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PPT on microbialogical activites

1. MICROBIOLOGY ACTIVITIES

2. Media Preparation and Sterilization Various types of media are required to carry out microbiology analysis. To fulfill the requirements of microbiology activities, the following media shall be prepared and sterilized as per the validated load pattern, stored, tested, and decontaminated/disposed of as per the respective operating procedures. Soya bean casein digest agar (SCDA) Soyabean casein digest medium (SCDM) Fluid Thioglycolate medium (FTGM) Sabourauds dextrose agar(SDA) MacConkey agar (MA) MacConkey Broth (MB) Xylose Lysine Deoxycholate Agar (XLDA) Cetrimide Agar (CA) Mannitol Salt Agar(MSA) After media preparation, all in-house media plates/bottles shall undergo pre-incubation. Pre-incubate the bacterial media at 30-35ºC for 48hrs and fungal media at 20-25ºC for 72hrs. After pre-incubation check all plates/bottles for any contamination and store them at 20-25ºC. Carryout the pH and Growth promotion test, after successful results the media shall be used for regular analysis.

3. Growth Promotion Test Growth promotion test shall be performed for each autoclave cycle prepared media and each received lot of Dehydrated media and ready to use media to check the media is promoting growth or Not by using Standard cultures. Maintenance of stock cultures is important to the consistency of microbiological test results. Cultures used for microbiological tests should be acquired from a national culture collection or a qualified secondary supplier. The cultures are available in frozen, freeze-dried, on slants, or in ready-to-use forms. Working cultures can be prepared, the seed lot technique is useful for the storage of stock cultures. Purity testing shall be carried out before usage in microbiological tests by using an automated identification system. All the stock cultures can be stored in a cryoprotectant medium and frozen at -30ºC or below, until use. For Growth promotion testing of in-house media use NMT 100cfu of culture suspension. All the media shall be checked for Growth promotion properties with the respective organisms mentioned in respective monographs . After completion of GPT Successful results media shall be used for regular analysis. Reference USP Chapters: USP<71> : USP<61> : USP<62> :

4. Isolate Identification Microorganisms, if detected in drug substances, excipients, water for pharmaceutical use, the manufacturing environment, intermediates, and finished products shall undergo identification up to the species level . Initially, many isolates may be characterized and identified to establish a database of the microorganisms found in the area. Periodic identifications should be performed on routine monitoring to check for changes in predominant groups of microflora. A change in the microbial flora might signify a change in a system that should be investigated. Prinmary screening: 1.Gram staining 2.Fungal stain Reference USP chapters USP <1113 >:Microbial characterization,Identification,and strain Typing.

5. Pick the isolates for identification as per the criteria mentioned below in Table

6. ENVIRONMENTAL MONITORING The primary purpose of environmental monitoring is to provide meaningful interpretable data that can help identify actual or potential contamination problems associated with specific procedures, equipment, materials, and processes. E nvironmental monitoring shall be carried out by using 4 methods Settle plate Air sampling. Surface monitoring by swab method, contact plate method, personnel monitoring . A sampling plan, Sites, and frequencies for viable monitoring should be established. 1. Settle Plate method Plates shall be exposed for NLT 4 hours, mark the first and last exposed plate with the start time and upon collection mark the first and last plate with the end time. If the batch is completed less than of 4 hours of exposure time, exposed plates shall be removed and send for incubation. Incubate the plates at 20-25ºC for 72hrs and transfer the plates to 30-35ºC for 48hrs. Wherever the plates are exposed on the plate exposure stand in the room keep the stand upright that is 90 º angles and near riser, the stand shall be bent around 45º Angle. While opening the plates for exposure, first keep the plates on stand then gently lift and slide open the lid of the plate and keep the lid facing down position to avoid contamination of the media. Never bend over the plates while exposing or while collecting the plates.

7. ENVIRONMENTAL MONITORING 2.Active Air Sampling: Using an air sampler to draw in a specified, known volume of air through or over a particle collection device, such as an agar plate. Sample 1000Litres for 10mins in a single location. Incubate the plates at 20-25ºC for 72hrs and transfer the plates to 30-35ºC for 48hrs. 3. Surface sampling: Surface sampling can be accomplished by the use of contact plates or by the swabbing method. Contact plates filled with SCDA are used for sampling of flat surfaces and are directly incubated for the appropriate time The swabbing method can be used for sampling of irregular surfaces, in the range of 24-30cm2. After sample collection, the swab is placed in an appropriate diluent or transport medium. Filter the diluent onto a nutrient-rich medium and incubate at suitable conditions for the recovery of microorganisms.

8. ENVIRONMENTAL MONITORING Settle plate frequencies and limits in different grades Sr.No. Grade Frequency CFU/Plate At rest In Operation # Action limit 1 Grade A Twice in a Week During Batch filling every four hours <1 2 Grade B Twice in a Week During Batch filling every shift NMT 5 3 Grade C Once in a Week During Batch filling once in a day NMT 50 4 Grade D Once in a Month During Batch filling once in a day NMT 100

9. ENVIRONMENTAL MONITORING Air sampling frequencies and limits Sr.No. Grade Frequency CFU/m³ At rest In Operation # Action limit 1 Grade A Twice in a Week During Batch filling once in a shift <1 2 Grade B Twice in a Week During Batch filling once in a shift NMT 10 3 Grade C Once in a Week During Batch filling once in a day NMT 100 4 Grade D Once in a Month During Batch filling once in a day NMT 200

10. ENVIRONMENTAL MONITORING Surface sampling frequencies and limits Sr.No. Grade Frequency CFU/Swab/55 mm Plate/90 mm Plate At rest In Operation # Action limit 1 Grade A NA During Batch filling end of the batch <1 2 Grade B NA During Batch filling end of the batch NMT 5 3 Grade C Once in a Week During Batch filling once in a day NMT 25 4 Grade D Once in a Month During Batch filling once in a day NMT 50

11. ENVIRONMENTAL MONITORING Personnel monitoring frequencies and limits Sr.No. Grade Frequency Limit Locations At rest In Operation CFU/55 mm Plate/90 mm Plate 1 A Hand gloves** NA During batch filling after Machine parts assembling < 1 2 B Gown* NA During batch filling every exit NMT 5 3 Hand gloves** NMT 3 Gown* Hand gloves** Forehead (FH) Left hand gloved finger tips (LHFT) Right hand gloved finger tips (RHFT)   Chest (CH) Left hand Elbow (LHE) Right hand Elbow (RHE)

12. ENVIRONMENTAL MONITORING References ISO 14644 EU Annex 1: Manufacture of Sterile medicinal products PDA USP<1116> WHO GMP

13. Microbial Limit Test By Pour-plate method : Total Aerobic Microbial Count (TAMC): Add 1 ml of the final dilution (Solution A) to each Petri dish then add approximately 15 to 20ml of sterile Soyabean Casein Digest Agar, into two Sterile Petri dishes of 90mm and mix the contents of Sterile Petri dishes by rotating and tilting the plate and allow the medium to solidify. The temperature of media during transfer should be approximately 45°C. Incubate these Sterile Petri dishes at 30 to 35°C for 3 to 5 days. Keep the plates in an inverted position. Total Combined Yeasts and Mold Count (TYMC): Add 1 ml of the final dilution (Solution A) to each Petri dish.dd approximately 15 to 20ml of sterile Sabouraud Dextrose Agar, in two Sterile Petri dishes of 90mm. Mix the contents of Sterile Petri dishes by rotating and tilting the plate, and allow the medium to solidify. The temperature of media during transfer should be approximately 45°C. Incubate these Sterile Petri dishes at 20 to 25°C for 5 to 7 days. Keep the plates in an inverted position. After completion of the incubation period, take the arithmetic mean of the count per medium, and calculate the number of CFU per g. or ml of the product.

14. Microbial Limit Test Negative Control: To verify testing conditions, a negative control shall be performed using the chosen diluents in place of the test preparation. There must be no growth of microorganisms. A failed negative control requires an investigation as per  SOP . Positive Control: Inoculate portions/plates of Soya bean Casein Digest Agar / Sabouraud-dextrose agar with a small number (not more than 100 CFU) of the micro-organisms. Incubate in the conditions as stated above.

15. Microbial Limit Test Test for specified Microorganisms Test For staphylococcus aureus on MSA The possible presence of S. aureus is indicated by the growth of yellow or white colonies surrounded by a yellow zone. References: USP <61>: Microbiological examination of Non-sterile pharmaceutical products USP<62>: Test for specified pathogens.

16. Microbial Limit Test Test for specified Microorganisms Test For Pseudomonas aeruginosa on CA Growth of colonies indicates the possible presence of P. aeruginosa.

17. Microbial Limit Test Test for specified Microorganisms Test For E.coli on MA Colonial Morphology: Brick-Red; may have the surrounding zone of precipitated bile.

18. Microbial Limit Test Test for specified Microorganisms Test For Salmonella on XLDA The possible presence of Salmonella is indicated by the growth of well-developed, red colonies, with or without black centres. References: USP <61>: Microbiological examination of Non sterile pharmaceutical products USP<62>: Test for specified pathogens.

19. Sterility Sterility testing is a GMP microbiology testing requirement used to confirm sterile products do not contain viable microorganisms before release and patient administration. The test for sterility is carried out under aseptic conditions. The following culture media have been found to be suitable for the test for sterility. Fluid Thioglycollate Medium is primarily intended for the culture of anaerobic bacteria. However, it will also detect aerobic bacteria. Soybean–Casein Digest Medium is suitable for the culture of both fungi and aerobic bacteria. The medias used for the sterility test must undergo GPT and after a successful release, the medias must be used for sterility. Direct inoculation: Sterile Gloves, Sterile rubber stoppers,Sterile vials Membrane filtration : 1.Open method-Finished product 2.Closed Method-Finished product

20. Sterility Incubation: NLT 14 Days SCDM: Incubation at 22.5±2.5 for 14 days FTGM : Incubation at 32.5±2.5 for 14 days Results: The test sample shall not show turbid. Negative controls shall not show turbid . Positive control shall show growth.

21. Sterility Minimum quantity to be used for each medium Quantity per container Minimum Quantity to be Used (unless otherwise justified and authorized) Liquids Less than 1 mL The whole contents of each container 1–40 mL Half the contents of each container, but not less than 1 mL Greater than 40 mL, and not greater than 100 mL 20 mL Greater than 100 mL 10% of the contents of the container, but not less than 20 mL Antibiotic liquid 1 mL Insoluble preparations, creams, and ointments to be suspended or emulsified Use the contents of each container to provide not less than 200 mg Solids Less than 50 mg The whole contents of each container 50 mg or more, but less than 300 mg Half the contents of each container, but not less than 50mg 300 mg–5 g 150 mg Greater than 5 g 500 mg

22. Sterility Number of Articles to Be Tested Number of Items in the Batch* Minimum Number of Items to be Tested for Each Medium (unless otherwise justified and authorized)* Parenteral preparations Not more than 100 containers 10% or 4 containers, whichever is the greater More than 100 but not more than 500 containers 10 containers More than 500 containers 2% or 20 containers, whichever is less

23. Sterility OBSERVATION AND INTERPRETATION OF RESULTS Observe the canisters daily for up to 14 days of incubation The test is considered invalid only if one or more of the following conditions are fulfilled: The data of the microbiological monitoring of the sterility testing facility show a fault. A review of the testing procedure used during the test in question reveals a fault. Microbial growth is found in the negative controls. After determination of the identity of the microorganisms isolated from the test, the growth of this species (or these species) may be ascribed unequivocally to faults with respect to the material and or the technique used in conducting the sterility test procedure. If the test is declared to be invalid, it is repeated with the same number of units as in the original test. If no evidence of microbial growth is found in the repeat test, the product examined complies with the test for sterility. If microbial growth is found in the repeat test, the product examined does not comply with the test for sterility . References USP <71>:Sterility Tests.

24. Bacterial Endotoxin Test The Bacterial Endotoxins Test (BET) is to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). Endotoxin (lipopolysaccharide), is a pyrogenic substance that is found in the cell wall of Gram-negative bacteria Pyrogenic substance (or pyrogen) can induce fever when injected into the blood or cerebrospinal fluid It is associated with injectable products Sterilization does not remove the endotoxin It is heat stable There are three techniques to perform this test: 1. gel-clot technique - which is based on gel formation; 2. Turbidimetric technique - based on the development of turbidity. 3. Chromogenic technique, based on the development of color . Apparatus: Depyrogenate all glassware in a hot air oven using a validated process. A commonly used minimum time and temperature is 30 min at 250°

25. LBPC For the determination of particulate matter, there are two methods Light Obscuration Particle Count Test Microscopic Particle Count Test Method 1 is applied for examining injections and parenteral infusions for subvisible particles. Method 2 is applied, in the case of preparations having reduced clarity or increased viscosity, Emulsions, colloids, and liposomal preparations are examples. Similarly, products that produce air or gas bubbles when drawn into the sensor may also require microscopic particle count testing. Carry out the test in a Laminar Air Flow Unit. Prepare required amount of Particle free water by filtering purified water through 0.2um ffilters . Carefully wash the glassware with particle-free water. In order to check that the environment is suitable for the test, that the glassware is properly cleaned, and that the water to be used is particle-free, the following test is carried out. Environment Test: Determine the particulate matter in five samples of particle-free water, each of 5mL by using liquid borne particle counter for particles of 10um or greater size exceeds 25 for the combined 25mL. Sample Testing: Take 10 number of test vials Reconstitute each vial with the required amount of particle-free water. Sonicate each container for 30 seconds.

26. LBPC Pool the contents of 10 vials in a cleaned, dried particle-free container. Allow it to stand for 2minuts and run the sample by using LBPC machine. Acceptance criteria: Solutions for parenteral infusion or solutions for injection supplied in containers with a nominal content of less than 100 mL: The preparation complies with the test if the average number of particles present in the units tested does not exceed 6000 per container equal to or greater than 10 µm and does not exceed 600 per container equal to or greater than 25 µm. References USP <788>:Particulate matter in injections.

27. Cleaning , Sanitization and Disinfection A sound cleaning and sanitization program is needed for controlled environments used in the manufacture of Pharmacopeial articles to prevent microbial contamination of these articles . Disinfectant —A chemical or physical agent that destroys or removes vegetative forms of harmful microorganisms when applied to a surface. Disinfection: It is a process of killing the microbes. Disinfectants are often categorized as high-level, intermediate-level, and low-level by medically oriented groups based upon their efficacy against various microorganisms. Chemical disinfectants are classified by their chemical type. This includes aldehydes, alcohols, halogens, peroxides, quaternary ammonium compounds, and phenolic compounds . When selecting a disinfectant for use in a pharmaceutical manufacturing area, the following points should be considered: the number and types of microorganisms to be controlled; the spectrum of activity of commercially available disinfectants; the claims as a sterilant ; the disinfectant or sanitizer supported by the EPA registrations; the concentration, application method, and contact time of the disinfectant; the nature of the surface material being disinfected and its compatibility with the disinfectant; the number of organic compounds on the surface that may inactivate a disinfectant;

28. Cleaning , Sanitization and Disinfection The disinfectant efficacy can be tested by 2 methods. Use dilution method screening disinfectants for their efficacy at various concentrations and contact times against a wide range of standard test organisms and EM isolates 2. Surface challenge test Using standard test microorganisms and EM isolates, applying disinfectants to surfaces at the selected use concentration with a specified contact time, and determining log reduction of microorganisms. Acceptance criteria: 2 log reduction must be observed in case of spores 3 log reduction must be observed against vegetative bacteria.