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 can’t 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 β-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 Testing-

The 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

Parenteral preparations LiquidsLess than 1 ml

1 ml or more

SolidsLess than 50 mg

50 mg or more but less than 300 mg

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

300 mg or more 150 mg

Ophthalmic and other non-injectablePreparations

Aqueous solutions

Other preparations soluble in wateror isopropyl myristate

Insoluble preparations, creams andointments to be suspended oremulsified

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

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

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

Catgut and other surgicalsutures 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, catalase-

positive bacterium commonly found in soil. A member of the genus Bacillus, B. subtilis is rod-

shaped, 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

Brewer’s 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 Brewer’s 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 Vol. of medium Sample vol. Organism (ml) results

ANC 10ml 0 0.0 _

APC 10ml 0 0.1 +

AT

AC

10ml

10ml

1ml

1ml

0.0

0.1

++

+

Anaerobic test

Test tube Vol. of medium Sample vol. Organism (ml) Results

NNC 10ml 0 0.0 --

NPC 10ml 0 0.1 +

NT 10ml 1ml 0.0 ++

NC 10ml 1ml 0.1 +

Fungi test

Test tube Vol. of medium Sample vol. Organism (ml) Results

FNC 10ml 0 0.0 --

FPC 10ml 0 0.1 +

FT 10ml 1ml 0.0 ++

FC 10ml 1ml 0.1 ++

Aerobic test - penicillin

Test tube Vol. of medium Sample vol organism Results

ANC 10ml 0 0.0 -

APC 10ml 0 0.1 +

AT 10ml 1ml 0.0 -

AC 10ml 1ml 0.1 ++

Anaerobic

Test tube Vol. of medium Sample vol. organism Results

NNC 10ml 0 0 -

NPC 10ml 0 0.1 +

NT 10ml 1ml 0 ++

NC 10ml 1ml 0.1 +

Fungi

Test tube vol. of medium Sample vol. organism results

FNC10ml 0 0 _

FPC10ml 0 0.1 +

FT10ml 1ml 0 _

FC10ml 1ml 0.1 +

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 (brewer’s) 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 can’t 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 can’t 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,