28 Pharmaceutical Microbiology Section name: Efficiency of Sterilization Methods and Sterility Test Prepared by/ Mohamed Eladawy Assistant Lecturer of Microbiology and ImmunologyWei cecil e Physical methods include automatic process control, thermocouple and temperature chart recorder. e Chemical methods include Browne’s tube No.1 (black spot) and Succinic acid (whose melting point is 121C) and Bowie Dick tape. e Biological methods include a paper strip containing 10° spores of Geobacillus stearothermophilus. Soa Tanseyee scent - To ensure that potentially infectious agents are destroyed by adequate sterilization regimes - Three levels: (Physical, Chemical, Biological) 1) Physical: measuring device control (temp, time, pressure) 2) Chemical: substances that undergo a color change or have melting points within the sterilizing range - Browne's tubes, Bowie Dick tape - give an immediate indication of a successful or non successful sterilization.A) Browne's tubes are glass tubes that contain heat sensitive dyes. HRECTIONS See LEAFLET NG erFec POM Tae Mtwent PRODUCE THESE COI jas at | 25 and over | 118 16 120 " | 98 190"a «These change color after sufficient time at the desired temperature. *Before heat exposure, the contents of the tube appear red. As heating progresses, the color changes to green. *Only when the tube is green, sterilization conditions can be considered adequate. see Te SOSei cnitilnier B) Bowie Dick Tape: is applied to articles being autoclaved. . = SN Before heat exposure, the tape is EW ied buff in color. Myvi adequate heating, the tape develops dark brown strips. ferrea Seen ee ameSa * The indicator inside the test pack should show a uniform color change to pass. esa) Ee Ee fecreaSo *Bacillus__stearothermophilus__spores__(10*-10° organisms) - survives steam heat at 121°C for 5 min. and is killed at 121°C in 13 min. - validate and determine the adequacy of steam or chemical vapor sterilization. + Bacillus subtilis spores - validate and determine the adequacy of ethylene oxide or dry heat sterilization. forrea eee ee a—— Sterility testeed * Sterile product: Product which is free from all forms of living beings as viruses, fungi, bacteria and parasites. So sterility test should be done to detect its sterility (absence or presence of microorganisms). «Sterilized product: product that passes sterilization process but it is not sure whether it is sterile or not. (eons Pat en eam——— -Practically, the sterility test carried on a significant, statistically random sampling of the batch (which has been prepared as a unit) to detect bacteria, fungi or spores which are the most resistant. *So the product is confirmed as sterile product when the product is subjected to one process of sterilization with successful sterility testing. fern aed «Objects which pass the test are free from living M.Os (but not pyrogen); so if they also pass the pyrogen testing they are presumed safe for use. *Since the term sterile is an absolute term it is preferable to label such product sterile. (ecresa ided *Batch: number of units subjected to the same sterilization conditions. Ex: penicillin powder batch. *Batch number: indicates which method applied for sterilization of the product.a * Significance of Sterility Test Sterility test is performed to test absence or presence of M.O (Sterility of the object) (ecresa idime needs: 1- Specialized media inoculated with specific amount of material under test and incubated at specific temperature. 2- Test carried on: bacteria (aerobic and anaerobic), fungi and yeasts. 3- Results of the test may show: a) No growth and so material passes the test. b) Growth and in such case the material fails the test.——— Media for sterility testing: No medium is available for detecting both bacteria and fungi. But each has its characteristic medium. ¢ Fluid thioglycolate medium for bacteria *Fluid sabouraud’s medium for fungi *The composition of each medium and condition of growth seen in the following table: Soe eS amMedia for bacteria Media for fungi 1-According to U.S.P: Fluid thioglycolate medium which can be used for both aerobic and anaerobic bacteria. It's composed of (cystine as reducing agent — agar — dextrose — NaCl — tryptone - yeast extract — water - rosazurine dye or methylene blue as indicator). pH=7. Temperature = 30-32c. Incubation period: 7days. 2-According to B.P: Nutrient broth medium for aerobic bacteria. Cooked meat medium for anaerobic bacteria Fluid sabouraud’s medium which composed of: Water - 2% glucose - 1% peptone. pH = 5.2-5.7 to inhibit bacterial growth. Temperature = 22 - 25c. Incubation period: 10 days. eres’ eee enon«It is made in order to avoid false results and this includes two main items: 1- Positive control. 2- Negative control. fore aNegative control Positive control Test for sterility of the media. Test the suitability of the medium to allow the growth of M.Os or not Tubes with out any inoculate Two tubes of Fluid thioglycolate: - One inoculated with aerobic bacteria (ex: E. coli). - The other one inoculated with anaerobic bacteria (ex: Clostridium histolyticum). Tube of Fluid sabouraud’s medium inoculated with fungi or yeast (ex: Candida albicans). It should show no growth They should show growth of these M.Os. Title teal Microbiology 20ed Sampling for sterility testing: «Number of units taken from the produced batch: Number of units taken for sterility testing: (two cases): 1- If total no 1000 2% of the first 1000 units are taken and additional 2 units foreach 1000 units beyond the first 1000 are also taken. Or using the equation: No. of units taken = 0.4 V (no of units in the batch). ieee nen oada x each unit: ° 1- For solid units: (Ex: Powder) batch>300mg Weight of unit Minimum of unit | Volume of medium The weight of the|/Entire sample is}30ml or more batch<300mg taken If the weight of the}300mg are taken |40ml or more ferrea sat en ren ega 2- For liquid units: (Ex: Water for injection) Volume of unit Minimum of unit Volume of medium If the batch 1ml or|/All amount is taken. |15ml less. If the batch 1-10ml. Take 1ml. 15m! If the batch 10-50ml. | Take Sml. 40ml If the batch >50ml. Take 10ml. 60m! ferea Pet enoneey * You are provided with sterile water for injection and required to test its sterility * Equipment: 1. Two test tubes, one containing Fluid thioglycolate medium and the other one Fluid sabouraud’s medium. 2. iml pipette.Set steps 1- Under aseptic conditions transfer 1ml of the sample to the thioglycolate media, and transfer 1 ml of the sample to the sabouraud’s media. 2- Incubate the thioglycolate media for 7 days at 30-35 °C and incubate the sabouraud’s media for 10 days at 22-25 °C and determine the M.Os growth.SF posuits — If thioglycolate media tubes are clear and transparent: sterile sample. — If thioglycolate media tubes are turbid: bacteria present. — If sabouraud's media tubes are clear: sterile sample. — If sabouraud's media tubes are turbid: fungi present. [ecresa id Pen en OsaSl cenit ¢ Preparation of —-ve and +ve controls as: -ve controls contain media only to ensure sterility of the media. +ve controls contain media and M.O to ensure suitability of media to M.O. « Example of fungi used to inoculate Sabouraud’s media: Candida albicans. * Example of aerobic bacteria used to inoculate thioglycolate media: E. coli or Staphylococcus aureus. * Example of anaerobic bacteria used to inoculate thioglycolate media: Clostridium species.