director at AAI Pharma,
was formerly in charge of production of bacterial
endospores at AMSCO, and has been the President
of the Pharmaceutical Microbiology Forum (PMF)
since 1994. Ms. Valdes-Mora can be reached by
phone at 850-763-5453, by fax at 850-913-9735, or by
e-mail at EMSource@aol.com.
Pamela Hardt-English, is President of PhF Specialists. She has M.S. degrees in Food Science and
Agricultural Engineering from the University of California (Davis) and a B.A. in Computer Science from the
University of California (Berkeley). For the last 23
years at PhF Specialists and FMC Corporation, Ms.
Hardt-English has served as a recognized FDA and
USDA process authority, instituted HACCP programs,
conducted thermal processing studies, and validations of autoclaves, incubators, and other chambers.
She has designed and taught numerous food processing courses. Ms. Hardt-English can be reached
by phone at 408-275-0161, by fax at 408-280-0979,

or by email at phfspec@pacbell.net.
Suggested Reading
The following publications were consulted during the preparation of the
protocol, execution of the work, and preparation of the final report.
Cloud, P.A., Validating a Laboratory Incubator, Biopharm 30,

pp. 39-42. November (1997).
Phil Cloud, Pharmaceutical Equipment Validation: The
Ultimate Qualification Guidebook (Interpharm Press, Buffalo
Grove, IL1998).
Cloud, P., How to Develop and Manage Qualification Protocols for FDACompliance. (Interpharm Press, Buffalo Grove,
IL 1999).
References
1.
Institute of Validation Technology. Journal of Validation

Technology. Vol. 8 No. 1, November 2001. pp. 63-72.
Article Acronym Listing

IQ:
LC:
OQ:
PQ:
RTD:
SOP:
TC:
UPS:
TSA:
TSB:
Installation Qualification
Wireless Loggers
Operational Qualification
Performance Qualification
Real Time Datalogger
Standard Operating Procedure
Thermocouple
Uninterrupted Power Supply
Tryptic Soy Agar
Tryptic Soy Broth
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Validation of a Laboratory

Incubator Using Wireless

he following article describes

ber 31487. This is a gravity convection unit model number 6M with an

Description of the Incubator Unit

The overall dimensions of the

The incubator is a conventional unit where fresh

Installation Qualification (IQ)

Journal of Validation Technology

Laura Valdes-Mora & Pamela Hardt-English

Cord and Plug NEMA Designation: 5-15 P

Notes: The ground wire is connected. Non-load

Laura Valdes-Mora & Pamela Hardt-English

In the diagrams seen for the top and lower shelf,

Relative Position of the

Empty Chamber Set-Up

Journal of Validation Technology

Tests for Temperature Setting

Laura Valdes-Mora & Pamela Hardt-English

Top Shelf Back Left

shown in Figure 4. It appears that the chart recorder

Top Shelf Back Left

Bottom Shelf Center

be 26 hours, 44 minutes, and 39 seconds.

Laura Valdes-Mora & Pamela Hardt-English

Results of 24-Hour Empty

Results of 24-Hour Empty

Top Shelf Back Left

Bottom Shelf Center

In order for this to be a 24-hour run, the readings

Journal of Validation Technology

Top Shelf Back Left

Bottom Shelf Back Left

temperature setting test was completed immediately

Laura Valdes-Mora & Pamela Hardt-English

All temperature devices were checked, and all were

Two (2) 2000 mL bottles were removed from

Loading Pattern Diagrams: The purpose of the

Laura Valdes-Mora & Pamela Hardt-English

Relative Position of the Various

Picture of Fully Loaded Chamber

Loaded Chamber Picture

Journal of Validation Technology

the loaded chamber by two 2-liter flasks, based on

Laura Valdes-Mora & Pamela Hardt-English

Top Back Left

03:04 03:14 03:24 03:34

Top Front Right

Following the power start-up, it took one hour

Top Back Left

TC 6 Bottom Back Left

of the double doors was opened, not both, for the

Laura Valdes-Mora & Pamela Hardt-English

Results of 24-Hour Loaded

Top Back Left

TC 6 Bottom Back Left

shortage is considered acceptable as this is a 24-hour

Journal of Validation Technology

Top Back Left

Bottom Back Left

The lowest temperature recorded was 34.3C found on

Comparisons Between Data Collected by

Laura Valdes-Mora & Pamela Hardt-English