What are alert , action and acceptance limit?

Alert limit, action limit and acceptance limits play very important role in different testing. Alert and action limits are defined to have better control on the system or process. 

Alert Limit: Alert limits are defined to check any change in the results as per trend data. Basically alert limits provide alert when results are crossing specific threshold. If results are higher than alert limit, it mandatorily not required any action but taking controlled measures at alert limit can control any future failure.  

Action Limit: Action limits are defined to prevent the any system failure. Action limits are the limits which when crossed required action. If specific action not taken then limits can pass the acceptance limits which leads to failure of the process.

Acceptance limits: Acceptance limits are defined as final limits. These limits when crossed results in system failure and operations needs to be stopped.

Alert, action and acceptance limits are defined to control any process. For example in case of purified water. The microbial acceptance limit of purified water is 100 cfu per mL. If we get results higher than 100 cfu per mL then water system needs to be shut down because purified water is used in the cleaning of equipment, manufacturing of pharmaceutical products etc.. Contaminated water can contaminate the product. So, alert and action limits shall be defined to have better control on the system and process. Alert and action limits shall be defined based on the trend data.

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Explaining the conditions when Sterility test may be considered invalid?

 Hi Friends,

Today, I am going to explain you very important point of sterility testing. I ask this question in interview but a very few candidates can give clear answer. So, I think I have to explain this here on the blog so that it can be helpful for everyone. 

We know that when we have growth in any of SCDM and FTM media in sterility analysis, we consider that product is fail. But there are certain condition in which we can consider that the test is invalid.

Here important thing is understanding the meaning of invalid. In any case, if test is fail we can not repeat the tests but if sterility test is considered as invalid it means we can repeat the sterility test with same number of units as used in original test. On the basis of below mentioned conditions we can invalid the test.

1. The data of microbiological monitoring of the sterility testing facility show a fault.

Explanation: If environment monitoring results of sterility testing area on the day of sterility testing show a fault means results found fail then sterility testing can be considered as invalid.

2. A review of testing procedure used during the test in question reveals a fault.

Explanation: It means after getting sterility failure results we have to initiate the investigation. During investigation we need to review the procedure. During review of sterility testing procedure if we found any fault in sterility test then sterility test can be considered as invalid.

3. Microbial growth is found in the negative control.

Explanation: During sterility testing, we use media negative control (NC) and negative diluting fluid control (NDFC). Media negative control is used to check any contamination in the media after sterilization and media negative control incubated for 14 days along with test sample. Negative diluting fluid control is used to verify the testing conditions in which we only filter the diluent used during the sterility testing. NDFC also incubated for 14 days along with test sample. If growth is found in any of negative control or negative diluting fluid control, the test may be considered as invalid because there may be problem with the media used during sterility testing or problem with the testing conditions.

4. After determination of the identity of the microorganisms isolated from the test, the growth of the species may be ascribed unequivocally to faults with respect to the material or technique used in conducting sterility test procedure.

Explanation: After identification of the contaminating microorganism up to species level, if identified microorganisms species is same as identified from the material used in sterility testing or from the environment of sterility testing area or personnel monitoring then sterility test considered as invalid.

With proper investigation and based on scientific justification, we can repeat the sterility test by using same number of units as we used in the original test. If no evidence of microbial growth is found in repeat test then sample complies for the sterility test. If growth is found in the repeat test then sample does not comply for test of sterility.

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How sterility test method validation performed and what is the purpose of sterility test method validation?

In sterility testing, we test different types products like general products and antibiotics etc. Every product is having different type of nature with respect to its mode of action. In case of antimicrobial product they can kill the contamination if present in the product and we are not able to detect the contamination inside the product. We may get false negative results and fail product can be released into the market. It may become threat to patient safety.

To avoid this situation we first need to perform the sterility test method validation. In sterility test method validation, we perform the sterility of the product, positive product control (PPC) and positive control (PC). In sterility testing of product, product is reconstituted by rinsing fluid A,D or K as per product requirement and filter through 0.45 micron filter. Rinsing shall be given with the diluent. Basically 100 mL of pre-wetting and 3x100 mL final rinsing shall be given. After that cut the filter paper into two equal halves and one half shall be inoculated in SCDM media and another half shall be inoculated in FTM media. Sample shall be incubated for up to 14 days. In case of PPC, sample shall be tested as in case of product by giving 2x100 mL rinsing of fluid A,D or K depending upon the product and in last 1x100 ml of rinsing not more than 100 cfu per 0.1 mL microorganisms specified in the pharmacopoeia shall be inoculated. Membrane filter shall be cut into two equal halves and incubated in respective media as per the used microorganisms. PPC sample shall be incubated for not more than 5 days. In case of PC, product is not reconstituted and filtered. In PC, 2x100 mL rinsing of fluid A,D or K depending upon the product shall be given and in last 1x100 ml of rinsing not more than 100 cfu per 0.1 mL microorganisms specified in the pharmacopoeia shall be inoculated and filtered.  PC also incubated for not more than 5 days. No growth shall be observed in product up to 14 days of period. Comparable growth of PPC with PC shall be observed. If comparable growth observed it means either product does not contain any antimicrobial property or antimicrobial property of the product has been successfully removed. 

In sterility method validation we need to validate the number of rinsing to remove any antimicrobial property of the product so that it does not inhibit the growth of microorganisms which may be transferred into the product through the process. 

If antimicrobial property of the product can not be removed by rinsing only then neutralizing agents by proving its efficacy and toxicity or different rinsing fluid can be used.

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Which microorganism is used for container closure integrity testing?

Brevundimonas diminuta is used for container closure integrity testing because of its very small size. 

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Tryptone Soya Agar with neutralizers for Surface and finger dab monitoring

Finger dab monitoring and surface monitoring is performed to check bioload on finger dab and on clean room floor and other surfaces. Gloved hands are frequently sanitized with 70% Isopropyl alcohol and clean room surfaces are disinfected with routine disinfectants. Frequent and regular use of disinfectant can results in false negative results. These disinfectants can inhibit the growth of microorganisms on sampled media plates. To prevent these false negative results Tryptone soya agar with neutralizers can be used to neutralize the residues of disinfectants and actual results can be obtained.

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I am very thankful for all readers for your support and valuable comments. Keep reading and learning...

Hello Friends,

I hope you are enjoying my post and expanding your knowledge. I am very thankful for your support and valuable comments. I have seen that many people just copy and paste my posts on their blog. Please don't copy and paste. This information is available for everyone and anybody can learn the things by understanding the concept and enhance their knowledge. My way of sharing knowledge is very simple and that's why it is easy for everyone to understand the concept. Keep reading and learning.

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Why fungus is considered as a threat in clean rooms as compare to bacteria whereas both are part of human microbial flora?

Both bacteria and fungus are part of normal human microbial flora. Bacteria are prokaryotic microorganisms whereas as fungi are eukaryotic. Bacteria are single celled microorganisms and fungus are multicellular microorganisms. 

In guidelines, acceptance limits are given grade wise for Settle plate, Volumetric air sampling, Surface monitoring and Personnel monitoring. But one thing is common in all guidelines that limits are given in form of total aerobic microbial count (TAMC) or viable particle contamination and it is not differentiating between bacteria and fungus. Then why fungus is considered as more critical than bacteria?

The reason is that fungus can contaminate the area in very destructive way as compare to bacteria. Fungus grow rapidly where is gets moisture and high RH. Fungus can grow on the surfaces easily. Fungus can easily spread through their spores and can contaminate the other areas of the facility. Fungus spores can become dormant for very long time and whenever it get favorable conditions it can grow easily. Fungal spores are more resistant then the vegetative cells. Whereas bacterial contamination can be controlled easily as compare to fungus. For all these reasons fungus is considered as a threat in clean rooms as compare to bacteria. 

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Why UV lights are not used for sterilization?

In Microbiology, there is great importance of UV lights. UV lights are available in pass boxes, laminar air flow benches, biosafety cabinets etc. There is a misconception that UV lights are used for sterilization of material kept inside pass box, laminar air flow bench or biosafety cabinet. UV lights are only used for surface decontamination not for sterilization. 

                                                        UV Light

Here surface decontamination means, removal of bio-load/contamination from the surface only which is in direct contact with the UV light but sterilization means complete removal of all form of microorganisms from the surface or inside the material. UV light can only kill the microbes present on surface because penetration power of UV light is low so it is not able to penetrate inside the material in order to sterilize them. Sterilization can be done by moist heat, dry heat, chemical treatment, gas sterilization etc. In sterilization, sterilant penetrate inside the material or article and kill all forms of living microbes present on surface and inside the material. 

Conclusion: UV light is only used for surface decontamination not for sterilization

Why bacterial endotoxin test is also called LAL test?

Bacterial endotoxin test (BET) is very important test and one of the important release criteria of injectable finished product into the market. Earlier bacterial endotoxin toxin test was called as LAL test.

 

What is the meaning of LAL test?

Here, LAL means Limulus amoebocyte lysate. Amoebocyte cells are purified from the blood of the horse shoe crab. These amoebocyte cells have special property that when they comes in contact with the endotoxin released by gram negative bacteria, it forms a clot. This concept is used in bacterial endotoxin test. There are currently four species of horse shoe crab.

a) Limulus polyphemus is found in the eastern coast of North and Central America. The size of the limulus polyphemus is big as compare to other three species. The name of LAL test come from the use of amoebocyte cell of limulus polyphemus which is also called lysate and here comes the name "LAL".

                                               Limulus Polyphemus

b) Carcinoscorpius rotundicauda is found in Indo Pacific region. Amoebocyte cells are also used from this species and it is also called CAL test.

                                             Carcinoscorpius rotundicauda

c) Tachypleus tridentatus is also found in Indo Pacific region. Amoebocyte cells are also used from this species and it is also called TAL test.

                                                  Tachypleus tridentatus

d) Tachypleus gigas is also found in Indo Pacific region. 

                                                      Tachypleus gigas

These are four species of horse shoe crab. But most commonly species of horse shoe carb is Limulus polyphemus which is widely used in India as well. However TAL (Tachypleus amoebocyte lysate) is generally used in China.

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What is the difference between Oven and Dry heat Sterilizer (DHS) used in pharmaceutical industries?

In pharmaceutical companies, both Oven and DHS plays very important role. Specially in microbiology Oven is used as an alternative of dry heat sterilizer (DHS) as both can maintain very high temperature inside the chamber which is required for depyrogenation (removal of endotoxin at high temperature). 

Hot Air Oven

But what is the difference between Oven and Dry heat sterilizer (DHS)?

In Oven surrounding air is thrown into the chamber and hot air circulated inside the chamber. But in DHS filtered air is coming through the HEPA filter and hot air circulated inside the chamber. HEPA filter is not available in Oven.

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How to calculate the bacterial endotoxin limit of the product?

Bacterial endotoxin test (BET) is very popular test in pharmaceutical industry. Bacterial endotoxin limit is given for most of the pharmacopoeial products. But what to do in case bacterial endotoxin limit is not given for a particular product? 

Horse Shoe Crab

It's become difficult to calculate endotoxin limit of the products. Though formula is already given in the pharmacopoeia for calculation of bacterial endotoxin limit but still people are not able to understand the calculations.

Today, I am explaining how to calculate the bacterial endotoxin limit of product for which bacterial endotoxin limit is not given in pharmacopoeia in my own simple way. 

I bet you, from today onwards you will be able to calculate the bacterial endotoxin limit of any product.

In pharmacopoeia, formula is given for calculation of bacterial endotoxin limit of any product:

Endotoxin limit (EL) = K/M

Where K is threshold pyrogenic dose of endotoxin per kg of body weight

M is maximum dose administered in a single hour period.

For calculation of endotoxin limit, weight of healthy person considered is 70 kg and maximum allowable endotoxin per person (70 kg body weight) will be 5 USP EU/Kg of body weight for any route of administration other than intrathecal for which K is 0.2 USP EU/kg of body weight.

For a person maximum allowable endotoxin will be 70x5= 350 EU (endotoxin unit).

Here K will be 350 EU. 

For maximum dose please refer product leaflet or literature in which detail of maximum dose per day will be mentioned. Ensure maximum recommended dose of product per day shall be considered for calculation. For example for any drug, if the maximum dose is 500 mg per day.

Calculate the endotoxin limit (EL) = K/M = 350 EU/500 mg = 0.7 EU/mg. 

The calculated endotoxin limit will be 0.7 EU/mg.

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Can 70% v/v Isopropyl alcohol is effective against all forms of bacteria and fungus?

70% v/v Isopropyl alcohol is very effective sanitizer. However can 70% Isopropyl alcohol is effective against all forms of bacteria and fungus? Let's understand this concept.

Mode of action of 70% v/v Isopropyl alcohol 

70% v/v Isopropyl alcohol penetrate into the cells of microorganism and cause denaturation of protein present in the cell wall of microorganisms.

70% v/v Isopropyl alcohol is very effective against all forms of vegetative cells of bacteria and fungus. During disinfectant validation studies, it has been found  that 70% v/v Isopropyl alcohol is very effective and can kill microorganisms in few seconds. If 70% v/v Isopropyl alcohol is so much effective against the microorganisms then why there is need of filtration of 70% v/v Isopropyl alcohol through 0.2 micron filter?

The answer is the limitation of 70% v/v Isopropyl alcohol. Though, 70% v/v Isopropyl alcohol is very effective against the bacterial and fungal vegetative cells but it is not effective against bacterial and fungal spore cells. Spore cells are the dormant stage of the bacteria or fungus to pass the unfavorable conditions. Bacillus subtilis is the example of spore forming bacteria.  A thick protective layer is made by bacterial or fungal cells out side their cell wall and it become difficult to kill the bacterial or fungal spore cells. Even it become hard to kill bacterial and fungal spore by disinfectants also. High level disinfectants are also available in the market and they can kill the bacterial and fungal spore cells. That's why it is recommended to filter the 70% v/v Isopropyl alcohol through 0.2 micron filter to protect 70% v/v Isopropyl alcohol from bacterial and fungal spores contamination.

Conclusion:

70% v/v Isopropyl alcohol is effective against bacterial and fungal vegetative cells but not effective against bacterial and fungal spore cells. So. filter 70% v/v Isopropyl alcohol through 0.2 micron filter before use.

If you like this post, please comments and share to create awareness in microbiology community.

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If sample is fail in Bacterial endotoxin test, does it mean it will be fail in sterility testing or vice versa?

 As a microbiologist question arise in mind that if a sample is fail in bacterial endotoxin test, does it fail in sterility test also?

The answer is big NO!

Bacterial endotoxin test is the test to detect the amount of endotoxin present in the sample. Endotoxin is release by the gram negative bacterial cells. Endotoxin is basically lipopolysaccharide (LPS) in nature and only release after the death of the bacterial cell. It means a dead gram negative bacterial cell only can release the endotoxin. 

               Gel clot formation in bacterial endotoxin test showing presence of endotoxin

Whereas in sterility testing, only viable (living) contamination is detected by checking the contamination of product in media (SCDM and FTM). If growth is observed in the media, it means viable (living) contamination is present in the sample. In sterility testing only viable contamination can be detected. 

Conclusion:

Based on the above discussion, it can be concluded that if any sample is failing in bacterial endotoxin test, it is not necessary that it will be fail in sterility test also and vice versa because bacterial endotoxin test only detect endotoxin released by dead bacterial and sterility only detects viable contamination. 

Comments if you like this post.

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Understanding of Bacterial Endotoxin Test Calculation

Hello Friends,

This particular blog is about calculation in BET testing. In microbiology, BET is a type of test which requires lot of calculation. It become difficult to understand the calculation as every person has their own style of calculation. I have also done many efforts during the beginning of my carrier to understand the calculation in BET. I know what kind of difficulty a fresher/microbiologist face during the beginning of their carrier.

But such time has gone as I am here to help you with this blog. In this blog we will understand how to prepare the particular potency of endotoxin with labelled claim lysate sensitivity.

Let's start to understand the calculation of BET.

In BET you have requirement of three types of reagents.

1. Lysate

2. Controlled Standard Endotoxin

3. Lal Reagent Water

When you receive the material, you will get a CoA of Lysate and seperate CoA for LRW. In the CoA of lysate you will get the lot no. of lysate, its labelled claim sensitivity and direction of storage, direction of reconstitution. In the same CoA you will get the detail of endotoxin like its lot no., reconstitution volume of CSE, its potency etc. First thing, you should know the labelled claim lysate sensitivity and potency of controlled standard endotoxin. Both are mentioned on the same CoA.

For example if we have a vial of CSE and in CoA it is mentioned that one vial contains 100 EU (endotoxin unit). Reconstitution volume also mentioned in CoA and consider this 5.0 mL here. So, after reconstituting the vial of endotoxin you will get 20 EU/mL potency. 

Your labelled claim lysate sensitivity is 0.125 EU/mL. Labelled claim lysate sensitivity is also denoted by lembda (λ) and if it is 50 test vial then you have to reconstitute the vial with 5.2 mL LRW and if it is 10 test vial then you have to reconstitute the vial with 1.2 mL LRW. Please refer vendor CoA for that.

Preparation of 4 λ endotoxin concentration:

Here 4λ means 4 multiplied by λ (which is lysate sensitivity)

4 X 0.125 =0.5 EU/mL. So you required 0.5 EU/mL concentration of endotoxin which is equivalent to 4λ.

Your initial vial concentration is 20 EU/mL and you have to prepare 0.5 EU/mL. Here is a simple technique. What you have divided by what you required?

You have 20 EU/mL and you required 0.5 EU/mL. So, divide 20 EU/mL with 0.5 EU/mL and you will get the dilution factor.

20 EU/mL = 40
0.5 EU/mL

Here 40 is the dilution factor which means if you dilute 20 EU/mL endotoxin 40 times you will get the concentration of 0.5 EU/mL.

Now, how to dilute the 20 EU/mL concentration to 40 times?

You have to take one part of controlled standard endotoxoin and rest part lal reagent water.

In case of 40 take 1.0 mL Controlled standard endotoxin and 39 mL LRW. Further to reduce the consumption as controlled standard endotoxin is only 5.0 mL available we can do like this, take 0.1 mL controlled standard endotoxin and 3.9 mL LRW and you will get the concentration of 0.5 EU/mL which is also called 4λ.

Preparation of 20 λ endotoxin concentration:

Here 20λ means 20 multiplied by λ (which is lysate sensitivity)

20X 0.125 =2.5 EU/mL. So you required 2.5 EU/mL concentration of endotoxin which is equivalent to 20λ.

Your initial vial concentration is 20 EU/mL and you have to prepare 2.5 EU/mL. Here is a simple technique. What you have divided by what you required?

You have 20 EU/mL and you required 2.5 EU/mL. So, divide 20 EU/mL with 2.5 EU/mL and you will get the dilution factor.

20 EU/mL = 8
2.5 EU/mL

Here 8 is the dilution factor which means if you dilute 20 EU/mL endotoxin 8 times you will get the concentration of 2.5 EU/mL.

Now, how to dilute the 20 EU/mL concentration to 8 times.

You have to take one part of controlled standard endotoxoin and rest part lal reagent water.

In case of 8 take 1.0 mL Controlled standard endotoxin and 7.0 mL LRW. Further to reduce the consumption as controlled standard endotoxin is only 5.0 mL available we can do like this, take 0.1 mL controlled standard endotoxin and 0.7 mL LRW and you will get the concentration of 2.5 EU/mL which is also called 20λ.

Hope this calculation is clear to everyone!

Thanks!!!

Frequently asked questions for microbiology interview

Q 1 : What is the microbial limit of raw water, purified water and water for injection?
Ans: Raw water limit is 500 cfu/mL, Purified water limit is 100 cfu/mL and water for injection limit is 10 cfu/100 mL.

Q 2: What is the TOC limit of purified water and water for injection?
Ans: Not more than 500 ppb.

Q 3: What is the inoculum size used for growth promotion test?
Ans: Not more than 100 cfu

Q 4: What is the inoculum size used for growth inhibition test?
Ans: More than 100 cfu

Q 5: What is biological indicator?
Ans: A biological indicator is well characterized preparation of specific microorganism with a known resistance to a specific sterilization process.

Q 6: How many biological indicators are used for total viable spore count?
Ans: At least four

Q 7: Which microorganism is used for the validation of steam heat sterilizer?
Ans: Geobacillus stearothermophillus

Q 8: What is the acceptance criteria for disinfectant efficacy validation?
Ans: Not less than 3 log reduction in vegetative cells and not less than two log reduction in spore cells.

Q 9: What is endotoxin?
Ans: Endotoxin is a lipopolysaccharide or LPS. LPS consists of the lipid A portion containing fatty acids and disaccharide phosphates, core polysaccharides and the O-antigen. Endotoxin is a type of pyrogen and is a component of the exterior cell wall of Gram-negative bacteria, like E. coli.

Q10: Why we expose the settle plate for four hours?
Ans: Settle plate method is one on the technique of environment monitoring to check the contamination level of the air. Microorganisms are not freely movable as they are attached with particles. We monitor the particles of 0.5-5.0 micron. The settlement of particles depends upon the size of the particle. The heavier the particle the more easily it settles down and the lighter the particle the more difficult for it to settle down. For these range of particles, it generally takes 4 hours for their settlement. The another factor is the dryness of the plate. If we expose the plates for more time, chances of its dryness become higher. However, validation study is required to prove that plates are not dried during or after completion of incubation time. By considering all these factors plates are exposed for 4 hours.

Q 11: Why 0.5-5.0 micron particles measured by non viable particle counter?
Ans: The product contamination is mainly caused by this range of particles because this size of particles are not easily settle down and become threat to the product.






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Frequently asked questions for microbiology interview

Hello everyone,

My today's post is quite different, interesting and very helpful for all industrial microbiologists. Here, I am posting few frequently asked questions as knowledge booster for interview preparation of microbiology/quality assurance. The level of questions will be such that it will helpful for fresher as well as experienced microbiologist also.

Q 1: What is the acceptance criteria for growth promotion test?

Ans: ±factor of two (for example if we have inoculum size of 50 cfu then it could be 25-100 cfu)

Q 2: How many pathogens are tested as per USP?

Ans: Total seven pathogens (Escherichia coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridia, Candida albicans and test for bile tolerant gram negative bacteria).

Q 3: Why Salmonella (pathogen) testing requires 10.0 g sample? 

Ans: Due to non uniform distribution of salmonella in sample more sample is required for identification.

Q 4: Why MacConkey broth is incubated at 42-44°C and MacConkey agar further incubated at 30-35°C?

Ans: Click on the link

Q 5: How many passage of cultures are acceptable?

Ans: Not more than five passage from the original culture.

Q 6: What is the difference between disinfectants, antiseptic and sanitizers?

Ans: Clink on the link

Q 7: Why 70% IPA is more effective then 90%,95% or 100%?

Ans: 70% IPA is more effective than other percentage of IPA because IPA destroy the bacterial cell by denaturing the protein content present in their cell wall. There is balance in penetration of IPA into interior of the bacterial cell and denaturation of proteins. If percentage of IPA will be high then protein quickly denatures and it form a protective covering outside the cell wall and IPA not able to penetrate into interior of the cell wall to completely kill the bacterial cell. However, if concentration remains low then it is not able to denature the protein content present in the cell wall of bacterial. That's why 70% IPA is more effective than 90%, 95% or 100% concentration.

Q 8: Which chemical is used for fogging and what is its concentration?

Ans: Hydrogen peroxide (H2O2) with silver ions. The combination of these two ingredients gives a synergistic broad spectrum of activity on all kinds of viruses, bacteria, fungi, yeast, mold, protozoa and algae. Generally 20% v/v concentration is used for fogging.

Q 9: What is the role of gram's iodine in gram's staining?

Ans: Gram's iodine is used as a mordant to form crystal violet-iodine complex so that the dye can not be removed easily.

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What is the difference between Bioburden and Microbial limit testing (MLT)?

Dear Friends,

In microbiology, we generally use two terms, bioburden and Microbial limit test (MLT). Do we know the difference between Bioburden and Microbial limit testing? Here is a little explanation about Bioburden and MLT testing which you can understand very easily.

Bioburden: The term bioburden means total bioload (living microorganisms) on the surface or within the material. It is a combination of bacterial count and fungal count and generally TVAC (total viable aerobic count) term is used for that. Bioburden is performed for non sterile material, unfiltered or filtered solutions. Bioburden is a quantitative testing in which we only detect number of colony forming units (cfu).

Membrane filter showing colony forming units

Microbial Limit Testing (MLT): Microbial limit testing comprises of detection of total aerobic microbial count (TAMC) and total yeast and mold count (TYMC) seperately in the material. MLT is performed for non sterile materials. MLT also includes detection of specified pathogens which means MLT is quantitative as well as qualitative testing. Quantitative testing is related to only the counting of number of cfu's and qualitative testing is related to detection of specified microorganisms in which we only detect whether the specified microorganism is present or absent.

Mannitol salt agar showing growth of Staphylococcus aureus

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"I am a Microbiologist"

Dear Friends,

I am a Microbiologist

Being a microbiologist, We surround ourselves with lot of reading, doubts and confusion. What if, we  get our answers in one search. Being a microbiologist, I understand the concerns of microbiologist. It doesn't matter if person is fresher or experienced because what matters is sharing knowledge. To strengthen your knowledge I will guide you what I have learnt from my professional experience. So, everyone is invited here to learn and experience the power of knowledge. I bet you, you will definitely love and enjoy different topics of microbiology.

Thanks and Regards,

Sandeep Dhiman

Professional Microbiologist 

What is 50:50 and hot spike method in BET testing and their importance?

Bacterial Endotoxin Testing is very important test for parenteral preparation. In BET testing generally two methods are followed as mentioned below.

1. 50:50 method
2. Hot spike method


1) 50:50 Method: 

Let's discuss about 50:50 method. In 50:50 method, in negative product control (NPC) sample volume and LRW volume are taken as 50 µL. If we prepare solution of MVD/2 then final results will be given at MVD because after adding same volume of LRW e.g 50 µL and the concentration reduced to half. That's why in 50:50 method we generally perform the BET at MVD/4 or MVD/2 dilution to get the results at MVD/2 or MVD because sample can not be diluted beyond MVD.

2) Hot Spike Method:

In hot spike method,  100 µL sample is required for analysis in negative product control (NPC). Sample is not diluted in hot spike method as there is no addition of LRW in negative product control (NPC). It means if we take MVD/2 sample then testing will be performed at MVD/2. If we take MVD/4 sample then final results will be given at MVD/4. Hot spike method is generally used in the water sample analysis. 

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Wet fogging and Dry fogging in Pharmaceutical Industry

Clean room disinfection is a major concern in pharmaceutical industry. In clean room of pharmaceutical industries the contamination is generally caused by air, water and equipment etc. But major role is played by personnel. Personnel are the major source of bio-contamination in clean rooms so as to control the contamination effective disinfection method is required.  

          

During clean room disinfection, area is cleaned and wiped by disinfectant solutions and another way to reduce the contamination is fogging.

There are two types of fogging.

(1) Wet fogging                        (2) Dry fogging

Wet Fogging:

In wet fogging, basically hydrogen peroxide solution with silver ions is used. ULV (ultra low volume) fogger are used to spread the solution in form of fog and average particle size ranges from 20-30 micron in size. That disinfectant solution is bactericidal, fungicidal and sporicidal. The result of the fogging is leaving the room wet with high RH and chances of corrosion. Post sanitization and manual cleaning activity is required in wet fogging. These are the few disadvantages of this fogging method.

ULV Fogger

Dry Fogging:

As compare to wet fogging, dry fogging is more reliable and effective. There are number of benefits of dry fogging. In dry fogging basically peracetic acid and hydrogen peroxide solution is used. This combination is also bactericidal, fungicidal and sporicidal. The main advantage of this fogging is particle size. The average particle size ranges around 7.5 micron in size. During dry fogging the particles of light weight bounce off the solid surfaces and resist the excessive condensation. These small particles when evaporates, the vapour penetrate in inaccessible areas and cause effective disinfection by killing the microorganisms. Dry fogging can cause much higher log reduction of Geobacillus stearothermophilus spores as compare to wet fogging. There is low risk of corrosion and  lesser material compatibility issue with dry fogging. It also eliminates post sanitization and manual cleaning activity.

Dry Fogger

Conclusion:

On the basis of above discussion it can be concluded that wet fogging and dry fogging both area effective but dry fogging has more advantages than wet fogging.

Thanks!