Sterility testing of pharmaceutical products Microbiology

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Sterility testing of pharmaceutical products Microbiology

Sterility testing of pharmaceutical products Introduction

A sterility testing of pharmaceutical products is defined as a test that critically evaluates whether a sterilized pharmaceutical product is free from infecting microorganisms.

According to Indian Pharmacopoeia IP (1996) the sterility testings are proposed for detecting the presence of possible forms of microorganisms in or on the pharmaceutical preparations.

Sterility testing Principle

In definite practice, one always comes across certain absolutely vital guidelines and Important precautionary measures that must be followed to strictly to achieve the maximum accuracy and precision of the whole concept of sterility testing for life saving secondary pharmaceutical products (drugs).

A few such fundamental factors, guidelines, and necessary details are as numbered under:

Sterility testing of pharmaceutical products

What is sterility testing?

(a) Sterility testing, due to its characteristic nature, is closely associated with a statistical process wherein the percentage of a batch is sampled almost randomly; and, therefore, the chance of the specific batch (lot) accordingly passed for actual usage (consumption) exclusively depends upon the ‘sample’ having passed the severe sterility test.

What is Sterility Testing in Microbiology?

(b) Sterility tests should be performed under conditions designed to avoid accidental contamination of the product (under investigation) during the test. Nevertheless, such particular precautions precisely taken for this purpose must not, in any case, adversely affect any microbes that should be revealed in the test ultimately.

(c) Working environment wherein the sterility tests are meticulously carried out must be adequately monitored at regular intervals by sampling the air and the surface of the working area by performing necessary control tests.

Sterility testing of water for injection

(d) Sterility tests are completely based upon the principle that in case the bacteria are deliberately placed in a particular medium that provides for the necessary nutritive material and water, and maintained suitably at a favourable temperature (37 ± 2°C), the microbes have a tendency to grow, and their genuine presence may be clearly indicated by the appearance of a turbidity in the formerly clear medium.

(e) Extent of probability in the detection of viable microorganisms for the tests for sterility usually increases with the actual number supposedly present in a given quantity of the preparation under examination, and is found to vary according to the species of microorganisms present. However, extremely low levels of contamination cannot be detected conveniently on the basis of random sampling of a batch.

(f) In case, observed contamination is not quite uniform throughout the batch, random sampling cannot detect contamination with absolute certainty. Therefore, agreement with the tests for sterility independently cannot certify absolute guarantee of freedom from microbial contamination. However, greater declaration of sterility should invariably originate from reliable rigorous manufacturing procedures both having same characteristics strict agreement with Good Manufacturing Practices (GMPs).

(g) Tests for sterility are adequately designed to reveal the presence of microorganisms in the ‘samples’ used in the tests. However, the clarification of results is exclusively based upon the assumption that the contents of each and every bottle in the batch, that had been tested actually, would have obeyed with the tests. As it is basically not possible to test every container, an enough number of containers must be inspected to give a suitable degree of confidence in the final results obtained of the tests.

(h) It has been duly observed that there exists no definite sampling plan for applying the tests to a specified proportion of discrete units selected carefully from a batch is capable of demonstrating that almost all of the untested units are in fact sterile absolutely.

Therefore, it is indeed quite pertinent that while determining the number of units to be tested, the manufacturer must have adequate regard to the environment parameters of manufacture, the volume of preparation per bottle together with other special concerns particular to the preparation under examination.

For this records the supervision on the exact number of items suggested to be tested with regard to the number of items in the batch on the hypothesis that the preparation has been suitably manufactured under definite strict parameters designed exactly to exclude any annoying contamination.

Number of Items in a Batch Vs Minimum Number of Items Recommended to be Tested

Sr.noProduct VariantsNumber of items in a BatchMinimum Number of Items
Recommended to be Tested
(a) Not more than 100 containers.
(b) More than 100, but not more than 500 containers.
(c) More than 500 containers.
Either 10% or 4 containers whichever
is greater.
10 containers
Either 2% or 20 containers whichever
is less.
2Ophthalmic and other Non-Injectable preparation(a) Not more than 200 containers.
(b) More than 200 containers.
Either 5% or 2 container whichever is greater.
10 containers
(a) Not more than 100 packages.
(b) More than 100, but not more than 500 packages.
(c) More than 500 packages.
Either 10% or 4 packages whichever is greater
10 packages.
Either 2% or 20 packages which ever is less.
4Bulk Solids(a) Less than 4 containers.
(b) 4 containers, but not more than 50 containers.
(c) More than 50 containers.
Each container.
Either 20% or 4 container whichever is greater.
Either 2% or 10 containers whichever is greater.

Sterility Testing In Microbiology

In a wider perception the wide spectrum of the pharmaceutical products, both pure and dosage forms, may be achieved by adopting any one of the following two well accepted, time tested,

Sterility testing Methods

(a) Membrane Filtration.

(b) Direct Inoculation.

These 2 methods specified above shall now be treated independently in the sections that follows:

sterility testing

1. Sterility testing Membrane Filtration

The membrane filtration method has gained and maintained its outstanding traditional recognition to not only avoid but also to overcome the activity of antibiotics for which there exist practically little deactivating agents. However, it may be accordingly extended to embrace rightfully a host of other relevant products as and when believed fit.

Importantly, the method definitely requires the following characteristic features, specifically;

  • an exceptional skill,
  • an in depth specific knowledge, and
  • Severe routine usage of positive and negative controls.

As a classic example of a suitable positive control with respect to the suitable usage of a known contaminated solution basically comprising a few microorganisms of overall different nature and types.

Features Of Membrane Filtration

(1) The solution of the product under examination is carefully filtered through a hydrophobic-edged membrane filter that would exactly retain any possible contaminating microorganisms.

(2) The resulting membrane is appropriately washed in situ to get free of any possible ‘traces of antibiotic’ that would have been sticking to the surface of the membrane closely.

(3) Finally, the separated microorganisms are methodically transferred to the suitable culture media under perfect aseptic environment.

Microorganisms for Positive Control Tests: There are, in fact, 4 typical microorganisms that are being used completely for the positive control tests beside with their respective type of definite enzymatic activity stated in parentheses:

  • (a) Bacillus cereus: [Broad spectrum];
  • (b) Klebsiella aerogenes : [Penicillinase + Cephalosporinase]
  • (c) Staphylococcus aureus : [Penicillinase]
  • (d) Enterobacter species: [Cephalosporinase].

Interestingly, the microorganisms invariably engaged for the positive control tests together with a particular product containing fundamentally an ‘antimicrobial agent’ must be, as far as possible, expressly sensitive to that agent, in order that the ultimate growth of the microbe solely indicates three vital and important information’s, viz.:

a. satisfactory inactivation,

b. satisfactory dilution, and

c. satisfactory removal of the agent.

Particular Instances of Pharmaceutical Products: Virtually all the Official Compendia namely., Indian Pharmacopoeia (IP); British Pharmacopoeia (BP), United States Pharmacopoeia (USP) ;Sterility testing European pharmacopoeia (EUR.P), and International Pharmacopoeia (Int. P.).

Have suitably provided comprehensive and particular details with regard to the ‘tests for sterility’ of parenteral products (e.g., IV-intravenous and IM-intramuscular injectables), ophthalmic preparations.

(e.g., eye-drops, eye suspension, eye-ointments, eye-lotions etc.); also a surplus of non-injectable preparations, such as catgut, dusting powder, and surgical dressings.

Sterility Testing Procedures: In a broader perspective, the membrane filtration is to be preferred exclusively in such instances where the substance under investigation could any one of the following four

Classes of pharmaceutical preparations:

(i) an oil or oil-based product,

(ii) an ointment that may be put into solution,

(iii) a non-bacteriostatic solid that is insoluble in the culture medium quickly, and

(iv) a soluble powder or a liquid that basically possesses either integral bacteriostatic or inherent fungistatic characteristic features.

The membrane filtration must be used for such products where the volume in a container is either 100 ml or more.

One may, however, select the exact number of samples to be tested from Table and later on use them for the various culture medium suitably selected for microorganisms and the culture medium appropriately selected for fungi.

Precautionary Measures: In actual practice, however, the tests for sterility must always be carried out under extremely special experimental parameters to avoid any least possible accidental contamination of the product being examined, such as;

(a) Sterile airflow cabinet of a sophisticated laminar is provided with effective HEPA filters,

(b) essential precautionary measures is taken to avoid contamination that they do not affect any microbes which must be discovered suitably in the test.

(c) succeeding environment (i.e., working conditions) of the Lab or laboratory where the ‘tests for sterility’ is performed must always be monitored at a certain periodical interval by:

Sterility testing laboratories

  • Sampling the air of the working area,
  • Sampling the surface of the working area, and
  • Perforating the stipulated control tests.

Method: In common practice, it is perfectly urgent and necessary to first clean carefully the exterior surface of ampoules, and closures of vials and bottles with an appropriate antimicrobial agent.

Sterility testing laboratories

And later on, the actual access to the contents should be collected carefully in a perfect aseptic manner.

However, in a situation where the contents are duly packed in a particular container under vacuum, insertion of ‘sterile air’ must be done by the help of an appropriate sterile device, for instance a needle suitably attached to a syringe barrel with a non-absorbent cotton.

Apparatus: The most suited unit comprises a closed reservoir and a receptacle between

which a decently supported membrane of suitable porosity is placed strategically.

  • A membrane normally found to be quite appropriate for sterility testing basically bears a nominal pore size should be about 0.45 μm, and diameter of about 47 mm, the effectivity which in the retention of microbes has been established adequately.
  • The whole unit is most rather assembled and sterilized with the membrane in place preceding to use.
  • In case, the sample happens to be an oil, sterilize the membrane separately and, after thorough drying, set up the unit, adopting suitable aseptic precautionary measures.

Diluting of Fluids: In the ‘test for sterility’ one invariably comes across with two different types of fluids which will be treated individually in the sections that follows:

(a) Fluid A-Digest 1gram of peptic digest of animal tissue or its equivalent in water to make up the volume up to 1Litre, filter or centrifuge to clarify, and then adjust to pH 7.1 ± 0.2, makeup into flasks in 100 ml quantities, and finally sterilize at 121 °C for 20 minutes (in an ‘Autoclave’).

Note: In a specific instance, where Fluid A is to be used in carrying out the tests for sterility on a specimen of the penicillin or cephalosporin class of antibiotics, aseptically incorporate an amount of sterile penicillinase to the Fluid A to be engaged to rinse the membrane(s) sufficient to inactivate any residual antibiotic activity on the membrane(s) after the solution of the specimen has been duly filtered.

(b) Fluid B: In a particular instance, when the test sample normally contains either oil or lecithin, use Fluid A to each litre of which has been added 1 ml of Polysorbate 80, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilize at 121° C for 20 minutes (in an Autoclave).

Note: A sterile fluid does not contain either antimicrobial or antifungal properties if it is to be considered appropriate for dissolving, diluting or rinsing a preparation being analyse for sterility.

Quantity of Sample Used for Tests for Sterility: In fact, the perfect and precise quantities of sampling to be used for determining the ‘Tests for Sterility’ are quite different for the injectables and ophthalmics + other non-injectables; and, therefore, they would be discussed individually as under:

(a) For Injectable Preparations: As a common regular practice and wherever possible always utilization of whole contents of the container; however, in any case not less than the quantities duly stated in table diluting wherever needed to 100 ml with a befitting sterile diluent e.g., Fluid A.

(b) For Ophthalmic and other Non-injectable Preparations: In this peculiar instance exactly take an amount lying very much within the range prescribed in Column (A) of table, if needed, making use of the contents of more than one container, and mix thoroughly.

For each specific medium use the amount suitably specified in column (B) of table, taken carefully from the mixed sample.

Quantities of Liquids/Solids per Container of Injectables Vs Minimum Quantity Recommended for Each Culture Medium

Sr.noTypes of
Quantity in each container
of Injectables
Minimum Quantity Recommended
For Culture Medium
1For Liquid (a) Less than 1 ml
(b) 1 ml or more but < 4 ml
(c) 4 ml or more but < 20 ml
(d) 20 ml or more but < 100 ml
(e) 100 ml or more
Total contents of a container
Half the contents of a container 2 ml.
10% of the contents of a container unless otherwise specified duly in the ‘monograph’.
Not less than half the contents of a container unless otherwise specified in the ‘monograph’.
2For Solid(a) Less than 50 mg
(b) 50 mg or more but < 200 mg
(c) 200 mg or more
Total contents of a container.
Half the contents of a container.
100 mg.

Type of Preparation Vs Quantity to be Mixed and Quantity to be Used for Each Culture Medium

Sr.noTypes of
Quantity to be
Quantity to be Used
For each Culture
Medium (B)
1Ophthalmic Solutions : Other non-
injectable liquid preparations.

1-100 ml5-10 ml
2Other Preparations : Prepara-
tions soluble in water or appropri-
ate solvents ; insoluble prepara-
tions to be suspended or emulsi-
fied duly (e.g., creams and
1-10 grams0.5-1 grams
3Absorbent cottonNo less than 1 gram

Method of Real Test: In reality, the method of actual test may be later re-divided into the following 4 categories, which are;

(i) Aqueous Solutions,

(ii) Liquids Immiscible with Aqueous Vehicles and Suspensions

(iii) Oils and Oily Solutions, and

(iv) Ointments and Creams.

These three types of pharmaceutical preparations shall be treated individually as under:

(I) Aqueous Solutions:

The following steps may be followed consecutive,

(1) Prepare all membrane by collecting and transferring aseptically a small amount which is sufficient to get the membrane moistened suitably, of fluid A on to the membrane and filtering it carefully.

(2) For every medium to be engaged, transfer aseptically into 2 separate membrane filter funnels or 2 separate sterile pooling vessels before transfer of not less than the quantity of the preparation being analyse which is duly prescribed.

(3) Simultaneously, transfer aseptically the added quantities of the preparation being analysed prescribed strictly in the two media onto one membrane exclusively.

(4) Suck in the liquid quickly by the membrane filter with the help of a negative pressure that is under vacuum.

(5) In case, the solution being analysed has important antibacterial characteristic features, wash the membrane by filtering through it for not less than three consecutive quantities, each of approximately 100 ml of the sterile fluid A.

(6) Exactly, the quantities of fluid actually engaged must be sufficient to permit the satisfactory growth of a small inoculum of microorganisms about (nearly 50) sensitive to the antimicrobial substance in the existence of the residual inhibitory material preserved duly on the membrane.

(7) Once the filtration is accomplished, aseptically remove the membranes from the holder, cut the membrane into half portion, if only one is used, then immerse the membrane or 1/2 of the membrane, in 100 ml of the Fluid Soyabean-Casein Digest Medium, and incubate at 20–25 °C for a time period of seven days.

(8) Similarly, carefully perforate the other membrane, or other half of the membrane, in 100 ml of Fluid Thioglycollate Medium, and incubate carefully at 30–35° C for a time period of seven days.

[II] Liquids Immiscible with Aqueous Vehicles and Suspensions:

For this process the test as condition under [I] Aqueous Solutions, but add an adequate amount of fluid A to the pooled sample to execute fast and rapid rate of filtration.

Special Features: These are as stated below:

(1) Sterile enzyme preparations, for instance:

(2) Penicillinase

(3) Cellulase

can be integrated to fluid A to help in the dissolution of insoluble substances.

(2) In a situation when the substance under test normally contains lecithin, always make use of fluid B for dilution.

Fluid Soyabean-Casein Digest Medium

1Pancreatic digest of casein 17.0
2Papaic digest of soyabean meal3.0
3Sodium Chloride5.0
4Diabasic potassium phosphate K2HPO4.2.5
5Dextrose monohydrate
[C6H12O6 . H2O]
6Distilled water1000 ml

Fluid Thioglycollate Medium

2Sodium chloride2.5
Dextrose [C6H12O6 . H2O]
4Granular agar [moisture < 15%
5Yeast-extract (water-soluble)5.0
6Pancreatic digest of casein 15.0
7Sodium thioglycollate0.5
8Thioglycollic acid0.3 ml
Resazurin [0.1% fresh solution]
1.0 ml
10Distilled water 1000 ml

[III] Oils and Oily Solutions:

The different steps that are basically involved in treating oils and oily solutions for carrying out the sterility tests are as given below;

(1) Filter oils or oily solutions having enough low viscosity as such i.e., without any dilution via a dry membrane.

(2) It is perfectly essential to dilute viscous oils as necessary with a proper sterile diluent e.g., isopropyl myristate which has been verified beyond any reasonable doubt not to exhibit any antimicrobial activities under the prevalent parameters of the test.

(3) Permit the oil to perforate the membrane, and carry out the filtration by the application of gradual suction with the help of a vacuum pump.

(4) Wash the membrane and then filter through it for at least 3 to 4 successive quantities, each of about 100 ml of sterile fluid B or any other suitable sterile diluent.

(5) Complete the test as mentioned under [I] Aqueous Solutions from step (7) onwards.

[IV] Ointments and Creams:

The various steps involved are as given under:

(1) Dilute ointments carefully either in a fatty base or in emulsions of the water in oil (i.e., w/o) type to obtain a fluid concentration of approximately, 1% w/v, by applying mild heat.

If essential, to not more than 40 °C with the aid of a befitting sterile diluent e.g., isopropyl myristate previously adequately sterilized by filtration via a 0.22 μm membrane filter which has been proved not to possess antimicrobial activities under the prevailing conditions of the test.

(2) Carry out the filtration as quickly as possible as per details given under oils and oily Solutions [Section III] from step (4) onwards.

(3) However, in certain extraordinary instances, it would be perfectly necessary to heat the substance at about 45 °C, and to make use of warm solutions for washing the membrane effectively.

[V] Soluble Soids:

For each respective culture medium, dissolve a specific quantity of the substance being analysed, as recommended in an appropriate sterile solvent e.g., fluid A, and perform the test described under Section (I) i.e.,

Aqueous Solutions, by employing a membrane suitable for the selected solvents.

[VI] Sterile Devices:

Pass cautiously and aseptically an adequate volume of fluid B via each of about 20 devices so that not less than 100 ml is recovered finally from each device.

Collect the fluids in sterile containers, and filter the whole volume collected by the membrane filter funnels as mentioned under Section (I), Aqueous Solutions.

2. Direct Inoculation [Direct Inoculation of Culture Media]

The three general methods which is used for performing the tests for sterility are as mentioned below;

(a) Nutrient Broth,

(b) Cooked Meat Medium and Thioglycollate Medium, and

(c) Sabouraud Medium.

These methods shall now be treated separately;

sterility testing methods

(a) Nutrient Broth

Significantly, it is entirely suitable for the aerobic microorganisms.

Oxidation-reduction potential (Eh) quantity of this medium happens to be quite high to change the growth of the anaerobes specifically.

Significantly, such culture media that especially allow the growth of festidious microorganisms, such as, soyabean casein digest broth, Hartley’s digest broth.

(b) Cooked Meat Medium and Thioglycollate Medium

These two contrasting types of media which is discussed briefly below;

(a) Cooked Meat Medium: It is generally suited for the cultivation and growth of clostridia.

(b) Thioglycollate Medium: It is peculiarly suited for the growth of anaerobic microbes.

It fundamentally comprises the following ingredients, Glucose and Sodium thioglycollate that invariably function as:

  • an inactivator of mercury compounds,
  • to augment and promote reducing parameters, and
  • an oxidation-reduction indicator.

Agar to cause reduction of the succeeding convection currents.

(c) Sabouraud Medium

It is a medium generally meant for fungal species. It basically bears two essential and important characteristic features, such as;

  • an acidic medium,
  • comprise a rapidly fermentable carbohydrate e.g., glucose or maltose.

The direct inoculation method shall now be distributing consecutive manner following three categories,

  • Quantities of sample to be employed,
  • Method of test, and
  • Observation and Interpretation of Results.

Quantities of Sample to be used: In real practice, the precise quantum of the substance or pharmaceutical preparation under inquiry, that is required to be used for inoculation in the respective culture media.

Usually varies with reason as per the amount present in each particular container, and is stated clearly together with the precise volume of the culture medium to be employed.

Method of Test: The method of test alters according to the substance to be analysed, for instance:

(a) Aqueous Solutions and Suspensions: The effective tests for microbial contamination are invariably performed on the same sample of the preparation under research by making use of the above stated media.

In certain particular instance when the amount present in a single container is quite deficient to carry out the qualify tests, the combined contents of either 2 or more containers may be employed to inoculate the preceding stated media.

Methodology: The various consecutive steps involved are as given under;

(1) Liquid from the test containers must be separate carefully with a sterile pipette or with a sterilized syringe or a needle.

(2) Transfer aseptically the needed to be prescribed volume of the substance from all container to a vessel of the culture medium.

(3) Mix the liquid with the medium cautiously taking care not to aerate excessively.

Why sterility test for 14 Days?

(4) Incubate the inoculated media for 14 days at 30–35°C for Fluid Thioglycollate Medium, and 20-25°C for Soyabean Casein Digest Medium.

Special Points: The favorable special points may be noted as given below;

(i) In case, the substance under research renders the culture medium opaque whereby the presence or absence of the actual microbial growth may not be discovered conveniently and readily by sheer visual investigation.

It is always recommended and suggested that a suitable transfer of a definite portion of the medium to other fresh container of the same medium between the third and seventh days after the said test actually commenced.

(ii) Later on, continue the incubation of the said transfer vessels for 7 additional days after the transfer, and for a total of 14 days.

(b) Oils and Oily Solutions: For carrying out the needed tests for the bacterial impurity of oils and oily solutions it is recommended to make use of culture media to which have been incorporated suitably:

The requisite test must be carried out as already mentioned under Section (a) above i.e., Aqueous Solutions and Suspensions.

Preventative Measures: The following two precautionary measures should be taken adequately;

(i) Cultures fundamentally comprising of oily preparations should be shaken mildly every day.

(ii) Significantly, when one employs the fluid thioglycollate medium for the supreme detection of the anaerobic microorganisms, shaking or mixing must be restricted to a bear minimum level to maintain complete anaerobic experimental parameters.

(c) Ointments: The following steps may be native in a consecutive manner:

(1) Cautiously prepare the test sample by diluting ten times in a sterile diluent, for instance:

Fluid B or any other appropriate aqueous vehicle which is capable of discharge the test material consistent throughout the fluid mixture.

(2) Mix 10 ml of the fluid mixture thus acquire with 80 ml of the medium, and later on continue as per the method given under Section (a) that is aqueous solution and also suspensions.

(d) Solids: The various steps involved are as stated under:

(1) Transfer cautiously the requisite amount of the preparation under investigation to the quantity of culture medium as specified and mix thoroughly.

(2) Incubate the inoculated media for 14 days, unless differently mentioned in the monograph at 30-35 °C in the peculiar instance of fluid thioglycollate medium, and at 20-25 °C in the specific case of soya bean casein digest medium.

(e) Sterile Devices: For section of such size and shape as allow the absolute immersion in 1 litre of the culture medium test the intact article, using the suitable media; and incubating as declared under Section (a) i.e., Aqueous Solutions and Suspensions.

(f) Transfusion or Infusion Assemblies: For introduction or infusion assemblies or where the size of an item almost renders immersion unfeasible, and exclusively the liquid pathway should be sterilized by all means.

Flush cautiously the lumen of each of twenty units with an adequate quantum of fluid thioglycollate medium and the cavity of each of 20 units with a sufficient quantum of soyabean casein digest medium to give an eventual recovery of not less than 15 ml of each medium.

Finally, incubate with 100 ml of each of the two media as official under Section (a) i.e., Aqueous Solutions and Suspensions.

Exception: Such medical devices wherein the cavity is so small such that fluid thioglycollate medium will not pass through easily, suitably substitute secondary thioglycollate medium

alternatively of the usual fluid thioglycollate medium and incubate that suitably inoculated medium anaerobically.

Note: In such determine where the presence of the sample under investigating, in the culture medium majorly interferes with the test by precision of result of the consecutive bacteriostatic or fungistatic action, wash the article completely with the naked minimal quantum of fluid A.

Eventually recover the rinsed fluid and carry out the test as declared under Membrane Filtration for sterile devices.

Observation and Interpretation of Results: In the case of direct inoculation the different observation and representation of results may be accomplished by taking into consideration the pursuing cardinal factors, such as,

(1) Both at intervals during the incubation period of time, and at its completion, the media may be analysed thoroughly for the critical macroscopic indication of the bacterial growth.

(2) In the event of a negative evidence, the sampling under investigation passes the tests for sterility.

(3) If positive evidence of microbial growth is found, modesty the containers exhibiting this, and unless it is richly proved and adequately demonstrated by any other means that their (microorganisms) presence is on account such causes unrelated to the sample being analysed, and, therefore, the tests for sterility are noticeable invalid.

In such cases, it may be suggested to carry out a retest employing an identical number of samples and volumes to be tested, and the media as in the primary test.

(4) Even then, if no evidence of microbial growth is duly determined, the sample under inquiry precisely passes the tests for sterility.

(5) In case, sensible evidence of bacterial growth is determined, one may go ahead with the isolation with help of sterility testing Isolator and consequent identification of the organisms.

(6) If they are found to be not readily differentiable from those (microbes) growing in the containers reserved in the very First Test, the sample under enquiry fails the tests for sterility.

(7) In case, the microorganisms are readily differentiable from the ones really growing in the containers reserved in the First Test, it is very much recommended carrying out a Second Retest by employing about twice the number of samples.

(8) Importantly, if no indication of bacterial growth is observed in the Second Retest, the sample under investigation legitimately passes the tests for sterility.

(9) Perversely, if evidence of growth of any microorganisms is duly discovered in the second retest, the sample under enquiry obviously fails the tests for sterility.


Sampling refers to the process of selecting a part or portion to represent the whole.

In regular practice, a sterility test attempts to infer and determine the state (sterile or non-sterile) of a peculiar batch; and, therefore, it designates predominantly a statistical operation.

Let us consider that p punctually refers to the proportion of infected containers in a batch, and q the proportion of related non-infected containers. Then, we may have:

we have, p + q = 1 or q = 1 – p.

Further, we may presume that a specific sample comprising two items is suitably withdrawn from a comparatively large batch containing 10% infected containers. Thus, the chance of a single item taken at random contracting infection is usually given by the pursuing expression: p=0.1q.

whereas, the probability of such an item being non-infected is invariably portrayed by the following expression:

q = 1 – p = 1 – 0.1 = 0.9

Probability Status-The chances of status of the said two items may be obtained virtually in three different forms, such as:

(a) When both items get contaminated: p2 = 0.01

(b) When both items being non-contaminated: q2 = (1-p)2

= (0.9)2

= 0.81, and

(c) When one item gets infected and the other one non-infected: 1 – (p2+ q2)

Or = 1 – (0.01 + 0.81) = 1 – (0.82)

r= 0.18

i.e., = 2pq

Assumption: In a peculiar sterility test having a sample size of ‘n’ containers, the succeeding probability p of suitably accomplishing ‘n’ consecutive ‘sterile s’ is represented by the following aspect:

qn = (1 – p)n

Consequently, the succeeding values for various levels of ‘p’ having basically a constant sample size are as furnish in the following.

Sampling in Sterility Testing

Percentage of Infected Items in Batch       
3Probability p of drawing 20 consecutive sterile itemsA1 0.98

Sterility testing Sop

That evidently elaborate that the sterility test fails to discover rather low levels of impurity contracted/present in the sample.

Likewise, in a situation whereby various sample sizes were actually used it may be definitely demonstrated that as the sample size enhances, the probability component of the batch

being passed as sterilised also gets decreased accordingly.

In actual practice, however, the additive tests, suggested by BP (1980), enhances substantially the very chances of passing a particular batch essentially comprising of a proportion or part of the contaminative items.

However, it may be safely deducted by making use of the pursuing mathematical formula:

(1 – p)n [2 – (1 – p)n]

That provides sufficient change in the First Re-Test of passing a batch containing of a proportion or part ‘p’ of the contaminative containers.


The different techniques described essentially make a sincere and sympathetic attempt.

To accomplish to a reasonably large extent, the rigorous control and continuous monitoring of a particular sterilization process.

Yet, it is relevant to state here that the sterility test on its own disappoint to provide any guarantee with respect to the particular sterility of a batch.

However, it categorically accounts for an extra check, likewise a continued compliance and offer enough cognizable confidence concern to the degree of an aseptic process or a sterilization technique being adopted.

Interestingly, an absolute non-execution of an official stated (as per the Official Compendia) sterility test of a particular batch, despite the ambiguous major criticism and objection of its gross inability and limitations to detect other than the gross contamination, could equivalent to both moral consequences and essential legal requirements.

US FDA promulgates and strongly advocator the adhesion of USP official requirements for the sterility test for parenteral as the most trustworthy, reliable, and trustworthy guide for testing the official sterile products.

On a wide perspective, it may be observed that the sterility test is not entirely intended as a thoroughly evaluative test for a product suitably subjected to a known sterilization method of unknown effectiveness.

However, it is entirely meant primarily as an intensive check test on the consecutive probability that:

  • A previously validated sterilization process has been repeated duly, and
  • To provide adequate assurance vis-à-vis its continued effectiveness legitimately.

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