INCUBATORS

In biology, an incubator is a device used to grow and maintain microbiological cultures or cell
cultures. The incubator maintains optimal temperature, humidity and other conditions such as
the carbon dioxide (CO2) and oxygen content of the atmosphere inside. Incubators are essential
for a lot of experimental work in cell biology, microbiology and molecular biology and are used to
culture both bacterial as well as eukaryotic cells.

What are laboratory incubators used for?

Laboratory incubators provide a controlled, contaminant-free environment for safe, reliable work with cell and
tissue cultures by regulating conditions such as temperature, humidity, and CO2. Microbiological incubators are
used for the growth and storage of bacterial cultures.

Types and sizes abound, including dry bath incubators with single or dual blocks, biological oxygen demand
(BOD) units ideal for insect or plant studies, shaking incubators, hybridization ovens, bioreactors, and a wide
variety of laboratory test chambers. Finding the correct size for your particular application is an easy task, with
sizes ranging from small tabletop units to room-size. Laboratory incubators are essential for cell and tissue
culture, biochemical and hematological studies, pharmaceutical work, and food analysis.

How do I choose a laboratory incubator?

The size and capacity of the incubator is the first, most basic consideration. Estimating the number of samples
that will be incubating at any one time will give a general idea of the proper internal volume. What temperature,
humidity, and CO2 ranges will be necessary for your work, and will a water source be required?

To ensure even heat distribution and elimination of cold spots, incubators can be water-jacketed or air-jacketed,
or another source of direct heat can be used. For CO2 incubators, controller options include thermal conductivity
detection (TCD) or infrared sensors.

Incubators offering design features that ensure regulatory compliance are well suited for use in accredited or
certified laboratories, where meeting accuracy and reproducibility requirements is essential.

Reducing contamination is one of the main goals in incubator design. To this end, manufacturers offer high-
temperature decontamination cycles, HEPA filtration, and the use of antimicrobial copper components in the
chamber. Some units feature inner glass doors that permit the contents to be viewed without disrupting the
atmosphere of the incubator.

Integrated touch-screen displays, programmable alarms, data storage, and removable shelves are additional
design improvements that focus on making laboratory incubators easy and more convenient to use.
 SOP FOR OPERATION AND MAINTENANCE

OBJECTIVE
To ensure that the incubator performs satisfactorily and maintain accurate temperature
for Microbial growth.

SCOPE
The SOP covers procedure for operation and calibration of incubator and is applicable
to Quality Control Department formulation Units

RESPONSIBILITY
Microbiologist.

ACCOUNTABILITY
Department Head.

PROCEDURE

1. Ensure that the power supply to the incubator is switched ‘OFF’.

2. Dedust the incubator daily externally with a clean dry cloth.
3. Once a week remove adhered dust by wet mopping using detergent solution.
4. Afterwards wipe the surface with a clean dry cloth to
remove traces of detergent.
5. Once in a month clean the interior surfaces with 2.5 % savlon solution using a clean
cloth.

OPERATING PROCEDURE
1. Ensure that the incubator is properly connected to the power supply.
2. Switch on the main .
3. Turn on the red colour power knob towards 0-1.
4. Turn on the cooling knob towards 0-1.
5. To set the incubator at 22°C , set the lower temperature 21 OC by pressing the
‘SET POINT -1’ and simultaneously adjust the
temperature with the help of screw of SET and RST by screw driver.
6. Set the higher temperature 23 OC by pressing the ‘SET POINT -2’ and
simultaneously adjust the temperature with the help of screw of
SET and RST by screw driver.
7. In the same manner the incubator can be set to 37°, 44° and 55°C whenever
required by setting the lower temperature to 36°,43° and 54° C respectively and by
setting the higher temperature to 38°,45° and 56° C respectively.
8. Record the temperature twice daily. i.e. in the morning and in the evening. The
temperature should not differ ± 2° C from the set
temperature.
Trouble shooting problems and remedial action :
1. The temperature display is not glowing. Check for power supply and proper
electrical connections of instruments with power point.
2. Temperature is not even in the incubator. The air circulating fan may not be
functioning. Check for it.
3. Report any discrepancy observed during operation or temperature monitoring to
Q.C. Executive and notify the defect to Maintenance Department.

CALIBRATION
1. Set the incubator temperature to 22°C. Wait till the set temperature is reached.
2. Take a calibrated thermometer and dip it in a 500 ml beaker filled to 3/4 of the
volume with Glycerol AR grade.
3. Keep the beaker inside, at the center of the incubator. close the incubator door.
Allow the temperature to equilibrate for 30 minutes.
4. Observe the temperature shown by the thermometer. The temperature shown by the
display and by thermometer shall not differ by more than 0.5°C.
5. Record the temperature at two time intervals ( with a gap of 6 hours) in the
temperature record.
6. By following the same procedure as above carry out calibration by setting the
incubator temperature to 37°C, 44°C and 55°C.
7. Record any discrepancy observed during operation or during temperature
monitoring to Quality Control Executive and notify the defect to technical assistant
for rectification. Affix “BREAK DOWN” label on the instrument.

FREQUENCY OF CALIBRATION :
1. Once a month and after each maintenance job.
2. Record the calibration record in the format prescribed.
AUTOCLAVE

Auto calve is a process of sterilization by saturated steam under reduced pressure above
100oc. Steam sterilization is carried out in a pressure chamber called an autoclave.

DESCRIPITION
Moist heating is done in an autoclave. It consists of a strong metallic chamber usually made
of satin less steel. It has a cover fitted with a steam vent, a pressure and a safety value.
Rubber gasket is fitted on the inner side of the lid, in order to make autoclave air tight.
The cover is closed with wing nuts and bolts. The electrically heated element is fitted at the
bottom to heat the water to convert in to the steam. The perforated inner chamber is placed on
the stand. The matreial is sterilized is loosely packed in to it.

PRINCIPLE
The principle of the autoclave or steam sterilizers is that water boils when its vapour pressure
equals that of the surrounding atmosphere.
When pressure is inside the closed vessel increases, the temperature at which water boils also
increases. it is a better sterilizing agent than dry heat because the staturated steam has
penetrative power.
Steam condensed to water and gives up to latent heat to that suraface when it comes in to
contact with the cooler surface.
The energy available from this latent heat is considerable. For example 1600 ml steam at
100oc and realese 518 cal of heat. The large reduction in the volume sucks in more steam in
the area and the process continuous till the temperature of the surface is raised to that of the
steam.
The water of the condensation is ensures moister conditions for killing of the exposed micro
oraganisms.
Bacteria is intrincally more suspectible to moist heat as bacterial proteins coagulates rapidly
and condensed water ensures moist conditions for killing the microbes present.

SOP FOR CALIBRATION

PURPOSE:
This SOP provides an authorised procedure to carry out calibration & validation of
Autoclave.

SCOPE:
This SOP provides the relevant methodology for the calibration and validation
of Autoclave.

RESPONSIBILITY :
Q.C. Microbiologist.

ACCOUNTABILITY:
Manager-Quality Control
 DEFINITIONS:
A chamber for sterilizing with steam under pressure. Theoriginal autoclave was
essentially a pressure cooker. The steam tightened the lid. Thedevice was called an
autoclave (from the Greek auto, self + clavis, key) meaningself-locking.

FREQUENCY:
1. Chemical Indicator - Every load.
2. Biological Indicator - Every Month.

Procedure :

Calibration:
1. Calibrate the temperature indicator, pressure gauge and probe once in a year by external
agency or whenever any replacement or
maintenance done on the apparatus.
2. Place the Benzoic acid AR grade as a chemical indicator for temperature calibration in
regular sterilisation cycle . It melts at 121°C.
3. Maintain the records, and abort the particular load used for calibration.

Validation:
1. Perform the validation of autoclave during installation of Equipment and revalidation.
2. Heat distribution studies on empty chamber 3 times by using a multi-point data logger.
3. All probes must reach temperature 121°C to 124°C. Pressure must be with in 15 to 18 lbs
maintain the same for 15 minutes cycle.
4. Heat distribution studies on minimum load of a load pattern one time by using a multi-
point data logger.
5. All probes must reach temperature 121°C to 124°C. Pressure must be within 15 to 18 lbs
maintain for 15 minutes, & find the lag time.
6. Heat distribution studies on maximum load of a load pattern three time by using a multi-
point data logger.
7. All probes must reach temperature 121°C to 124°C. Pressure must be within 15 to 18 lbs
maintain for 15 minutes, and find the lag time.
8. Heat penetration studies on minimum load of a load pattern three times by using a
multi-point data logger.
9. All probes must reach temperature 121°C to 124°C. Pressure must be within 15 to 18 lbs
maintain for 15 minutes, and find the lag time.
10. Heat penetration studies on maximum load of a load pattern three times by using a multi-
point data logger.
11. All probes must reach temperature 121°C to 124°C. Pressure must be within 15 to 18 lbs
maintain for 15 minutes, and find the lag time.
12. With the above heat penetration study the maximum lag time plus 15 minutes is regular
operating cycle of the validated load pattern.

13. Microbial Challenge Test:

1. Keep spores suspension of Bacillus Stearothermophilus of having population 10 6 at
various location of the autoclave along with probes during maximum load heat penetration
study.
2. Sterilised spore ampoules incubate at 55° to 60°C for 120 hours.
3. Spore ampoules should not show any colour change at the end of the incubation.
4. Maintain the records.
Cleaning:
1. Ensure that the equipment is isolated from the power before starting the cleaning activity.
2. The outside of the autoclave should be wiped with a wet sponge and allow to dry.
3. Clean the chamber with 0.1% SLS solution using the sponge.
4. Wash thoroughly with purified water till get free from the detergent.
5. Enter the cleaning details in the log book.

SOP FOR OPERATION AND CLEANING

Purpose :
This SOP provides an authorised procedure for operating and
cleaning of an Autoclave

Scope :
This procedure applicable to Vertical Autoclave used for media and
Culture destruction in microbiology department.
(Identification number)

Responsibility :
Microbiologist QC , Q.C.Manager.

Procedure :

OPERATION:
1. Ensure that the equipment is clean.
2. Connect the equipment to the main power supply.
3. Open valve and fill with purified water up to the indicator level.
4. Loosen the butterfly knobs and lift up the lid of the autoclave using leg operated
paddle and slide it on its one side.
5. Load the items to be destructed and close the lid and tighten any two opposite
knobs evenly until cover is securely fastened
6. Switch on the autoclave.
7. Allow the pressure to reach upto 5 lbs, then open the pressure valve and release the
vapour along with the water droplets.

8. Close the pressure release valve allow the pressure to reach upto 15 to 18 lbs and it
maintains the pressure range constantly by safety valve.
9. Allow the process till the validated time required for destruction.
10. After sterilisation, as per the holding time the autoclave switch OFF.
11. Open the safety valve to release the steam.
12. After the pressure gauge shows ‘0’ reading, open the lid and remove the
destructed items.
13. Close the lid properly and disconnect the equipment from the power supply.
14. Enter the details in the media destruction log book.
a) Load number.
b) Autoclave holding time ( From - To).
c) Pressure ( 15lbs to 18lbs).
d) Sign.

Cleaning:
1. Ensure that the equipment is isolated from the power before starting the cleaning
activity.
2. The outside of the autoclave should be wiped with a wet sponge and allow to dry.
3. Clean the chamber with 0.1% SLS solution using the sponge.
4. Wash thoroughly with water till it is free from the SLS and rinse with distilled
water.
5. Enter the cleaning details in the log book.
6. Frequency: Once in a week.