Sterility test and Rapid microbiological methods

BY/ MOHAMMED FAWZY

MICROBIOLOGIST AT NODCAR

Agenda Sterility test

Introduction

Definition

Media in sterility test

Methods

Preparation of test products

Incubation period

Growth promotion tests

Interpretation of results

Rapid microbiological methods Introduction

ATP-bioluminescence

Colorimetric growth detection

Autofluorescence detection

Cytometry systems

Sterility testintroduction

The test is applied to substances, preparations, or articles which, according to the Pharmacopeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating microorganism has been found in the sample examined under the conditions of the test

Sterility test is applied only for sterile, non-pyrogenic products

Example as parenteral, ophthalmic products, Pulmonary drug delivery system, and sterile medical devices as ocular inserts.

Introduction All products labeled sterile must be pass sterility test This test is suitable to reveal the presence of viable form of

bacteria, fungi and yeasts in pharmaceutical and medical devices

Sterile product must be free fromMicroorganismsSpores PyrogensPathogens

Definition Sterility is define as freedom from the presence of

viable microorganisms, is a strict, uncompromising requirement of an injectable dosage form.

Sterility test is define as:Microbiological test applied to sterile product

to show are products manufactured and processed under specification guided by cGMP.

Or to confirm the products either sterile or non. sterility test is a destructive test thus, it is impossible to

test every item for sterility.

Media in sterility test

USP and EP describe two primary types of culture media to be used in the sterility testing of parenteral products.

Fluid thioglycollate media (FTM)Soybean casein digest broth

FTM used for detection of aerobic and anaerobic bacteria.

SCDB used for detection of molds and molds.

Media in sterility test

FTM1. Detect aerobic and anaerobic

bacteria.

2. Contain O2 sensitive substance (resazurin sodium)

3. The top part should not consume more than 1/3 of tube.

4. Neutralize the bacteriostatic properties of mercuric compounds.

SCDB

1-Detect molds and yeasts

2-possesses a higher pH

3- a better nutrient for fungal contaminants

4-Suitable for aerobic bacteria

FTMCONPONANTS FUNCTION

L-Cysteine 0.5 g Antioxidant

Agar, granulated (moisture 0.75 g Nutrient and viscosity content _15%)

 

inducer 

Sodium chloride 2.5 g Isotonic agent 

Dextrose 5.5 g Nutrient

Yeast extract 5.0 g Nutrient 

Pancreatic digest of casein 15.0 g Nutrient 

Sodium thioglycollate or 0.5 g Antioxidant

thioglycollic acid 0.3 ml 

Nutrient 

Resazurin sodium solution 1.0 ml (1:1000), freshly prepared

 

Oxidation indicator

Purified water QS 1000 ml 

pH after sterilization 7.1 _ 0.2. 

SCDBComponents Function

 Pancreatic digest of casein 17.0 g

 Nutrient

Papaic digest of soybean meal 3.0 g Nutrient

Sodium chloride 5.0 g  

Isotonic agent

 Dibasic potassium phosphate 2.5 g  

 Buffer

Dextrose 2.5 g Nutrient 

Purified watera QS 1000 ml 

Solvent

pH after sterilization 7.3 _ 0.2. 

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Minimum Quantity to be used for Each Medium

Quantity per ContainerMinimum Quantity to be Used

(unless otherwise justified and authorized)

Liquids (other than antibiotics)

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 liquids 1 mL

Other preparations soluble in water or in isopropyl myristate The whole contents of each container to provide not less than 200 mg

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 50 mg

300 mg–5 g 150 mg

Greater than 5 g 500 mg

Devices

Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30-cm long)

Surgical dressing/cotton/gauze (in packages) 100 mg per package

Sutures and other individually packaged single-use material The whole device

Other medical devices The whole device, cut into pieces or disassembled

Methods

I. Direct transfer method It is most usable method and carried out by transfer the test product after preparation to the medium and incubate for not less than 14 days.

II. Membrane filtration method It is usually used in sterility test of antimicrobial agents as antibiotic and other. It is carried out by using a membrane filter having a pore size about (0.45) micrometer that retain most of bacteria, after filtration of the test product, the membrane is aseptically transfer and washed with a diluting fluid such as fluid A,D and K. then aseptically transfer to the medium and incubate for not less than 14 days.

Preparation of test products

a- Oily liquid

Added the tween 80 as 10gm.to 1000 ml of media for emulsification and to be easily dispersion to media.

b- Ointment and creams:

Preparing by taking about 1 gm. of test product and aseptically transfer to 10 ml of diluting fluid as fluid A, then transfer to a medium not contain an emulsifying agent. And incubate for not less than 14 day. Observe the cultures several times during the incubation period. Shake cultures containing oily products gently each day. However, when thioglycollate medium or other similar medium is used for the detection of anaerobic microorganisms, keep shaking or mixing to a minimum in order to maintain anaerobic conditions.

Preparation of test products

c- devices with pathways labeled sterile Aseptically pass not less than 10 pathway volumes of Fluid D through each device tested. Collect the fluids in an appropriate sterile vessel, and proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies.

d- solids:

Transfer the quantity as described before in the previous table either directly in a dray form or after making a suspension by adding sterile diluent to the immediate container.

Preparation of test products

e-Sterile devices: Articles can be immersed intact or disassembled. To ensure that device pathways are also in contact with the media, immerse the appropriate number of units per medium in a volume of medium sufficient to immerse the device completely, and proceed as directed above. For extremely large devices, immerse those portions of the device that are to come into contact with the patient in a volume of medium sufficient to achieve complete immersion of those portions.

Preparation of test productsf-For catheters:

where the inside lumen and outside are required to be sterile, either cut them into pieces such that the medium is in contact with the entire lumen or fill the lumen with medium, and then immerse the intact unit.

g-Surgical dressing: Three sample each of about 1 gm. or 10 cm are taken from each dressing after opening the wrappers. They are chosen from different places including regions where the contamination is more likely (center and outside). If dressing is less than 1 gm. Or 10 cm2, it is used entire or divided in to equal pieces for inoculation in the bacterial and fungal medium. OR each sample is shaken for 10 minutes in 50 ml. of sterile broth and then the liquid is pass through a membrane filter, the membrane is washed with diluting fluid and then incubated in to medium.

Incubation period

For bacteria:Temp 30-35°C.Time NLT 14 days.

For molds and yeasts:Temp 20-25°CTime NLT 14 days.

Growth promotion tests

Aerobic bacteria Staphylococcus aureus

Bacillus subtilis

Pseudomonas aeruginosa

Anaerobic bacterium Clostridium sporogenes

Fungi Candida albicans

Aspergillus brasiliensis (Aspergillus Niger)

Growth promotion

Inoculate media Not more than 100 CFU

Incubate Bacteria

Not more than 3 days

Fungi Not more than 5 days

Clear growth of microorganism: Suitable for use

Interpretation of result

examine the media for macroscopic evidence of microbial growth.

If the material being tested renders the medium turbid so that the presence or absence of microbial growth cannot be readily determined by visual examination

14 days after the beginning of incubation transfer portions (each not less than 1 mL) of the medium to fresh vessels of the same medium, and then incubate the original and transfer vessels for not less than 4 days.

Positive and negative control

Positive control For validation of nutrient

medium.

For testing the growth ability of media.

Done by inoculation of NMT 100 CFU.

Incubate at 30-35 for 14 days.

If growth where obtain →used in sterility test.

Negative control For validation of nutrient

medium.

For testing the sterilization of media.

Done by exposure to the same environmental condition as test exposure.

Incubate at 30-35 for 14 days.

If growth where obtain →sterilization failed or improper storage of media

Interpretation of result

If no evidence of microbial growth is found, the product to be examined complies with the test for sterility.

If evidence of microbial growth is found, the product to be examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product to be examined.

Interpretation of result

The test may be considered invalid only if one or more of the following

conditions are fulfilled:

a. The data of the microbiological monitoring of the sterility testing facility

show a fault.

b. A review of the testing procedure used during the test in question reveals

a fault.

c. Microbial growth is found in the negative controls.

Interpretation of result

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

RMMIntroduction

Current harmonized compendial sterility test methods using either membrane filtration or direct inoculation require at least 14days of incubation.

In cases where drug products either possess an intrinsic turbidity, or because of their formulation become opaque or cloudy during the incubation period, identification of microbial contamination based on visual confirmation of turbidity of growth media becomes difficult. In such instances, at the end of the 14-day incubation, a portion of the sample is sub-cultured into fresh medium for an additional 4–5 days to allow detection, further extending the incubation period.

Introduction The replacement or the supplement of the conventional

sterility test by a rapid microbiology test will have significant benefits.

A rapid method has the potential to produce test results much faster at enhanced sensitivity.

a test based on current technologies can increase throughput and allow better data handling.

a greater assurance of product safety.

Time saving.

Characters of ideal method: simple

economic

relatively easy to implement.

ATP Bioluminescence

Adenosine triphosphate (ATP) bioluminescence is a well established rapid method utilizing a specific substrate and enzyme combination, luciferin/luciferase, to break down microbial ATP from growing cells and produce visible light, which can be measured using a luminometer. Several commercial systems have been developed for a range of pharmaceutical test applications, including sterility testing, especially for filterable samples where non-microbial ATP in the sample is less of a concern. The test time can be reduced considerably because detection of microbial growth in culture media is accomplished by ATP-bioluminescence, rather than by visible turbidity. Typically, results equivalent to those of compendial tests are available within 7 days or less.

Celsis Rapid Detection Advance luminometer system, AkuScreen™ reagents

which use the enzyme adenylate kinase to increase the quantity of microbial ATP produced and reduce detection times by 25-50%.

ATP Bioluminescence

ATP (adenosine triphosphate) act as energy source for all a live organisms That includes microorganisms, and it is for the detection of bacteria, yeasts, and molds that ATP bioluminescence has been applied most successfully.

ATP bioluminescence is well-established technique in food industry

Used for hygienic monitoring and finished product testing ATP bioluminescence today used in cosmetic industry and

pharmaceutical industry

ATP Bioluminescence

Bioluminescence is an enzymatically catalyzed reaction that generates light.

based on a reaction that occurs naturally in the North American firefly, Photinus pyralis.

The reaction was famously first described by McElroy 1947.

ATP Bioluminescence

ATP bioluminescence reaction depend on production of light that can be detected by an instrument after magnification .

The amount of emitted light is proportionally to amount of ATP.

SO. that there is a linear relationship between the amount of ATP in a sample and the amount of light generated.

Working mechanism luciferin-based bioluminescence reaction makes use of

the firefly enzyme luciferase (luciferin 4-monooxygenase, EC 1.13.12.7),which catalyzes the oxidation of D-luciferin in the presence of ATP, magnesium ions and molecular oxygen.

The reaction yields a quantum of yellow light at 564 nm for each molecule of the luciferin substrate oxidized.

ATP + Luciferin + O2 →AMP + Oxyluciferin + CO2 + PPi + Light (Bioluminescence). In presence of luciferase

Light produced from the luciferin–luciferase reaction is proportional to the amount of ATP used and can be measured in Relative Light Units (RLU) by a luminometer.

ATP Bioluminescence-based Pallchek™

Milliflex Rapid Microbiology Detection and Enumeration SystemThe Milliflex® Rapid Microbiology Detection and Enumeration

system from Millipore also uses ATP-bioluminescence to detect microbial cells and is designed specifically for monitoring microbial contamination in filterable samples. It is automated, employing image analysis technology to detect microcolonies growing directly on the surface of a membrane filter after the addition of bioluminescence reagents. The system is designed to be quantitative, but a method has been developed and validated to use it for a rapid sterility test with an incubation time of just five days

Milliflex Rapid Microbiology Detection and Enumeration Systemprons

Equivalent to compendial tests

Results in 7 days

consNon-microbial ATP

detection

Colorimetric Growth Detection

Colorimetric growth detection methods rely on a colour change being produced in a growth medium as a result of microbial metabolism during growth, often as a result of CO2 production.

Colorimetric Growth Detectionmechanism

1. Media (two different media are used) as SCDB supplemented with complex amino acids and carbohydrates and are designed both to support growth and to ensure optimal C02 production.

2. CO2 sensor :a C02 sensor is bonded to the bottom of each bottle and is separated from the broth medium by a semipermeable membrane.

3. The semipermeable membrane is permeable to CO2 and impermeable to H2 ion and most ion are exist in the media

Colorimetric Growth Detectionmechanism

4. Carbon dioxide produced by growing organisms diffuses across the membrane into the sensor and dissolves in the water, thereby generating hydrogen ions according to the following equation:

C02 + HO2 +H2CO3 -> H+ + HCO3-5. Free hydrogen ions can interact with the sensor, which is blue to dark green in the alkaline state. As C02 is produced and dissolves in the water, the concentration of hydrogen ions increases and the pH decreases. This causes the sensor to become lighter green and eventually yellow, which results in an increase of red light reflected by the sensor.

BacT/ALERT® 3D Dual-T Microbial Detection System

The system is automated and employs sensitive colour detection and analysis technology to produce a result in as little as three days. It can detect both aerobic and anaerobic bacteria, as well as yeasts and molds. 

1-Results 3 days.2-Aerobic/anaerobic bacteria.3-Yeasts and molds.

Autofluorescence detection

All living cells produce a small amount of fluorescence (autofluorescence) and this can be used to detect microbial colonies growing on a solid surface long before they are visible to the naked eye.

particularly useful for filterable samples.

where a membrane filter can be incubated on a conventional nutrient medium and scanned using highly sensitive imaging systems to detect microcolonies.

Automatic detection. uses a large area CCD imaging system without magnification to detect developing microcolonies.

detect both aerobic and anaerobic bacteria, as well as yeasts and molds.

 produce a result in as little as three days.

BacT/ALERT® 3D Dual-T Microbial Detection System from bioMerieux.

Cytometry systems Cytometry does not rely on microbial growth to detect contamination. but instead uses cell labelling techniques to detect viable microorganisms. detect a wide range of organisms, including yeasts and molds, within minutes. the cells are labelled using a fluorescent dye or a non-fluorescent substrate,

which is converted to a fluorochrome in viable cells. Detection of the labelled cells occurs by laser scanning in either a flow cell

(flow cytometry), or on a solid phase platform such as a membrane filter (solid phase cytometry).

AES Chemunex has developed solid phase cytometry detection systems. The company’s Scan®RDI (also known as ChemScan RDI) system is capable of detecting 1 CFU per sample and has been evaluated as a possible RMM for sterility testing. The technology has been developed for the Stereal®-T sterility testing system.

Rapid Sterility Methods

AES Chemunex Scan®RDI (ChemScan RDI) system

BD Diagnostic Systems BD FACSMicroCount™

bioMérieux (industrial applications) BacT/ALERT® 3D Dual-T Microbial Detection System

Celsis Rapid Detection Advance luminometer system, AkuScreen™ reagents

Rapid Sterility Methods

Merck KGaA Milliflex® Rapid Microbiology Detection and Enumeration System

Pall Life Sciences Pallchek™ Rapid Microbiology System

Rapid Micro Biosystems Growth Direct™ System