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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. 

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