VALIDATION OF INCUBATORS

 1.0      OBJECTIVE

           To lay down a procedure for validation of incubator in microbiology laboratory.

2.0        RESPONSIBILTY

            Microbiologist / Executive.

3.0        ACCOUNTABILITY

            Quality  Assurance Control

4.0               PROCEDURE

4.1       DETAILS OF the Standard thermometer used for validation of  Lab thermometer.

Ø      Type    : Liquid - Glass lab Thermometer.

Ø      Place the standard thermometer dipped in glycerin in incubator at various locations as mentioned in annexure-II.

Ø      The incubator is set for desired temperature with the knob on control panel of incubator e.g 32.5 ⁰C,  35 ⁰C & 22.5 ⁰C.

Ø      Wait till the temperature reaches at set point & note down the temp. displayed on the small screen of incubator and compare this temp. with standard thermometer kept near the RTD probe.Likewise check the temperature after every 15 minutes up to one hour and note down the readings on the format.

Ø      Record any difference between the displayed temp. & temp. showed by standard thermometer and set the incubator accordingly

Ø      Frequency of validation :  once in a year.

Ø      The observations are noted in the format of annexure-I

             

4.2       PROCEDURE FOR VALIDATION OF D.H.S (HOT AIR OVEN):

Ø      Keep all the apparatus wrapped thrice with aluminum foil inside DHS as locations shown in the diagram.                

Ø      Keep the Spore loaded strips (having spore of B subilis) & Endotoxin indicator indicators having 10,000EU/vial in DHS(hot air oven) kept in 30ml vial wrapped thrice with aluminum foil at locations shown in the diagram keep one vial unbaked as PPC.   

Ø      Set the hot air oven at 250°C ,wait till the temperature reaches upto the set temperature.

Ø      After reaching the set temperature, note the time & temperature, hold for one hour

Ø      After completion of depyrogenation cycle ,switch off the power supply and take out Indicators for testing.

4.3       Procedure for testing reduction of endotoxin as under.

Ø      Take out the baked endotoxin loaded vial from the DHS.

Ø      Reconstitute 1ml of LRW in the baked vial and transfer 100ml of sample to the depyrogenated reaction tube kept in heating block at 37⁰ C and add 100ml  of LAL in the same tube in duplicate.

Ø      Prepare the dilutions for PPC for confirming 10,000EU/vial .

Ø      Note the gel clot after incubation of one hour

4.4.       Procedure for testing of reduction of prepared spore loaded strip(B subtilis):

Transfer (spore loaded) strip from backed vials and are inoculated in100ml sterile SCD media and incubate at 30 – 35⁰C for 7 days to observe for any turbidity, if any, report to Manager QC

5. 0          REFERENCES:         

USP 25 page no.-1890 & 2251

 Annexure 1. Formats for Instrument validation

DILUTION FOR PPC (Use depyrogenated glass-wares for testing as per SOP no.:K/QC/052)

 CSE contains 1,00000 EU Reconstituted  with  1000  ml of LRW distributed 100ml each in 10ml vials.

Keep one vial as positive.

Dilution for PPC
10,000 EU / vial--> 0.1 ml of CSE + 4.9 ml of LRW---> 0.1 ml of above + 3.9 ml of LRW---> 
0.1 ml of above + 4.9 ml of LRW (0.5 EU / ml) --->  500ml of above  +  500ml of LRW (25 EU / ml)-->
500ml of above  +  500ml of LRW (0.125 EU / ml) --> 500ml of above  +  500ml of LRW (0.06 EU / ml)
--> 500ml of above  +  500ml of LRW    (0.03 EU / ml)

FOR SAMPLE : Reconstitute 1 ml in each backed vial vortex for at least ten minutes and take 100ml of sample  + 100ml of Limulus amoebocyte lysate in the reaction tube and incubate for one hour at 37 °C  ± 1°C and record the results.

HEATING BLOCK TEMPERATURE: 37°C ± 1°C    TIME FROM:           TO:           HRS

S. no.	DETAILS	GEL	S. NO.	DETAILS	GEL
1.	Blank	-ve     -ve	10.	Sample (NPC) in duplicate	-ve     -ve
2.	2 l dilution in duplicate (PPC)	+ve    +ve	11.	Sample (NPC) in duplicate	-ve     -ve
3.	2 l dilution in duplicate (PPC)	+ve     +ve	12.	Sample (NPC) in duplicate	-ve      -ve
4.	l dilution in duplicate (PPC)	+ve    +ve	13.	Sample (NPC) in duplicate	-ve      -ve
5.	l dilution in duplicate (PPC)	+ve    +ve	14.	Sample (NPC) in duplicate	-ve       -ve
6.	l/2  dilution in duplicate (PPC)	-ve     -ve	15.	Sample (NPC) in duplicate	-ve       -ve
7.	l/2  dilution in duplicate (PPC)	-ve     -ve
8.	l/4  dilution in duplicate (PPC)	-ve     -ve
9.	l/4  dilution in duplicate (PPC)	-ve     -ve

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OBSERVATION OF SPORE LOADED STRIP OF B.subtilis

NAME OF MEDIA	SCD media              INCUBATION TEMP. 30-35°C
NO. OF DAYS  ®	1	2	3	4	5	6	7
GROWTH OBSERVED Location:- B.subtilis