What is Calibration, Verification and Validation in pharmaceuticals?

In pharmaceutical industries these three terms are commonly used. Sometimes there is a confusion between these terms. Here I would like to explain these terms in a simple way which will be easy to understand.

Calibration: It is simply a process of comparing the unknown with a reference standard and adjusting the instrument response as close to the values shown by reference standards and reporting the results. In calibration we can adjust instrument response as per standards values. For example during pH meter calibration, we set the pH values of standard buffers in pH meter as 4.0,7.0 and 9.2 in three point calibration which means after dipping electrode in a particular buffer we set pH value as per buffer used like 4.0, 7.0 or 9.2 irrespective of values shown by the standard buffers. Another example is if applied voltage = 1.50 v, Indicated = 1.45 v then error= -0.05 v then we can adjust the instrument response by removing that error to 0.00. It is called calibration.

Verification: Verification is the comparison of results against specifications. For example if as per specification limit is 4.0-4.5 and value shown by pH meter is 4.6 then it is fail result against specification. Another example is error= -0.05 v, specification = ±0.03 v and results is fail. This is called verification.

Validation: Validation is the procedure of establishing documented evidence that provide high degree of assurance that the specific process will consistently produce a product meeting its pre-determined specification and quality attributes. This is called validation.


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Suitable temperature for the growth of fungus is 20-25°C but why in MLT 30-35°C temperature is mentioned in pharmacopoeia for incubation of C.albicans?

Fungus is mainly divided in to two categories yeasts and molds and C.albicans is a type of fungus under the category of yeast. The difference between yeast and mold is that yeasts are unicellular (made of single type of cell) and molds are multicellular (made of different types of cells). Low temperature and humidity is the primary requirement for the growth of fungus that's why 20-25°C temperature is recommended for the proper growth of fungus by different pharmacopoeias. But in case of MLT (Microbial limit test), when we perform test for specified microorganisms 30-35°C temperature is mentioned in pharmacopoeia for incubation of C.albicans. But why 30-35°C temperature is mentioned in pharmacopoeia while 20-25°C temperature is suitable for the growth of fungus?

The reason is that at 30-35°C temperature rapid differentiation of Candida strain takes place. This high temperature suits for the growth of C.albican strain which grow properly at this temperature while other strains of Candida couldn't grow at this high temperature so by using high temperature C.albican strain is differentiated from the other closely ralated strains of candida species. That's why Sabouraud dextrose broth and Sabouraud dextrose agar media are incubated at 30-35°C for identification of C.albican strain as per pharmacopoeias.

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In MLT why MacConkey broth is inbubated at 42-44°C while MacConkey agar is incubated at 30-35°C for E.coli testing?

During microbial limit testing MacConkey broth and MacConkey agar are two media used for identification of E.Coli which is a pathogen. But MacConkey broth is incubated at 42-44°C and MacConkey agar is incubated at 30-35°C temperature for the identification of same pathogen. Why elevated temperature of 42-44°C is used initially and then 30-35°C is used for incubation? The reason is that there are different strain of E.coli present in the environment. E.coli is gram negative bacteria and is an indicator of fecal contamination. Such contamination could arise from poor hygiene of operators, contamination from animals like cats, birds or a low quality water supply. E.coli is capable of causing diarrhoea and sickness also. So initially, 42-44°C temperature is provided for the incubation which is suitable for growth of fecal E.coli strain which we need to identify but other strains of E.coli couldn't grow at this high temperature. Once the enrichment of fecal E.coli has been performed in MacConkey broth then sample is streaked on MacConkey agar media and then incubate at 30-35°C which is suitable temperature for the growth of fecal E.Coli. That's why initially elevated temperature is used for the selection of fecal E.coli.

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Pathogens mentioned in different pharmacopoeias with ATCC number and morphological characters.

In pharmaceuticals, pathogen testing is very important and critical testing in which different media are used to identify whether specified pathogen is present in the product or not. This pathogen identification is part of Microbial limit testing (MLT) in which first enrichment of sample is performed and then enriched sample is streaked on different media to identify the pathogens. Pathogens should be absent from the sample as per pharmacopoeia. If pathogen is present in the sample then product is not suitable for use. Different pharmacopoeias ask for testing of different pathogens. Below is the table showing list of pathogens as per IP,BP and USP.

ATCC number and morphological characteristics are also mentioned in this table.

S.No.	Name of Pathogens to be tested as per different Pharmacopoeia			ATCC No.	Morphological characters
	IP	BP	USP
1	Bile tolerant gram negative bacteria	Bile tolerant gram negative bacteria	Bile tolerant gram negative bacteria	NA	Gram negative rod shaped
2	Escherichia coli	Escherichia coli	Escherichia coli	8739	Gram negative rod shaped
3	Salmonella	Salmonella	Salmonella	14028	Gram negative rod shaped
4	Staphylococcus aureus	Staphylococcus aureus	Staphylococcus aureus	6538	Gram positive round shaped
5	Pseudomonas aeruginosa	Pseudomonas aeruginosa	Pseudomonas aeruginosa	9027	Gram negative rod shaped
6	Clostridia	Clostridia	Clostridia	19404	Gram positive drum stick shaped
7	Candida albicans	Candida albicans	Candida albicans	10231	Gram negative rod shaped
8	Shigella	Not mentioned	Not mentioned	12022	Unicellular yeast

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Why A.niger has been replaced by A.brasiliensis in different pharmacopoeias?

In different pharmacopoeias A.niger was mentioned as a challenge microorganisms for used in different testing like Sterility validation, MLT and Disinfectant validation etc. But recently in different pharmacopoeias A.niger has been replaced by A.brasiliensis. ATCC announced that the recent molecular and genotyping testing clearly indicates organism previously designated as A.niger ATCC no. 16404 belongs to the A.brasiliensis species and ATCC no. 16404 will remain same. Hence A.niger has been replaced with A.brasiliensis in pharmacopoeias as well. But it is not possible to differentiate A.niger from A.brasiliensis on the basis of their morphological characteristics. Both colonies look like same but with the help of advance testing at molecular and genotypic level it is possible to differentiate. 

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In microbiology why petriplates are incubated in inverted positon?

In microbiology, petriplates are used for different testings like MLT of products and water etc. After completion of the test petriplates are incubated in an inverted conditions in incubators. What is the reason behind incubation of petriplates in an inverted  position and what are the benefits of incubation of of these petriplates in inverted position? Here I would like to explain this.

Petriplates incubated in inverted position

First thing is that, media should be poured in to petriplates at specific temperature which means media should not be very hot during testing to cause damage of microorganisms and media should not be cool to become solidified before pouring. That's why media should be poured in to petriplates when temperature is at about 45° C. During incubation, media contains moisture and if petriplates are incubated in upright position then moisture will evaporate from the media and that moisture condense on the lid in form of droplets.

Moisture on lid of the Petriplate 

When condensed moisture fall on the media plates then it can cause spreading of microbial colonies and it would be difficult to count the number of colonies properly. So, to prevent this petriplates are incubated in an inverted position. There are also other reason as well like handling of petriplates would be more easy in case  of inverted position because in upright position chances of opening of petriplates would be more during handling. That's why petriplates are incubated in inverted positions.

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Why round shaped petriplates are most commonly used in microbiology?

Julius Richard petri was a german bacteriologist who is generally credited with inventing petridish while working as an assistant to pioneering bacteriologist Robert Koch.

Different shapes of petriplates are available in market but most commonly round plates are used in microbiology because liquids are less likely to conglomerate around smooth surfaces than corners. During sample testing, sample should be properly mixed and distributed in the media in petriplate for proper colony counting and if petriplates are round then you can easily swirl around liquids without having sample catch around the corners or agitating the liquid too much. Another reason is that round shape petriplates are easy to handle and clean. That's why round petriplates are most commonly used in microbiology.

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Why 70% Isopropyl alcohol is used as disinfectant in pharmaceutical industries?

Iso-propyl alcohol (IPA) is widely used as a disinfectant in pharmaceutical companies . IPA is very effective on gram positive as well as gram negative bacteria. Most of the hand disinfectants available in the market contains IPA as an active component just because of its effectiveness. IPA is very effective and kill microbial cell with in a few seconds. But one limitation of IPA is that it is not effective against bacterial and fungal spores. Disinfectant efficacy depends upon two factors. First is concentration of disinfectant solution and second is contact time. Disinfectant validation activity also depends upon these two factors. In pharmaceuticals that's why validation activity must be performed to validate its effective concentration and contact time. Different guidelines and pharmacopoeias recommend 70% IPA solution because at this particular concentration, IPA is very effective. But why 70% IPA is effective and recommended by pharmacopoeias and guidelines why not 100 %? The reason is behind the mode of action of 70% IPA. Bacterial cells have proteins in their cell wall and when this protein comes in contact with the 70% IPA during disinfectant application, coagulation of proteins takes places in which denaturation of proteins occurred and after that IPA penetrate in the interior of the cell which cause lysis or death of the cell. Protein coagulation also happens in case of 100% IPA but with very fast rate and because of this very fast protein coagulation process denatured protein forms protective layer out side of the cell. When this happens, 100% can not penetrate inside the cell and not able to kill the microbe. Microorganisms become dormant in that conditions. In case of 70% IPA protein coagulation takes place with a slow rate and due to this 70% IPA get enough time to penetrate in the cell and kill the microorganism. Another factor is contact time, 70% IPA takes longer time to evaporate from any surface hence get enough contact time and in this mean time it show its efficacy but in case of 100% IPA, evaporation will be very fast, contact time will be less and it will not be so effective against microbes. That's why 70% IPA solution is used as disinfectant in pharmaceuticals industries.

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Why saline solution is used for the preparation of serial dilution of microorganisms in microbiology?

Saline solution is used for the preparation of culture suspension or serial dilution in microbiology. Saline is 0.9 % sodium chloride solution. Culture suspensions are prepared in saline solution because 0.9 % sodium chloride solution is isotonic in nature. In isotonic solution the concentration of solutes remains the same both inside and outside of the microbial cell and cells remains at their usual osmotic pressure. But water is hypotonic in nature in which the concentration of solute is greater inside the cell than outside and due to this water try to enter inside the bacterial cell. The osmotic pressure develop on the bacterial cell which can damage the bacterial cells during serial dilution. That's why serial dilution is prepared in 0.9% saline solution.

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Non Viable Particle Monitoring in Aseptic Processing Area

In pharmaceuticals, non viable particle monitoring is as important as viable particle monitoring. In non viable particle monitoring particles of 0.5 µm and 5.0 µm are measured. Particle counter is used for particle monitoring in aseptic area. Basically 0.5-5.0 µm particle range are main source of product contamination because this range of particles carry microorganisms with them. These particles are not settled down easily so these are the main threat to product contamination. If particle contamination increased from the defined limits in the aseptic area then number of particles could also increases in the product as well and could contaminate the products. Different classes has different particle limits defined by EU GMP guideline. So control over these particles is very important to maintain the aseptic area. On the basis of both viable and non viable particles, area is classified. In aseptic area, different factors are responsible for increase of particle contamination. Manufacturing area, equipments, utilities and personnels are main source of particle contamination in aseptic area. For example particles are generated through the movement in aseptic area so movement of the personnels should be controlled and rhythmic in aseptic area.Good manufacturing practises should be followed to control the particle contamination in aseptic area. As per EU GMP 1m³ air should be sampled per location for particle monitoring. Particle contamination depends upon the activity in aseptic area. If there is no activity in aseptic area then particle contamination would be low and if activity is in progress in aseptic area then particle contamination would be increased. That's why guidelines has defined different limits for at rest and in operation conditions. In pharmaceuticals, online and continuous monitoring of particles are also performed in which particle counter gives results in form of print out during production hours. This is very important tool to have great control over the aseptic area during production hours. During monitoring any deviation from the specified limits could results in investigation. Below is the chart showing maximum number of permitted particles in different classes of area as per EU GMP guideline.

Maximum permitted number of particles per m3 equal to or greater than the tabulated size
Grade	At Rest		In operation
	0.5 µm	5.0 µm	0.5 µm	5.0 µm
A	3520	20	3520	20
B	3520	29	352000	2900
C	352000	2900	3520000	29000
D	3520000	29000	Not defined	Not defined

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Viable Particle Monitoring in Aseptic Processing Area

In pharmaceutical companies products are manufactured in classified area. Different guidelines use different terms for classification of area. Basically four classes are there in pharmaceuticals. These are class A,B,C and D as per EU GMP and Drug and cosmetic act (schedule M) and class 5,6,7,8 as per ISO guideline. For environment monitoring most of the companies follow EU GMP guidelines because this guideline has most stringent limits for viable count. In these classified area different product like injections, tablets, capsules, dry syrups, liquid syrups, ointments are manufactured. Product manufacturing and related activities performed in a particular class of area depending upon the criticality of the product. For example if sterile product are manufactured then filling operations must be performed under class A environment surrounded by class B and class B surrounded by C and then C by class D and if other less critical products are being manufactured like liquid syrups, tablets and capsules then class C and D area are suitable. Guidelines have defined different limits for viable particles for different classes. So, control over the environment is very important and critical. Any deviation from the specified limits could results in failure of the product hence rejection of batch which would be a big loss for the company. So, to prevent contamination in the product, control over the manufacturing environment is critical factor. We can't perform manufacturing activities if viable count in the area is not with in the specified limit. Different microbiological methods are used in pharmaceuticals to check viable contamination as per different guidelines and regulatory authorities as mentioned below.

1. Settle Plate exposure ( Passive air sampling)

2. Volumetric Air Sampling (Active air sampling)

3. Surface monitoring

4. Personnel monitoring

1. Settle Plate exposure ( Passive air sampling): Settle plate exposure method is also called passive air sampling because in this method no mechanical means are used for the sampling of air. In this method 90 mm soyabean casein digest agar (SCDA) media plates are used with addition of glycerol depending upon the validation study. Glycerol prevent dehydration of the media during exposure. In this method SCDA media plates are prepared and after pre-incubation of media plates are exposed in the classified area for 4 hours as per EU GMP. After completion of exposure time, plates are collected form the particular area and incubated for total 5 days at 20-25° C for 72 hours and 30-35° C for further 48 hours. After completion of 5 days incubated plates are monitored for viable count. In aseptic processing areas where sterile products are manufactured continuous monitoring should be preferred as per different guidelines in which if filling time is extended then fresh plates should be exposed to cover the whole filling time. The positive point of this method is that viable contamination could be checked for long hours. But drawback is also there with this method as it could only check viable contamination which collect or deposit over the media plate during exposure hours. So, locations for plate exposure should be properly selected and justified based on the risk assessment studies.

2. Volumetric Air Sampling (Active air sampling): This is also called active air sampling because in this monitoring method an instrument (air sampler) is used to sample the air. As per guidelines 1 m3 or 1000 litres air should be sampled per location for volumetric air sampling. Pre-incubated SCDA media plates are used for air sampling and after completion of sample plates are incubated for 5 days at 20-25° C for 72 hours and 30-35° C for further 48 hours. The positive point of this method is that it can give more accurate data of viable count as compare to settle plates exposure because air is forcefully sucked by the air sampler during sampling. But sample duration time of this method is very less and can not be used for continuous hours like settle plate method.

3. Surface monitoring: Surface monitoring is performed by two methods. 

(a) Surface monitoring by swab

(b) Surface monitoring by contact plates

In surface monitoring different surface of the manufacturing area and equipments are sampled by using swab sticks and 55 mm contact plates. If surface is irregular then swab sticks are preferred. Most commonly 25 cm² area is sampled by swab stick and if surface is flat then 55 mm contact plates are preferred for monitoring. To cover 25 cm² area we can use templates for accuracy and one more thing should be considered during surface monitoring by swab is that whole sampling area should be covered during sampling.

4. Personnel monitoring: Personnel monitoring is very critical in aseptic area. Personnels are the main source of contamination in aseptic area. Contact plates of SCDA media are used for the monitoring of personnels working in aseptic area. Depend upon the criticality and handling of products personnels are monitored for different locations like finger dabs, forehead, chest, armpits, elbows, abdomen, booties. etc. For personnel monitoring 55 mm contact plates are used but for finger dab monitoring 90 mm  SCDA plates can be used because 55 mm plates are small to cover five finger dabs properly on single plate. Personnel monitoring locations should be finalised wisely based on the product handling and criticality. All personnels should be monitored during the exit from the aseptic area.

Below is the table showing viable count limits by different methods in different grades of areas as per EU GMP guideline.

Recommended limits for microbial contamination (a)
Grade	Settle Plate cfu/4 hours (b)	Air Sampling (cfu/m3)	Surface monitoring (cfu/plate)	Personnel monitoring
A	<1	<1	<1	<1
B	5	10	5	5
C	50	100	25	-
D	100	200	50	-

Notes

(a) These are average values.

(b) Individual settle plates may be exposed for less than 4 hours.

We can see that different methods are used to check microbial contamination in manufacturing area. Each method has its own positive points and limitations. A sound environment monitoring plan to check viable contamination should include all these methods to have proper control over the manufacturing area.

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Qualitative, Quantitative and Semi quantitative testing in Microbiology.

In microbiology different tests are performed like Microbial limit tests (MLT), Sterility, Bacterial endotoxin testing (BET) and LBPC etc. In these tests some are qualitative, some are quantitative and some are semi quantitative tests. 

Qualitative tests: Qualitative tests are those tests in which results are only given in form or either pass or fail or present or absent. In these type of tests results are not given in form of counting or calculations. Sterility test is one of example of qualitative test because in sterility test, results would be either pass or fail. In sterility testing we could check either contamination is present or absent in the sample.

Quantitative tests: Quantitative tests are those tests in which results are given in form of counting or calculations. MLT is one of the example of quantitative test in which results are given in form of number of cfu's for total aerobic microbial count or total yeast and mold count. But when we perform pathogen testing in MLT, that pathogen identification part is qualitative testing because in pathogen testing we couldn't determine number of cfu's of pathogens but we give the results in form of pass or fail whether pathogens are present or absent. Colony counting is another example of quantitative testing in which results are given in form of number of cfu's.

Semi quantitative tests: BET is one of the example of semi-quantitative test. In BET if it is performed by gel clot method then it is semi quantitative because by this method we could either give results in form of less than or greater than the specified limits. For example if product A is having BET limit of not more than 0.20 EU/mg then results would be either less than 0.20 EU/mg or greater than 0.20 EU/mg. In this, less than 0.20 EU/mg would be pass result and more than 0.20 EU/mg would be fail result. That's why it is called semi quantitative test. But now a days different advance methods are being used for BET in which actual level of endotoxin in the product  could be determined very easily. If BET is performed by these advance methods then exact level of endotoxin could be determined in the products hence it would be included in quantitative tests.

In simple language if I ask you Do you have oranges? Your answer would be either yes or no this is called qualitative and if I ask how many oranges you have? then your answer would be either 1,2,3,4.... that could be any number, this is called quantitative. But if I ask you whether you have less than or more than 5 oranges then your answer would be either less then 5 oranges or more than 5 oranges, then this is called semi quantitative.

Hope you understand the difference.

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What are alert and action limits and why they are so important in pharmaceuticals?

In pharmaceuticals alert and action limits are very important. These limits are used to have effective control over the process. 

Alert Limits: Alert limits are in house limits which are defined based on the trend analysis. These limits are like a barrier before the final action limits, given by different regulatory agencies. We have to be alert if microbial count reach to this alert level. It is not necessary to take any action when count reach to alert limit level but we have to be alert as this is a trigger that something is going wrong within the environment and microbial contamination is increasing in the area.

Action Limits: Action limits are also in house limits which are defined based on the trend analysis. When microbial contamination reach to these action limits then action is mandatory to control the contamination in area otherwise if not controlled, we might get area failure. These alert and action limits must be less then the final limits defined by different guidelines and regulatory agencies. For example in case of  settle plate exposure, 5 cfu's is the final limit for class B area defined by EU GMP and Drug and Cosmetic act (Schedule M). If we work on this final limit of 5 cfu's then there are chances that we may get more than 5 cfu's in class B and this results in area failure because there is no limit barrier before this final limit. But if we define in house limits like 3 cfu's for alert limit and 4 cfu's for action limits (based on trend data) then we have two barriers before the final limit and there would not be any chance of area failure because if microbial count increases and reach at this in house alert and action limit then we have lot of time to control the area by taking effective action. But if we work with final limits mentioned in guidelines then area results would be either less then 5 cfu's or more than 5 cfu's which means results would be either pass or fail. So, its better to set alert and action limits based on the trend data.

Why spore cells are very resistant and ineffective against most of the disinfectants?

Microbial cells are basically of two types, vegetative cells and spore cells. Vegetative cells are actively growing cells of microorganisms under favourable conditions. But conditions are not always favourable for microorganisms. Some microorganisms under unfavourable conditions (non availability of food, temperature etc.) transformed in to spore cells. Not all microbes are spore forming but some bacteria like bacillus and clostridium are spore forming bacteria and in case of fungus A.brasiliensis is spore forming. Spore cells are the dormant form of microorganisms in which metabolic activities are reduced. Water content is very low in spore cells. Spore cells have very thick outer coat. This coat is mainly composed of calcium and pectin which provide resistant to these cells. But when these spore cells get favourable conditions again they converted in to the vegetative cells and grow actively. Most of general disinfectants like alcohols are ineffective against spore cells. Outer coat of these spore cells are very hard and impermeable to disinfectants that's why some disinfectants like alcohol can't penetrate inside these spore cells and not able to kill spores. Sporocidal disinfectants are used to kill spore cells. These sporocidal disinfectants penetrate inside the spore cells and easily kill these cell.

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What is the meaning of false positive and false negative results?

In pharmaceuticals false positive and false negative results are commonly used terms. What are false positive and false negative results? I would like explain this. 

False positive results: False positive results means actual result would be negative but because of the error (problem in testing method/sampling/handling) positive results found. This is called false positive result. For  example in case of Microbial limit testing (MLT), in actual no contamination was present in the sample but because of error in practise, the analyst contaminated the product during analysis and growth found in the product after incubation.

False negative results: False negative results means actual result would be positive but because of error negative results found. For example in case of Microbial limit testing (MLT) of product, contamination was present in the product but because of pouring very hot media in the product petriplates no growth found in product after incubation. 

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Why 0.5 and 5.0 micron particles are measured in pharmaceuticals during non viable particle monitoring ?

Particles measurement is very important in classified area. Different guidelines like ISO, EU GMP has defined certain limits for these particles for a particular class in classified area. Particle counters are used for monitoring of non viable particles. If number of these particles increases in particular area then they may cause product contamination, so control over particles in classified area is very important. But why all guidelines talk about 0.5 and 5.0 micron particles. Why these particles particles are so important to measure and what about the particles smaller than 0.5 micron and particles greater than 5.0 micron, aren't they important? 

In classified area non viable particles are generated in the environment through system, process and movement but how viable particles are generated in the classified area? Personnels are the main source of viable particle contamination.Viable particles are carried by the personnels working inside the classified area.  Millions of bacteria are shed by the body of personnels on continous basis. But how microbes move in classified area, are they freely movable or require any source for movement? Microbes can't move on its own, first they require a source for attachment and this source is particle. Non viable particles are main source for movement of viable particles because microbes first attach to the non viable particles and after attaching to these particles they are carried from one area to another area through air movement. During non viable particle monitoring 0.5 and 5.0 micron particles are measured because most commonly found bacteria range is in between 0.5 to 5.0 micron so this range of particles are the main source of product contamination and can be carried from one area to another area very easily with the air movement. Size less than 0.5 micron is not very critical because microbes are less likely to attach to this size of particles and greater than 5.0 micron particles become heavy as size of these particles increases and can be easily settled down as compared to 0.5 and 5.0 micron particles. That's why 0.5 and 5.0 micron partilces are measured in pharmaceuticals.

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Class 1 indicator tapes with or without lead

In one of my previous blog, I discussed about different classes of chemical indicators. Class 1 indicators are also called process indicators. Class 1 indicator tapes are available in the market with different brand names with or without lead.

Lead is very poisonous metal. It affect almost every organ and system of the body. Lead is neurotoxic and can cause brain disorders. In pharmaceutical companies, class 1 indicator tapes with or without lead are commonly used. Indicator tapes which contains lead basically converted in to light colour to dark colour after processing in autoclaves. Disposal of these tapes containing lead is very important. These tapes can't be disposed off anywhere because it can cause health risk to living beings. These lead containing tapes should be disposed off as hazardous chemical waste by taking precautions. But now a days,there is another option available in the market that is class 1 indicator tapes without lead. These tapes are very safe to use and doesn't come under the category of hazardous waste. One more thing is that you can check whether lead free is mentioned on the tape or not. If lead free is not mentioned then these tapes are likely to contain lead. You can also check material safety data sheet (MSDS) for verification.  

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