Silvia_84

I have a protocol but it says just to add 40 ul of 10% SDS . The SDS I have is in powder.. So, I can suppose that I have to weight 10 g and dissolve them in 100 ml, haven't I?

Hi all, do you know a protocol I can use to do PFGE on Klebsiella Pneumoniae? Thank you very much. Silvia

Hi all, can you tell me how I prepare a stock solution of 10% SDS? What does 10% mean in this case? 10mg in 100 ml?10 g in 100 ml ? Thank you very much. Silvia

Hi all, just a quick question because I am new with laboratory stuff and calculations. If I have to prepare a stock solution of 10mg/ml of lysozyme, don't I need to know the molecular weight of lysozyme, do I? I just weight 10 mg and I dissolve them in 1 ml of water..right? Can I also weight 5 mg and dissolve it in 500 ul..it is the same, isnt'it (so that I don't waste it in case something goes wrong)? Thank you very much. Silvia

Hi all, I am new with Microbiology laboratory stuff and I am doing some MICs with microbroth dilution method. I have a problem with that because I obtain MIC values for my control strains higher than expected (2-3 fold higher) and I can't understand what does not work in my experiment. I thought that it could be a diminished antibiotic potency but the drug is not out of date. I thought that it could depend on the solvent I use to make the antibiotic dilutions..I am doing them in Iso Sensitest broth. The procedure I used so far for my MIC is always the same and it worked with antibiotics like Ampicillin and Meropenem. Now I changed antibiotic and I am using Amikacin and Gentamicin and for both the drugs I always have MIC higher than expected. Could you suggest me what might be the problem? Thank you very much in advance. Best, Silvia

Hi all, please I really need your help. I am doing some MIC with Amikacin and the values I obtained so far for the reference controls are totally out of range and higher than expected. Can you tell me if the procedure I am following for the antibiotic diluition is correct? Just to be sure that it does not depend on me. I prepare an amikacin stock solution of 5 mg in 500 ul. I prepare my antibiotic dilutions that are in the range : 128-0.125 ug/ml. The final volume in each eppendorf (I have one for each antibiotic dilution) should be 1.4 ml and the dilution should be made two-fold . So what I do is to prepare the solution of antibiotic most concentrated (128 ug/ml) with this calculation: I apply the formula CiVi=CfVf Initial volume to be taken from the stock solution is = 128 x 1.4/ 5000/500 .This is 17.9 ul of stock So the Volume of diluent is 1400 ul- 17.9 ul= 1382ul From this dilution I take 700 ul and I pour it in the lower dilution (64 ug/ml) where already 700 ul of diluent are. And so on to halve the concentration everytime. Is it correct? Thank you very much in advance. Silvia

Hi all, I am aware of the difference between a laminar flow hood and a biosafety cabinet and I am using these days the first one to pour agar into my plates. Even so, the laminar hood is always busy this period and I was wondering if in your opinion I can use also the biosafety cabinet for this activity. Thank you very much, Silvia

Hey all, have you ever done MIC with meropenem? which solvent did you use to dissolve it in order to prepare the antibiotic stock solution? I have a bottle of meropenem provided by "MELFORD" supplier and on the bottle the meropenem is reported to be soluble in 5% of monobasic potassium phosphate. Anyway I checked in internet and I found that meropenem is soluble in water. So what would you suggest to use ? and which concentration should I have for my stock? Can you also give me some info about the storage modalities?and how long can I keep my stock? I am not sure about that. Thank you in advance. Silvia

well, I think that the third condition you mentioned is that affecting the measurements most because of some residuals possibly left in the cuvette by the other higher concentrated sample. then the second condition and lastly the first you mentioned..this latter should be that affecting the higher concentrated sample least.. so, the ranking in terms of potential measurement errors should be like this -measure high then low concentrated samples - measure samples with relatively close concentrations - measure low concentrated sample then one with high concentration Is it right?

So, you say that if I use a more concentrated sample I can even re-use the same cuvette...cant' I? Thanks, Silvia

Hi all, I have a very stupid question. I have an overnight culture and I want to measure its absorbance at spectrophotometer. Since I want to adjust the density of the culture to a specific value of absorbance, I need to diluite or concentrate my samples with water depending on which absorbance value I get. My question is: do I need to change the cuvette everytime even if it is the same sample diluited or concentrated? If no, how do I wash the cuvette? I use the plastic cuvettes. Thanks in advance. Silvia

Hi all, I am encountering difficulties trying to figure out some calculations I have to do in order to diluite (or concentrate) my 2 bacterial solutions for a MIC test. I should adjust the density of my overnight cultures with water to equal that of the 0.5 McFarland Standard, which is roughly equivalent to OD600 0.08-0.1 (107-108 cfu/ml). I diluted my original overnight cultures like this: 1900 ul of water and 100 ul of overnight culture (total: 2 ml) and I take 1 ml of this solution for the measurements at the spectrophotometer. One culture has an OD of 1.10, so I have to diluite it and the other 0.06 so I should concentrate it. My question is : how do I diluite the first ? and viceversa..for the second..how do I concentrate it? which calculation should I do? Any suggestion would be really appreciated. Thank you in advance. Silvia

Hi all, I need to make MIC with imipenem. We have the 500 mg/500 mg Imipenem/Cilastatin Powder for Solution for Infusion. My question is: how do I calculate the quantity of powder for the antibiotic stock solution?and the volume of this? I need to test 5 isolates of Klebsiella pneumoniae with broth microdiluiton. Since I need to use the Iso-Sensitest Broth to diluite my antibiotic , do I need to grow also my cells in this same broth overnight before doing the MIC? If I choose to use the agar dilution plate method, can I test different isolates of the same bacterium in the same diluited plate in your opinion? Sorry for my stupid questions but I am completely new in that. Thank you in advance. Silvia

Thank you very much for your reply. Well, as far as I know, they come from pharmaceutical companies. For example we have Meropenem bought from ZenecaPharma. My issue is that I don't know how to proceed for the first steps of MIC. for example I need to do MIC with Meropenem whose suggested range for MIC determinations (mg/L) is 0.015-4 and I don't have an idea of how to do the antibiotic stock solution and which volumes I should use both for the stock and for the working solutions.. I thank you again, Silvia

Hi all, I am new with experimental stuff in Microbiology. I need to do some MICs (first time I do it) and I am trying to read up a little bit. I saw that the first step to do is making antibiotic stock solutions. The protocols that I saw suggests to get the purity of the antibiotic and calculate the amount of it to be weighted with the following formula: volume x concentration/potency. My question is: how can I get the potency of antibiotic? and which volume is better to use? Any suggestion would be really appreciated. Thank you in advance, Silvia

Hi all, I am new in the field of epidemiology and microbiology. Could someone explain to me the difference between an endemic and epidemic plasmids and which are the medical implications do they involve? Thank you very much. Silvia