EQUIPMENT LINE CLEARANCE BOTTLE WASHING MACHINE

1.0 SCOPE:
To provide guidelines to ensure effective line clearance of the area and equipment.
2.0 RESPONSIBILITY:
Operator, Production Officer.
3.0 PROCEDURE:
3.1 Ensure that the area is cleaned as per SOP: PRGN:xxx.
3.2 Ensure that Bottle washing machine is cleaned as per the SOP: PRCL:xxx/A; Point No: 3.1 for Batch change over/At the end of shift or is cleaned as per the SOP: PRCL:xxx/A; Point No: 3.2, for Product change over/Expiry of use before date.
3.3 Ensure that all containers, documents and labels of previous product / batch are removed and status board is updated.
3.4 Ensure that all the cervices and near by area is free from previous product.
3.5 Ensure that all the gloves and accessories of previous product/batch are removed.
3.6 Check the Temperature, Relative Humidity and the Differential Pressure of the cubicle and enter the same in the Batch Manufacturing Record.
3.7 Line clearance is given by Production Officer to start the next batch, after checking the above-mentioned points, which is to be recorded in the Batch Manufacturing Record (BMR).
3.8 After checking all above points and the BMR, Final Line Clearance should be given by In Process Quality Assurance Officer.
3.9 Record the observations in the format PRLC:xxx:F1.
4.0 ENCLOSURE:
4.1 Line Clearance Checklist for Bottle washing machine, format PRLC:xxx:F1.

EQUIPMENT LINE CLEARANCE BOTTLE DRYING MACHINE

1.0 SCOPE:
To provide guidelines to ensure effective line clearance of the area and equipment.
2.0 RESPONSIBILITY:
Operator, Production Officer.
3.0 PROCEDURE:
3.1 Ensure that the area is cleaned as per SOP: PRGN:xxx.
3.2 Ensure that Bottle drying machine is cleaned as per the SOP: PRCL:xxx/A; Point No: 3.1 for Batch change over/At the end of shift or is cleaned as per the SOP: PRCL:xxx/A; Point No: 3.2, for Product change over/Expiry of use before date.
3.3 Ensure that all containers, documents and labels of previous product / batch are removed and status board is updated.
3.4 Ensure that all the cervices and near by area is free from previous product.
3.5 Ensure that all the gloves and accessories of previous product/batch are removed.
3.6 Check the Temperature, Relative Humidity and the Differential Pressure of the cubicle and enter the same in the Batch Manufacturing Record.
3.7 Line clearance is given by Production Officer to start the next batch, after checking the above-mentioned points, which is to be recorded in the Batch Manufacturing Record (BMR).
3.8 After checking all above points and the BMR, Final Line Clearance should be given by In Process Quality Assurance Officer.
3.9 Record the observations in the format PRLC:xxx:F1.
4.0 ENCLOSURE:
4.1 Line Clearance Checklist for Bottle drying machine, format PRLC:xxx:F1.

EQUIPMENT LINE CLEARANCE BOPP TAPE DISPENCER

1.0 SCOPE:
To provide guidelines to ensure effective line clearance of the area and equipment.
2.0 RESPONSIBILITY:
Operator, Production Officer.
3.0 PROCEDURE:
3.1 Ensure that the area is cleaned as per SOP: PRGN:xxx.
3.2 Ensure that the BOPP tape dispenser is cleaned as per SOP: PRCL:xxx/A
3.3 Ensure that all containers, documents and labels of previous product / batch are removed and status board is updated.
3.4 Ensure that all the cervices and near by area is free from previous product / batch.
3.5 Ensure that accessories of previous product/batch are removed.
3.6 Check the Temperature and Relative Humidity of the Packing hall and enter the same in the Batch Packing Record.
3.7 Line clearance is given by Production Officer to start the next batch, after checking the above-mentioned points, which is to be recorded in the Batch Packing Record (BPR).
3.8 After checking all above points and the BPR, Final Line Clearance should be given by In Process Quality Assurance Officer.
3.9 Record the observations in the format PRLC:xxx:F1.
4.0 ENCLOSURE:
4.1 Line Clearance Checklist for BOPP tape dispenser, format PRLC:xxx:Fx.

EQUIPMENT LINE CLEARANCE BLISTER PACK MACHINE (PHARMAPACK 240 SS)

1.0 SCOPE:
To provide guidelines to ensure effective line clearance of the area and equipment.
2.0 RESPONSIBILITY:
Operator, Production Officer.
3.0 PROCEDURE:
3.1 Ensure that the area is cleaned as per SOP: PRGNxxx
3.2 Ensure that the Blister Pack Machine (240 SS) as per the SOP: PRCL:xxx/A; Point No: 3.1,for batch change over/at the end of shift or as per SOP: PRCL: xxx/A; Point No: 3.2,for product change over/expiry of use before date.
3.3 Ensure that all containers, documents and labels of previous product / batch are removed and status board is updated.
3.4 Ensure that all the cervices and near by area is free from previous product.
3.5 Ensure that all the PVC/PVDC/Aluminum foils, gloves and accessories of previous product/batch are removed.
3.6 Check the Temperature, Relative Humidity and the Differential Pressure of the cubicle and enter the same in the Batch Packing Record.
3.7 Line clearance is given by Production Officer to start the next batch, after checking the above-mentioned points, which is to be recorded in the Batch Packing Record (BPR).
3.8 After checking all above points and the BPR, Final Line Clearance should be given by In Process Quality Assurance Officer.
3.9 Record the observations in the format PRLC:xxx: F1.
4.0 ENCLOSURE:
4.1 Line Clearance Checklist for Blister Pack Machine (Pharmapack 240 SS), format PRLC:xxx: Fx.

OPERATION OF AF-90T CAPSULE FILLING MACHINE

1.0 SCOPE:
Applicable to AF-90T capsule-filling machine manufactured by Pam Pharmaceutical and Allied Machinery Company Limited.
2.0 RESPONSIBILITY:
Operator, Production officer.
3.0 PROCEDURE:
FOR MACHINES HAVING CODE NUMBERS PRxxx AND PRxxy.
3.1 Verify the cleanliness of the area and the capsule-filling machine (AF-90T). Confirm that ‘CLEANED’ label is affixed to the machine.
3.2 Remove the ‘CLEANED’ label and affix the ‘EQUIPMENT IN USE’ label on the equipment. Affix the ‘CLEANED’ label in the BMR.
3.3 Switch ‘ON’ the main supply.
3.4 Release the ‘EMERGENCY’ switch.
3.5 Switch ‘ON’ the control panel by rotating the key provided in the control panel.
3.6 Open the compressed air valve and adjust the pressure, it should be 5 to 7 kg/sq.cm.
3.7 Check the vacuum on vacuum gauge; it should be 20 to 24 inch/Hg.

3.8 Press ‘ MAIN’ key, screen will display the following:

3.9 Press ‘Next screen’, screen will display the following:

3.10 Press ‘COUNT RESET’ prior to start a new batch.

3.11 Press ‘Next screen’, screen will display the following:

3.12 Press ‘Pass word’ key and enter the supervisor level password using the keys provided in the control panel.
3.13 Press ‘Enter’ key twice.
3.14 Press ‘Next screen’, the screen will display:

3.15 Enter the details as per BMR using the keys provide in the control panel.
3.16 Press ‘Main’ to return to the main screen.
3.17 Load the drug blend by operating PCS-200 as per SOP: PROP:029 and empty capsule by operating SE-100 as per SOP: PROP:014 to the respective hoppers.
3.18 Enter the details in the ‘Equipment usage log’
3.19 Press ‘Green’ push button to start the machine.
3.20 Run the machine approximately for 20 strokes to form a uniform powder bed.
3.21 Place the empty capsule releasing lever below the releasing pin and run the machine for eight to ten strokes and check the group weight of 20 capsules as per weight mentioned in the BMR.
3.22 Press ‘Red’ push button to stop the machine.
3.23 Adjust the required weight by varying the height of tamping pins.
3.24 Higher weight capsules and lower capsules produced during weight setting to be open manually and powder will be taken for filling by through 20# sieve.
3.25 Set the required locked length as per product requirement.
3.26 After required weight setting, run the machine with standard speed.
3.27 During filling carry out the in process checks as given in batch manufacturing record.
3.28 Re-use the powder collected in powder collection box as well as powder collected at machine platform and intermediate powder collection box. Powder to be passed through 20#.
3.29 Stop the machine after completion of batch or at end of shift.
3.30 Press ‘Red’ push button to stop the machine.
3.31 Switch ‘OFF’ the main power supply to the machine.
3.32 If batch is not completed at end of shift then remove the balance powder from powder hopper and keep along with balance powder in tightly closed container. Do not remove the powder from powder tub and keep silica bag around the powder tub, remove the balance capsules from the capsule hopper and keep along with empty capsules in closed condition. Remove the silica bag prior to re-start the machine.
3.33 Remove the ‘EQUIPMENT IN USE’ label and affix ‘TO BE CLEANED’ label to the machine. Destroy the ‘EQUIPMENT IN USE’ label.
3.34 Enter the details in the ‘Equipment usage log’
Note: Powder collected in ADU 100 should be destroyed by adding water.
In case any abnormal sound heard then stop the machine and restart the
machine only after rectification.

FOR MACHINE HAVING CODE NUMBER PRxxy.
3.1 Verify the cleanliness of the area and the capsule-filling machine (AF-90T). Confirm that ‘CLEANED’ label is affixed to the machine.
3.2 Remove the ‘CLEANED’ label and affix the ‘EQUIPMENT IN USE’ label on the equipment. Affix the ‘CLEANED’ label in the BMR.
3.3 Switch ‘ON’ the main supply.
3.4 Release the ‘EMERGENCY’ switch.
3.5 Open the compressed air valve and adjust the pressure, it should be 5 to 7 kg/sq.cm.
3.6 Check the vacuum on vacuum gauge; it should be 20 to 24 inch/Hg.
3.7 Enter the details in the ‘Equipment usage log’
3.8 Press ‘Green’ push button to start the machine.
3.9 Run the machine approximately for 20 strokes to form a uniform powder bed.
3.10 Place the empty capsule releasing lever below the releasing pin and run the machine for eight to ten strokes and check the group weight of 20 capsules as per weight mentioned in the BMR.
3.11 Press ‘Red’ push button to stop the machine.
3.12 Adjust the required weight by varying the height of tamping pins.
3.13 Higher weight capsules and lower capsules produced during weight setting to be open manually and powder will be taken for filling by through 20# sieve.
3.14 Set the required locked length as per product requirement.
3.15 After required weight setting, run the machine with standard speed.
3.16 During filling carry out the in process checks as given in batch manufacturing record.
3.17 Re-use the powder collected in powder collection box as well as powder collected at machine platform and intermediate powder collection box. Powder to be passed through 20#.
3.18 Stop the machine after completion of batch or at end of shift.
3.19 Press ‘Red’ push button to stop the machine.
3.20 Switch ‘OFF’ the main power supply to the machine.
3.21 If batch is not completed at end of shift then remove the balance powder from powder hopper and keep along with balance powder in tightly closed container. Do not remove the powder from powder tub and keep silica bag around the powder tub, remove the balance capsules from the capsule hopper and keep along with empty capsules in closed condition. Remove the silica bag prior to re-start the machine.
3.22 Remove the ‘EQUIPMENT IN USE’ label and affix ‘TO BE CLEANED’ label to the machine. Destroy the ‘EQUIPMENT IN USE’ label.
3.23 Enter the details in the ‘Equipment usage log’
Note: Powder collected in ADU 100 should be destroyed by adding water.
In case any abnormal sound heard then stop the machine and restart the
machine only after rectification.

EQUIPMENT LINE CLEARANCE BOTTLE WASHING MACHINE

1.0 SCOPE:
To provide guidelines to ensure effective line clearance of the area and equipment.
2.0 RESPONSIBILITY:
Operator, Production Officer.
3.0 PROCEDURE:
3.1 Ensure that the area is cleaned as per SOP: xxxxx
3.2 Ensure that Bottle washing machine is cleaned as per the SOP: xxxxx; Point No: 3.1 for Batch change over/At the end of shift or is cleaned as per the SOP: xxxxx; Point No: 3.2, for Product change over/Expiry of use before date.
3.3 Ensure that all containers, documents and labels of previous product / batch are removed and status board is updated.
3.4 Ensure that all the cervices and near by area is free from previous product.
3.5 Ensure that all the gloves and accessories of previous product/batch are removed.
3.6 Check the Temperature, Relative Humidity and the Differential Pressure of the cubicle and enter the same in the Batch Manufacturing Record.
3.7 Line clearance is given by Production Officer to start the next batch, after checking the above-mentioned points, which is to be recorded in the Batch Manufacturing Record (BMR).
3.8 After checking all above points and the BMR, Final Line Clearance should be given by In Process Quality Assurance Officer.
3.9 Record the observations in the format PRLC:xxxx.
4.0 ENCLOSURE:
4.1 Line Clearance Checklist for Bottle washing machine, format PRLC:xxxxx.

EQUIPMENT LINE CLEARANCE BOTTLE DRYING MACHINE

1.0 SCOPE:
To provide guidelines to ensure effective line clearance of the area and equipment.
2.0 RESPONSIBILITY:
Operator, Production Officer.
3.0 PROCEDURE:
3.1 Ensure that the area is cleaned as per SOP: xxxxx.
3.2 Ensure that Bottle drying machine is cleaned as per the SOP: xxxxx; Point No: 3.1 for Batch change over/At the end of shift or is cleaned as per the SOP: xxxxxx; Point No: 3.2, for Product change over/Expiry of use before date.
3.3 Ensure that all containers, documents and labels of previous product / batch are removed and status board is updated.
3.4 Ensure that all the cervices and near by area is free from previous product.
3.5 Ensure that all the gloves and accessories of previous product/batch are removed.
3.6 Check the Temperature, Relative Humidity and the Differential Pressure of the cubicle and enter the same in the Batch Manufacturing Record.
3.7 Line clearance is given by Production Officer to start the next batch, after checking the above-mentioned points, which is to be recorded in the Batch Manufacturing Record (BMR).
3.8 After checking all above points and the BMR, Final Line Clearance should be given by In Process Quality Assurance Officer.
3.9 Record the observations in the format xxxxxx.
4.0 ENCLOSURE:
4.1 Line Clearance Checklist for Bottle drying machine, format xxxxxx.

EQUIPMENT LINE CLEARANCE BOPP TAPE DISPENCER

1.0 SCOPE:
To provide guidelines to ensure effective line clearance of the area and equipment.
2.0 RESPONSIBILITY:
Operator, Production Officer.
3.0 PROCEDURE:
3.1 Ensure that the area is cleaned as per SOP: xxxxxx
3.2 Ensure that the BOPP tape dispenser is cleaned as per SOP: xxxxxx
3.3 Ensure that all containers, documents and labels of previous product / batch are removed and status board is updated.
3.4 Ensure that all the cervices and near by area is free from previous product / batch.
3.5 Ensure that accessories of previous product/batch are removed.
3.6 Check the Temperature and Relative Humidity of the Packing hall and enter the same in the Batch Packing Record.
3.7 Line clearance is given by Production Officer to start the next batch, after checking the above-mentioned points, which is to be recorded in the Batch Packing Record (BPR).
3.8 After checking all above points and the BPR, Final Line Clearance should be given by In Process Quality Assurance Officer.
3.9 Record the observations in the format xxxxxx
4.0 ENCLOSURE:
4.1 Line Clearance Checklist for BOPP tape dispenser, format xxxxxx

EQUIPMENT LINE CLEARANCE BLISTER PACK MACHINE (PHARMAPACK 240 SS)

1.0 SCOPE:
To provide guidelines to ensure effective line clearance of the area and equipment.
2.0 RESPONSIBILITY:
Operator, Production Officer.
3.0 PROCEDURE:
3.1 Ensure that the area is cleaned as per SOP: xxxxxx
3.2 Ensure that the Blister Pack Machine (240 SS) as per the SOP: xxxxaa; Point No: 3.1,for batch change over/at the end of shift or as per SOP: xxxxbb; Point No: 3.2,for product change over/expiry of use before date.
3.3 Ensure that all containers, documents and labels of previous product / batch are removed and status board is updated.
3.4 Ensure that all the cervices and near by area is free from previous product.
3.5 Ensure that all the PVC/PVDC/Aluminum foils, gloves and accessories of previous product/batch are removed.
3.6 Check the Temperature, Relative Humidity and the Differential Pressure of the cubicle and enter the same in the Batch Packing Record.
3.7 Line clearance is given by Production Officer to start the next batch, after checking the above-mentioned points, which is to be recorded in the Batch Packing Record (BPR).
3.8 After checking all above points and the BPR, Final Line Clearance should be given by In Process Quality Assurance Officer.
3.9 Record the observations in the format xxxxxx.
4.0 ENCLOSURE:
4.1 Line Clearance Checklist for Blister Pack Machine (Pharmapack 240 SS), format PRLC xxxxcc

SOP FOR PREPARATION AND STANDARDIZATION OF 1.0 M SODIUM HYDROXIDE

PREPARATION:
Dissolve 42 g of Sodium hydroxide in sufficient carbon dioxide-free water to produce 1000 ml.
STANDARISATION:
Weigh accurately about 0.5g of potassium hydrogen phthalate previously powdered and dried at 120°C for 2 hours. Dissolve it in 75 ml carbon dioxide free water. Add 0.1 ml phenolphthalein solution and titrate with prepared sodium hydroxide solution until a permanent pink colour is produced.
Each ml of 1 M sodium hydroxide ≡ 0.2042 gm of potassium hydrogen phthalate.
Perform a duplicate and calculate the molarity factor (MF).
Note down the average value
Calculation
MF =W/ 0.2042 x V
Where
V = Volume of Sodium hydroxide consumed in ml.
W = Weight of potassium Hydrogen phthalate in gram.
Note :
Restandardise before use.
Store in bottle with well fitted suitable stoppers, which prevents access to atmospheric carbon dioxide.
Discard the solution after 30 days.
For storage and labeling follow the general procedure.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.05M EDTA

PREPARATION:
Dissolve 18.6 g of disodium edetate in sufficient water to produce 1000 ml.
Standardisation
Weight accurately about 0.8 g of Granulated Zinc, dissolve by gentle warming in 12ml of diluted Hydrochloric acid and 0.1ml of bromine solution boil to remove the excess bromine, cool, add sufficient water to produce 200 ml. Pipette 20 ml in to a conical flask and nearly neutralize with 2 M sodium hydroxide. Add about 125 ml water and sufficient ammonia buffer pH 10 to dissolve the precipitate and add 5 ml in excess. Add 50 mg of mordant black II mixture and titrate with the prepared disodium edentate solution until the solution turns to green point.
Each ml of 0.1M disodium edetate solution ≡ 0.00654g of Zinc
Perform a duplicate and calculate the molarity factor (MF)
Note down the average value
Calculation
MF =W x 20/200 x 0.00654 x V
Where
W = Weight of Zinc in gram.
V = Volume of EDTA consumed in ml.
Note:
1) Discard the solution after 30 days.
2) Restandardise before use
Store in amber bottle with well fitted suitable stoppers which prevents access to atmospheric carbon dioxide.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.004M BENZETHONIUM CHLORIDE

PREPARATION:
Dissolve 1.792 g of benzethonium chloride, previously dried to constant weight at 100° to 105°, in sufficient water to produce 1000 ml.
Standardisation
Dissolve 0.35 g of the dried substance in 30 ml of anhydrous acetic acid, add 6 ml of mercury(II) acetate solution and carry out potentiometric titration with 0.1M perchloric acid using 0.5ml of crystal violet solution as an indicator to the full colour change of indicator. Carry out a blank titration.
Each ml of 0.1M perchloric acid VS is equivalent to 44.81 mg of C27H42ClNO2.
Calculation
MF = W/0.04481 x V
Where
W = Weight of Benzethonium chloride in g.
V = Volume of perchloric acid consumed in ml.
Note:
1) Discard the solution after 30 days.
2) Restandardise before use
3) For storage and labeling follow the general procedure

SOP FOR PREPARATION AND STANDARDIZATION OF 0.02M MERCURIC NITRATE

PREPARATION:
Dissolve 6.85 g of mercury nitrate in 20 ml of 1M nitric acid and add sufficient water to produce 1000 ml.
Standardisation
Dissolve 15 mg of sodium chloride in 50 ml of water and titrate with the mercury nitrate solution determining the end point potentiometrically, using a platinum or mercury indicator electrode and a mercury-mercury(I) sulphate reference electrode. Each ml of 0.02M mercury(II) nitrate VS is equivalent to 2.338 mg of NaCl.
Calculation
----------------
Note:
1) Discard the solution after 30 days.
2) Restandardise before use
3) For storage and labeling follow the general procedure.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.1M ZINC SULPHATE

PREPARATION:
For a 0.1M solution Dissolve 29 g of zinc sulphate in sufficient water to produce 1000 ml.
Standardisation
To 20 ml add 5 ml of 2M acetic acid in a 500 ml conical flask dilute the prescribed solution to 200 ml with water and add about 50 mg of xylenol orange triturate and sufficient hexamine to produce a violet-pink colour. Add a further 2 g of hexamine and titrate with 0.1M disodium edetate VS until the colour changes to yellow. Each ml of 0.1M disodium edetate VS is equivalent to 6.54 mg of Zn.
Calculation
---------------
Note:
1) Discard the solution after 30 days.
2) Restandardise before use
3) For storage and labeling follow the general procedure.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.1M SODIUM THIOSULPHATE

Preparation
Dissolve 25 g of sodium thiosulphate and 0.2 g of sodium carbonate in sufficient carbon dioxide-free water to produce 1000 ml.
Standardisation
To 20 ml of 0.0167M potassium bromate VS add 40 ml of water, 10 ml of potassium iodide solution and 5 ml of 7M hydrochloric acid. Titrate with the sodium thiosulphate solution using 1 ml of starch solution, added towards the end of the titration, as indicator. Each ml of 0.1M sodium thiosulphate VS is equivalent to 2.784 mg of KBrO3.
Perform a duplicate and calculate the molarity factor (MF)
Note down the average value
Calculation
-----------
Note:
1) Restandardise before use
2) Discard the solution after 30 days.
3) For storage and labeling follow the general procedure.

SOP FOR PREPARATION AND STANDARDIZATION OF 0. 1M SODIUM HYDROXIDE

PREPARATION:
Dissolve 4.2 g of Sodium hydroxide in sufficient carbon dioxide-free water to produce 1000 ml.
STANDARISATION:
Weigh accurately about 0.5g of potassium hydrogen phthalate previously powdered and dried at 120°C for 2 hours. Dissolve it in 75 ml carbon dioxide free water. Add 0.1 ml phenolphthalein solution and titrate with sodium hydroxide solution until a permanent pink colour is produced.
Each ml of 0.1 M sodium hydroxide ≡ 0.02042 gm of potassium hydrogen phthalate.
Perform a duplicate and calculate the molarity factor (MF).
Note down the average value .
Calculation
MF = W/ 0.02042 x V
Where
V = Volume of Sodium hydroxide consumed in ml.
W = Weight of potassium Hydrogen phthalate in gram.
Note :
Restandardise before use.
Store in bottle with well fitted suitable stoppers which prevents access to atmospheric carbon dioxide. Discard the solution after 30 days.For storage and labeling follow the general procedure

SOP FOR PREPARATION AND STANDARDIZATION OF 0.1M SILVER NITRATE

Preparation
Dissolve 17g of silver nitrate in sufficient chloride free water to produce 1000 ml.
Standardisation
Weigh accurately about 0.1g sodium chloride, previously dried at about 110º C for 2 hours and dissolve in 5 ml of water. Add 5 ml of glacial acetic acid, 50 ml of methanol and 0.15 ml of eosin solution. Titrate with silver nitrate solution with constant stirring to purple end point.
Each ml of 0.1 M silver nitrate ≡ 0.005844 g of sodium chloride.
Perform a duplicate and calculate the Molarity factor (MF)
Note down the average value
Calculation
MF = W/0.005844 X V
Where
W = Weight of sodium chloride in gm.
V = Volume of silver nitrate solution consumed in ml.
Note:
1) Restandardise before use
2) Discard the solution after 30 days.
Store in amber bottle with well fitted suitable stoppers which prevents access to atmospheric carbon dioxide.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.1M PERCHLORIC ACID

PREPARATION:
To 900 ml of glacial acetic acid add 8.5 ml of perchloric acid, mix, add 30 ml of acetic anhydride, dilute to 1000 ml with glacial acetic acid, mix and allow to stand for 24 hours.
STANDARISATION:
Wigth accurately about dissolve 0.35 g of potassium hydrogen phthalate in 50 ml of anhydrous acetic acid, warming gently if necessary. Allow to cool protected from the air and titrate with the perchloric acid solution using 0.05 ml of crystal violet solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 20.42 mg of C8H5KO4.
Perform a duplicate and calculate the molarity factor.(MF)
Note down the average value .
Calculation
MF = W/ 0.02042 x V
Where
V = Volume of perchloric acid consumed in ml.
W = Weight of potassium hydrogen phthalate in g.
Note :
Restandardise before use.
Store in amber bottle with well fitted suitable stoppers which prevents access to atmospheric carbon dioxide.
Discard the solution after 30 days.
For storage and labeling follow the general procedure.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.1M PERCHLORIC ACID

PREPARATION:
To 900 ml of glacial acetic acid add 8.5 ml of perchloric acid, mix, add 30 ml of acetic anhydride, dilute to 1000 ml with glacial acetic acid, mix and allow to stand for 24 hours.
STANDARISATION:
Wigth accurately about dissolve 0.35 g of potassium hydrogen phthalate in 50 ml of anhydrous acetic acid, warming gently if necessary. Allow to cool protected from the air and titrate with the perchloric acid solution using 0.05 ml of crystal violet solution as indicator. Each ml of 0.1M perchloric acid VS is equivalent to 20.42 mg of C8H5KO4.
Perform a duplicate and calculate the molarity factor.(MF)
Note down the average value .
Calculation
MF = W/ 0.02042 x V
Where
V = Volume of perchloric acid consumed in ml.
W = Weight of potassium hydrogen phthalate in g.
Note :
Restandardise before use.
Store in amber bottle with well fitted suitable stoppers which prevents access to atmospheric carbon dioxide.
Discard the solution after 30 days.
For storage and labeling follow the general procedure.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.01M LEAD NITRATE

PREPARATION:
Dissolve 3.312 g of Lead nitrate in sufficient water to produce 1000 ml.
STANDARISATION:
Pipette 50 ml of the solution into a flask,add 50 mg of xylenol orange mixture and sufficient hexamine to produce a violet pink colour and titrate with 0.1 M disodium edetate to a lemon yellow end point.
Each ml of 0.1M disodium edetate is equivalent to 0.03312 g of Lead nitrate
Molarity factor:0.03312 x 50 x V/1000 x W
Where
V= Volume of 0.1M disodium edetate consumed in ml
W= Weight of lead nitrate in g
Note:
1) Restandardise before use
2) Discard the solution after 30 days.
3) For storage and labeling follow the general procedure.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.01M LEAD NITRATE

PREPARATION:
Dissolve 3.312 g of Lead nitrate in sufficient water to produce 1000 ml.
STANDARISATION:
Piptte 50 ml of the solution into a flask,add 50 mg of xylenol orange mixture and sufficient hexamine to produce a violet pink colour and titrate with 0.1 M disodium edetate to a lemon yellow end point.
Each ml of 0.1M disodium edetate is equivalent to 0.03312 g of Lead nitrate
Molarity factor : 0.03312 x 50 x V/1000 X W
Where
V = Volume of 0.1M disodium edetate consumed in ml.
W = Weight of lead nitrate in g
Note:
1) Restandardise before use.
2) Discard the solution after 30 days.
3) For storage and labeling follow the general procedure.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.1M FERROUS AMMONIUM SULPHATE

PREPARATION:
Dissolve 40g of ferrous ammonium sulphate in a previously cooled mixture of 40 ml of sulphuric acid and 200ml of water,dilute with sufficient freshly boiled and cooled water to produce 1000ml .
STANDARISATION:
Pipette accurately 25.0ml of the solution into a flask,add 2 drops of 1,10-phenanthroline solution and titrate with 0.1M ceric ammonium sulphate until red colour is changed to pale blue.Each ml of 0.1M ceric ammonium sulphate is equivalent to 0.03921g of Fe(NH4)2(SO4)2,6H2O .
Note :
The molarity factor of volumetric solution should not differ by more than ± 5% of the Prescribed strength and the molarity should be determined with a precision of 0.2%.
Restandardise after 15 days.
Storage in amper bottle with well fitted suitable stoppers which prevent access to atmospheric carbon dioxide.
Discard the solution after 30 days.
For storage and labeling follow the general procedure.

SOP FOR PREPARATION AND STANDARDIZATION OF0.1M EDTA

PREPARATION:
Dissolve 37.2 g of disodium edetate in sufficient water to produce 1000 ml.
Standardisation
Weight accurately about 0.8 g of Granulated Zinc, dissolve by gentle warming in 12ml of diluted Hydrochloric acid and 0.1ml of bromine solution boil to remove the excess bromine, cool, add sufficient water to produce 200 ml. Pipette 20 ml in to a conical flask and nearly neutralize with 2 M sodium hydroxide. Add about 125 ml water and sufficient ammonia buffer pH 10 to dissolve the precipiate and add 5 ml in excess. Add 50 mg of mordant black II mixture and titrate with the prepared disodium edentate solution until the solution turns to green point.
Each ml of 0.1M disodium edetate solution ≡ 0.00654g of Zinc
Perform a duplicate and calculate the molarity factor (MF)
Note down the average value
Calculation
MF = W x 20/200 x 0.00654 x V
Where
W = Weight of Zinc in g.
V = Volume of EDTA consumed in ml.
Note:
1) Discard the solution after 30 days.
2) Restandardise before use
Store in amber bottle with well fitted suitable stoppers which prevents access to atmospheric carbon dioxide.

SOP FOR PREPARATION AND STANDARDIZATION OF 0.1M AMMONIUM THIOCYANATE

Preparation
Dissolve 7.612 g in sufficient water to produce 1000 ml
Standardisation
To 20 ml of 0.1M silver nitrate VS add 25 ml of water, 2 ml of 2M nitric acid and 2 ml of ammonium iron(III) sulphate solution R2 and titrate with the ammonium thiocyanate solution until a reddish yellow colour is obtained.
Each ml of 0.1M silver nitrate VS is equivalent to 7.612 mg of NH4SCN.
Perform a duplicate and calculate the molarity factor (MF)
Note down the average value
Calculation :--------------

Note:
1) Restandardise before use
2) Discard the solution after 30 days.
3) For storage and labeling follow the general procedure.

OPERATION OF AF-90T CAPSULE FILLING MACHINE

1.0 SCOPE:
Applicable to AF-90T capsule-filling machine manufactured by Pam Pharmaceutical and Allied Machinery Company Limited.
2.0 RESPONSIBILITY:
Operator, Production officer.
3.0 PROCEDURE:
FOR MACHINES HAVING CODE NUMBERS xxxxx xxxxx
3.1 Verify the cleanliness of the area and the capsule-filling machine (AF-90T). Confirm that ‘CLEANED’ label is affixed to the machine.
3.2 Remove the ‘CLEANED’ label and affix the ‘EQUIPMENT IN USE’ label on the equipment. Affix the ‘CLEANED’ label in the BMR.
3.3 Switch ‘ON’ the main supply.
3.4 Release the ‘EMERGENCY’ switch.
3.5 Switch ‘ON’ the control panel by rotating the key provided in the control panel.
3.6 Open the compressed air valve and adjust the pressure, it should be 5 to 7 kg/sq.cm.
3.7 Check the vacuum on vacuum gauge; it should be 20 to 24 inch/Hg.

3.8 Press ‘ MAIN’ key, screen will display the following:

3.9 Press ‘Next screen’, screen will display the following:

3.10 Press ‘COUNT RESET’ prior to start a new batch.

3.11 Press ‘Next screen’, screen will display the following:

3.12 Press ‘Pass word’ key and enter the supervisor level password using the keys provided in the control panel.
3.13 Press ‘Enter’ key twice.
3.14 Press ‘Next screen’, the screen will display:

3.15 Enter the details as per BMR using the keys provide in the control panel.
3.16 Press ‘Main’ to return to the main screen.
3.17 Load the drug blend by operating PCS-200 as per relavant SOP and empty capsule by operating SE-100 as per relavant SOP to the respective hoppers.
3.18 Enter the details in the ‘Equipment usage log’
3.19 Press ‘Green’ push button to start the machine.
3.20 Run the machine approximately for 20 strokes to form a uniform powder bed.
3.21 Place the empty capsule releasing lever below the releasing pin and run the machine for eight to ten strokes and check the group weight of 20 capsules as per weight mentioned in the BMR.
3.22 Press ‘Red’ push button to stop the machine.
3.23 Adjust the required weight by varying the height of tamping pins.
3.24 Higher weight capsules and lower capsules produced during weight setting to be open manually and powder will be taken for filling by through 20# sieve.
3.25 Set the required locked length as per product requirement.
3.26 After required weight setting, run the machine with standard speed.
3.27 During filling carry out the in process checks as given in batch manufacturing record.
3.28 Re-use the powder collected in powder collection box as well as powder collected at machine platform and intermediate powder collection box. Powder to be passed through 20#.
3.29 Stop the machine after completion of batch or at end of shift.
3.30 Press ‘Red’ push button to stop the machine.
3.31 Switch ‘OFF’ the main power supply to the machine.
3.32 If batch is not completed at end of shift then remove the balance powder from powder hopper and keep along with balance powder in tightly closed container. Do not remove the powder from powder tub and keep silica bag around the powder tub, remove the balance capsules from the capsule hopper and keep along with empty capsules in closed condition. Remove the silica bag prior to re-start the machine.
3.33 Remove the ‘EQUIPMENT IN USE’ label and affix ‘TO BE CLEANED’ (LBPR:001) label to the machine. Destroy the ‘EQUIPMENT IN USE’ label.
3.34 Enter the details in the ‘Equipment usage log’ (SOP: xxxx).
Note: Powder collected in ADU 100 should be destroyed by adding water.
In case any abnormal sound heard then stop the machine and restart the
machine only after rectification.
FOR MACHINE HAVING CODE NUMBER xxxx.
3.1 Verify the cleanliness of the area and the capsule-filling machine (AF-90T). Confirm that ‘CLEANED’ label is affixed to the machine.
3.2 Remove the ‘CLEANED’ label and affix the ‘EQUIPMENT IN USE’ label on the equipment. Affix the ‘CLEANED’ label in the BMR.
3.3 Switch ‘ON’ the main supply.
3.4 Release the ‘EMERGENCY’ switch.
3.5 Open the compressed air valve and adjust the pressure, it should be 5 to 7 kg/sq.cm.
3.6 Check the vacuum on vacuum gauge; it should be 20 to 24 inch/Hg.
3.7 Enter the details in the ‘Equipment usage log’ .
3.8 Press ‘Green’ push button to start the machine.
3.9 Run the machine approximately for 20 strokes to form a uniform powder bed.
3.10 Place the empty capsule releasing lever below the releasing pin and run the machine for eight to ten strokes and check the group weight of 20 capsules as per weight mentioned in the BMR.
3.11 Press ‘Red’ push button to stop the machine.
3.12 Adjust the required weight by varying the height of tamping pins.
3.13 Higher weight capsules and lower capsules produced during weight setting to be open manually and powder will be taken for filling by through 20# sieve.
3.14 Set the required locked length as per product requirement.
3.15 After required weight setting, run the machine with standard speed.
3.16 During filling carry out the in process checks as given in batch manufacturing record.
3.17 Re-use the powder collected in powder collection box as well as powder collected at machine platform and intermediate powder collection box. Powder to be passed through 20#.
3.18 Stop the machine after completion of batch or at end of shift.
3.19 Press ‘Red’ push button to stop the machine.
3.20 Switch ‘OFF’ the main power supply to the machine.
3.21 If batch is not completed at end of shift then remove the balance powder from powder hopper and keep along with balance powder in tightly closed container. Do not remove the powder from powder tub and keep silica bag around the powder tub, remove the balance capsules from the capsule hopper and keep along with empty capsules in closed condition. Remove the silica bag prior to re-start the machine.
3.22 Remove the ‘EQUIPMENT IN USE’ label and affix ‘TO BE CLEANED’ (LBPR:001) label to the machine. Destroy the ‘EQUIPMENT IN USE’ label.
3.23 Enter the details in the ‘Equipment usage log’ .
Note: Powder collected in ADU 100 should be destroyed by adding water.
In case any abnormal sound heard then stop the machine and restart the
machine only after rectification.

OPERATION OF AIR DISPLACEMENT UNIT (ADU-100)

1.0 SCOPE:
Applicable to Air Displacement unit manufactured by Pam Pharmaceutical and Allied Machinery Company Limited.
2.0 RESPONSIBILITY:
Operator, Production officer.
3.0 PROCEDURE:
3.1 Verify the cleanliness of the area and the equipment. Confirm that CLEANED’ label is affixed to the air displacement unit
3.2 Remove the ‘CLEANED’ label and affix the ‘EQUIPMENT IN USE’ (LBPR:004) label on the equipment. Affix the ‘CLEANED’ label in the BMR.
3.3 Connect the equipment to the power supply point.
3.4 Ensure that the seal filter is fitted properly.
3.5 Enter the details in the ‘Equipment usage log’
3.6 Switch ‘ON’ the starter.
3.7 In case of clogging of the filter bag. Switch of the machine and press the lever down and lift up for shaking of the filter bag.
3.8 At the end of batch / shift switch ‘OFF’ the machine.
3.9 Remove the ‘EQUIPMENT IN USE’ label and affix the ‘TO BE CLEANED’ label to the equipment. Destroy the ‘EQUIPMENT IN USE’ label.
3.10 Enter the details in the ‘Equipment usage log’

HANDLING OF OUT OF SPECIFICATION RESULTS

1.0 OBJECTIVE
To lay down a procedure for handling of In-Process Samples and Finished Products out-of-specifications test results [except Microbiological Analysis]
2.0 SCOPE
This SOP is applicable to Quality Assurance Department.
3.0 RESPONSIBILITY
Head of Respective Departments
4.0 ACCOUNTABILITY
Manager - QC
5.0 PROCEDURE
5.1 Any out-of-specification test result shall be investigated and documented. The investigation report shall include,
5.1.1 Errors in sampling, handling and storage of sample.
5.1.2 Error in Analysis.
5.1.3 Error in manufacturing (process related, non-process related and operator related).
5.2 In case investigation reveals out of specification result is due to manufacturing error, the batch shall be rejected.
5.3 In case investigation reveals that sampling is one of the reason for out of specification result, the repeat analysis shall be done on re-sample.
5.4 In case investigation reveals that out-of-specification results is solely due to analytical error, the repeat analysis shall be performed on same sample.
5.5 If repeat analysis result is found within specification, following step 5.3 and 5.4 then the sample shall be approved. If the analysis result is found out of specification, the sample shall be rejected.
5.6 When investigation does not reveal any abnormalities, due to assignable cause, then repeat analysis shall be performed (except uniformity of Dosage Units and Dissolution Test) by following procedure :
a. Same sample shall be re-analyzed. (i.e., Analysis shall be done on a second aliquot from the same portion of sample that was the source of the first aliquot or Analysis shall be done on portion of the same larger sample previously collected for Laboratory purpose.)
b. Analysis shall be done in triplicate by two analysts . Out of two analysts one may be the original analyst.
5.7 The original out-of-specification test result shall be ignored if,
a. Each test result is within specification.
b. Each set of triplicate results show RSD less than 3%.
c. Difference between average value by the two analysts is less than 3%.
5.8 The original out-of-specification test result shall not be averaged along with repeat analysis results.
5.9 The average of average values reported by individual analyst shall be reported on Analytical Report.
5.10 If repeat analysis results do not meet the specification the batch shall be rejected.
5.11 Annexure - II shall be used for documentation of investigation.
5.12 USP procedure shall be followed for out-of-specification test results for uniformity of dosage units testing if the cause is non-assignable.
5.13 USP procedure shall be followed for out-of-specification test results for Dissolution testing of 6 units if the cause is non-assignable.
5.14 In case of rejection of samples, thorough investigation shall be done involving Production Manager, Product Development Manager and General Manager - Quality Assurance / Senior Group Leader and the same shall be documented.
5.15 The investigation shall be completed within 30 days after QC rejection of the sample. Investigation report shall outline the corrective actions necessary to save batch to prevent similar reoccurrence.
5.16 The investigation shall be extended to other batches and products possibly affected due to process related error.
5.17 The investigation shall also be extended to other batches and products possibly affected due to operator error or malfunctioning of equipment.

SPRING CLEANING OF MICROBIOLOGY LAB

1.0 OBJECTIVE
To lay down procedure for spring cleaning of microbiology Lab.
2.0 SCOPE
This SOP covers the procedure for spring cleaning of microbiology lab and is applicable to the Quality Control Department
3.0 RESPONSIBILITY
Housekeeping supervisor
4.0 ACCOUNTABILITY
Department Head
5.0 PROCEDURE
5.1 With the help of non-shedding mop or vacuum cleaner, clean the area in the following order,
a. Ceiling , fixtures and walls.
b. Outer surface of equipment’s i.e, LAF, incubator, autoclave, etc.,
c. Media preparation room
d. Table top and chairs
e. Flooring
5.2 Subsequently swab the area in the order given above with any of the following disinfectant solutions 2.5% Dettol solution or 2.5 % Fairgenol solution or 2.5 % Savlon solution or with 2.5 % Phenyl solution .
5.3 Clean the area in following order.
a. Critical (LAF room)
b. Change rooms
c. Incubator room
d. Media preparation room
e. Autoclave room
5.3 Complete the entire procedure of cleaning (sweeping and moping) of critical area before proceeding to other areas. Record the Cleaning details in Annexure
Note -Use sterile water for making disinfectant solution for cleaning in critical area (LAF room).
6.0 FREQUENCY
6.1 Once in a month.

TEST FOR BACTERIAL ENDOTOXINS

PRINCIPLE
The bacterial endotoxin test or LAL test is the most sensitive and specific means available to detect and measure bacterial endotoxins, a fever producing by-product of gram negative bacteria commonly known as Pyrogen. The gram negative bacteria causes blood coagulation in Americal Horse Shoe Crab (Limulus Polyphemus) and clotting phenomenon was an enzyme-mediated response to endotoxin by components in the intercellular fluid of the circulating cells, the amebocyte. This phenomenon is exploited for estimating the concentration of bacterial endotoxins that may be present in or on the sample. The rate of reaction depends on the concentration of endotoxin, the pH and the temperature. The reaction requires the presence of certain bivalent cations, a proclotting enzyme system and clottable protein; these are provided by the lysate.
As per BP
Before carrying out the test for endotoxins on the preparation being examined, it is necessary to verify.
1. that the equivalent used does not adsorb endotoxins,
2. the sensitivity of the lysate and
3. the absence of interfering factors.
Carry out the test in a manner that avoids microbial contamination. If necessary, treat equipment to eliminate Endotoxins.
Reagents
Limulus Amoebocyte Lysate
A lysate of amebocytes from the horse shoe crab, Limulus Polyphemus. Reconstitute the lysate as stated on the label. For each batch, confirm the stated sensitivity as prescribed under sensitivity of the lysate. The sensitivity of the lysate is defined as the lowest concentration of
Endotoxin that yields a firm gel in the test conditions and is expressed in Units per milliliter.
Water BET
Water that gives a negative result in the conditions prescribed in the test for bacterial Endotoxins on the preparation being examined. It may be prepared by distilling water three times in an apparatus fitted with an effective device to prevent the entrainment of droplets or by other means that give water of the requisite quality.
0.1M Hydrochloric Acid BET
0.1 M Hydrochloric acid that has been prepared using water BET. After adjustment to pH 6.5 to 7.5 with 0.1 M Sodium Hydroxide BET it gives a negative result in the conditions of the test.
0.1 M Sodium Hydroxide BET
0.1 M Sodium Hydroxide that has been prepared using water BET. After adjustment to pH 6.5 to 7.5 with 0.1 M Hydrochloric acid BET it gives a negative result in the conditions of the test.
Standard Endotoxin Preparation
The standard preparation is the 1st International Standard for Endotoxin , established in 1986, consisting of freeze-dried Endotoxin from Escherichia coli 0113:”H10:K negative with trehalose (supplied in ampoules containing 14000 units), or another suitable preparation the activity of which has been determined in relation to the Internaltional Standard using a gelatin method.
Procedure
Unless otherwise prescribed, prepare the solutions an dilution’s used in the test using Water BET
1. If necessary, adjust the solution being examined to pH 6.5 to 7.5 using 0.1 M Hydrochloric acid BET, 0.1 M Sodium Hydroxide BET or a suitable buffer.
2. Add a volume of the lysate appropriate to the chosen receptacle (for example a slide or a tube) to each of the requisite number of such receptacles maintained at 36 - 38 OC .
3. At intervals that will permit the examination of each receptacle and the recording of each result, add to each receptacle an equal volume of the solution being examined and immediately mix gently with the lysate.
4. Incubate, the reaction mixture without vibration and avoiding loss of water by evaporation, for a constant period that has been found suitable in the chosen experimental conditions (usually 20 to 60 min.,), examine the receptacle and record the result.
5. A positive results is indicated by the formation of a firm gel that does not disintegrate when the receptacle is gently inverted.
6. A result is negative is such gel is not formed.
Sensitivity of the Lysate
1. Prepare not fewer than four replicate series each of not fewer than three dilution’s of the Standard Preparations such that at least the final dilution in each series gives a negative result.
2. Examine the dilution’s and a negative control solution consisting of water BET, as described under procedure.
3. Calculate the average of the logarithms of the lowest concentration of Endotoxin in each series of dilutions for which a positive result is found.
4. The antilogarithm of this average gives the estimated lysate sensitivity.
5. The estimated lysate sensitivity is confirmed if it does not differ by more than a factor of 2 from the stated sensitivity.
6. The estimated lysate sensitivity is the used in all tests performed using this lysate.
Interfering Factors
1. Operate as prescribed under sensitivity of the lysate but to prepare the dilutions of the Standard Preparation use the preparation being examined at the maximum valid dilution calculated from the expression :

= maximum allowable endotoxin concentration/ sensitivity of lysate

2. If the sensitivity of the lysate determined in the presence of the preparation being examined does not differ by more than a factor of 2 from that determined in the absence of the preparation being examined, the latter does not contain factors that interfere in the experimental conditions and it may be examined without further treatment.
3. If the sensitivity of the lysate determined in the presence of the preparation being examined differs by more than a factor of 2 from that determined in the absence of the preparation being examined, the preparation being examined acts as an inhibitor or an activator.
4. The interfering factors must be eliminated by suitable treatment such as dilution, filtration, neutralization, dialysis or addition of substances that displace absorbed Endotoxins.
5. The use of a more sensitive lysate permits the use of a greater dilution of the preparation being examined and this may contribute to the elimination of interference.
6. Ultrafiltration may be used when the interfering factor passes through a filter with a nominal separation limit corresponding to a molecular weight of 10,000 to 20,000 daltons.
7. Assymmetrical membrane filters of cellulose triacetate of polysulphone have been found to be suitable .
8. The material retained on the filter, which contains the Endotoxins, is rinsed with water BET or a suitable buffer and endotoxins are recovered in water BET or a suitable buffer.
9. The test volume and the final volume used to recover the endotoxins are determined for each preparation being examined.
10. Establish that the chosen treatment effectively eliminates interference without removing Endotoxins by repeating the test for interfering factors using the preparation being examined to which the Standard Preparation has been added and which has then been submitted to the chosen treatment.
Test for Bacterial Endotoxins in the Preparation.
1. Carry out the method described under procedure in duplicate using the maximum valid dilution of the preparation being examined which has been treated if necessary to eliminate interfering factors.
2. Examine at the same time a negative control consisting of water BET and two positive controls each of which contains the standard preparation at a concentration corresponding to twice the stated sensitivity of the lysate and one of which contains the preparation being examined (treated if necessary to eliminate interfering factors after the addition of the standard preparation) at the concentration being used in the test.
3. The test is not valid unless the negative and both positive controls give the appropriate results.
INTERPRETATION
1. The preparation being examined complies with the test if a negative result is found for both test mixtures.
2. The preparation being examined does not comply with the test if a positive result is found for both test mixtures.
3. If a positive result is found for one test mixture and a negative result for the other, repeat the test; the preparation being examined complies with the test if a negative result is found for both test mixtures.
As per USP
Reference Standard and Control Standard Endotoxins
1. The reference standard endotoxin (RSE) is the USP Endotoxin reference standard, which has defined potency of 10,000 USP Endotoxin Units (EU) per vial.
2. Constitute the entire contents of vial of the RSE with 5 mL of LAL Reagent Water, mix intermittently for 30 min. using a vortex mixer and use this concentrate for making appropriate serial dilutions.
3. Preserve the concentrate in a refrigerator, for making subsequent dilutions, for not more than 14 days.
4. Mix vigorously, using a vortex mixer for not less than 3 min. before use.
5. Mix each solution for not less than 30 sec. before proceeding to make the test solution.
6. Do not store dilutions, because of loss of activity by absorption, in the absence of supporting data to the contrary.
7. A control standard endotoxin (CSE) is an endotoxin preparation other than the RSE that has been standardized against the RSE.
8. Standardize each new lot of CSE prior to use in the test calibration of a CSE in terms of RSE must be with the specific lot of LAL reagent and the test procedure with which it is to be used.
9. Standardization of a CSE against the RSE using an LAL reagent for the gel-clot procedure may be effected by assaying a minimum of 1 vial of the CSE and 1 vial of the RSE, as directed under Test procedure, but using 4 replicate reaction tubes at each level of the dilution series of the RSE and 4 replicate reaction tubes similarly for each vial or aliquot of the CSE.
10. The antilog of the difference between the mean log10 endpoint of the RSE and the mean log10 endpoint of the CSE is the standardized potency of the CSE, which is to be converted to and expressed in Endotoxin Units per mg under stated drying conditions for the CSE, or in Endotoxin units per container, whichever is appropriate.
11. A suitable CSE has a potency of not less than 2 Endotoxin units per mg and not more than 50 Endotoxin Units per mg.
Preparatory Testing
1. Use LAL reagent of confirmed label sensitivity.
2. Treat any containers or utensils employed so as to destroy extraneous surface endotoxins that may be present, such as by heating in an oven at 250 OC or above for sufficient time.
3. The validity of test results for bacterial endotoxins requires an adequate demonstration that specimens of the article or of solutions washings, or extracts thereof to which the test is to be applied do not of themselves inhibit or enhance the reaction or otherwise interfere with the test.
4. Validation is accomplished by performing the inhibition or Enhancement Test as described below.
5. Appropriate negative controls are included.
6. Validation must be repeated if the LAL reagent source or the method of manufacture or formulation of the article is changed.
Test for Confirmation of Labeled LAL Reagent Sensitivity
1. Confirm the labeled sensitivity using at least one vial of the LAL reagent lot.
2. Prepare a series of two fold dilutions of the RSE (CSE) to give concentrations of 2l , l , 0.5 l, and 0.25 l, where l is the labeled sensitivity of the LAL reagent in Endotoxin Units per mL.
3. Perform the test on the four standard concentrations in quadruplicate and include negative controls.
4. The geometric mean endpoint concentration (see Calculation and Interpretation) must be greater than or equal to 0.5 l and less than or equal to 2.0 l.
5. Confirm the labeled sensitivity of each new lot of LAL reagent prior to use in the test.
Inhibition or Enhancement Test
1. Perform the test on aliquots of the specimen, or a dilution not to exceed the Maximum Valid Dilution, in which there is no detectable endotoxin.
2. Perform the test on the specimen without added endotoxin and with endotoxin added to give final concentration of 2 l, l , 0.5 l, 0.25 l.
3. Perform the test as directed under Test Procedure, but using not less than replicate tubes for the untreated specimen to which Endotoxin has been added.
4. In parallel with the above, test in duplicate the same endotoxin concentrations in water and untreated negative controls.
5. Calculate the geometric mean endpoint endotoxin concentration for the specimen as described under Calculation and Interpretation.
6. The test is valid for the article if the geometric mean endpoint concentration in the specimen is greater than or equal to 0.5 l and less than or equal to 2.0 l.
7. If the result obtained for the specimens to which endotoxin has been added is outside the specified limit, the article is unsuitable for the Bacterial Endotoxins Test.
8. Repeat the test for inhibition or enhancement after neutralization, inactivation, or removal of the interfering substances or after the specimen has been diluted by a factor not exceeding the Maximum Valid Dilution.
9. Use a dilution, not exceed the Maximum Valid Dilution sufficient to overcome the inhibition or enhancement of the known added endotoxin, for subsequent assays of Endotoxin in test specimens.
10. If endogeneous Endotoxin is detectable in the untreated specimens under the conditions of the test, the article is unsuitable of the Inhibition or Enhancement Test, or, it may be rendered suitable by removing the Endotoxin present by ultra-filtration, or by appropriate dilution.
11. Dilute the untreated specimen (as constituted, where applicable for administration or use), to a level not exceeding the maximum valid dilution, at which no endotoxin is detectable.
12. Repeat the test for Inhibition or Enhancement using the specimens at those dilution’s.
MAXIMUM VALID DILUTION (MVD)
1. The Maximum Valid Dilution is appropriate to Injections or to solutions for parental administration in the form constituted diluted for administration, or where applicable, to the amount of drug by weight if the volume of the dosage form for administration could be varied.
2. Where the endotoxin limit concentration s specified in the individual monograph in terms of volume (in EU per mL), divide the limit by l, which is the labeled sensitivity (in EU per mL) if the lysate employed in the assay, obtain the MVD factor.
3. Where the endotoxin limit concentration is specified in the individual monograph in terms of weight or Units of active drug (in EU per mg or in EU per Unit), multiply the limit by the concentration in mg per mL or in Units per mL of the drug in the solution tested or of the drug constituted according to the label instructions, whichever is applicable, divide the product of the multiplication by l, to obtain the MVD factor.
4. The MVD factor so obtained is the dilution limit for the preparation for the test to be valid.
Test Procedure
1. In preparing for and applying the test, observe precautions handling the specimens in order to avoid gross microbial contamination.
2. To quantify the amount of endotoxin in a specimen, an assay is performed on decreasing concentrations of specimens prepared by serial dilution.
3. Select dilution’s so that they correspond to a geometric series in which each step is greater than the next by a constant ratio.
4. Include negative and positive controls and a positive product control.
5. Use not less than 2 replicate reaction tubes at each level of the dilution series for each specimen under test.
6. A standard endotoxin dilution series involving not less than 2 replicate reaction tubes is conducted in parallel.
7. A set of standard endotoxin dilution series is included for each block of tubes, which may consist of a number of a racks for incubation together, provided environmental conditions within blocks are uniform.
8. Minimum Valid Concentration (MVC) : The lowest drug specific concentration that will permit detection of endotoxin contamination within the endotoxin limit.
9. Positive Product Control, (PPC) : An aliquot of test sample spiked with a known amount of endotoxin (2l). This control serves as the inhibition control for get clot assay.
10. Negative Product Control (NPC) : It is the actual test where in 0.1 mL of diluted sample is added to 0.1 mL of lysate.
Calculating the Endotoxin Limit :
Endotoxin Limit = K/M ,

where K = 0.5 EU/Kg/hr. for all parentals

M = Max. dose of product/Kg/hr.

Calculating MVD & MVC :
Lambda = Lysate Label Claim Sensitivity
Potency of the product = Concentration of the product in Units / mL
MVC = Lambda x M (dosage)/ K (constant)

OR

MVC = Lambda / Endotoxin Limit

Eg. Fluconozole MVC = 0.125 EU/mL x 200 mg/60 Kg/ 5.0 EU/Kg == 0.083 mg/mL

MVD = (potency of product) x Endotoxin Limit / Lambda

or

MVD = Potency/ MVC

Eg. Flucanozole MVD = [2 mg/mL] / [0.83 mg/mL] = 24
I.e., the final formulation of Zolstan infusion can be diluted 24 (1 + 23) times for endotoxin test, when lysate of 0.125 EU/mL sensitivity is used.
Preparation And Storage Of Reagents
1. Since the form and amount per container of standard endotoxin and of LAL reagent may vary, constitution and / or dilution of contents should be as directed in the labeling.
For Lysate of 0.125 EU/mL sensitivity : If CSE contains 100 EU/vial, which can reconstituted with 5 mL of LRW, each 1 mL will have 20 EU. To prepare 4 lambda solution, mix 1.95 mL of LRW and 0.05 mL of reconstituted CSE. Dilute this four times (1+3) to get a solution of lambda strength (0.125 EU/mL).
2. The pH of the test mixture of the specimen and the LAL reagent is in the range 6.0 to 8.0 unless specifically directed otherwise in the individual monograph.
3. The pH may be adjusted by the addition of endotoxin - free Sodium Hydroxide or Hydrochloric acid or suitable buffers to the specimen prior to testing.
4. Collect LAL powder into the bottle of the vial. Rehydrate with LAL by gentle mixing. Do not vortex. Discord the vial if the vacuum is absent or if color or opacity is present. Lyophilized LAL reagent is stored at 2 - 8 OC . Rehydrated LAL can be frozen (below -20 OC ) and used upto four weeks after reconstitution. LAL may be frozen and thawed only once. While in use LAL should be kept at 2 - 8 OC.
Procedure For Testing The Samples
1. Preparation of control curve / positive & water controls :
Prepare a control standard endotoxin series to get 2 l, 0.5 l , and 0.25 l as per the directions provided in the Certificate of Analysis for the specified lot of Lysate. A control series must be run with first set of tests each day. Otherwise test only a 2l endotoxin level, in there is no change in test parameters. Prepare a series of endotoxin control in the following way in 10 x 75 mm test tube

2. Preparation of test solution and controls :
i. Asepticaly withdraw sample solution. Calculate MVD for the sample and dilute to get a dilution which is equal to half MVD. Vortex for 15 seconds. Label as ‘STOCK’.
ii. Negative Product Control : Take 50 µl of half MVD solution in 10 x 75 mm test tube.
iii. Positive Product Control : Take 50 µl of half MVD solution in 10 x 75 mm test tube and add 50 µl of 4 l CSE solution to give a concentration of 2 l.
iv. Negative Water Control : Take 100 µl of LAL reagent water in 10 x 75 mm test tube (blank, NWC).
Incubation
Immediately prior to testing, rehydrate or thaw out the LAL frozen under validated conditions for storage and rehydration.
Inoculate all the test specimens and controls, beginning with the Negative Control and ending with Positive Control (with the greatest endotoxin concentration), with 100 µl of LAL reagent and mix. Promptly incubate the reaction tubes at 37 OC for 60 ±2 minutes. Observe and record the results.
Interpretation Of Results :
1. A positive reaction is characterized by the formation of a firm gel that remains when inverted through 180 OC.
2. Record such a result as positive (+).
3. A negative result is characterized by the absence of such a gel or by the formation of a viscous gel that does not maintain its integrity.
4. Record such a result as negative ( - ) .
5. Handle the tubes with care, and avoid subjecting them to unwanted vibrations, or false negative observation may result.
6. A valid test yields :
I. A positive gel clot in the positive product control and in 2 l and l CSE control.
ii. A negative gel (no gel) reaction in the negative controls of product (NPC) and water (NWC).
iii. A negative gel is half l and in 0.25 l (one forth lambda conc.,)
iv. the following results are also accepted
Test Tube No. Result
1,2 - -
3,4 + +
5,6 + +
7,8 + -
9,10 - -
v. A negative gel with the negative product control, means that there is less than the USP limit of endotoxin.
vi. If the PPC does not gel, then there is product inhibition. In this case product must be diluted and the test should be repeated.
vii. If the Ppc gels and the product and pyrogen free water do not, then the product is considered to have less than the endotoxin than the labeled Lysate sensitivity.
7. The test is invalid if the positive product control is negative or the endotoxin standard does not show the endpoint concentration to be within ±1 two-fold dilutions from the label claim sensitivity of the LAL reagent or if any negative control is positive.
8. Proceed to Endotoxin content Calculation to determine the amount of endotoxin present in the test specimen.
VALIDATION OF LAL TEST / Geometric Mean Calculation
Prepare serial two fold idltuions of endotoxin bracketing lysate sensitivity using the 1 EU/mL dilution and pyrogen free water. After each dilution vortex tube for one full minute.

Label test tubes in a quadruplicate and add 0.1 mL of each dilution and a PFW negative contro Add 0.1 mL of LAL to each tube in incubator block and incubate at 37 OC for one hour and observe for the clot formation. Calculate the Geometric Mean End Point to determine lysate sensitivity. Acceptable variation is one half to two times the labeled lysate sensitivity.
Eg. Table for Assay Results of GEL CLOT method.

Each end point value is converted to Log10 . The individual log10 values are averaged and lysate sensitivity is taken as the antilog10 of this average log value.
Eg. In the above case the mean Log10 end point is -0.996
Antilog10 means = 0.10 EU/mL
if the label claim of endotoxin is 0.125 EU/mL the results met the validation test.
INTERPRETATION
The article meets the requirements of the test if the concentration of endotoxin is not more than that specified in the individual monograph.
NOTE: Use the method as per the Pharmacopoeial status (grade) of the material. In case of In-house specifications, follow the method as per IP or as specified .

SWAB TEST

OBJECTIVE
Swab test is provided to determine compliance with the requirements given in the individual monograph / specifications.
PRINCIPLE
Swab test is the counting of total number of aerobic bacteria, yeasts and molds on any surface.
PROCEDURE
1. A cotton Swab of Johnsons and Johnsons make is moistened with normal saline( 0.9 % NaCl) and placed in a suitable test tube or screw cap test tube. The mouth of the test tube is closed with a cotton plug and rapped with aluminum foil.
2. The test tube containing the swab is then sterilized by autoclaving at 121 OC , 15 psi, pressure for 15 min.
3. Alternatively pre sterilised cotton swab of HI- media make can also be used.In this case, moisten the swab with sterile normal saline before using the swab.
4. Wear handgloves and take out the swab carefully from the test tube and swab the surface to be checked.
5. The area of the swab should be approximately 10 Sq. cm.
6. Replace the swab immediately in the test tube and close.
MICROBIOLOGICAL TESTING
1. Add accurately 10 mL of sterile phosphate buffer pH 7.2 to the test tube containing the Swab aseptically.
2. Shake well and transfer 1 mL to each of two sterile petridishes and proceed for test for Total Bacterial Count and yeast and mould as per the respective Standard Testing Procedures.
3. Also determine the presence of pathogens if necessary (E. coli, Salmonella, S. aureus and Pseudomonas ) by transferring 1 ml to each of the enrichment medium as per respective Standard Testing Procedures.

IDENTIFICATION OF PSEUDOMONAS AERUGINOSA

OBJECTIVE
The test for Identification of Pseudomonas aeruginosa is provided to determine compliance with the requirements given in individual monograph / specifications.
PRINCIPLE
Identification of Pseudomonas aeruginosa is the identification or detection of a pathogenic bacteria which may cause infection to human body. It can be identified by using specific differential media which support only the growth of Pseudomonas aeruginosa.’
REAGENT AND MEDIA
A. 1 % w/v solution of N,NN’N’-Tetramethyl-p-phenylene diammonium dichloride (Reagent A)
B. Cetrimide Agar
Pancreatic Digest of Gelatin 20.0 g
Magnesium Chloride 1.4 g
Potassium Sulfate 10.0 g
Cetrimide 0.3 g
Agar 13.6 g
Glycerol 10.0 mL
Water 1000 mL
Heat to boiling for 1 minute with shaking. Adjust the pH 7.0 +0.2. Sterilize the media an autoclave at 121 OC for 15 min.
C. Cetrimide Broth
Prepare the broth with the addition of all the ingredients specified in Cetrimide agar except the agar.
D. Pseudomonas Agar Medium for detection of Hourescein
Pancreatic Digest of Casein 10.0 g
Peptic Digest of Animal Tissue 10.0 g
Anhydrous Dibasic Potassium Phosphate 1.5 g
Magnesium Sulfate (MgSO4. H2O) 1.5 g
Glycerin 10.0 mL
Agar 15.0 g
Water 1000 mL
pH after sterilization : 7.2 ± 0.2
Dissolve the solid components in the water before adding the Glycerin. Heat, with frequent stirring, and boil for 1 minute.
E. Pseudomonas Agar Medium for detection of Pyocyanin
Pancreatic Digest of Gelatin 20.0 g
Anhydrous Magnesium Chloride 1. 4 g
Anhydrous Potassium Sulfate 10.0 g
Agar 15.0 g
Glycerin 10.0 mL
Water 1000 mL
pH after sterilization : 7.2 ± 0.2
Dissolve the solid components in the water before adding the glycerin. Heat, with frequent stirring, and boil for 1 minute .
F. Fluid Soyabean Casein Digest Medium
Pancreatic Digest of Casein 17.0 g
Papacy Digest of Soybean Meal 3.0 g
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose (C6H12O6. H2O) 2.5 g
Distilled Water 1000 mL
Final pH after Sterilization 7.3 ± 0.2
Dissolve the solids in the water, warming slightly to effect solution. Cool to room temperature and adjust the pH with 0.1 N Sodium Hydroxide or phosphoric acid and . Filter, if necessary. Distribute the media into suitable containers and sterilize in an autoclave at 121 OC for about 15 min.
Incubate all the above media for 24 h at 37 OC before use to check any external contamination.
Prepare media by using different ingredients as above or use readymade dehydrated media of Hi media / Difco make. Rehydrate the required quantity as per instructions on the bottle label and sterilize at 15 psi at 121 OC for 15 min.
PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
Water-Soluble Products :
1. Dissolve or dilute 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in Lactose broth or another suitable medium shown not to have anti-microbial activity under the conditions of the test and adjust the volume to 100 mL with the same medium.
2. If necessary, adjust the pH to about 7.0
Non-Fatty Products Insoluble in Water :
1. Suspend 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in Lactose or another suitable medium shown not to have anti-microbial activity under the conditions of the test and dilute to 100 mL with the same medium.
2. If necessary divide the preparation being examined and homogenize the suspension mechanically.
3. A suitable surface-active agent such as 0.1 % Polysorbate 80 may be added to assist the suspension of poorly wettable substance.
4. If necessary, adjust the pH of the suspension to about 7.0
Fatty Products :
1. Homogenize 10 g or 10 mL of the preparation being examined, unless otherwise prescribed with 5 g of Polysorbate 20 or Polysorbate 80.
2. If necessary, heat to not more than 40 OC . Mix carefully while maintaining the temperature in a water bath or in an oven.
3. Add 85 mL of Lactose broth or another suitable medium shown not to have anti-microbial activity in the conditions of the test, heated to not more than 40 OC, if necessary.
4. Maintain this temperature for the shortest time necessary for formation of an emulsion and in any case for not more than 30 min.
5. If necessary, adjust the pH of the emulsion to about 7.0
For a Fluid Specimen in Aerosol Form :
1. Chill the container in an Alcohol-dry Ice mixture for approx. 1 h, cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible and transfer the quantity of test material required for the procedures specified in one of the two proceeding paragraphs, as appropriate.
2. Where 10 g or 10 mL of the specimen, whichever is applicable,. cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residues.
As per IP
Primary Test
1. Place the prescribed quantity in a sterile screw-capped jar containing 100 mL of Fluid Soyabean Casein Digest Medium and incubate at 35 - 37 OC for 18 - 24 h.
2. Subculture on a plate containing a layer of Cetrimide agar and incubate at 35 -37 OC for 18 - 24 h.
3. Examine the resulting growth by Gram’s stain and apply the oxidase test; Gram-negative bacilli giving oxidase test indicate the presence of Pseudomonas.
Confirmatory Tests
Oxidase Test: Place 2 to 3 drops of a freshly prepared 1 % w/v solution of reagent A on a piece of filter paper (Whatman No.1 is suitable) and smear with the suspect colony. if a purple color is produced within 5 to 10 seconds, the test is positive.
Pigments Test: please refer Page 7 of this SOP from point1 - 5 for pigment test.
As per BP
Primary Test
1. Pre - treat the preparation being examined as described above but using buffered Sodium Chloride - Peptone solution pH 7.0, or another suitable medium shown not to have anti-microbial activity under the conditions of the test, in place of Lactose Broth.
2. Inoculate 100 mL of Casein soybean digest broth with a quantity of the solution, suspension or emulsion thus obtained containing 1 g or 1 mL of the preparation being examined.
3. Mix and incubate at 35 - 37 OC for 24 - 48 h.
4. Subculture on a plate of Cetrimide agar and incubate at 35 - 37 OC for 24 - 48 h.
5. If no growth of micro-organisms is detected, the preparation being examined passes the test.
Confirmatory Test
1. If growth of colonies of Gram-negative rods, usually with a greenish fluorescence occurs, apply an oxidase test and test the growth in casein soybean digest broth at 42 OC.
2. Place 2 or 3 drops of a freshly prepared 1 % w/v solution of Reagent A on filter paper (Whatman No. 1 is suitable) and smear with the suspect colony; if a purple color is produced within 5 - 10 seconds., the test is positive.
3. The preparation being examined passes the test if cultures of the type described do not appear or if the confirmatory biochemical test is negative.
As per USP
Primary Test
1. To the specimen add Fluid Soybean-Casein digest medium to make 100 mL, mix, and incubate.
2. Examine the medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of cetrimide agar plated on petridishes. Cover and invert the dishes, and incubate.
3. If upon examination, none of the plates contains colonies having the characteristics listed in Table for the media used, the test specimen meets the requirements for freedom from Pseudomonas aeruginosa.’
Confirmatory Test
Oxidase and Pigment Tests :
1. With the aid of an inoculating loop, streak representative suspect colonies from the agar surface of Cetrimide agar medium on the agar surfaces of Pseudomonas agar medium for detection of Fluorescent and pseudomonas agar medium for detection of pyocyanin contained in petridishes.
2. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be inoculated from a separate colony.
3. Cover and invert the inoculator media, and incubate at 35 ±2 OC for not less than 3 days.
4. Examine the streaked surface under ultraviolet light.
5. Examine the plates to determine whether colonies having the characteristics, listed in Table are present.
6. Confirm any suspect colonial growth n one or more of the media as pseudomonas aeruginosa by means of the oxidase test.
7. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with reagent A; if there is no development of a pink color, changing to purple, the specimen meets the requirements of the test for absence of pseudomonas aeruginosa.
8. The presence of pseudomonas aeruginosa maybe confirmed by other suitable cultural and biochemical tests, if necessary.
Morphologic Characteristics of Pseudomonas aeruginosa on Selective And Diagnostic Agar Media

NOTE:
1. Carry out a control test by repeating the test adding the prescribed quantity and a volume of broth containing 10 to 50 pseudomonas aeruginosa (NCTC 6750), prepared from a 24 h. culture in Nutrient broth, to a sterile, screw-capped jar containing 100 ml of Cetrimide broth or Fluid Soyabean Casein Digest Medium.
2. The test is invalid if the results do not indicate that the control contains Pseudomonas.

IDENTIFICATION OF ESCHERICHIA COLI

OBJECTIVE
The test for Escherichia coli is provided to determine compliance with the requirements given in individual monograph / specifications.
PRINCIPLE
This includes identification of Escherichia coli, a bacteria pathogenic to human body and causes infection of stomach. It is detected by using specific, differential media which supports growth of Escherichia coli.
REAGENTS AND MEDIA
1. Kovac’s Reagent
Dissolve 5 g of 4-Dimethylaminobenzaldehyde in 75 mL Amyl alcohol by warming on a water bath at 50 - 55 OC , cool and add 25 ml of Conc. Hydrochloric acid. Store between 2 - 10 OC protected from light or use readymade reagent.
2. Nutrient Broth
Beef Extract 10.0 g
Peptone 10.0 g
Sodium chloride 5.0 g
Water 1000 mL
Dissolve with the aid of heat. Adjust to pH 8.0 - 8.4 with 5M Sodium Hydroxide and boil for 10 min. Filter, adjust to pH 7.2 - 7.4 and sterilize by maintaining at 115 OC for 30 min.
3. MacConkey’s Broth
Peptone 20.0 g
Sodium Chloride 5.0 g
Sodium Taurocholate 5.0 g
Bromocresol purple 10.0 mg
Lactose 10.0 g
Water 1000 mL
Dissolve the peptone, the sodium chloride and the sodium taurocholate in the water with the aid of heat. Adjust to pH 8.0 and boil for 20 min. Cool, filter and adjust to pH 7.4, Add the lactose and the indicator solution, mix and distribute in tubes containing inverted Durham’s tubes. Sterilize by maintaining at 115 OC for 30 min.
4. Peptone Water ( Buffered sodium chloride-peptone solution pH 7.0)
Potassium dihydrogen orthophosphate 3.56 g
Disodium hydrogen orthophosphate 7.23 g
Sodium chloride 4.30 g
Peptone (meat or casein) 1.0 g
Water 1000 mL
0.1 to 1.0% w/v of Polysorbate 20 or Polysorbate 80 may be added. Sterilize by heating in an autoclave at 121 OC for 15 min.
5. Lactose Broth
Beef Extract 3.0 g
Pancreatic digest of gelatin 5.0 g
Lactose 5.0 g
Water 1000 mL
Adjust the pH so that after sterilization it is 6.7 to 7.1. Sterilize by heating in an autoclave at 121 OC for 15 min. and cool immediately.
6. Levine Eosin-Methylene Blue Agar Medium
Pancreatic digest of Gelatin 10.0 g
Dibasic potassium phosphate 2.0 g
Agar 15.0 g
Lactose 10.0 g
Eosin Y 400 mg
Methylene blue 65 mg
Water 1000 mL
PH 7.1 ± 0.2
Dissolve the pancreatic digest of Gelatin, the Dibasic potassium thiosulphate and the agar in the water, with warming and allow to cool. Just prior to use, liquefy the gelled agar solution, add the remaining ingredients, as solutions, in the following amounts, and mix; for each 100 mL of the liquefied agar solution 5 mL of Lactose solution (1 in 5) , 2 mL of the Eosin Y solution (1 in 50) , and 2 mL of Methylene blue solution (1 in 30) . The finished medium may not be clear.
All the above media should be incubated for 24 h at 37 Oc before use. Any contaminated media should be discarded. Prepare media by using different ingredients as above or use readymade dehydrated media of Hi media / Difco make.and use required quantity as per instructions on the bottle label,. dispense in required quantities and sterilize at 15 lbs and 121 OC for 15 min.
PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
Water - Soluble Products :
1. Dissolve or dilute 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in Lactose broth or another suitable medium shown not to have anti-microbial activity under the conditions of the test and adjust the volume to 100 mL with the same medium
2. If necessary, adjust the pH to about 7.
Non - Fatty Products Insoluble in Water :
1. Suspend 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in Lactose broth or another suitable medium shown not to have anti-microbial activity under the conditions of the test and dilute to 100 mL with the same medium.
2. If necessary divide the preparation being examined and homogenize the suspension mechanically.
3. A suitable surface-active agent such as 0.1 % w/v Polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust the pH of the suspension to about 7.
Fatty Products :
1. Homogenize 10 g or 10 mL of the preparation being examined, unless otherwise prescribed with 5 g of Polysorbate 20 or Polysorbate 80. If necessary, heat to not more than 40 OC.
2. Mix carefully while maintaining the temperature in a water bath or in an oven.
3. Add 85 mL of Lactose broth or another suitable medium shown not to have anti-microbial activity in the conditions of the test, heated to not ore than 40 OC if necessary.
4. Maintain this temperature for the shortest time necessary for formation of an emulsion and in any case for not more than 30 min.
5. If necessary, adjust the pH of emulsion to about 7.
For a Fluid Specimen in Aerosol Form :
1. Chill the container in an Alcohol-dry ice mixture for approx. 1 h., cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible and transfer the quantity of test material required for the procedures as appropriate.
2. Where 10 g or 10 mL of the specimen, whichever is applicable, cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residue.
PROCEDURE
As per IP
A. Enrichment
1. Transfer aseptically 10 g or 10 mL of sample to 100 Nutrient broth and incubate at 37 OC for 24 h.
2. After incubation, examine the tube for growth, and if present, mix by gentle shaking.
B. Primary Test
1. Pipette 1 mL of the enrichment culture into tubes containing 5 mL MacConkey’s broth and incubate at 37 OC for 48 h.
2. If the contents show acid and gas carry out the secondary test.
C. Secondary Test
1. After incubation, if the tube shows presence of acid and gas transfer 0.1 mL from the tube to each of two tubes containing
i. 5 mL MacConkey’ s broth
ii. 5 mL Peptone water.
2. Incubate both tubes in a water bath at 43.5 - 44.5 OC for 24 h. After incubation examine tube (a) for acid and gas and (b) for Indole.
3. To test for Indole production add 0.5 mL of Kovac’s reagent, shake well and allow to stand for one min. , if a red color is observed in the reagent layer, indole is present.
4. The presence of acid and gas and of Indole indicates presence of Escherichia coli.
As per BP
A. Enrichment
Transfer a quantity of the homogenate in Lactose broth containing 1 g or 1 mL of the preparation being examined, prepared and incubated as of the test for Enterobacteriaceae and certain other Gram-negative bacteria, to 10 mL of MacConkey’s broth and incubate at 43 - 45OC for 18 - 24 h.
B. Primary Test
1. Sub-culture on a plate of MacConkey agar and incubate at 43 - 45 Oc for 18 - 24 h.
2. Growth of red, generally non-mucoid colonies of Gram negative rods, sometimes surrounded by a reddish precipitation zone, indicates the possible presence of Escherichia coli.
C. Secondary Test
1. This may be confirmed by the formation of Indole at 43.5 - 44.5 OC and by other biochemical reactions.
2. The preparation being examined passes the test if such colonies are not seen or if the confirmatory biochemical reactions are negative.
As per USP
A. Enrichment
1. to the specimen contained in a suitable vessel, add a volume of Fluid Lactose medium to make 100 mL and incubate at 37 OC for 48 h.
2. Examine the medium for growth, if growth is present mix by gentle shaking.
B. Primary Test
1. by means of an inoculating loop, streak a portion from the remaining Fluid Lactose Medium on the surface o f MacConkey agar medium.
2. Cover and invert the plate and incubate at 37 OC for 48 h.
3. Upon examination, if none of the colonies conforms to the description given in the Table below for this medium, specimen meets the requirements of the test for absence of Escherichia coli.
If colonies matching the description in Table given below are found, proceed with further identification.
Morphological Characteristics of Escherichia coli
On MacConkey Agar Medium
Characteristic Colonial Morphology-Brick-red; may have surrounding zone of precipitated bile
Brick-red; may have surrounding zone of precipitated bile-Negative rods
(Cocco-bacilli)
C. Secondary Test
1. Transfer the suspect colonies individually, by means of an inoculating loop, to the surface Levine Eosin-Methylene blue agar medium, plated on petridishes.
2. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be seeded from a separate colony.
3. Cover and invert the plates and incubate at 37 OC for 48 h.
4. Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light, the specimen meets the requirements of thet est for the absence of Escherichia coli.
5. The presence of Escherichia coli may be confirmed by further suitable cultural and biochemical tests.
NOTE:
Carry out a control test by repeating the primary and secondary tests adding 1 mL of the enrichment and culture a volume of broth containing 10 to 50 Escherichia coli (NCTC 9002), prepared from a 24 h, culture in Nutrient broth, to 5 mL of MacConkey’s broth. The test is invalid if the results do not indicate that the control contains Escherichia coli.
NOTE: Use the method as per the pharmacopoeial status (grade) of the material. In case of In-house specificaiton, follow the method as per IP or as specified.

IDENTIFICATION OF SALMONELLA SPECIES

OBJECTIVE
The test for Identification of Salmonella species is provided to determine compliance with the requirements given in individual monograph / specifications.
PRINCIPLE
Identification of Salmonella species which is pathogenic bacteria causes disease in human body. It can be identified by using specific, differential media which only supports growth of Salmonella.
REAGENTS AND MEDIA
1. Selenite F Broth
Peptone 5.0 g
Lactose 4.0 g
Disodium Hydrogen Orthophosphate 10.0g
Sodium Hydrogen Selenite 4.0 g
Water 1000 mL
Dissolve, distribute in sterile containers and sterilize by maintaining at 100 OC for 30 min.
2. Fluid Lactose Medium
Beef Extract 3.0 g
Pancreatic Digest of Gelatin 5.0 g
Lactose 5.0 g
Water 1000 mL
Cool as quickly as possible after sterilization. pH after sterilization 6.9 ± 0.2
3. Fluid Selenite - Cystine Medium
Pancreatic Digest of Casein 5.0 g
Lactose 4.0 g
Sodium Phosphate 10.0 g
Sodium Acid Selenite 4.0 g
L - Cystine 10.0 g
Water 1000 mL
Final pH 7.9 ± 0.2. Mix and heat to effect solution. Heat in flowing steam for 15 minutes.
Do not Sterilize.
4. Fluid Tetrathionate Medium
Pancreatic Digest of Casein 2.5 g
peptic Digest of Animal Tissue 2.5 g
Bile Salts 1.0 g
Calcium Carbonate 10.0 g
Sodium Thiosulphate 30.0 g
Water 1000 mL
Heat the solution of solids to boiling. On the day of use, add a solution prepared by dissolving 5 g of Potassium Iodide and 6 g of Iodine in 20 mL of water. Then add 10 mL of a solution of brilliant green (1 in 1000) and mix. Do not heat the medium after adding the brilliant green solution.
5. Tetrathionate Broth
Beef Extract 0.9 g
Peptone 4.5 g
yeast Extract 1.8 g
Sodium Chloride 4.5 g
Calcium Carbonate 25.0 g
Sodium Thiosulphate 40.7 g
Water 1000 mL
Mix, bring to the boil, cool to below 45 OC , and add a solution of 6 g of Iodine and 5 g of Potassium Iodide in 20 mL of water. Mix, and distribute in sterile containers.
Do not heat after Iodine has been added. The medium with Iodine should be used on the day it is prepared. The base medium without Iodine may be stored indefinitely after sterilization.
6. Nutrient Broth
Beef Extract 10.0 g
Peptone 10.0 g
Sodium Chloride 5.0 g
Water 1000 mL
Dissolve with the aid of heat. Adjust to pH 8.0 to 8.4 with 5 M Sodium Hydroxide and boil for 10 min. Filter, adjust to pH 7.2 to 7.4, and sterilize by maintaining at 115 OC for 30 min.
7. Brilliant Green Agar
Peptone 10.0 g
Yeast Extract 3.0 g
Lactose 10.0 g
Sucrose 10.0 g
Sodium Chloride 5.0 g
Brilliant Green 12.5 g
Agar 12.0 g
Water 1000 mL
Mix, allow to stand for 15 min. sterilize by maintaining at 115 OC for 30 min. and mix before pouring.
8. Desoxycholate Citrate Agar
Beef Extract 5.0 g
Peptone 5.0 g
Lactose 10.0 g
Trisodium Citrate 8.5 g
Sodium Thiosulphate 5.4 g
Ferric(III) Citrate 1.0 g
Sodium Desoxycholate 5.0 g
Neutral Red 0.02 g
Agar 12.0 g
Water 1000 mL
Mix and allow to stand for 15 min. with continuous stirring, bring gently to the boil and maintain a boiling point until solution is complete. Cool to 80 OC , mix, pour and cool rapidly.
Care should be taken not to overheat Desoxycholate citrate agar during preparation. It should not be re-melted and the surface of the plates should be dried before use.
9. Bismuth Sulphite Agar
1.
Beef Extract 6.0 g
Peptone 10.0 g
Agar 24.0 g
Ferric(III) Citrate 0.4 g
Brilliant Green 10.0 mg
Water 1000 mL
Dissolve with the aid of heat and sterilize by maintaining at 115 OC for 30 min.
2. Ammonium bismuth Citrate 3.0 g
Anhydrous Sodium Hydrogen Orthophosphate 5.0 g
D-Glucose Monohydrate 5.0 g
Water 100 mL
Mix , heat to boiling, cool to room temperature, add 1 volume of solution (2) to 10 volumes of solution (1) previously melted and cooled to a temperature of 55 OC and pour.
Bismuth sulphite agar should be stored at 2 - 8 OC for 5 days before use.
10. Triple Sugar Iron Agar
Beef Extract 3.0 g
Yeast Extract 3.0 g
Peptone 20.0 g
Lactose 10.0 g
Sucrose 10.0 g
D-Glucose Monohydrate 1.0 g
Iron (III) Sulphate 0.2 g
Sodium Chloride 5.0 g
Sodium Thiosulphate 0.3 g
Phenol Red 24.0 mg
Agar 13.0 g
Water 1000 mL
Mix, allow to stand for 15 min. bring to and maintain at boiling point until solution is complete, mix, distribute in tubes and sterilize by maintaining at 115 OC for 30 min. Allow to set in a sloped form with a butt about 2.5 cm long.
11. Urea Broth
Potassium Dihydrogen Orthophosphate 9.1 g
Anhydrous Disodium Hydrogen Orthophosphate 9.5 g
Urea 20.0 g
Yeast Extract 0.1 g
Phenol Red 0.01 g
Water 1000 mL
Mix, sterilize by filtration and distribute aseptically in sterile containers.
12. Xylose-Lysine-Desoxycholate Agar Medium
Xylose 3.5 g
Lysine 5.0 g
Lactose 7.5 g
Sucrose 7.5 g
Sodium Chloride 5.0 g
Yeast Extract 3.0 g
Phenol Red 18.0 mg
Agar 13.5 g
Sodium Desoxycholate 2.5 g
Sodium Thiosulfate 6.8 g
Ferric Ammonium Citrate 800.0 mg
Water 1000 mL
Final pH : 7.4 ± 0.2
Heat the mixture of solids and water, with swirling, just to the boiling point. Do not overheat or sterilize. Transfer at once to water batch maintained at about 50 OC and pour into plates as soon as the medium has cooled.
Incubate all the above media for 24 h at 37 OC before use. Discard any contaminated media.
Instead of preparing media, one can also use dehydrated media of Himedia/Difco and rehydrate the required quantity as per instructions on the bottle label, dispense in required quantities and sterilize at 15 lbs and 121 OC for 15 min
TABLE - I


PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
Water Soluble Products :
1. Dissolve or dilute 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in sterile Lactose broth or another suitable medium shown not to have anti-microbial activity under the conditions of the test and adjust the volume to 100 mL with the same medium.
2. If necessary, adjust the pH to about 7.0
Non-Fatty Products in Water :
1. Suspend 10 g or 10 mL of the preparation being examined, unless otherwise prescribed, in sterile Lactose medium shown not to have anti-microbial activity under the medium broth or another suitable
2. If necessary divide the preparation being examined and homogenize the suspension mechanically.
3 . A suitable surface-active agent such as 0.1 % w/v of Polysorbate 80 may be added to assist the suspension of poorly wettable substances.
4. If necessary, adjust the pH of the suspension of about 7.0.
Fatty Products :
1. Homogenize 10 g or 10 mL of the preparation being examined, unless otherwise prescribed with 5 g of Polysorbate 20 or Polysorbate 80.
2. If necessary, heat to not more than 40 OC.
3. Mix carefully while maintaining the temperature in a water bath or in an oven.
4. Add 85 mL of sterile Lactose broth or another suitable medium shown not to have anti-microbial activity in the conditions of the test, heated to not more than 40 OC if necessary.
5. Maintain this temperature for the shortest time necessary for formation of an emulsion and in any case for not more than 30 min.
6. If necessary, adjust the pH of the emulsion to about 7.0.
For a fluid specimen in Aerosol form :
1. Chill the container in an Alcohol-dry ice mixture for approx. 1 h., cut open the container, allow it to reach room temperature, permit the propellant to escape , or warm to drive off the propellant if feasible and transfer the quantity of test material required of the procedures as appropriate.
2. Where 10 g or 10 mL of the specimen, whichever is applicable, cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residue.
PROCEDURE
As per BP
A. Enrichment
Incubate the solution, suspension or emulsion obtained by appropriately pre-treating the preparation being examined as described above at 35 - 37 OC for 5 - 24 h, as appropriate for enrichment.
B. Primary Test
1. Transfer 10 mL of the enrichment culture to 100 mL of Tetrathionate bile brilliant green broth and incubate at 42 - 43 OC for 18 - 24 h.
2. Subculture on at least two of the following three agar media : Desoxycholate citrate agar; Xylose, Lysine, Desoxycholate agar and Brilliant Green Agar.
3. Incubate at 35 - 37 OC for 24 - 48 h.
4. If any colonies conforming to the description in Table II are produced, carry out the secondary test.
C. Secondary Test
1. Subculture any colonies showing characteristics given in Table I on Triple sugar iron agar using surface and deep inoculation. (This can be done by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle and incubating at 35 - 37 OC for 18 - 24 h.)
2. The presence of Salmonella is provisionally confirmed if, in the deep culture but not in the surface culture, there is a change of color from red to yellow and usually a formation of gas, with or without production of Hydrogen sulfide in the agar.
3. Precise confirmation may be carried out by appropriate biochemical and serological tests.
4. The preparation being examined passes the test if, in the primary test, cultures of the type described do not appear or if, in the secondary test, the confirmatory biochemical and serological tests are negative.
As per IP’96
A. Enrichment
1. Place the prescribed quantity in a sterile screw-capped jar, add 100 mL (unless otherwise directed) of Nutrient broth, allow to stand for 1 h (4 h for Gelatin ), and shake again.
2. Loosen the cap and incubate at 35 - 37 OC for 24 h.
B. Primary Test
1. Add 1 mL of the enrichment culture to each of two tubes containing
a) 10 mL of Selenite F broth and
b) 10 mL of Tetrathionate broth and incubate at 36 - 38 OC for 48 h.
2. From each of these two cultures inoculate three plates containing a layer of
a) Brilliant Green Agar
b) Desoxycholate Agar
c) Bismuth Sulphite agar
3. Incubate the plates at 36 - 38 OC for 18 - 24 h.
4. If nay colonies conforming to the description on Table I are produced, carry out the secondary test.
C. Secondary Test
1. Subculture any colonies showing the characteristics given in Table I in Triple sugar iron agar, by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle and at the same time inoculate a tube of Urea broth.
2. Incubate at 36 - 38 OC for 18 - 24 h.
3. The formation of acid and gas in the stab culture (with or without concomitant blackening ) and the absence of acidity from the surface growth in the triple sugar iron agar, together with absence of a red color in the urea broth, indicates the presence of Salmonella.
4. If acid but no gas is produced in the stab culture the identity of the organisms should be confirmed by agglutination tests.
As per USP
A. Enrichment
1. To the specimen contained in a suitable vessel, add a volume of Fluid Lactose Medium to make 100 mL and incubate at 37 OC for 48 h.
2. Examine the tubes for growth and if growth is present, mix by gentle shaking.
3. Pipette 1 mL portions into tubes containing, respectively, 10 mL Fluid Tetrathionate medium and 10 mL Fluid Selenite-Cysteine medium, mix and incubate at 37 OC for 12 - 24 h.
B. Primary Test
1. After incubation streak, by means of sterilized wire loop, portions from both the media on the surfaces of.
i. Brilliant Green Agar Medium
ii. Xylose-Lysine-Desoxycholate Citrate Agar Medium
iii. Bismuth Sulphite Agar Medium contained in petridishes.
2. Cover and invert the petridishes and incubate at 37 OC for 24 h.
3. After incubation, examine the colonies.
4. If none of the colonies conforming to the description given in table are obtained, Then the sample passes the test for absence of genus Salmonellae.
C. Secondary Test ( Conformatory test)
1. If colonies of gram negative rods conforming to the description given in table are obtained proceed with further identification by transferring representative suspect colonies individually, by means of an inoculating wire, to a butt-slant tube of Triple sugar-iron medium by first streaking the surface of the slant and then stabbing the wire, well beneath the surface. Incubate at 37 OC for 24-38 h.
2. If examination discloses no evidence ou tubes having alkaline (red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from Hydrogen Sulphide production), the specimen meets the requirements of the test for the absence of the genus salmonellae.
NOTE :
1. Carry out control test by repeating the primary and secondary tests using 1 mL of the -enrichment culture and a volume of broth containing 10 to 50 Salmonella abony (ATCC 6017) prepared from a 24 h culture in Nutrient broth, for the inoculation of tubes (a.) and (b.)
2. The test is invalid if the results do not indicate that the control contains salmonellae.
Note : Use the method as per the Pharmacopoeial status ( grade ) of the material. In case of In-house specification, follow the method as per IP or as specified.