It normally should not have any adverse effects , but we have seen effect of gentamycin on the expression of some genes of our interest so we changed to regular Pen-Strep. You should definitely evaluate this before you begin your assay namely looking at if it affects on the genes of your interest. It is also a good idea to fumigate the cell culture room using formaldehyde ( To potassium permanganate add formaldehyde in a petri dish and keep room closed for 3-4 days- this is toxic to eyes , so beware!!), Also there is a product called VIRKON which you can use to decontaminate your lab. It is not advisable to use cultures in which you are seeing fungal growth with out amphotericin.

Dear Asha

The antibotics have always effect on cell culture conditions depending type of the cells and the dossage of antibiotics.

Antibiotics such as gentamicin and fungizone (amphotericine B) working differently.

Mammalian and fungal membranes both contain sterols, a primary membrane target for amphotericin B, this is one mechanism by which amphotericin B causes cellular toxicity.

Regarding Gentamicine although its action is inhibiting protein synthesis by binding to 30S ribosomal RNA it can induce ER stress and necrosis in some type of mamalian cells see review paper :

TOXICOLOGICAL SCIENCES 119(2), 245–256 (2011), doi:10.1093/toxsci/kfq267: An Integrative Overview on the Mechanisms Underlying the Renal Tubular Cytotoxicity of Gentamicin.

Regarding cells you are using I advice you to read following paper carefully which may answer your question.

Gut 2010;59:496-507 doi:10.1136/gut.2008.169805, Restoration of APC gene function in colorectal cancer cells by aminoglycoside- and macrolide-induced read-through of premature termination codons

So always be carefull using any antibiotics.

Good Luck

Thank you very much for all of your kind reply. Though we fumigated still we observed fungal colony in PDA media. we are adding antibiotics as per guidelines,so hope it wont have any adverse effect on results. I will try to collect mentioned papers.

Have a great day.........

Might be depend on the cell response you used.

antibacterial and anti fungal agent disrupt the microbes cell wall through primarily detergent properties. But it has a role to promote immunity by adding immune protein

Dear Asha,

I don't use Gentamicin, but I found a decreased transfection efficiency using Pen/Strep.

When I add mycoplasma agents to my culture I don't do any experiments because cells growing a little bit slowlier, so it has an influence on cell growth and I'm sure, there are a lot of other side effects.

You need to fumigate/sanitize your incubators too. The level of contamination you are seeing is too high, to begin with. You also need to sanitize the cell culture hoods. And use closed mouth FLASKS with air-vents instead of culture dishes for some time until you have the problem resolved. Once you get Amphotericin resistance you will be in trouble. So my opinion is to spend more time on getting the issue sorted, where you don not have to use the antibiotic so continuously, simply because resistant organisms when they appear will ruin everything for later.

Amphotericin B alters cell surface properties and can affect their adhesive characteristics and thus motility etc.

It depends of your analyse system,but we use antibiotic in our cuture media ! finally if you need verified it we can use a control test !

Dear Asha

I have used gentamycin and fungizone in my culture system. I cultured Spermatogonial stem cells on sertoli cells in vitro. After using antibiotics my cells grew up more than before. I think these antibiotics don't have reverse effect on cell culture.

It completely depends on the cell type you investigate... Fibroblasts for example show unchanged phenotype in presence of AmphetericinB. In contrast, primary hippocampal neurons are highly sensitive and respond with disturbed maturation and changed electrophysiological properties as a consequence of AmphoB-treatment.

To sum up: It depends and should be tested with "untouched" cells as a control.

Yes, will try to check above mentioned aspects......thank you all for valuable suggestions

Any idea where the contamination is coming from? Is it in your equipment or your cell lines?

It is from incubator, we tried to sterilize using alcohol, but its of no use,we don't have procedure to fumigate incubator. Can we fumigate incubator?If we fumigate means how can we remove fumes from incubator after fumigation?

I. Try to clean the the incubator with a 3% solution of bleach powder in water. This should kill mild infections.

2. leave for 10-15 mins.

3. Rinse thoroughly with 70% isoproponal.

II. A procedure for fumigation using formaldehyde and potassium permanganate: FROM USDA.

1. PLEASE USE EXTREME CAUTION TO AVOID EXPOSURE OF ANY PERSONS TO THE FUMES AT ANY TIME.

2. AS LITTLE AS A COUPLE OF MINUTES EXPOSURE CAN OVERCOME A PERSON.

3. THIS IS FROM THE USDA.

4. PLEASE READ ALL STEPS BEFORE TRYING.

** For something like a hood, or incubator, it is relatively safe IF common sense precaution is used.

a) Concentration A

53 ml formalin (37.5%) and 35 g potassium permanganate per m³ of space.

This can be expressed as:

5.25 oz by volume (148.5 ml) formalin (37.5%) and 3.5 oz by weight (98 g) potassium

permanganate per 100 ft³ (2.8 m³) of space.

VERY IMPORTANT: Use a Stainless steel metal container or earthenware container. Plastic will melt. Glass will crack - due to the rapid heat generated.

** TELL ALL PEOPLE OF THE FUMIGATION SO NO ONE OPENS THE INCUBATOR.

1. I STRONGLY SUGGEST THIS STEP IF YOU CAN: Try a very small reaction under a good chemical fume hood. In a good metal container (see step 5), mix 1/100 volume of reactants.i.e. 1.5 ml of formalin and 1 g of permanganate. Observe what happens so you have an idea of what goes on. YOU MUST DO UNDER A CHEMICAL FUME HOOD.

2. Calculate the amount of formaldehyde and permanganate.

3. BLOCK ALL HOLES IN THE INCUBATORS. LOOK VERY CAREFULLY, AND PLUG OR TAPE WITH VERY STRONG TAPE ALL HOLES. USE DUCT TAPE IF YOU HAVE IT, it should not corrode with formaldehyde exposure.

4. TURN OFF ALL FANS IN INCUBATOR. The formaldehyde will kill spores in the filters also, so dont' worry about removing them. Since it is safe for HEPA filters it should be safe for incubators.

5. Use a METAL container - the water pan in the hood can be used - with 4 times the volume of the mixture. A WIDE MOUTH container is a MUST. This prevents pressure build up.

6. Put permanganate powder first.

7. When ready, add formaldehyde and IMMEDIATELY CLOSE incubator. Fumes will form immediately.

8. IMMEDIATELY LEAVE THE ROOM. YES, LEAVE THE ROOM. DO NOT ALLOW ANYONE IN THE ROOM. STAY AWAY FOR 24 HRS. Yes, 24 hrs. The formaldehyde will decompose to formic acid and CO2, but dont take chances.

9. Neutralise the residual formaldehyde using either ammonium bicarbonate or ammonium hydroxide.

** Place a heating stir plate in the incubator letting the wire come out.

** Place at least 60% of volume or weight of ammonium carbonate or ammonium hydroxide and turn on the heat plate so that ammonium hydroxide/carbinate can vaporize.

** Leave for 2 hours.

** Ammonia also is a strong irritant, so please seal the door where the wire of the stir plate is coming out.

I hope this helps !!!!!!!!!!!!

Thank you very much.........yes will try to follow all precautions .

Have a nice time......

While fumigating please take out the hepa filter as the fumes may damage the filter. Clean the hood and incubator with 70% ethanol and 30% methanol followed by autoclave. you can keep some agar plates to monitor the contamination.