Why Sterility test require 14 days of long incubation time?

Sterility testing is the method to check viable contamination (bacteria, fungus, spores etc.) in the product. This testing is very important but it is time consuming. Once you tested the product, it require 14 days of long incubation time.As we know that bacteria require 3-5 days for the growth and fungus require 5-7 days for growth then why sterility incubation require 14 days? 

                                        Sterile products are manufactured in aseptic environment and that environment must be suitable for the sterile product manufacturing. We perform environmental monitoring to check viable contamination in the area and to control viable contamination we perform disinfection, sanitization and fogging in the area. Personnel are also involved in the aseptic manufacturing activities and in aseptic area personnel are the main source of product contamination. Sterility testing require 14 days of long incubation time because there are some bacteria which are very slow growing like Propionibacterium acne. P.acne is gram positive, rod shaped, slow growing bacteria which is found in the acne of humans. This bacteria is associated with the humans and very slow growing and it could be the source of product contamination. So, for the recovery of these type of slow growing microorganisms, 14 days are enough to support the growth of these slow growing microorganisms if present in the product. Another reason is that in aseptic environment microorganisms could be in damaged or in injured form so it require long time for the recovery of these microorganisms in media. That's why sterility testing require 14 days of long incubation time.

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Why we use 0.45 micron pore size filter in Sterility testing but 0.22 micron pore size filter during filtration?

There are different grade and pore size filters are available in the market. But for sterility testing only 0.45 micron pore size filter is recommended in different pharmacopoeias. First question comes in mind that many bacteria are present in the environment which are smaller than 0.45 micron pore size like Brevundimonas diminuta then why don't we use 0.22 micron pore size filter which can retain these small size bacteria instead of 0.45 micron? Can bacteria smaller than 0.45 micron size pass through the filter paper of 0.45 micron if yes then why we are using 0.45 pore size? Can't we use 0.22 micron pore size filter for sterility testing? There are different questions comes in mind and create confusion.

     

Sterility test 

Sterility test

When we perform sterility testing we use 0.45 micron filter but when we prepare and filter the disinfectant solution we use 0.22 micron filter. During batch preparation of SVP, LVP and liquid injections we also use 0.22 micron pore size filter for filtration of batch. The reason behind these questions is the purpose of using these filters. Being a microbiologist, I will explain you every aspects of this concept. First let we understand the morphology of the filter paper with particular pore size. Actually membrane filters are not having the single uniform sized holes passing from top to bottom , but they are ramification of channels through their whole thickness. So, because of morphology of filters its not possible for microorganism to pass thorough the filter. Therefore a filter of 0.45 micron size can retain large number of microbial cells smaller than the 0.45 micron.

In microbiology we perform sterility testing to check any viable contamination in product/sample by observing turbidity in the SCDM (Soyabean casein digest medium)  or FTM (Fluid thioglycollate medium) media. If viable contamination is present in the sample then it could be retained on the filter paper during sample filtration and during incubation we could detect that contamination. But what happen if we can't detect the contamination which is present in the product or during sterility testing if we unintentionally destroy the contaminating microorganisms which were already present in the sample. It will leads to false negative results which means contamination was present in the original sample but because of our testing problem or errors we couldn't detect the contamination in the product. That might cause release of sterility failure batch in market. Here in sterility testing our priority is to detect the contamination if present in the product. That is also mentioned in the pharmacopoeia that the test must be carried out under aseptic conditions designed to avoid accidental contamination of the product during testing. For achieving these conditions, a grade A laminar airflow cabinet or an isolator is recommended. The test environment has to be adapted to the way in which the tests are performed. Precautions taken for this purpose should not adversely affect any microorganisms, which are to be revealed in the tests. The test is designed to reveal the presence of microorganisms in the samples used in the test. During sterility we apply vacuum for filtration of sample, if we use 0.45 micron filter then microorganisms could be easily retained on the filter paper and the applied vacuum will not have impact on the viability of the microorganism involved in the test but if we use 0.22 micron filter paper stringent pore size and applied vacuum leads to damage of microbial cell which will not further recover during the incubation time. That's why we use 0.45 micron pore size filter for sterility testing.
                                                                                           

0.22 micron filter

0.45 micron filter

But during filtration of disinfectants or any liquid batch we use 0.22 micron pore size filter because in that our main purpose is to get sterile solution by removing the contamination from the solution whether live or dead it doesn't matter but the thing matters is that the solution must be sterile. Filtration is one of the method of sterilization. So, by filtering through 0.22 micron filter all form of contamination could be removed. That's why we use 0.22 micron pore size filter for filtration.

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Differences between endotoxin and exotoxin

There are so many differences between endotoxin and exotoxin. Endotoxin are released by gram negative bacterial cell from their cell wall and released only after the cell lysis. Living gram negative bacteria can't release endotoxin. That's why they are called endotoxin.

Exotoxin are mainly produced by the gram positive bacteria and released outside the cell by living bacterial cell. That's why they are called exotoxin. Differences between both are mentioned below.

Properties	Endotoxin	Exotoxin
Chemical Nature	Lipopolysaccharides	Proteins
Molecular weight	10 k Da	50-1000 k Da
Effect of heat	Heat stable not denatured	Denatured by heat
Relationship to cell	Part of outer membrane	Extracellular
Specificity	Low	High
Pyrogenicity	Yes	Occasionally
Source	Gram negative bacteria like E.coli, P.aeruginosa, Salmonella	Commonly gram positive bacteria like S.aures, B.subtilis
Can be toxoid	No	Yes
Antigenicity	Poor	Very good
Potency	Low	High
Mode of detection	Limulus amebocyte lysate	Neutralization, Precipitation etc.

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During viable particle monitoring which sampling method is more effective - Passive air sampling or Active air sampling?

During viable particle monitoring we perform passive air sampling and active air sampling. Which sampling method is more effective during monitoring?

Actually both these methods are equally important. We can't say that a particular method is more effective. During viable particle monitoring all these methods are part of good environmental monitoring programme . This environment monitoring programme would be effective if all these methods are used together which will give us reliable data with respect to the count observed in a particular area. Both these methods have their own importance and we can't relay on only one method. Let's have a look on both these methods.

Settle plate or passive air sampling: In this method, SCDA media plates are exposed on different predefined locations for particular time like 4 hours as per EU GMP. During this time particles are settled down on the media plate by gravitational force. After that these plates are collected and incubated. Then cfu's observed after completion of incubation time. The limitation of this method is that the viable particles which are settled only on the media plate during that 4 hours of exposure time will be counted but the particles which didn't settle on the plate couldn't be observed. But good thing about this method is that, area could be monitored for long hours like we can cover the area by exposing the plates at 4 hours intervals.

Passive Air Sampling

        

              Passive Air Sampling

Volumetric air Sampling or active air sampling: This is one of the method we use during the environment monitoring. In this method we use the instrument which is called air sampler. That air sampler sucks the air forcefully and the impact of the air would be on the agar media plate. That's why this method is called active air sampling because we use any mode (air sampler) to sample the air from the environment.In this method we generally sample 1000 liters of air per location as per mentioned in guidelines. In this method sampling could be done in very short span of time depending upon the flow rate of the air sampler. For example if flow rate of air sampler is 100 liters/minute then it will take about 10 minutes to sample the 1000 liters air and if flow rate is 75 liters/minute then it will take 13.33 minutes to sample 1000 liters air. So, sampling time is depend upon the flow rate of the air sampler. In active air sampling we check the viable particles in the air by sampling the air rather than the waiting for the particles to settle down on media plates by gravitational force. But we can't use this method for long hours like it will take few minutes to sample the air and it will tell us the quality of air for that particular time of sampling. But how environment is behaving for long hours could not be detected by this method.

Active air sampling by using microbial air sampler

So, if we use both these methods then we can monitor the environment effectively for long hours by passive air sampling as well as by forcefully sucking air from the environment with air sampler to get reliable data.



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Biological Indicators and their role in validation.

Biological Indicators: As per ISO, test system containing viable microorganisms providing a defined resistance to a specific sterilization process is called biological indicator (BI's). Biological indicators are bacterial spore suspension used to check efficacy of any sterilization process. There are different types of biological indicators with different strain of microorganisms for different sterilization process.

  

Self contained Biological Indicator

         Spore Strip

Spore Strip

A complete biological indicator pack includes:

(1) Spores 
(2) Carrier material 
(c) Primary pack 

Spores are used in biological indicators because spores are the most resistance form. By using spores suspension we challenge the particular sterilization process.Its very difficult to kill the spores as compare to vegetative cells. Spores contain very little amount of water and contains minerals like calcium, magnesium etc. That's why they are more resistant and stable. Carrier material is that material on or in which spores are present. There are different types of carrier material available like paper, liquid, stainless steel etc. Carrier material should be non reactive and should not have any impact on the spores during processing. Then primary packing material is there. The primary packing material holds the carrier so it is also very important. Primary packing material must allow the sterilizing agent to penetrate and come in contact with the spores. During validation of particular sterilization process two types of studies are performed. 

(A) Heat distribution study (B) Heat penetration study

In heat distribution study, we check the temperature uniformity within the sterilization chamber and define the hot and cold spot. In that study we only use chemical indicator. But in case of heat penetration study biological indicators are used. Heat penetration means biological indicators should be placed with in the load to check the efficacy of sterilization process. For example if we sterilize garments load then these indicators should be placed with in the garments load to check the efficacy and if liquid loads are used then biological indicators should be placed with in the liquid load. One more important thing is that for porous load like garments, glassware etc. we should use biological indicator with paper carrier and if we sterilize liquid load like media then self contained liquid biological indicators ampules should be used. So choose your biological indicators wisely depending upon the sterilizing load. There are different sterilization process  used in pharmaceutical industries and different strains of microorganisms used as BI's. Perform the verification of spore count before using the spores in sterilization process. Chart is given below for use of particular strain for particular sterilization process.

Name of Sterilization Process	Microorganism Used
Steam sterilization (121°C)	Geobacillus stearothermophilus (ATCC 7953)
Steam sterilization (<121°C)	Bacillus subtilis (ATCC 5230)
Dry heat sterilization	Bacillus atrophaeus (ATCC 9372)
Gas sterilization	Bacillus atrophaeus (ATCC 9372)
Ionizing radiations sterilization	Bacillus pumilus (ATCC 27142)
Vapour phase hydrogen peroxide (VPHP) sterilization	Geobacillus stearothermophilus (ATCC 7953)

After completion of heat penetration study, these BI's collected and incubated at particular temperature for particular time as per certificate. If no contamination found during or completion of incubation period then it means sterilization process is effective to kill the known concentration of spores. But if contamination found during or completion of incubation period then sterilization process must be reviewed to check any failure. Discard biological indicators properly after use.

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Reference microorganism used for Growth Promotion Test for Sterility testing media.

Sterility testing is one of the most important and critical testing in microbiology. It is a method by which we check the viable contamination in sterile items. In pharmaceuticals, sterility testing is performed to check viable contamination in sterile drugs like Liquid injections, Dry injections, SVP, LVP etc. In sterility testing basically two media are used.

(1) Soyabean casein digest medium (SCDM) used to detect aerobic bacteria and fungus.

(2) Fluid  thioglycollate medium (FTM) used to detect aerobic, facultative and anaerobic bacteria.

Sterility is one of the important parameter of release of any sterile product into the market. So, sterile product must pass the sterility testing before its release into the market. Growth promotion test is very important and in pharmacopoeia different microorganisms are mentioned for the growth promotion test of sterility testing media. List of microorganisms is mentioned below.

Microorganisms used for Growth Promotion Test of Sterility testing media

Medium	Test Microorganism	Incubation
		Temperature (°C)	Duration	Type of Microorganism
FTM	Clostridium sporogenes (ATCC' 19404)	30-35	3 days	Anaerobic
	Staphylococcus aureus (ATCC 6538)	30-35	3 days	Aerobic
	Pseudomonas aeruginosa (ATCC 9027)	30-35	3 days	Aerobic
SCDM	Aspergillus brasiliensis (ATCC 16404)	20-25	5 days	Aerobic (Mold)
	Candida albicans (ATCC 10231)	20-25	5 days	Aerobic (Yeast)
	Bacillius subtilis (ATCC 6633)	30-35	3 days	Aerobic

Inoculate a portion of Fluid Thioglycollate Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Clostridium sporogenes, Pseudomonas aeruginosa, and Staphylococcus aureus.

Inoculate portion of Soybean–Casein Digest Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Aspergillus brasiliensis, Bacillus subtilis, and Candida albicans. We can use ATCC or equivalent strains for growth promotion test.

Form the above statement it is clear that we have to use each of the above mentioned microorganisms for the GPT. Incubation time is not more than 3 days in case of bacteria and not more than 5 days for fungus which means if copious growth found in the media before 3 days or 5 days then we can remove that media form the incubator and note down the observation. Each autoclaved load of each lot of the medium should be tested for the GPT. The media are suitable if clear and copious growth of the microorganisms occurs.

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Why only 10-100 cfu's or not more than 100 cfu's are required for growth promotion test of media?

During growth promotion test, We check whether media is capable of promoting growth of inoculated microorganisms or not.So, in GPT culture suspension of 10-100 cfu's or not more than 100 cfu's required because first thing is that we can easily count the number of recovered microorganisms on the media plate.

Growth Promotion test using not more than 100 cfu's

Another important thing is that we are challenging the media with the microorganisms and the microorganisms which we use for GPT should not be more than 5 passage removed form the original culture. So, we are working with the healthy microorganisms and these microorganisms should grow on the media which we are testing. This is like a challenge to use low number of microorganisms to check the growth promotion properties of the media. For this reason we use 10-100 cfu's or not more than 100 cfu's for growth promotion test.

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What is the difference between Growth promotion test and Positive control?

In Microbiology, Growth promotion test and positive control is common terminology. Sometime these terms are used interchangeably but there is difference between these two terms.

                                                 E.coli on MacConkey agar

Growth promotion test is used to qualify the media and growth promotion test has to be performed before using that media in any testing. As per pharmacopoeia, growth promotion test has to be performed for every new lot/batch of the media and on the basis of GPT of one container , whole lot/batch of the media could be approved. But in pharmaceuticals different companies have different practices like some perform GPT for every new container or every autoclaved load of the media and this is a good practice though. GPT is just to check whether media is supporting the growth of microorganism or not by inoculating not more than 100 cfu's of particular microorganism. Another term is positive control, which is used to check any positive outcome. For example if we are testing any product for E.coli. Then during observation of characteristic growth on MacConkey agar plate, another plate should be there which is having growth of E.coli streaked on the media for comparison purpose. As we know that E.coli gives brick red coloured colony on MacConkey agar media so by observing both product plate and positive plate (which is having characteristic growth of E.coli) we can differentiate whether the product is having E.coli or not by matching both plates. If characteristic growth matches in both plates then it means product is having E.coli contamination and if characteristic growth does not match then E.coli is absent form the tested product.

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Growth promotion, Inhibition, Sterility and Indicative test and its acceptance criteria.

Growth Promotion test: Growth promotion test is used for the qualification of the media. We use different media for microbiology testing. But before using that media in any testing media must qualify GPT. In growth promotion test, media are inoculated with different kinds of microorganisms as mentioned in pharmacopoeia. For example general purpose media like Soyabean casein digest agar (SCDA) and Nutrient agar (NA) are inoculated with different cultures. But for selective media particular microorganism is used for inoculation as mentioned in pharmacopoeia. Not more than 100 cfu culture is required for the growth promotion test. Spread plate method is used for solid agar media.

Duration of incubation is very important in GPT. Media should be incubated for the shortest period as the time mentioned in the pharmacopoeia. For example if 24 to 48 hours time is mentioned then for GPT, media should be incubated for 24 hours. As per pharmacopoeia, acceptance criteria for GPT is ±factor 2. Meaning of ±factor 2 is, standard inoculum size is multiplied by 2 and divided by 2. For example if we have standard  size of 50 cfu's then acceptance criteria would be 25 cfu's to 100 cfu's. But 70% recovery is also good option for in house acceptance criteria for GPT. But in case of liquid broth media copious or luxuriant growth should be there in form of turbidity and should be comparable to the previous tested and approved lot of the media. 

Inhibition test: In pharmacopoeia, in addition to growth promotion test, inhibition test is also mentioned. Inhibition test is used for selective media which can support the growth of particular microorganism and inhibit the growth of other type of microorganisms. In inhibition test, particular microorganism is inoculated in the media in a concentration of not less than 100 cfu's and that media shouldn't support the growth of that microorganism. For example in case of Cetramide agar media E.coli is used for inhibition and that media should not support the growth of E.coli and it should inhibit the growth of E.coli. This is called inhibition test. In inhibition test, media should be incubated for the longest period. For example if 24 to 72 hours are mentioned then media should be incubated for 72 hours and no growth should be found on the media at the end of incubation time. Spread plate method is used for solid agar media.

Sterility test: In media sterility test, prepared plate of media should be incubated with the other GPT and inhibition test plates. No microorganism added in that case. Sterility test plates should be incubated till the end of the test and no growth should be there on the media till the end of the test.

Indicative test: In indicative test, we check the characteristic growth and indication reaction on the media. For indicative test observation, we can consider GPT plates. Not more than 100 cfu's culture suspension is used for that. Spread plate method is used for solid agar media. Indicative test plates should be incubated for a period of time within the range specified in the test. For example if 24 to 48 hours incubation time is mentioned then we can release the plates in between 24 to 48 hours. Colonies should be comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch of medium.

As per pharmacopoeia, every new received batch of the media should be tested for GPT and in that, on the basis of one container GPT we can approve the whole batch of the media. For example if we receive 10 container of media of same batch then we can perform GPT for only one container and can approve whole 10 containers of the media but in pharmaceuticals depending upon the criticality of the testing, every container of media should be qualified for GPT and every autoclaved lot of the media should be tested for Growth promotion test also.

 Table I: Growth Promotion Test requirement for Test for Specified Microorganisms

Medium	Name  of Organism	Properties	Temperature	Incubation Period
Enterobacteria enrichment broth mossel	E.coli	Growth Promotion	30-35oC	24 hours
	P.aeruginosa	Growth Promotion	30-35oC	24 hours
	S.aureus	Inhibitory	30-35oC	48 hours
Violet red bile glucose agar	E.coli	Growth Promoting & Indicative (purplish red colonies)	30-35oC	Growth-18 hours Indicative-18-24 hours
	P.aeruginosa	Growth Promoting & Indicative (colourless  colonies)	30-35oC	Growth-18 hours Indicative-18-24 hours
MacConky Broth	E.coli	Growth Promoting	42-44oC	24 hours
	S.aureus	Inhibitory	42-44oC	48 hours
MacConky Agar	E.coli	Growth Promoting & Indicative (Pink   colonies)	30-35oC	Growth-18 hours Indicative-18-24 hours
Rappaport vassiliadis salmonella enrichment broth	S.enterica	Growth Promoting	30-35oC	18 hours
	S.aureus	Inhibitory	30-35oC	24 hours
Xylose lysine deoxycholate agar	S.enterica	Growth Promoting & Indicative (red  colonies) with or without black centers	30-35oC	Growth-18 hours Indicative-18-48 hours
Cetrimide agar	P.aeruginosa	Growth Promoting	30-35oC	18 hours
	E.coli	Inhibitory	30-35oC	72 hours
Mannitol salt agar	S.aureus	Growth Promoting	30-35oC	Growth-18 hours Indicative-18-72 hours
	E.coli	Inhibitory	30-35oC	72 hours
Reinforced medium for     clostridia	C.sporogenes	Growth Promoting	30-35oC	48 hours (Anaerobic conditions)
Columbia agar	C.sporogenes	Growth Promoting	30-35oC	48 hours (Anaerobic conditions)
Sabouraud dextrose agar	C.albicans	Growth Promoting & Indicative (white  colonies)	30-35oC	Growth-24 hours Indicative-48 hours
Sabouraud dextrose broth	C.albicans	Growth Promoting	30-35oC	3 days

Table II: Growth Promotion Test requirement for Sterility Test

Medium	Name of Organism	Temperature	Incubation Period
Fluid thioglycollate medium	C.sporogenes	30-35oC	≤3 days
	S.aureus	30-35oC	≤3 days
	P.aeruginosa	30-35oC	≤3 days
	B.subtilis	30-35oC	≤3 days
Alternative Fluid thioglycollate medium	C.sporogenes	30-35oC	≤3 days
Soyabean casein digest medium	A.brasiliensis	20-25 oC	≤5 days
	C.albicans	20-25 oC	≤5 days
	B.subtilis	30-35oC& 20-25oC	≤3 days

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