1.

Procedure for air sampling (environmental monitoring) in sterile pharmaceutical manufacturing area 2 Maintenance of Primary Standards in Pharmaceutical Industries

Tapped Density Tester Setup, Operation, and Cleaning Guide

3

4. Growth Promotion Test 5. Identification of Environmental Flora

Procedure for air sampling (environmental monitoring) in sterile pharmaceutical manufacturing area

Procedure for air sampling in pharmaceuticals: 1 Prepare SCDA medium and aseptically pour approximately 15 20 ml of sterile molten cooled (400C) SCDA agar into sterile 90 mm petri plates. 2 Allow to solidify the plates at room temp, after solidification label all the plates with name of media, preparation batch No. and date of preparation. 3 Invert and incubate the plates at 30 to 350C for 24 - 48 hrs. After incubation check the plates for any contamination, if there is contamination discard the plates.

4 After pre-incubation, label all the plates with date of sampling, location and Shift with the help of marker pen and wrap with aluminium foil and then place in clean SS container. 5 Transfer the container and Air sampler which is sanitized and wrapped in aluminium foil, to sterile area through pass box and personnel must be entered through Air locks by proper entry and gowning procedure for sterile area. 6 Place the pre incubated SCDA plates in Air sampler holder and operate the air sampler for time to sample 1000 ltrs of air. 7 After complition of Air sampling, remove the plates from Air sampler, close the lid immediately and place aside. 8 Immediately clean the Air sampler with 70% sterile IPA solution and carry out the air sampling for other specified locations. 9 After air sampling collect all the plates in clean SS container and send to microbiology laboratory through hatch box. Follow the exit procedure to come out from sterile area. 10 Prepare positive control by streaking Bacillus subtilis and negative control as it is without streaking 11 Invert all the plates and incubate at 20 to 250C for 72 hrs and then 30 to 350C for 48 hrs. 12 After incubation, count the number of colony forming units and with the help of colony counter and express the result cfu/m3. 13 Record the result as per the following formula X = Pr X 1000/V Where X = cfu/m3 r = colony forming units counted on 90 mm plates Pr = Probable count obtained by positive hole correction from table I against r value V = Volume of air sample Precaution

1 Maintain aseptic condition during air sampling 2 Sanitize Air sampler with 70% IPA solution for each location sampling 3 Result must be expressed as per the given formula 4 All pre-incubated plates should be rejected if a single plate shows evidence of microbial contamination.

Maintenance of Primary Standards in Pharmaceutical Industries

OBJECTIVE: To provide a procedure for maintenance of primary standards. SCOPE: Applicable to all primary standards used for standardization of volumetric solutions and calibration of instruments in the quality control laboratory. RESPONSIBILITY: Chemist PROCEDURE: Procure primary standard substances with the certificate of analysis. Use these certified substances exclusively for the volumetric analysis or for calibration of instruments or for appropriate critical tests only. Transfer the substances from their original container into the glass vial of required size & label it as follows. Name: Write name of the primary standard. B.No.: Write batch number of the primary substance as per the log book. Prepared on: Write the date of transfer of the substance from its original container to the vial. Use before: Write the use before date as per the logbook.

Sign: Chemist transferring the substance shall sign. Log book recording Log book for primary standard contains following columns: Prepared on B.No. Use before Prepared By Checked By

Prepared on: Write the date of transfer of the substance from its original container to the vial. B.No.: Write batch number as PX/nnn/yy, where X is serial number allotted for the primary substance starting from one (this number is allotted on the basis of first number for first in i.e. serial inclusion of the substance in use & the number will be permanently allotted through out the life cycle of the substance requirement). nnn is serial number in chronological order for the transfer of the substance, starting with 001. yy is year i.e. 04 for the year 2004, e.g. P1/001/04. Use before: All the primary standard substances which are remaining in the vial shall be discarded & replaced with the same substance from the original container every three months or prior if required. Put the date of three months period from the prepared on date. Prepared By: Chemist transferring the substance shall sign. Checked By: Chemist or designee cross checking the activity shall sign. Dry the primary standard substances at appropriate temperature before use, if required. Store the substance in well closed vials. Keep the vials in desiccators over silica gel. REFERENCE: Indian Pharmacopoeia.

Tapped Density Tester

OBJECTIVE: To provide a procedure for the operation of Tapped Density Tester. SCOPE: Applicable to the Tapped Density Tester in Quality Control Department. Model : ETD-1020 Manufacturer: Electrolab RESPONSIBILITY: Chemist. PROCEDURE: USER METHOD (this method is used only when no. of taps are specified in the method). Check that all the connections of the instrument are proper. Weigh the sample & fill in a measuring cylinder of Tapped Density Tester. If sample weight is about 100g or above, use 250 ml measuring cylinder otherwise use 100 ml measuring cylinder. Fix the measuring cylinder into the holder provided for holding the measuring cylinder on the instrument. Power ON the instrument. The system will initialize itself and the display will flash and show the start up screen. The system is now ready for Setting of the test Parameter and to run the Test. Using the Digit Scroll key (on front panel) set the mode of operation as USER (In this mode, the instrument will stop after the set value of taps). Press the SET key to set the test parameter. The taps are programmable from 1 to 9999 taps. By using the arrow keys set the tap value. After setting the tap value, press the START key to start the test. The display will now show the elapsed taps. After tapping is complete, check the volume, set the similar number of taps again & run the test once more.

Check the volume after second tapping cycle & calculate the difference between the two volumes observed after tapping. If this difference is not less than 2%, continue the tapping for one more cycle & this activity shall be repeated until the difference between succeeding measurements is less than 2%. After the test is complete, calculate the tapped density using final volume observed. USP METHOD: Using the Digit Scroll key, set the mode of operation as USP. Press the Method key to toggle between USP-I (drop of 14mm 2mm at speed of 300 drops per minute) & USP-II (drop of 3mm 10% at speed of 250 drops per minute) method & select required method. Weigh the sample & fill in a measuring cylinder of Tap Density Tester. If sample weight is about 100g or above, use 250 ml measuring cylinder otherwise use 100 ml measuring cylinder. Fix the measuring cylinder into the holder provided for holding the measuring cylinder on the instrument. Press start key & feed the weight of the sample & press enter. Fill the value of initial volume & press enter. Press start key to start the taps. Tapping will automatically be stopped after 500 taps. Fill the value of volume observed after tapping. Press start key. Tapping will automatically be stopped after 750 taps. Fill the value of volume observed after tapping & press enter. Difference between two succeeding measurements is displayed. If this difference is not less than 2%, press START to continue. Tapping will automatically be stopped after 1250 taps. Repeat steps 4.2.12 to 4.2.14. till difference between two succeeding measurements is less than 2%. Switch off the instrument after the test is over. Remove the cylinder, clean it & store in its box.

Tapped Density Tester Setup, Operation, and Cleaning Guide

1.1. Turn on the equipment with the switch on the left side of the back of the instrument. 1.2. Take out both 100 mL graduated cylinders with the lip on the top. 1.3. Prepare each cylinder 1.3.1. Record the weight of the empty cylinder. 1.3.2. Pour the powder into the cylinder (through a 1.00 mm sieve, if possible), preferably somewhere between the 90 and 100 mL marks. 1.3.3. Record the weight of the full cylinder. 1.3.4. Calculate the mass of powder by subtracting the weight of the empty cylinder from the weight of the full cylinder. 1.4. Record the initial volume of powder in the graduated cylinder. 1.4.1. Calculate the bulk density of the powder by dividing the mass of powder by the initial volume of powder. 1.5. Place each cylinder on the mounts, all the way underneath the prongs.

1. Setup

2. Operation

2.1. Choose how the taps will be done with the Time/Count button. Pressing this button will illuminate the corresponding light on the display. 2.1.1. Time: a desired amount of time for tapping can be entered in the format hh:mm:ss. Press Enter after putting in the time.

OR 2.1.2. Count: a desired number of taps can be entered, up to 999,999. Press Enter after putting in the number of taps. 2.1.2.1. One method is to start with one tap, and double the number of taps each time until the powder volume no longer changes. 2.2. Press Start/Stop to begin tapping. The machine will automatically stop when it has completed the specified time or number of taps. If you need to stop the test before it is complete, press the Start/Stop button again to immediately stop the tapping. 2.3. Shut the acoustic cabinet while tapping to reduce the noise. 2.4. After each set of taps, record the volume of powder. Divide the initial mass by the volume to determine the density. When this density no longer changes with increasing the number of taps, this is the tapped density of the powder. 2.4.1. One way to determine if you have reached the tapped density is to plot the density versus the total number of taps. The density where the curve levels off is the tapped density.

3. Cleaning

3.1. Turn off the instrument. 3.2. Remove the powder from the cylinders, and take the waste with you. 3.3. Wash the cylinders with water, and make sure they are completely free of powder. 3.4. Set the wet cylinders by the sink to dry.

Growth Promotion Test

1.0 Objective Objective of this Standard Operating Procedure is to provide guidelines for Growth Promotion Test and Inhibitory Properties of the Media. 2.0 Scope This procedure is applicable for all the dehydrated culture media to be used for microbiological testing. 3.0 Responsibilities Implementation : Microbiologist Execution : Dy. Manager QC Review & Approval : AGM QA / QC 4.0 Abbreviation SOP : Standard Operating Procedure

QA : Quality Assurance QC : Quality Control NA : Not Applicable WFI : Water for Injection LAF : Laminar Air Flow 5.0 Procedure 5.1 Equipments required LAF Colony Counter Incubators DHS Autoclave 5.2 Material Required Dehydrated culture media Sterile Saline solution Sterile Petri Plates Measuring Cylinder Test organism E. coli, Staph. aureus, Ps. aeruginosa, C. albicans, A. niger, Salmonella. B. subtilis, Cl. Sporogenes, S. epidermidis. 5.3 Utilities Required Power 5.4 Methodology 5.4.1 After receipt of dehydrated culture media, make a necessary entry in receipt register including B. No, Mfg date and use before date. 5.4.2 Collect minimum 5.0 g sample from each received packs (i.e. Batch wise) and mix properly. 5.4.3 Prepare culture media & sterilize as per SOP for preparation of culture media SOP. 5.4.4 After sterilization, transfer the media to microbiology analysis room and allow it to cool at 40 to 450C. 5.4.5 Start the LAF as per SOP and further proceed works under LAF. 5.4.6 Test For Growth Promoting Properties, Solid Media 5.4.6.1 Label the plates with culture name, Media B. No, Date of incubation on the base of petri plates 5.4.6.2 Add 1.0 ml suspension of specific culture containing 10 to 100 cfu/ml (as specified in Table I) into two sterile petri plates. 5.4.6.3 Aseptically pour the cooled media at 40 to 450C into both the labeled plates, mix the plates by gently rotating clock wise and anti-clock wise direction. 5.4.6.4 Allow the plates to solidify at room temperature under Laminar Air Flow. 5.4.6.5 Simultaneously run negative control to verify testing conditions, using the same procedure in place of the test organism use diluents i.e. 1.0 ml of saline solution

5.4.6.6 Incubate the plates at specified temperature and period as listed in Table 1, 5.4.6.7 Observe the plates for number of colonies, and count the cfu observed on both plates and express the result in cfu by following formula P1 + P2 / 2 Where P1 and P2 are plate 1 and plate 2 5.4.6.8 Calculate the Microbial recovery in percentage by following equation Mean cfu observed % Recovery = Inoculated cfu ml

X 100

5.4.6.9 Recovery should not less than 75% 5.4.7 Test For Growth

Inhibitory Properties, Solid Media 5.4.7.1 Label the plates with culture name, Media B. No, Date of incubation on the base of petri plates. 5.4.7.2 Add 1.0 ml suspension of specific culture containing 100 cfu/ml (as specified in Table I of Growth inhibitory property) into two sterile petri plates. 5.4.7.3 Aseptically pour the cooled media at 40 to 450C into both the labeled plates, mix the plates by gently rotating clock wise and anti-clock wise direction. 5.4.7.4 Allow the plates to solidify at room temperature under Laminar Air Flow. 5.4.7.5 Incubate at specified temperature and period as listed in Table 1, 5.4.7.6 Observe the plates for number of colonies, No growth of the test organism occurs. 5.4.8 Test For Growth Promoting and Inhibitory Properties, liquid Media 5.4.8.1 Prepare required quantity of liquid culture media, dispense 100 ml in test tubes and sterilize as per manufacturers instruction. 5.4.8.2 After sterilization, transfer the media to microbiology analysis room and allow it to cool at room temperature. 5.4.8.3 Start the LAF as per SOP and proceed further work under LAF 5.4.8.4 Add 1.0 ml of positive culture of Growth promoting properties, containing 100 cells (as per table 1) into broth tube and label with Media B. No, name of positive culture & date of inoculation. 5.4.8.5 For Growth Inhibitory Test, add 1.0 ml of positive culture of Growth inhibitory properties, containing 100 cells ( as per table 1) into broth tube and label with Media B. No, name of positive culture & date of inoculation. 5.4.8.6 Simultaneously run negative control to verify testing conditions, using the same procedure in place of the test organism use diluents i.e. 1.0 ml of saline solution

5.4.8.7 Incubate all the tubes at specific temperature as specified in table 1. 5.4.8.8 Daily observe the tubes for growth for turbidity. 5.4.8.9 Satisfactory growth should be observed within 3 days of incubation in the test. There should not be growth in growth inhibitory test & negative control. 5.4.8.10 In case the media passes the growth promotion test, a Approved label shall be affixed on the media container, then the same should be used for analysis. 5.4.8.11 In case the media fails for the growth promotion test then a rejected label shall be affixed on the container then the same shall be rejected and accordingly the rejection entry should be made in the stock register. 5.4.8.12 The rejected media should be discard or returned back to the supplier. 5.4.8.13 The specimens of the Receipt, Sampled, Approved and Rejected label are attached in Annexure 5.5 Precaution 5.5.1 The dehydrated culture media as well as their ingredients are highly hygroscopic and must be stored in a cool dry place away from bright light. These media are meant for laboratory use only and shall never be used for human or animal consumption. 5.5.2 Use fresh sterile pipette for each transfer. 5.5.3 The medium to be poured in Petri plates should have a temperature of 40 - 450C. 5.5.4 The plates should be incubated in an inverted position to prevent collection of condensation on the plate surface. 5.5.5 If any spillage of cultures, immediately wash with 70% IPA solution. 5.5.6 Entire operation inside the microbiology room should be carried out under the laminar airflow chamber using gas burner. 5.5.7 Examine the physical nature of the dehydrated medium. If any unusual colour, odour or physical appearance is noticed, discard the medium. 5.5.8 Always use a dry spoon or spatula for weighing the dehydrated media. The weighing operation shall be completed as quickly as possible to prevent absorption of moisture by the hygroscopic contents. Wear a face mask while weighing the dehydrated media to avoid inhalation of fine particles of media. 5.5.9 All dehydrated media must be retest after the release of three months interval and finally media must be discarded after release of one year. Table - 1

Name of Media

Fluid Thioglycollat e Medium

Positive Positive Expected Incubation Incubation culture to be culture to growth temperature period used for be used for characteristics Growth Growth of the test Promotion Inhibitory organism for Test Test the respective media B. subtilis NA -Growth 30 to 350C 72 hrs NCIM 2063 (Turbidity) Ps.aeruginosa Observed in the NCIM 2200 respective media S.aureus tubes NCIM 2079 NA -Growth (Turbidity) Observed in the respective media tubes 20 to 250C 72 hrs

Soybean Casein digest Medium

B. subtilis NCIM 2063 C.albicans NCIM 3471 A. niger NCIM 1196 Soybean E.coli NCIM Casein digest 2065 Agar Ps.aeruginosa NCIM 2200

MacConkey Agar

-Opaque white colonies on SCDA Plates -Large greyish colonies on SCDA Plates S.aureus NCIM -Tiny white 2079 colonies on SCDA Plates E.coli NCIM S.aureus - Brick red 2065 NCIM 2079 colonies on MacConkey Agar Plates E.coli NCIM 2065 S.aureus NCIM 2079 - Yellow colour change

NA

30 to 350C

48 hrs

30 to 350C

48 hrs

MacConkey broth

30 to 350C

48 hrs

Name of Media

Positive Positive Expected Incubation Incubation culture to be culture to growth temperature period used for be used for characteristics Growth Growth of the test

Promotion Test Mannitol salt Agar Cetrimide agar

Inhibitory Test

S.aureus NCIM E.coli NCIM 2079 2065 Ps.aeruginosa E.coli NCIM NCIM 2200 2065

Antibiotic assay Medium No. 11 Peptone water

S. epidermidis NCIM 2493

NA

E.coli NCIM 2065

NA

Sabouraud s Dextrose Agar Selenite F broth

C.albicans NCIM 3471

NA

Salmonella abony NCIM 2257 E.coli NCIM 2065

S.aureus NCIM 2079

EMB agar

S.aureus NCIM 2079

Brilliant green agar

Salmonella abony NCIM 2257

S.aureus NCIM 2079

Nutrient Broth

E.coli NCIM 2065

NA

organism for the respective media - Yellow colonies on Mannitol Salt Agar Plates - Greenish Colonies on Cetrimide Agar Plates - Good Luxuriant colonies on the respective media Plates Luxuriant (Turbid) growth in the respective media tubes -white colonies on Sabourauds Dextrose Agar Plates -Red ppt. observed in the Selenite F broth - Blue Black colonies with metallic sheen on EMB Agar Plates - Opaque pinkish to white colonies on Brilliant green agar Plates -Good Luxuriant (Turbid) growth Observed.

30 to 350C

48 hrs

30 to 350C

48 hrs

30 to 350C

24 hrs

30 to 350C

48 hrs

22 to 250C

120 hrs

30 to 350C

24 hrs

30 to 350C

24 hrs

30 to 350C

48 hrs

30 to 350C

24 hrs

Name of

Positive

Positive

Expected

Incubation Incubatio

Media

Triple sugar Iron agar Vogel Johnson Agar Baired Parker Agar

Pseudomon as agar(For Fluorescein ) Pseudomon as agar(For Pyocyanin) Urea broth

Tetrathiona te Brilliant Green Bile Broth Bismuth

culture to culture to growth be used for be used characteristi Growth for cs of the test Promotion Growth organism for Test Inhibitory the Test respective media Salmonella S.aureus -Red and abony NCIM NCIM 2079 Yellow butt 2257 (With or without blackening) S.aureus E.coli NCIM - Black NCIM 2079 2065 Colonies on Vogel Johnson Agar Plates S.aureus E.coli NCIM - Black NCIM 2079 2065 Colonies surrounded by a clear zone Ps.aeruginos S.aureus - Yellowish a NCIM 2200 NCIM 2079 Colonies on Pseudomonas Agar Plates Ps.aeruginos S.aureus - Greenish a NCIM 2200 NCIM 2079 Colonies on Pseudomonas Agar Plates Salmonella S.aureus -Luxuriant abony NCIM NCIM 2079 growth 2257 with no colour change Observed in the respective media tubes Salmonella S.aureus -White ppt. abony NCIM NCIM 2079 observed 2257 in the TBGB broth tubes Salmonella S.aureus - Black or

temperatu n period re

30 to 350C

48 hrs

30 to 350C

48 hrs

30 to 350C

48 hrs

30 to 350C

72 hrs

30 to 350C

72 hrs

30 to 350C

24 hrs

30 to 350C

24 hrs

30 to 350C

48 hrs

green Colonies on Bismuth Sulphite Agar Plates Xylose Salmonella S.aureus - Red Colonies 30 to 350C lysine abony NCIM NCIM 2079 on deoxychola 2257 Xylose lysine te agar deoxycholate Agar 5.6 Frequency 5.6.1 For each batch of consignment

sulphite agar

abony NCIM NCIM 2079 2257

48 hrs

Identification of Environmental Flora

1.0 Objective Objective of this Standard Operating Procedure is to provide guidelines for Gram Staining of in house isolates (Manufacturing Area Environmental Flora). 2.0 Scope This procedure is applicable for Gram Staining of in house isolates (Manufacturing Area Environmental Flora) of microbial flora observed during monitoring of manufacturing area. 3.0 Responsibilities Implementation : Microbiologist Execution : Dy. Manager QC Review & Approval : AGM - QA / QC 4.0 Abbreviation SOP : Standard Operating Procedure QA : Quality Assurance QC : Quality Control NA : Not Applicable 5.0 Procedure 5.1 Equipments required Microscope LAF 5.2 Material Required loop Slides Cover slip Immersion oil Gram stain Kit 5.3 Utilities Required LPG Electricity 5.4 Methodology 5.4.1 Cultural Characteristics: 5.4.1.1 Select the unknown culture plate (microbial flora observed in sterile area environment). 5.4.1.2 Examine the plate for Colony characteristic as per Table - I and record the characteristic. Table-I Colony Characteristics

5.4.1 Gram Staining 5.4.1.1 Prepare a thin smear of microbial culture observed in sterile environment, dry in air and fix by gentle heat. 5.4.1.2 Hold the slide in slide rack near to ss tray and flood with Grams Crystal Violet (HiMedia S012) for 1 minute. 5.4.1.3 Wash with water and flood with Grams Iodine (HiMedia S013) for 1 minute. 5.4.1.4 Wash with water and decolourize with Grams Decolorozer (S032) until no further violet colour comes off. 5.4.1.5 Wash with water and counter stain with 0.5% safranin (HiMedia S027) for about 1 minute. 5.4.1.6 Wash with water, dry and observe under oil immersion objective. 5.4.1.7 Those bacteria that appear purple or violet colour are referred as Gram Positive; those appearing pink colour are described Gram Negative. Table-II Morphological Characteristic

5.4.1.1 After observing morphological characteristics record the observation. 5.5 Precaution 5.5.1 All preparation should be carried out under LAF of Microbiology room. 5.5.2 Use nose mask and hand gloves while working with microbial cultures. 5.5.3 After completion of work wash your hand with disinfectant solution. 5.6 Frequencies Daily or as soon as colony observed in sterile area environment.