eBooks

Microbiological Culture Media in Pharmaceutical Industry

IbrahimSection Head Hashim Microbiology
Sabaa Pharma - Egypt

www.esciencecentral.org/ebooks

Ibrahim Hashim

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Guidance for Assuring Quality of Microbiological Culture Media in Pharmaceutical Industry

Table of Contents
A-Dehydrated media I.
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Receiving of Dehydrated Media

II. Storage of Dehydrated media Containers III. Physical Inspection of Dehydrated Media IV. Disposal of Expired/Deteriorated Dehydrated Media B-Prepared Media
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1. Preparation of Media 2. Storage of Prepared Media 3. Inspecton of Prepared Media 4. Disposal of Expired, Deteriorated and Contaminated Media 5. Testing of Media C-Assigning of Expiration Dates of Prepared Media 1. Approach-1 2. Approach-2 D-References
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MICROBIOLOGICAL CULTURE MEDIA IN PHARMACEUTICAL INDUSTRY

A. Dehydrated Media
I. Receiving of Dehydrated Media
Upon receiving a new lot of dehydrated medium, inspect & record each lot for the following items: 1. Assure the arrival of the certificate of analysis and material safety data sheet for each received lot. 2. Assure the presence of a neat, clear label on the package container. 3. Assure the correct name and correct formula on the label of container. 4. Assure the packages are sealed tightly and there is no cracking in the container. 5. Check the expiry date (Expiration dates vary depending upon the culture media). 6. Upon receipt, dehydrated media arriving at the laboratory shall be recorded in the log book of Microbiological media receipt for the following information: Name of media Lot number Quantity received, (i.e., size and number of containers) Date received Expiration date Storage location Date opened 7. Paste a label of identification on the container, record the name, Manufacturer, lot no., expiry date, date of receipt, date of container opening, storage conditions and signature of the employee.

II. Storage of Dehydrated Media Containers

1. Store at designated area away from potential contamination. 2. Store safely, protected from humidity or moisture (Dry area), light and heat (autoclaves, drying ovens, or other heat sources), which are the most frequent causes of their alteration. 3. Store at the specified temperature on the Package label (manufacturers instructions). 4. Store sealed tightly since dehydrated powders are hygroscopic particularly containers which are frequently opened and closed. 5. Arrange the containers according to FEFO rule (First Expired First Out).

III. Physical Inspection of Dehydrated Media

1. Upon opening a new container of dehydrated media it should be inspected for: Appearance Color Clarity Consistency Completeness of information on the label 2. Mark the opened container with a marker pen to be differentiated from other non-opened containers.
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IV. Disposal of Expired /Deteriorated Dehydrated Media

1. Discard the dehydrated medium in the following cases: If the container is expired If the appearance has changed from its original color If the powder is not free flowing 2. If the label information is not correRefer to Media MSDS for proper disposal (if available). 3. The expired dehydrated media are disposed into a trash receptacle unless otherwise indicated on the manufacturers label. 4. Upon disposal of dehydrated media, the following data shall be recorded (Name of disposed medium, manufacturer, lot number, quantity disposed, reason of disposal, date of disposal, analyst initials).

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B. Prepared Media
I. Preparation of Media
1.1 Equipment, Tools & Glassware used
Medium container Calibrated top loading balance Calibrated pH-meter Validated autoclave Hot plate Spatula Cleaned beakers-flasks (high quality, low alkali borosilicate glass) Marker pen Pipettes Sodium hydroxide solution 1M Hydrochloric acid solution 1M Dispensing bottles Petri-dishes Liquid media dispenser Identification labels Dissolving purified water (Deionized water of pH=5.5-7.5) 1.2 Reconstitutions of Dehydrated Media Directions for the preparation of culture media are provided on the label of each container. Follow these directions carefully, using the guidelines below: 1. Dehydrated culture media should be prepared with purified water, having a neutral pH 5.5-7.5. 2. Rinse all glassware before use to remove any trace substances, especially detergents. Prepare the medium in a flask that holds twice the final volume of the medium to allow adequate mixing. 3. Wear the safety protective equipment (Refer to MSDS). 4. Weigh out the appropriate amount of dehydrated medium quickly and accurately, avoid creating dust, do not inhale powder. Close the container as soon as possible to prevent contamination. 5. Add half of the required volume of purified water in the flask, followed by the weighed quantity of medium. Agitate briskly for a few minutes until a homogeneous suspension is obtained. Add the remaining water, ensuring any medium that has adhered to the wall of the flask is added to the liquid. 6. Culture media without agar or gelatin (broth formulations) can usually be dissolved with gentle heat and agitation. Culture media containing agar or gelatin must be heated with frequent agitation and boiled for few minutes to completely dissolve the medium.
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Caution: Overheating culture media containing agar will cause it to boil over very rapidly. Take care to not overheat. Note media that are very sensitive to overheating. Overheated media will frequently appear darker. Do not heat in a microwave. 7. Allow the medium to cool and reach the room temperature before adjusting its pH. 1.3 pH Adjustments

1. The pH of media shall be taken before and after sterilization. If applicable, pH adjustment shall be done with filter 005 sterilized acids and/or bases.

2. Commercial dehydrated media are designed to fall within the specified pH range after steam sterilization. The pH tends to fall approximately 0.2 units during steamsterilization. 3. After adequate cooling, the pH should be tested after the medium has cooled to 25C and solidified. 4. If required, the pH should be adjusted using HCL 1M or Na OH 1M to the specified limit according to the manufacturer Recommendation. 5. Avoid excessive pH adjustments because this can alter the chemical composition of the medium. 1.4 Dispensing of Prepared Media (Bottle Dispensing) 1. Dispense the liquid media (Broth) in the specified bottles according to the required volumes using the liquid media dispenser. 2. Dispense the agar containing media into the specified bottles; ensure gentle mixing during dispensing to maintain homogenous distribution of the agar into the poured bottles. 3. Recap the bottles tightly, prepare for the next step of sterilization. 1.5 Sterilization 1. The autoclave set-temperature should be 121C. 2. The recommended 15 minute sterilization assumes a volume of 1 liter or less. Larger volumes may require longer cycles. Check with your autoclave manufacturer for recommended load configurations. 3. Quantities of media in excess of two liters may require an extended autoclave time to achieve sterilization. Longer sterilization cycles can cause nutrient concentration changes and generation of inhibitory substances. 4. Some microbiological media are thermolabile so will not be autoclaved, only heat on hot plate till boiling and release immediately for pouring (Refer to manufacturer label). 1.6 Dispensing of Prepared Media (Plate Dispensing) 1. Medium should be cooled in a water bath to 50-55C prior to dispensing. 2. Gently swirl medium before and during dispensing to ensure that it is evenly mixed. Dispense quickly. 3. Immediately recap or recover plates to reduce the chance of contamination. Petri dish covers should be slightly ajar, for 12 hours to reduce moisture build-up on lids.

II.Storage of Prepared Media

1. The recommended expiration date of prepared culture media varies greatly. Screw-capped tubes can be stored for 6 months or longer at low to ambient temperatures. Plated media may be stored inverted in a plastic bag or other container in a refrigerator for 12 weeks or longer. 2. Store all media away from light. 3. Prepared media shall not be stored in areas of the laboratory where Biohazardus materials are kept. 4. Older stock of media and reagents should be used first. 5. It is good laboratory practice to establish expiration dates for all prepared media.

III. Inspection of Prepared Media

1. Upon Preparation and during storage of media, the medium tube/plates should be inspected for the following conditions: Uneven distribution of media in petri dishes (Affecting colony size) Variable amounts of medium in dishes/tubes/bottles Shelf-life Discoloration or hemolysis Integrity of packaging Broken or cracked petri dishes Quality and accuracy of labeling Condensation in petri dishes
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 Dehydration (split or retracted medium, dry surface) Sloped or uneven filling of petri dishes Contamination Gel strength Pitted surface or large bubbles Presence of leakage 2. Prepared media shall not be used if any of the previous conditions was deviated.

IV. Disposal of Expired-Deteriorated and Contaminated Media

1. Media shall be disposed after use or upon expiration date whichever is earlier. 2. Media is disposed of into a trash receptacle for disposal of biological waste.

V. Testing of Media
5.1 Sterility Check Incubate portions of microbiological media at 30-35C for 24 hours; the media must not appear any growth. 5.2 Growth Promotion Testing (GPT) Purpose To determine the suitability of microbiological media used in microbiological tests. Scope The GPT should be performed on each batch of dehydrated media/ready-prepared media. Basic requirements 1. The new batch of medium must be inoculated with a small number of M.Os (less than 100 cfu). 2. The media should be tested with the M.Os required by the pharmacopeias. 3. The M.Os must not be more than five passages removed from reference culture (original master seed lot). 4. In order to a new batch of medium to be approved for use, growth on new batch of medium must be comparable to growth obtained from a previously approved batch. 5. Test all media in duplicates. 6. Test each agar plate/broth tube with only one M.O at time. 5.3 Microbiological Examination of Non-Sterile Products Microbial enumeration tests Tested Medium Standard Organisms <100 cfu Staphylococcus aureus ATCC 6538 <100 cfu Pseudomonas aeruginosa ATCC 9027 TSA agar <100 cfu Bacillus subtilis ATCC 6633 <100 cfu Candida albicans ATCC 10231 <100 cfu Aspergillus niger ATCC 16404 Sabouraud dextrose agar <100 cfu Candida albicans ATCC 10231 <100 cfu Aspergillus niger ATCC 16404 Incubation temperature 3035C 3035C 3035C 3035C 3035C 2025C 2025C Incubation period 3 days 3 days 3 days 3 days 3 days 5 days 5 days
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Preparation of Inoculums 100 cfu Use standardized stable suspensions of test strains/not more than 5 passages removed from the original master seed lot Grow each bacterial or fungal strain separately Use buffered sodium chloride-peptone solution pH=7.0 or phosphate buffer solution pH=7.2 to make test suspensions In case of Aspergillus niger add (0.05%) of polysorbate 80 to the buffer Use the suspension within 2 hour or within 24 hours if stored at 2:8C Preparation of test strain Staphylococcus aureus SCD agar 3035C 18 : 24 hours Procedure 1. For each organism, label 2 plates .from the new batch of medium and 2 plates of the previously approved batch of medium. 2. Dispense 0.1 ml of the inoculums per plate ( 100 cfu/0.1 ml) to the 2 plates of the new batch and the previously approved batch. 3. Use the surface spread method, by spreader to disperse the inoculums and distribute it over the entire agar plate. 4. Incubate as indicated in table. 5. Make a negative control (sterility check ) for each batch of media, incubate the negative control at the same temperature as the M.Os tested and at the same time. It must have no growth. 6. Determine if the new batch of medium is suitable for use by using the following acceptance criteria. Acceptance criteria Average number of colonies from the plates of new batch Average number of colonies from the plates of previously approved batch If 1. There is growth in plates. 2. Not more than (100 cfu) on agar plates. 3. Average of new batch plates, must be within a factor of 2 of the average number of previously approved media. Pseudomonas aeruginosa SCD agar 3035C 18 : 24 hours Bacillus subtilis SCD agar 3035C 18 : 24 hours Candida albicans SDA agar 20-25C 23 days Aspergillus niger SDA agar 20-25C 23 days

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Growth promotion test (microbial enumeration test)

Prepare inoculums <100 cfu/0.1 ml Buffer

New Previous approved

Inculcate 2 plates new batch Inculcate 2 plates Previous approved batch

Spread inoculate and incubate Count colonies in Previous approved batch plates Count colonies in new batch plates Compare results Count should be within a factor of 2

Average

Average Average : Average Example a)

If previously approved batch plates average=40.

New batch plates Average must be between20:80. b) If previously approved batch plates average=60. New batch plates Average must be between (30:100) only not 120.

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5.4 Test for Specified Micro Organisms To indicate nutritive, indicative, inhibitory, properties of media Medium EE Broth Mossel Organism E. coli P. aeruginosa S. aureus E. coli P. aeruginosa E. coli S. aureus E. coli S. enterica S. aureus S. enterica P. aeruginosa E. coli S. aureus E. coli Candida albicans Candida albicans Property Growth promoting Growth promoting Inhibitory Growth promoting Indicative(purple red colonies) Growth promoting Indicative(colorless colonies) Growth promoting Inhibitory Growth promoting Indicative(Pink colonies) Growth promoting Inhibitory Growth promoting Indicative(red colonies)with or without black centers Growth promoting Inhibitory Growth promoting Indicative(yellow or white colonies)with yellow zone Inhibitory Growth promoting Growth promoting Indicative(white colonies) Incubation temp 3035C 3035C 3035C 3035C 3035C 42-44C 42-44C 3035C 3035C 3035C 3035C 3035C 3035C 3035C 3035C 3035C 3035C Incubation period 24 hours 24 hours 48 hours 18 h 18-24 h 18 h 18-24 h 24 h 48 h 18 h 18- 72 h 18 hours 24 hours 18 h 18-24 h 18 h 72 h 18 h 18-72 h 72 h 3 days 24 hours 48 hours

VRBG agar

Mac Broth Mac agar RVSE Broth (Rappaport) XLD agar Cetrimide agar

MS agar Sabouraud Dextrose Broth Sabouraud Dextrose Agar

Preparation of inoculums Use standardized stable suspensions of the test strains/not more than 5 passages removed from the original master seed lot Grow each bacterial or fungal strain separately Use buffer solution pH=7.2 to make test suspensions Use the suspension within 2 hour or within 24 hours if stored at 2:8C Strain E. coli, P. aeruginosa, S. aureus, S. enterica Preparation On SCD agar 3035C 18 : 24 hours On Sabouraud Dextrose Agar 20 : 25C 23 days

Candida albicans Procedure

2. Dispense 0.1 ml of the inoculums per plate (100 cfu/0.1 ml) to the 2 plates or tubes of the new batch and previous approved batch. 3. Use spreader to spread inoculum on solid media only. 4. Incubate as in table, make negative control. 5. Also test each M.Os individually on non selective control agar plates.

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1. For each organism, label 2 plates or tubes of the new batch of medium and 2 plates or tubes of the previous approved batch of medium.

Acceptance criteria Solid media: 1. There must be growth on (non selective control agar), growth promotion agar plate. 2. There must be less than 100 cfu on non selective control agar plate and growth promotion agar plate. 3. Average no. of colonies on new medium must be comparable to the average no. of colonies on the previously approved medium. 4. Usually compare the colonies of a previously approved batch to new batch in (appearance, reaction ). Liquid media: Visually compare the growth in the tubes of new batch to the 2 tubes of previously approved batch and must be: 1. There is should be growth in the tubes of new batch, previous approved batch and control agar plate. 2. Must be no more than 100 cfu on control agar plates. 3. Amount of turbidity in a new batch of liquid should be comparable to that of the previous approved batch.

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C. Assigning of Expiration Dates of Prepared Media

Approach-1
Prepare a batch of the medium to be shelf-life validated. This should be of a size that will allow testing with a number of different microorganisms per number of weeks. (10 organisms for 10 weeks=110plates/broths. Package and store the batch of medium as is the normal protocol of the laboratory, e.g. plates wrapped in cellophane or plastic, store at 2-8C in dark; broths caps tightened, packaged in cardboard or plastic/cellophane, stored as appropriate in low light or dark. Label packages week 0 to 10. In this example the batch of medium is constant but the operator and the standard techniques may have week to week variation. Using quantitative or semi-quantitative recovery testing procedures, inoculate test microorganisms onto media to be validated and a freshly made control/reference batch each week. Record all results: Growth, colony size, colonial morphology, biochemical responses, volume (can be determined by weight), gel strength, gas, turbidity, clarity, haemolysis etc. The test medium will progressively get older but the reference medium remains fresh (but not constant). Continue until test medium displays noticeable character changes such as reduction in colony size, reduction in amount of growth, media colour changes, drying of medium (cracking, loss of volume) etc. Determine at which week the last acceptable results were recorded. This then represents the upper limit of the shelf-life of that batch of medium. The laboratory may decide that an acceptable safety margin may need to be included in the shelflife. This is usually a reduction in the shelf-life expectancy. If the medium tested is acceptable at 10 weeks, the laboratory may decide to place an 8 week expiry date on the medium. Where media is to be transported, a simulated or real transport phase needs to be included in the testing protocol. This could be done either during the number of weeks testing period or after determining the shelf-life under ideal condition.

Approach-2
If a type of medium is made regularly i.e. weekly, collect a number of plates each week from the batch (if 10 organisms to be tested, collect 10 plates/broths) for the predicted shelf life number of weeks i.e. 10 weeks Ensure that test media is packaged and stored correctly as per laboratory protocol. When enough media has been collected, the testing protocol can begin. During this collecting phase, test media could be transported and returned to laboratory to be included in test. Oldest collected media could be 10 weeks and the youngest is fresh. Label all packages with week number. In this example, the test batch of medium is not constant, but the operator, inoculation techniques, incubation conditions, control/reference batch and recording of results are constant. Using quantitative or semi-quantitative recovery testing procedures, inoculate test microorganisms onto every weeks media to be validated and fresh control/reference batch. In this example all testing is completed in 1-2 days rather than progressively over the weeks as in Example 1. Record all results: Growth, colony size, colonial morphology, biochemical responses, volume (can be determined by weight), gel strength, gas, turbidity, clarity, haemolysis etc. It is important to note all changes and at which week they occurred. Determine at which week the last acceptable results were recorded. This then represents the upper limit of the shelf-life of that batch of medium. The laboratory may decide that an acceptable safety margin may need to be included in the shelflife. This is usually a reduction in the shelf-life expectancy. If the medium tested is acceptable at 10 weeks, the laboratory may decide to place an 8 weeks expiry date on the medium.

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D. References
1. Australian Society for Microbiology, Guidelines for Assuring Quality of Food and Water Microbiological Culture Media, August 2004. 2. Growth Promotion Test Guide, Microbiologics. 3. USP-NF.2009 (61,62) Microbiological Examination of non sterile pharmaceutical products, USP32-NF27. 4. USP-NF.2009 (71) Sterility Test,USP32-NF27. 5. The OXOID Manual, 9th Edition 2006.

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