KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCE

DEPARTMENT OF PHARMACEUTICS

MICROBIOLOGY PROJECT LAB

PROJECT TITLE: STERILITY TESTING

NAME: HENRY YAW DUFFOUR

INDEX NO.: 9538906

LAB GROUP: 3C3 - Henry Yaw Duffour

Mary Gidigasu

John Appenteng

Douglas Adjei

Victoria Aboagye

Date of submission: 25-03-09

INTRODUCTION

Sterile products are those that must be free from living micro-organisms. Products that are intended to be sterile must therefore be tested for the absence of any contaminating organism. Examples of products that must be sterile and hence must pass the test include ready made injections (including solutions and suspensions, both aqueous and oily), solids for injection (eg. heparin antibiotics), ophthalmic products, implants surgical dressing etc. This experiment uses dusting powder and penicillin as test samples

LITERATURE REVIEW The test is applied to substances, preparations or articles which, according to the Pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating micro-organism has been found in the sample examined in the conditions of the test. The probability of detecting micro-organisms by the test for sterility increases with their number present in the sample tested and varies according to the readiness of growth of micro-organism present. The probability of detecting very low levels of contamination even when it is homogenous throughout the batch is very low. The interpretation of the results of the test for sterility rests on the assumption that the contents of every container in the batch, had they been tested, would have given the same result. Since it is manifest that every container cannot be tested, an appropriate sampling plan should be adopted. In the case of aseptic production, it is recommended to include samples filled at the beginning and at the end of the batch and after significant intervention.

Sterility test must initiate and maintain the vigorous growth of 1. The aerobic and anaerobic bacteria that can be cultivated on artificial media. This includes bacteria that are pathogenic to man eg. pyogenic cocci and spore bearing bacteria. 2. The lower fungi i.e. yeast and mould that are responsible for spoilage. 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. Soya-bean casein digest medium was primarily intended for the culture of aerobic bacteria but is also suitable for fungi. Other media may be used provided that they have been shown to sustain the growth of a wide range of micro-organisms. DEALING WITH ANTIBACTERIAL SAMPLES Some preparations come with certain active components and excipients that have antibacterial properties. These components could affect results of the test. Hence inactivation of these compounds is carried out. Three methods may be employed 1. Dilution of the agent to concentrations below the MIC of the antibacterial agent 2. Neutralization of the antibacterial agent using another chemical 3. Separation of organisms from products by filteration Dilution The relationship between a bactericide and the rate at which it kills bacteria is given by the expression Cnt = a constant, where C is concentration of the bactericide, t is the time taken to kill the bacteria and n, the dilution coefficient. In this method the bactericide is diluted to a very low concentration that cant support its bactericide activity. Neutralization

For bactericides containing heavy metals, simple dilution will not inactivate them. Examples include mercurials and arsenicals. Thioglycollate broth is used for their cultivation. Penicillins are destroyed enzymatically by penicillinase, which attacks the -lactam ring to produce penicilloic acid. Hence samples containing penicillin is first neutralized using penicillinase solution The antibacterial action of sulphonamides is due to their interference with the utilization of para aminobenzoic acid, a growth factor for many bacteria. The activity is by competitive inhibition. The enzyme has greater affinity for PABA than the sulphonamide hence a small amount of PABA in the system will displace a large amount of the sulphonamide.

METHODS USED The USP describes three general methods for sterility testing: 1) Membrane Filtration 2) Direct Transfer (Product Immersion) 3) Product Flush Membrane Filtration Sterility TestingThe Membrane Filtration Sterility Test is the method of choice for pharmaceutical products. It is not the method of choice for medical devices. An appropriate use of this test is for devices that contain a preservative and are bacteriostatic and/or fungistatic under the direct transfer method. With membrane filtration, the concept is that the microorganisms will collect onto the surface of

a 0.45 micron pore size filter. This filter is segmented and transferred to appropriate media. The test media are fluid thioglycollate medium (FTM) and soybean casein digest medium (SCDM). FTM is selected based upon its ability to support the growth of anaerobic and aerobic microorganisms. SCDM is selected based upon its ability to support a wide range of aerobic bacteria and fungi (i.e. yeasts and molds). The incubation time is 14 days. Since there are many manipulations required for membrane filtration medical device sterility testing, the propensity for laboratory contamination is high. Therefore, in an open system, more sterility failures are expected when using this method. A closed system is recommended for drugs and small devices or combination products. Most pharmaceutical articles are tested using a closed system. In closed systems, the propensity for extrinsic contamination is very low.

Direct Transfer Sterility Testing

Combination products: This method is the method of choice for medical devices because the device is in direct contact with test media throughout the incubation period. Viable microorganisms that may be in or on a product after faulty/inadequate sterilization have an ideal environment within which to grow and proliferate. This is especially true with damaged microorganisms where the damage is due to a sub-lethal sterilization process. All microorganisms have biological repair mechanisms that can take advantage of environmental conditions conducive to growth. The direct transfer method benefits these damaged microorganisms. The entire product should be immersed in test fluid. With large devices, patient contact areas should be immersed. Large catheters can be syringe filled with test media prior to immersion. Cutting catheter samples to allow for complete immersion is the method of choice.

The method requires that the product be transferred to separate containers of both FTM and SCDM. The product is aseptically cut, or transferred whole, into the media containers. The test article should be completely immersed in the test media. The USP limits the media volume to 2500 ml. After transferring, the samples are incubated for 14 days.

Product Flush Sterility Testing

Combination products: The product flush sterility test is reserved for products that have hollow tubes such as transfusion and infusion assemblies where immersion is impractical and where the fluid pathway is labeled as sterile. This method is easy to perform and requires a modification of the FTM media for small lumen devices. The products are flushed with fluid D and the eluate is membrane filtered and placed into FTM and SCDM. This method is not generally used. SAMPLES The USP and other pharmacopoeia states certain quantities for certain dosage forms to be used for the sterility testing. The table below shows the various quantities Type of preparation Quantity per container Minimum quantity to be used for each medium, unless otherwise justified and authorised The whole contents of a container Half the contents of a container but not more than 20 ml The 100% contents of a container Half the contents of a container

Parenteral preparations

Liquids Less than 1 ml 1 ml or more Solids Less than 50 mg 50 mg or more but less than 300 mg

300 mg or more

150 mg

Ophthalmic and other noninjectable Preparations

Aqueous solutions

The whole contents of one or more containers to provide not less than 2.5 ml

Other preparations soluble in water or isopropyl myristate

The whole contents of one or more containers to provide not less than 0.25 g

Insoluble preparations, creams and ointments to be suspended or emulsified

The whole contents or one or more containers to provide not less than 0.25 g

Catgut and other surgical sutures for veterinary use

3 sections of a strand (each 30cm long)

ORGANISMS USED

Bacillus subtilis, known as the hay bacillus or grass bacillus, is a Gram-positive, catalasepositive bacterium commonly found in soil. A member of the genus Bacillus, B. subtilis is rodshaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions. Unlike several other well-known species, B. subtilis

has historically been classified as an obligate aerobe, though recent research has demonstrated that this is not strictly correct

B. subtilis is not considered a human pathogen; it may contaminate food but rarely causes food poisoning.[5] B. subtilis produces the proteolytic enzyme subtilisin. B. subtilis spores can survive the extreme heating that is often used to cook food, and it is responsible for causing ropiness a sticky, stringy consistency caused by bacterial production of long-chain polysaccharides in spoiled bread dough

Candida is a genus of yeasts. Many species of this genus are endosymbionts of animal hosts including humans. While usually living as commensals, some Candida species have the potential to cause disease. Clinically, the most significant member of the genus is Candida albicans, which can cause infections (called candidiasis or thrush) in humans and other animals, especially in immunocompromised patients. Many Candida species are members of gut flora in animals, including C. albicans in mammalian hosts, whereas others live as endosymbionts in insect hosts. Some Candida species are responsible for superficial infections such as oropharyngeal candidiasis (thrush) and vulvovaginal candidiasis (vaginal Candidiasis). In otherwise healthy individuals, these infections can be cured with antifungal medications. However, persistent and deep-seated yeast infections can be lethal in immunocompromised patients.

Candida are also responsible for a number of life-threatening opportunistic infections in AIDS patients and other immunocompromised persons - including patients treated in intensive care units (ICUs), cancer patients receiving chemotherapy, and organ transplant patients. Another common Candida infection is oral candidiasis caused by acrylic dentures, especially in elderly denture wearers. Colonization of the gastrointestinal tract by C. albicans may result from taking

antacids or antihyperacidity drugs. This colonization may interfere with absorption of Coenzyme Q10.

MATERIALS

Penicillin injection Penicillinase Dusting powder Candida albicans Bacillus subtilis Clostridium histolyticum Test tubes Nutrient broth Brewers agar Czapek Dox agar Loop

METHOD

10ml of penicillin injection was measured into a beaker and 10ml of penicillinase was added to it to neutralize the antibacterial activity. Test tubes were labeled ANC (aerobic negative control), APC (aerobic positive control), AT (aerobic test), AC (aerobic control). Each tube contained 10ml nutrient broth. No sample or organism was added to the ANC tube. 0.1ml of Bacillus subtilis was added aseptically to the APC test tube. 1ml of the neutralized penicillin was transferred into the AT test tube (with no organism). 1ml of the neutralized penicillin and 0.1ml of Bacillus subtilis were added to the AC test tube. The same process was used for the anaerobic test using Clostridium histolyticum as organism, heated Brewers medium and test tube labeled as in the first case but A replaced by N for anaerobic and in the fungal test Candida albicans
was used, Czapek Dox agar as the medium and test tubes labeled appropriately were A was replaced by F for fungal All the content of the powder was used. The procedure above was used were the powder was added to medium and shaken to ensure uniform distribution. The fungi test set up were incubatored at room temperature for 7days and the others (aerobic and anaerobic test) incubatored at 37degrees Celsius for 5days.

RESULTS Aerobic test dusting powder

Test tube code ANC APC AT AC

Vol. of medium 10ml 10ml 10ml 10ml

Sample vol. 0 0 1ml 1ml

Organism (ml) 0.0 0.1 0.0 0.1

results _ + ++ +

Anaerobic test Test tube NNC NPC NT NC Vol. of medium 10ml 10ml 10ml 10ml Sample vol. 0 0 1ml 1ml Organism (ml) 0.0 0.1 0.0 0.1 Results -+ ++ +

Fungi test Test tube FNC FPC FT FC Vol. of medium 10ml 10ml 10ml 10ml Sample vol. 0 0 1ml 1ml Organism (ml) 0.0 0.1 0.0 0.1 Results -+ ++ ++

Aerobic test - penicillin Test tube ANC APC AT AC Vol. of medium 10ml 10ml 10ml 10ml Sample vol 0 0 1ml 1ml organism 0.0 0.1 0.0 0.1 Results + ++

Anaerobic Test tube NNC NPC NT NC Vol. of medium 10ml 10ml 10ml 10ml Sample vol. 0 0 1ml 1ml organism 0 0.1 0 0.1 Results + ++ +

Fungi Test tube FNC 10ml FPC 10ml FT 10ml FC 1ml 0.1 + 1ml 0 _ 0 0.1 + vol. of medium 10ml Sample vol. 0 organism 0 results _

DISCUSSION From the results obtained using dusting powder, there was no growth in the ANC tube indicating that the nutrient broth was sterile and contained no organism which could interfere with the results. A growth in APC shows that the medium can initiate and maintain the growth of Bacillus subtilis used. AT had a lot of microbial growth, an indication that the powder was not sterile after all. Hence there could be the presence of some microbes in the powder. AC gave growth showing that the drug had no inhibitory action on Bacillus subtilis. In the anaerobic test for the powder, there were growth in NPC and NC, but not in NNC and NT. This means that the medium (brewers) is sterile and can support the growth of the anaerobes

(Clostridium histolyticum) and that the powder is also sterile and has no inhibitory action on the organisms The fungi test for the powder showed that the medium (Czapek Dox) was sterile and supported the growth of Candida albicans. The powder was however not sterile as growth was observed in FT
but it had no inhibitory action on the organism. A summary for the powder can be that the media used were all sterile hence any growth cant be attributed to them. Also the powder had no inhibitory action against any of the organisms. Two results indicated that the powder was not sterile. But that cant be the final conclusion as this experiment is affected by many factors (esp. environmental) such as the presence of contaminated air were experiment was undertaken, the lack of aseptic techniques etc. For the penicillin, there was no growth in any of the media used indicating sterility but also supported the growth of the various organisms. Only the anaerobic test gave the indication of non-sterility of penicillin as there was growth in NT. The rest gave no growth and here too the variation could be as a result of environmental factors.

The sterility test environment is described in USP General Informational, Chapter 1211. The environment should be as stringently controlled as a clean room. Such a room delivers laminar flow air which has been filtered through microbial retentive HEPA filters. The room is kept at a positive pressure and has specifications for room air changes per hour. An area used for sterility testing should be similar in design to a clean room. There should be an anteroom for gowning and a separate area for the actual sterility testing. Along with particulate testing in the environment, the laboratory must test for viable bacterial and fungal organisms. The sterility test technician must be suitably gowned in sterile garments

that prevent microbial shedding into the room. The room should be validated in terms of particulate and microbial levels. The laboratory must have a validation and training program for gowning and sterility testing.
CONCLUSION The powder and penicillin may be sterile (neglecting any external factors).

REFERENCES

1. The United States Pharmacopeia, 2008 30th Revision, The United States Pharmacopeial Convention Pg 142, 446 1129-1137

2. FDA Guidelines ,2004 "Guidance for Industry Sterile Drug Products by Aseptic Processing, Current Good Manufacturing Practices," September, 2004 Pg 12-45 3 . Kay J, Ray CG (2004). Sherris Medical Microbiology (4th edition). McGraw Hill pulishers Page 104-119 3. Hutton J and Ray E. M, 2000 World of Microbes,