Dear Blogger and Followers,

I had blogged "Critical issue: Ready to use 90mm Petriplates of Himedia not as per Standard."

I have withdrawn this blog because I had not done a complete and thorough investigation in this matter and quoted information .  All the guidelines have very clearly mentioned the diameter to be 90mm

I don’t have any substantial and validated data to make such clams that this affects the performance of microbiological analysis and this information was posted on my personal judgement.

I apologise for this this misinformation and have nothing to profit or benefit from this post. Also don’t carry any prejudice against any manufacturer or organisation.

I will be more meticulous in my future post and will not misquote or misinterpret guideline or standards. 

   

Vented Vs Non-Vented Plates

What is Vent in Petriplates?

Vented Petri dishes have a small lip on the top edge of the dish that allows the lid to sit a little up from the bottom, allowing for some air flow.  Non-vented Petri dishes allow the lid to sit more or less flat on the bottom.
                    Standard Petri Dishes are always vented, so if the don't say vented or non-vented, you should assume they are vented.  "Vented" means that the lid is slightly elevated above the base.  This allows for good, plentiful air exchange.  This is useful when you want to encourage evaporation, for example, when you want to use poured plates as soon as possible, and the plates themselves, or a liquid seeding solution, needs to dry beforehand.  The basic design of the dish tends to maintain sterility because particles would have to go up and over the dish's wall to get inside, and this is rare in normal airflow.

Types of Vent:

1.      Triple vented: aids gaseous exchange. Ideally suited for short term work

2.      Single vented: limits gaseous exchange, minimise evaporation and dehydration. Ideally suited for long term work

3.      Six Vented: Number of vent increases allows more gaseous exchange.

4.      Deep Vented: It allows more gaseous exchange, vent is slightly thick, it is useful plant and tissue cultures

5.      Non-vented: most suitable for anaerobic and long term work

Non Vented Plates:

.  With "non-vented" dishes, the lid fits quite flatly on the base.  While it is not a hermetic seal, the space between dish and lid is extremely small.  This results in even less potential for external contamination and a significantly reduced evaporation rate.  For example a 60mm vented Petri Dish containing 10ml of agar medium typically dries out in 2-3 weeks; whereas, a similar 60mm non-vented dish typically lasts 2-3 months. Except those in very humid climates, prefer the non-vented dishes.  Non-vented dishes provide sufficient air exchange for the worms to breath while greatly increasing the life of the dish.

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.

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. 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

How to do documentation in aseptic area?

Autoclavable Munising Paper

We all know ,that in documentation  is very important in  pharma industry  we use A4 size paper for documentation

"Paper should is main source of contamination in sterile area-

so how can we do documentation without contaminating our clean rooms "

Autoclavable munising paper is widely accepted for documentation in aseptic area

Stationery

The polymer impregnated substrate surrounds and bonds each individual cellulose fibre resulting in a cleanroom paper. The cellulose fibre core provides true paper characteristics giving problem free writing, printing and photocopying. Munising LP is auotoclavable, and benefits from extremely low static build-up and particulate generation.

Features:

• Munising LP is Latex Free
• The paper has a high degree of chemical resistance. The maximum loss of strength was 21% after soaking in the following 5% v/v H²SO4 2% w/w NaOH, Acetone, Ethanol and water. Tensile test on air-dried sheets.
• Munising LP can be sterilised by gamma irradiation, ETO or steam without seriously impairing the sheet. The particle count remains at the normal, low level.
• Microbe Testing - The yielded four colony forming units (CFU) according to micro pore filtration methodology and zero CFU according to Rodac plate methodology.
• The paper has a flammability rating of "moderately flammable" Classification 2, according to the National Fire Protection agency (NFPA-702)
• Using tests similar to those methods described in Federal Substances Act, CFR 16, Section 1500.41, it was determined that Munising LP would not be classified as primary skin irritant.
• Tests from NAMSA state that the paper is non-cytotoxic (MEM elution with l929 mice fibre blast cells).

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.

What is viable and non viable particle monitoring in pharmaceuticals?

In pharmaceutical companies monitoring of controlled area is performed by two methods. 1 Viable particle monitoring 2 Non- viable particle monitoring
In pharmaceutical companies drugs are manufactured in controlled or aseptic areas. So before going to manufacture any product we need to verify the manufacturing environment by monitoring viable and non viable particles. Viable particle monitoring: Viable particles includes bacteria, fungus and their spores and to check the number of these particles we have an environmental monitoring programme which includes Settle plate exposure (Passive air sampling), Active air sampling, Surface monitoring and Personnel monitoring methods. So monitoring of environment by these methods is called viable particle monitoring. By using these different methods we can determine the number of cfu's in particular class of area. Pharmacopoeias and regulatory agencies have defined certain limits of viable particles in these controlled area and number of colony forming units (cfu's) should be less than the final defined limits. If microbes are not with in the limits in the manufacturing area then they may cause product contamination. By using these methods and based on the observations we can assure that the number of cfu's are with in the limits and controlled area is safe for drug manufacturing. Non-viable particle monitoring: In non-viable particle monitoring we check the number of particles of 0.5 and 5.0 micron size. These particles also should be with in the limits. Particular counter is used for the non viable particle monitoring but there is a limitation of this instrument. This instrument can't differentiate between viable and non viable particle. Any particle either viable or non viable if detected by the sensor of this instrument, it will count this particle. If non viable particles increases in a particular area then there will be chance of increase in the viable particle count also because viable particles are not free floating they need a carrier to float from one area to another area. So these viable particles are carried by the non viable particles from one place to another place. So, to control a classified area we need to control both viable and non viable particles.

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