Science topic

Microbial Biotechnology - Science topic

The study and application of Microbes.
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I am a Microbial Biotechnology student and I am also interested in Bioinformatics, therefore, I want to know which programming language may help me in Microbial Biotechnology research, although, I know all programming languages have their own benefits, however, which language will be easier to learn for beginner student and may help in significant research work.
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I recommend you R & Python
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I am going to optimize the medium composition for enzyme production. there are many optimisation method such as RSM, Taguchi Orthogonal array etc... Which method is best for medium optimisation?
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Please, see also Dr. V. V. Biryukov 's work in the Attachement.
Greetings,
Sergey Klykov
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I am trying to get CRISPR/Cas9 working in my lab. I have a strain of Aspergillus that expresses the Cas9, and I need a way of introducing the gRNA.
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George Matthews thank you very much!
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Hi, all. I am now scaling up a GS-CHO cell culture process from lab scale to pilot scale for the first time. I don't know what will happened and what should I adjust. What problems have you encountered and how have you solved them when you scaled up a CHO cell culture process from 1L or 5L stired tank bioreactors to 200L,2000L or larger?such as metabolic flux shift? viable cell density decreased faster? peak viable cell density was higher but titer was low?
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kLa is a very important design parameter. I have studied the energetic optimization and adaptive control of the dissolved oxygen concentration in aerobic fermenters by numerical simulation. The parameters of the usual KLa correlation were estimated on-line and at real-time through the recursive least squares algorithm with forgetting factor, most effectively when a small sinusoidal disturbance was imposed to the manipulated variables (stirring rate and/or air flow). The power dissipated by agitation was accessed by a torque meter (pilot plant). This investigation was reported at (MSc Thesis):
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When determining titer of T7 phage by plaque assay, I get a gradient of plaques where one side of the plate is clearing and the other half of the plate is a lawn of bacteria.  Anyone experience this issue?
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AS YOU KNOW WELL THE T PHAJES ARE OF TWO TYPES
the ODD. T1, T3, T5 T7, and
THE EVEN ARE T2.T4, T6,
As I KNOW LOT OF WORK AND LITERATURE EXITS BECAUSE THE PLAQUE SIZE ID GOOD ENOUGH TO OBSERVE AND EASY TO WORK AND DETECT MUTATIONS
AND VARIOUS KINDS OF EXPERIMENTs have been done
With odd phages the plaque size is too small and little work has been done
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I would like to know any simple stain or test bu which i could stain or quantify only the EPS or biofilm present and not bacteria present in it.
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FilmTracer SYPRO Ruby biofilm matrix stain
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Could you please tell about the opportunities available at various Universities and Institutes which offers Post Doctoral Fellowship for Doctoral students of life science stream (Especially Ph. D's of Biotechnology, Microbiology and Biochemistry) from India.
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In Case of China, almost each University has its own provincial postdoctoral fellowship. So getting a proper supervisor is more than enough.
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I would like to quantify the OTR and the oxygen demand in different cultures and rpm of shaker by the method of gas in and gas out, but is not very clear which formulas used, and which measurement magnitude are replaces the variables in the formulas.
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I have studied energetic optimization and adaptive control of the dissolved oxygen concentration in aerobic fermenters having air flow and/or agitation rate as manipulated variable(s). The parameters from the usual KLa correlation were estimated through the recursive least squares algorithm with forgetting factor, thus allowing for on-line estimation of KLa in real-time. A sinusoidal disturbance was imposed to manipulated variables:
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What is the difference between bacteriocins and antibiotics? Can you give me some more examples or usages to distinguish between them?
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Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). ... Antibiotics or antibacterials are a type of antimicrobial used in the treatment and prevention of bacterial infection. They may either kill or inhibit the growth of bacteria. The differences between them are:
1- Bacteriocins are produced on the surface of ribosomes in microbial cells, while antibiotics are primarily secondary metabolites of the cell.
2- Bacteriocin producers are insusceptible to the bactericidal agents, unlike producers of antibiotics.
3- Bacteriocins can attach to the target cell wall anyplace on the surface, as no specific receptors on the target cell wall apparently exist.
4- The mechanism of bacteriocin on target cells is diverse and is associated with the method of pore formation in the outer cell membrane. Bacteriocins bind to cell walls of sensitive microbes, motive ionic imbalances, and produce pores. Inorganic ions leak the target cells through the created pores and thereby killing them. Antibiotics, on the other hand, can inhibit synthesis of the subcellular processes (cell wall synthesis, intracellular protein production, and DNA and RNA replication). Their bactericidal and bacteriostatic mechanisms are diverse and may comprise pore formation, degradation of cellular DNA, disruption via specific cleavage of 16S rRNA, and blockage of peptidoglycan synthesis.
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I am in need of a free on-line software for performing chemometric analysis and chromatographic alignment of retention time shifts. any suggestions!!
Also i have another inquiry about how the numerical data matrix of HPLC-MS/MS can be arranged? I mean how the data of time (min)/relative intensity (%) for MS, TIC monitored chromatograms can be composed in an excel spreadsheet file to be imported into the software?
Thanks in advance
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Hi, I am affraid it is way too late to answer this but for the record...
You should consider learning the fabulous statistical computing and graphical representation R. R is freely available for several operative systems and contains several libraries specifically designed to solve many data analysis questions. Here is a shortlist of some libraries you could use:
Chromatographic data treatment
Chemometrics
A good introduction to R may be found at https://stat545.com, https://www.statmethods.net/, and many resources more.
If you dive into a non-programing-like software you may need to spend some time learning how to use it anyway... If you spend that time learning R you can use it for many other purposes that will enrich the way you treat and visualize your research data so it is worth giving it a try.
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I try to optimize the conditions for both liquid cultures (in Bio reactors) and agar cultures.
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Hello,
If you want to grow them in rich media, YPD or synthetic wort media work fine. If you are after some physiological characterization in bioreactors where you would like to do carbon balances or other metabolic analysis, then you might go for a more defined media like YNB with maltose or glucose as carbon sources.
With regards,
Rosa
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Normally bacterial cellulose produced from fermentation was in the form of gelatinous layer. Almost 98% of their structure was water. If this gelatinous layer is dry in oven it will become a very thin layer film thus hard to be grind.
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I have tried two methods:
1. freeze dying BC followed by dipping in liquid nitrogen for a few seconds and grinding in a ball mill.
2. freeze dying followed BC by keeping in the refrigerator overnight and grinding in a blender. The powder is sieved and bigger particles are put back in the blender for another round. (modified method of Munair Badshah )
Both of the methods gave BC in powder or particle form.
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Is there any medium or visual method to determine the acid production of bacteria? if so, kindly send the literature and references.
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Agree with Rudy Situmeang
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it‘s because some of the enzyme(and which one, please?)show no activity in anaerobic condition, or it's because of the energy(NADH/NAD+) Problem? 
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There are (basically) two categories of anaerobic metabolism: (1) fermentation and (2) oxidative phosphorylation that does not use oxygen to make ATP. However, many people are incorrectly taught that fermentation is the only type of anaerobic metabolism. So, when folks say "anaerobic organisms do not have the TCA cycle" what they really mean is "fermentative organisms do not have the TCA cycle". In contrast, it is quite common for non-fermentative anaerobic organisms to use the TCA cycle, such as sulfate reducers, nitrate reducers, iron reducers, etc.
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Hi all,
I met a problem about recombinant protein production from yeast CEN.PK_alpha. The target enzyme should secrete into the medium.  First, the strains were cultured in 20 mL medium for around 30 h (OD ~ 0.5) and then they were transferred to 200 mL medium with an initial OD of 0.05. When OD reaches 1, the medium was obtained by centrifugation. After ammonium sulfate precipitation overnight, there is almost no pellet on the bottom.
Is there anything wrong here? Could anyone give me some suggestions?
Thank you very much. 
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Try this: may be you find some protocols and booklets.
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Hello, 
I am new to this field of microbiology. I need someone's help in helping me develop the crystal violet assay in my lab. I am trying to develop a biofilm layer in a drinking water system and I would like to measure the amount. I will use different types of bacteria like E.Coli and S. aureus.
What kind of equipment do I need? A plate reader spectrophotometer? Do I need to transfer the biofilm from the metal surface to a certain plate? Is the absorbance being measured in a liquid phase? How does the reader work?
Thanks 
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1- grow the bacteria 2- guarantee the pure culture 3- Next form the biofime, add 500 microLiters of bacteria into a 24 well microplate. 4- keep without agitation for 24 or 48 or 72 days until biofilm forms 5 - Then centrifuge with microplate rotor 6- discard the supernatant 7-Wash the plate carefully with buffer solution, and air dry 8-Biofilm cells adhered to the well were fixed by the addition of 1000 microliters methanol for 15 min (4 ° C) and then the methanol was removed and air dried at room temperature. 9-Subsequently, added 1000 microliters of 0.1% violet crystal for 15 min. After this time, the plates were washed and air dried again. 10- Then, 1000 microliters of ethanol (96%) were added to each well and allowed to act for 5 min. Finally, 200 microliters from each well will be transferred to a 96-well microplate. The fluorescence intensity of the violet crystal was measured by a microplate reader using a wavelength of 595nm. doubts, salomaobit@hotmail.com
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Dear scientist fellows, 
We would like to investigate the effect of various enzyme cocktails on the hydrolysis of a range of biomass and then quantify sugars by HPLC. However we are concerned with microbial contamination. Therefore we are looking for a robust method to prevent any microbial contamination while having minimum effect on the hydrolysis itself. For example, we would like to avoid to autoclave our biomass as it is expected to have strong effect on the polysaccharides. 
Our biomass is not sterile (harvested in nature), but has been dried (40°C) and grinded (coffee grinder) and then store at 5°C. 
We are thinking of using microbial inhibitor such as Lactrol, Pen Strep, or others. But we don't know which one to use. We are seeking on your advice on that matter ! 
Thanks,
Arthur 
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There are several contamination interferences and preventative means that NREL (https://www.nrel.gov/docs/fy15osti/63351.pdf ) have highlighted for enzymatic saccharification of lignocellulosic biomass, please read section 5. Interferences, point 5.9 and 5.10 in attached file. Hope this is of help. Cheers.
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I tried using Normal LB and YPD  still no success. I am conjugation using E.coli S17.1. 
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Yes, I know but things have changed a lot since :)
It's much easier now. I can send you the required tools if gene KO is what you are after...
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Does fumigating soil with aluminum phosphide sterilizes the soil effectively? is it as good as autoclaving?
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Following
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.
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Please have a look at this useful RG link.
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I need to produce Oxalate decarboxylase enzyme as a recombinant enzyme. I choosed my source organisim named bacillus subtilis 168 designed my primers (with doubt) and I want to express in pET-SUMO vector. Are there anyone who tried this vector to produce protein for enzymatic reaction ? OR Can you please suggest me a vector system to produce protein for enzymatic reaction ?
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It can be any vector of your choice, but I recommend to go for targeted expression in plant system (transient expression in N.benthamiana) could be helpful, If you are not able to achieve expression in E.coli
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I am seeing that microbial biotechnology field is having great work as compare to plant biotechnology.
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Hello, here the global challenges in plant biotechnology.
Increase crop productivity especially in adverse environments,
Management of herbicide tolerance,
Management of resistance to pests,
Management of resistance to diseases,
Improvement of genetic engineering technologies to enhance public perception etc.
What about microbial biotechnology the many topics related with plant biotechnology for example genetic engineering and transgenic crops, biopesticides, bioinsecticides, biofeltilisers, bioleaching, soil desalination etc.
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I am trying to identify some bacteria at species level and I read gyrB and rpoB are two good genes for that. Please, do you know if there are any universal primers?
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The following article may be helpful. i am attaching it for you.
All the best
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I want to know What´s the best method for microbial DNA extraction of banana roots because i need microbial root DNA of banana for metagenomic studies. 
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I have utilized CTAB with great success in extracting fungal DNA from leaf litter at a very low cost. The MOBIO powersoil kit has also worked very well with leaf litter.
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I am using a Nanodrop machine to measure cell density at 730 ( Cyanobacterial cell). But I have some doubt that sometimes this machine gives varied data in replication. Does anybody have experience to measure cell density in Nanodrop reliability?
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This document basically suggests that Nanodrop OD600 reads are not accurate for measuring bacterial cell density unless a conversion factor is determined by dividing the cuvette OD to Nanodrop OD, which can then be used in a decently wide range of ODs.
But that also means you need access to a cuvette spectrometer, at least once every time you need to calculate the conversion factor for a specific application/OD range.
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LacZ activity was assayed using a colorimetric assay based on the reaction of LacZ with ONPG (ortho-nitrophenyl--galactoside), with product concentration measured at 420 nm. To allow calculation of specific activities, the amount of protein in samples was determined by the method of Lowry (J. Biol. Chem. 1951, 193: 265-275).
Urgent help please, thanks.
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Can anyone answer this?
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Hi, I am characterizing the transcriptional regulator of Mycobacteirum tuberculosis .
Because we don't have facility for M.tuberculosis culture, I am trying to over express (using PVV16 which has Hsp60 promoter) it in M.smegmatis. but I never got colonies though the clone and vector back bone are fine. So I thought over expression might be toxic to M.smegmatis. so to check its toxicity I did frame shift mutations and found its over expression is not lethal.
However for the same protein Chip-seq data available in MTB Network Portal. By going through their publication in NAR, I realised they over expressed corresponding transcription factors in M.tuberculosis using Tet-inducible system and did Chip-seq analysis.
My question is, is over expression of my protein not toxic to M.tuberculosis but toxic to M.smegmatis or am I simply doing something wrong?
How can I get a over expression strain of M.smegmatis?
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Dear scientists,
I have a basic query, will bacteria express it genes only when the substrate is present, or by normal basic metabolic pathways and other genes controlled by an operon system?
Is this applicable to eukaryotes? If not, what is the condition for expression of a gene in eukaryotes?
In summary, I want to know, whether a gene identified in bacteria by PCR may or may not express phenotypically?
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I think everybody so far are giving their opinion but not straight to the point as per the question of Nandagopal. So I again reiterate that yes, some genes are controlled but not all.
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I was wondering whether you can express the following:
CMV Promoter - NeomycinResistance - viral2a - mitochondrial signal peptide - GFP - Terminator.
In theory the 2a will self-cleave and you get a NeoR protein and a mitochondrial tagged GFP.
However, the T2a ("viral 2a") leaves a proline attached to the N-Terminus.
Will this mess up the signal peptide, so the GFP will stay in the cytosol? Or will it still work and be imported into the mitochondria?
The same question is asked for chloroplast targeting peptides, ER localizaton signals, and extracellular secretion signals.
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I had the same question and found this Cold Spring Harbor protocol/review of 2A peptides, which comments that 2A generally does not affect the localization of downstream proteins except when the upstream protein is targeted to the ER lumen. 
Design and Construction of 2A Peptide-Linked Multicistronic Vectors
Andrea L. Szymczak-Workman, Kate M. Vignali, and Dario A.A. Vignali
Cold Spring Harbor Protoc; 2012; doi:10.1101/pdb.ip067876
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Hello, everybody!
I am trying to obtain axenic cultures from filamentous brown algae. In order to test axenic conditions, i m using agar marine sabouraud (for checking fungi) and agar marine (this is, solidified marine broth; for checking bacteria ) with an incubation time of 2 weeks at 25°C and 28°C, respectively, in the dark. I came with this idea after reading this paper "A new method for removing microflora from macroalgal surfaces" (Klentz et al. 2011). Previous papers in this topic used Zobell 2216 or half-PES with 1% glucose and tryptone as testing mediums under standard conditions (light and temperature according to each macroalgal species). 
Should i use Zobell or half-PES medium for texting axenic conditions or the mediums i ve been using are suitable for this?
Thanks in advance
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Dear José Avila-Peltroche,
I would like to emphasize that my intention was never to discourage your research. Although difficult, it is not impossible to obtain axenic cultures of algae. Good luck in your search.
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While I am preparing WEB (Water extract molecule from Streptococcus pneumoniae) after 6 hours shaking it supposed to centrifuge in 10000rpm. But my centrifuge machine was shown error that is why I could not centrifuge then. I kept the sample in -20 and after two days I shaked it again in 100rpm and centrifuge for getting bacterial surface protein. My question is after this hampering will I get my require surface protein? Is there any chance to spoil the protein?
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Thank you very much. I will try.
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I am using mixed culture of LAB and Yeast for cereal fermentation.For enumeration of each bacterium in a mixed culture, I need to add anilin blue to the MRS medium but I didn't found how the different strains will looks like on a plate. 
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Dear Khan 
The media uses for lactic acid mix depended on used isolates. Anilin blue with MRS medium is not selective 100%. you can used other media while The yeast  enumeration was used Chloramphenicol glucose yeast extract agar   
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I need to inoculate 50L of media to reach a final concentration of 10^5 CFU/ml. I have a stock culture at 10^7. I need to calculate how much of the stock I need to add and at what concentration the stock needs to be. 
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If you intent is to establish by inoculation 50 liters at 10^5/ml, think you'll need 500 ml of 10^7/ml inoculum in those 50 liters (i.e. 500ml inoculum in 49.5 liters).  Consider: concentration x volume = concentration x volume.
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if A136 is growing well n LB it mean it is active, then why it is not growing in AT media? can anybody recommend some changes in At media specification?
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Check following parameters..
1. composition that inhibit growth.
2. Expiry date of media.
3. Environmental temperature.
4. Contaminating organism.
5. Aerobic / anaerobic conditions.
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I have isolated the cellulose degrading bacteria on CMC media, bacteria showed hydrolysis zone, done colony PCR using 16S RNA universal primer and sent the product to compny. the company sent me sequences when I blast on NCBI I got Bacillus stratospheric strain. Now I have to find the gene and want to clone which degrades the cellulose. how can I find?
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Read the papers related to this bacteria. and find the probable cellulose, then get that protein sequence on NCBI and then blast together.
or you can read this paper I hope it will help you.
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Hello everyone, I am trying to determine Catalase activity (KatA) in B. subtilis from the exponential phase, cells were exposed to different concentrations of H2O2. However, when I determined catalase activity in the control and induced cells I see differences in the activity. How can this be interpreted just considering that I am working just with KatA? Considering that Catalases generally, being catalytically active enzymes do not follow Michaelis–Menten and follows Bonnichsen-Chance-Theorell (BCT) mechanism.
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The problem it´s that in the system it´s already overexpressed, I am comparing it in the same OD600, protein has been quantified, and actually when bacteria is exposed to H2O2, population starts decreasing
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I am working on Vibrio Parahaemolyticus biofilm , as Vibrio Parahaemolyticus is a pathogenic bacteria. So, i want to detect type III secretion gene .Can anyone help me to suggest some recognize primer with reference for detecting type III secretion gene ?
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Hi guys,
This question might be a stupid one but I had some argument on it. What I understand is that bacteria can not grow in 10% SDS solution. We generally use SDS to lyse them. Am I right? Some people from my University are claiming that Bacteria or fungus can grow in 10% SDS. Can you guys help me in this regard?
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SDS is used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. I advise you to read the following interesting paper:
Screening of SDS-degrading bacteria from car wash wastewater and study of the alkylsulfatase enzyme activity
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I would like to harvest Photobacterium and Morganella via centrifuge. Which rpm and time is better to avoid damage cells?
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Hi:
In order to pellet cells, people usually use around 5000 g (centrifugal acceleration 5000 times the gravity on earth) for 10 minutes.
Regards
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Hello everyone, I am having difficulty growing Listeria monocytogenes biofilms using the Calgary Biofilm Device (CBD) for the purpose of completing a MBEC assay.  It seems like the biofilms are not adhering to the pegs, as tested by a CV staining assay. I have tried using different broths (brain heart infused (BHI) broth, BHI broth with 0.1% glucose, BHI broth with 1% glucose, BHI broth with 2% glucose, Tryptic soy broth (TSB) and TSB with 0.6% yeast extract), different incubation temperatures (37 degrees celsius and 30 degrees celsius), different rotation speeds (110 rpm, 200 rpm and stationary) as well as different incubation times (24hrs, 48 hrs, & 72hrs (changing media every 24hrs)). Iv'e tried all of these different methods with no success. Does anyone have any suggestions or different methodology? Thank you! 
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Thank you Mert Sudagidan for your valuable input. You are right, this strain of Listeria monocytogenes is probably not a strong biofilm former. We will try with another strain for these experiments, thanks again!
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I was screening phenol utilising bacteria from the fecal isolates using MRS medium... Here I need to compare the isolates with positive control(phenol degrading or resistant strain) and negative control(sensitive strain). So please guide me how to find out the phenol sensitive strain from the identified lab pathogen(E.coli, Staphylococcus sp, Pseudomonas sp, Klebsiella sp, Streptococcus sp, Salmonella typhi)
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i am working on bacterium, Deinococcus radiodurans.
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i cant find any link down here... 
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I don't know, if competent cells and transformation protocols used for E. coli also applied to anaerobic bacteria such as Clostridium acetobutylicum?
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Thanks James.
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I need mass phage production to apply in aquaculture farm (same as phage production for industry application). Does anybody know method or protocol to do this?
Thank you very much.
Cheers,
Son
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Hi
you can see the following link on some experiences that are now commun in Cuba for  biologic controls production in field conditions, we call it CREE - Entomophagous and Entomopathogens Reproductive Centers-  . Best regards,
Redimio
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Mycobacterial curli pili dont have an operon of pili secretion and assembly factors and chaperones. Its a very different picture from other pili types that usually consist of the components of a pathway mapped on one or more operons. This makes it very difficult to design an experiment to discover and characterise these components. Where do I start? Any ideas?
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Thanks for your answer. This is a very unique protein. When a BLAST search is done on ncbi, the protein does not match any proteins known to assemble into pili in any other pathogens. The protein was characterised by Alteri 2006 upon purification of the pilin subunits by mass spec and was established as curli pili by biochemical characteriastics and appearance of the pili on electron micrographs. What are the steps taken to uncover a secretion and assembly pathways when the genes are unknown?
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since i am trying to grow deinococcus radiodurans in my lab, i encounter a lot of compititive bacteria like E. coli and Bacillus types. please suggest 
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The paper below uses 1 mM methionine, but, as the paper demonstrates, methionine is not necessary . . .
---
Not an experimentalist.
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I am expressing some proteases in E. Coli that works under acidic conditions (e.g. pepsin). I am trying to find out if there is a high-throughput way to qualitatively test the activity of my protease mutants on specific protein substrates. For example, after plating my e. coli cells that contain different mutations of pepsin on an agar plate that is made with casein (or other proteins), I would like to be able to see some changes around the colony (it could be a change in color etc.) to qualitatively access the activity of my pepsin mutant. Does anyone know of a way to do something like that?
I have seen papers where people bromocresol green dye on an agar plate to access secreted protease activity. However, my proteases are likely not going to be secreted. So I am also trying to find ways to lyze e. coli cells on an agar plate to release the proteases into the agar. 
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I agree with Steingrimur Stefansson answers
good luck
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I am working on the microbial degradation of a herbicide "Metribuzin" and I want to check the difference in protein expression of bacteria in Mineral salt Media (MSM) with higher metribuzin concentration and without metribuzin. Please, anyone guides me what carbon source I can add into the MSM having no Metribuzin for the growth of bacteria...?
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You should use a carbon source which directly enters the Krebs cycle, what means, to select one of its intermediates, such as acetate, or citrate. This helps to prevent the induction of additional genes for additional catabolic enzymes (including their transport systems) and, as a consequence, reduces the background for proteomics studies which you have in mind. In this sense, sugars, as mentioned by the colleagues above, are not the first choice.
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i have tried with heat shock method but the results are negative. there were no growth in positive control as well. 
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thank you
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I wish to know which microbe is best for Solid state fermentation, fungi or bacteria. What is your suggestions for selection of microbe? 
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Fungi are the most potent microorganisms to grow in solid state fermentation. Filamentous fungi are the best such as Aspergilli, Penicilli, Trichoderma, and so on.
Sidkey, N. M.; Ammar, M.S.; Ashmawy, A.M.; Rady, A.H.& El-Hagar,O. A.(2001): Regulation of the thermo -Amylase productivity by Thermomyce slanuginosus Tsiklinsky, Ferm-Bam,F4 while utilizing water hyacinth as the sole carbon source under solid state fermentation. Al-Azhar Bull. Sci. Vol. 12 (1) June 2001: pp. 243-262.
Younis,N. A.; Sidkey, N. M.; Ammar, M.S.& Ouda, S. M.(2001): Recycling of water hyacinth for production of cellulases by Aspergillus terreus, I5 and Penicilliumm oxalicum, I8 under solid state fermentation. Al-Azhar Bull. Sci. Vol. 12, No. 2, Dec.2001: pp. 353-373.
M.S. Ammar, H.M. Atta, A.T. Abul-Hamd & N. M. Sidkey (2002): Bagasse Recycling For-Lactam Antibiotic Production By Penicillium chrysogenum, Ferm-BAM-1 Under Solid State Fermentation Conditions. Al-Azhar Bull. Sci. Vol.13,No. 1 (June): pp.169-196
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Bioprocess engineers and Fermentation technologist. 
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Kla depends primarily on power input and medium. Reactor configuration and viscosity may lead to 'dead' zones reducing overall mass transfer. Also oxygen solubilty is important for mass transfer. So measurements are required or someone's else experience in a very similar reactor may give at least an indication.
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We select thousand of microorganisms on plant composts. We include the microorganisms (Bacteria and fungi) on pellets for the commercialization. The question is, What is the lowest temperature that kill the microorganisms at lag phase? I know that according to the microorganism specy this temperature will change; it s just to have an idea. Thanks
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Thanks you for your answer. My email adress is pblanquet@marcel-mezy-environnement.com
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I need to make a solution of phosphatidyl choline (DPPC) to use in simulated lung fluid. I generally dissolve in water for bioaccessibility tests and it never fully dissolves at pH 7.4. I need to make simulated lung fluid to incubate dust microorganisms. I need to filter sterilize the fluid and not autoclave. But I cannot figure out how to make a solution of DPPC without using chloroform or something that will kill microbes. If anyone can help me suggest something to solubilize DPPC (10g/L) so I can filter sterilize using 0.2 micron sterile filters, I would be very grateful.
Best regards
Farzana Kastury
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Thank you so much.Appreciate it a lot.
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Currently Im study the biodegradation of polystyrene. When i preparing my control (minimal salt media plate + cell inoculum without carbon source), i observed cell growth on the control plate. 
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Dear Kiang,
1. Some bacteria can grow without added carbon source. They exhibit an "autotrophic metabolism" and fix carbon in the form of CO2. Traces of CO2 could thus support them.
2. What can also happen is that organic buffers (TRIS, MOPS) are partly degraded as carbon source by some microbes.
3. Finally, chemicals such as salts contain small impurities of organics, depending on their purity. Even water can have some depending on purity. This could also deliver carbon for growth.
4. If you prepare an agar plate, you use a polymer to achieve the solid medium type, which can be degraded by some bacteria. If this is the case, the colonies will sink into the plate becuase they liquify the medium around them. we used this many years ago select for gellan degrading bacteria.
Best,
Christoph
 
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Dear all:
I have the following questions about aeromonas salmonicida¨s growth:
In my lab, actually I´m growing Aeromonas s. in bioreactor (5L VF) with filtered medium BHI (Brain Heart Infusion) + 0,5% v/v yeast extract and 1% NaCl (not autoclaved). For meausure the growth of this bacteria I´m doing growth Kinetics (D.O600 vs time) and we control pH at 6,5 once the culture reaches this value with NaOH 1M (normally the inicial pH before inoculate the bacteria is about 7,3+-0,2), but once the culture reaches 6,5 (after 2 hours approx. with NaOH control) the culture begins to alkalize the medium until 7,1 (peristaltic bomb of NaOH is off when the pH is above 6,5) also in flask assays with the same medium, the bacteria acidific until reach pH 5,8 aprox without pH control or raise in pH).
- May a bacteria like Aeromonas alkalize a medium like this according with the conditions showed up?
- Has someone observed this situation before with this bacteria or with another bacteria ?.
Thanks for you help
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Dear Felipe: Do you measure concentration of dissolvbed oxygen? According to my opinion if your culture is limited by oxygen (especially during log phase), it could produce some metabolites which alkalize the growth medium.
Best regards   Vit
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The medium gets precipitated when all the salts were added .Kindly suggest a good technique.
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thank you all 
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what method i can use to digest this material without disturb the bacteria?
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What for???
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Hi guys currently i am doing a subject using crystal violet assay to quantify s.aureus. The negative control is one non-biofilm forming e.coil, verified by sequencing. The absorbance at 600nm is 0.2 for e.coli, and the s.a. is normally 0.8. But after staining, i can still oberve some substances of e.coli around air-surface look like biofilm. I have tried this method for many times and it should be not containmination. Could anyone please tell me what is that? many thx.
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If you recollect your basic organic chemistry, then you will know that CV is no doubt a dye belonging to the pararosaaniline group with a phenolic group which makes it inhibitory to an organism like E. coli. It is always better to use a medium as a negative control rather than any organism. 
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When we inoculate any filamentous fungi to any biomaterial for its fermentation first it grow for few days until the substrate is being utilized.I wish to any is there any publication in this line which can give me information regarding the production of Aflatoxin when it is inoculated with any filamentous fungi for its nutrient enrichment.
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Thank you Sir. 
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If I inoculate the E. coli MG1655 in LB and incubate it on bench overnight, will the E. coli do anaerobic respiration or fementation? Since LB is a rich medium with all sorts of metabolites, it may have the electron acceptors for example, nitrate or nitrite. Anybody could help me with this question or direct me to the right literature? Thank you!
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 Thank you both for the answer. I understand that E. coli goes to fermentation if there is no electron acceptor anaerobically. Just to clarify my question, if there is electron acceptor in LB under anaerobic condition? Thanks again!
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I need to express new unknown phytase-like protein in some host to further study its properties (phytase activity, temp. tolerance etc), but have not much experience in protein expression work. I found paper by Huang (Env. Microbiol. 2011, 13(3), 747-757) in which authors made protein expression in E.coli BL21(DE3), but wonder if this strain would be good candidate because it contains phoA and AppA genes and in theory should produce own phytase which might affect the phytase expression level.
I would be extremely grateful if protein experts can advice me for or against using  E.coli BL21(DE3) strain for phytase expression work and/ or recommend  other expression strain (E.coli?) which could be used.
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Thank you, Nicole!
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Does anybody know, where we can find the sake to prepare the desired medium for Lactobacillus fructivorans ATCC 15435 ?
Is there any substitution medium for this strain !? Can we use the MRS ?
ATCC medium: 142 Lactobacillus sake medium
Sake.................700.0 ml
Yeast extract..........5.0 g
Agar..................13.0 g
Liver extract..........0.2 g
Distilled water......100.0 ml
Autoclave at 121C for 15 min.
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 Thank you so much, Mr. Udhia Hussain Jassim Al-Delemi. I will read the articles.
Dear Jameela Esmaeel, I am not sure this medium can be work for Lactobacillus fructivorans ATCC 15435.
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Hi all! We are working on lactic acid bacteria and we want to make the bacteriocin production more efficient. Any suggestions will be very much appreciated. Also, may we ask if you happen to know a protocol (cheapest) on how to isolate and purify bacteriocins? 
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Numerous methods are published
Note  Nettles and Barefoot,1993. J Food Protection
Enan etal 1996. Int J Food Microbiol
Hoover 1993
De Vuyst and Vandamme 1992 and others
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specially I am looking for mercury resistant bacteria. Will one able to find bacteria with such function (mercury reduction) MerA. Please help. Thanks.
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Dear Ma Reina,
Amplicon sequencing would not be useful due to is frecuently used to analyze diversity using universal genes (16S, ITS). With shotgun you can assess microbial traits with software and databases as GOTTCHA (Genomic Origins Through Taxonomic Challenge) and KEGG (Kyoto Encyclopedia of Genes and Genomes) 
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We are running a bioreactor with a chemically defined media (Riesenberg). Is there any downside to growing our starter cultures in a complex media like LB? Would the bugs need to take time adapting to the new environment?
The only way I can think to test is by experimentally trialing LB->RB vs RB ->RB but that would be time consuming. 
Is there a microbiologist or someone of that ilk who might know that step in a bit better detail?
PS: We are using LB for shake flasks as it is quick and easy. RB is a bit more involved but good for high density cell growth. 
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Dear Gabriel
To be more sure that your pre-culture contain an higher % of live bacteria and minimize the lag phase, generally you have to permorm inoculation when the preculture is still in the exponential growth phase or just at the beginning of the stationar.
For reach thisresults in my experience, i used on the basis of the situation 3 different approaches:
1) At 37°C in LB: Do a small preculture O/N from LBplate or glicerol and, use it for inoculate by diluting 1:100-1:50 the real preculture and once it reach an OD around 1 (3-5 h depending from the e.coli strain and plasmid antibiotic resistance) then inoculate your fermenter. OF course this shift all your fermentation timelines and depends if it is compatible with your time work.
2) Replace LB with a medium which allow to reach higher OD (e.g enpresso media of biosilta or also TB) which allow longer exponential phase and therefore after the O/N growth the most of your bacteria are still alive.
3) Use LB and growth it ON but shift down the growth temperature to decrease the bacterial growth rate and reduce the amount of dead cells.
Of course if you would like to be more precise  and carefully evaluate the amount of live and dead cells in your preulture, just the first time during the protocoll set up, you can check the cell viability using some probes (as live dead of thermo e looking it at the fluorescence microscope) but i tihnk that if in LB, you are able to stop your pre-culture  in a good phase ( for example in LB at OD around 1 maximun 1.2) the most of cells will be still viable. 
All this 3 approaches can allow you to minimize dead cells and make the OD a good follow up for the inoculation. 
good luck
Manuele
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What is the most important difference between these two processes?
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aerobic condition
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For example, when an inoculum is 5% in terms of v/v, what does the v/v percentage mean?
Also, how is this applied in industry?
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This term is very often used once you have standardized your inoculation procedure. For bacteria o yeasts for example, you may count cells and establish that you are going to use a 10^8cells/mL-inoculum, or xx CFU/mL, or a given O.D. inoculum. But then, you need to standardize how much inoculum of this N° or density you are going to add for starting your next culture. Normally, one uses 5-10% v/v, which means what Dr. Belaouni detailed above. If you are setting a1L-culture you may add 50-100 mL of inoculum (or starter). Being strict, quantitatively, you should discount this volume from the total volume of culture (as Belaouni said). In the practice, at pilot plant scale or industrially, many times you directly add this 5-10% without discounting from the whole culture. The main thing is standardization, which assures you the possibility to reproduce your results EVERY time. Other relevant thing is to keep your inoculum, not only in the same proportion, BUT always in the same growth stage and equally activated. In the case of fungi, sometimes the inoculum processing is slightly different. You may neither count (except for conidia) nor measure OD. But you can add the same number of agar plugs per volume, or standardize by dry weight. Hope it serves. Success!
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i have amylolytic bacteria strains purified upto ammonium sulphate precipitation
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Dear Kumar,
It depends what kind of enzyme activity you wish to study: some (most, probably) might be inhibited by high ammonium sulfate concentrations, other maybe fine. I would recommend that you test both crude extract and dialyzed extract and see if (1) you get any of your desired enzyme activity and (2) if dialysis improve activity. Further purification may be needed, but i would recommend you try that first.
Best regards,
Eric Gomès.
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I couldn't isolate the plasmid from E.coli.
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 Thank you for your correcting, Dr. Benedik. I am sorry for imperfect knowledge in my question. Now, I have realized that. On the other hand, I have overcome the problem about my research. Thank you so much again for your consideration.
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Glucose oxidase method is acceptable or not?
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Purpose:  The glucose tolerance test measures the clearance of an intraperitoneally injected glucose load from the body. It is used to detect disturbances in glucose metabolism that can be linked to human conditions such as diabetes or metabolic syndrome. Animals are fasted for approximately 16 to 18 hours, fasted blood glucose levels are determined before a solution of glucose is administered by intra-peritoneal (IP) injection. Subsequently, the blood glucose level is measured at different time points during the following 2 hours.
 
Scope: 
Minimum number of mutants: 7 mice for each sex
Age at test: 13 weeks
Sex: Both (sexually dimorphic)
Materials:
One Touch® Ultra2 glucose meter
Stainless steel disposable scalpels
Balance
Timer
Glucose solution 20% (0.9 NaCl)
Gauge needle (25 G 5/8)
Syringe 1 ml
One Touch® Ultra Test Strips or Generic Brand
Gauze and alcohol (70%)
Topical anesthetic cream (optional)
Mouse restraining device (optional)
Quality Control
Calibrate the glucometers each day.
Weighing of mice should be completed before the pre-bleed is taken.
Mice should only be housed in a duplex with animals of the same sex.
 
Set-up
Fast mice overnight for 16 hours by transferring mice to clean cages with no food. Ensure that they have access to drinking water at all times
Prepare an experiment record sheet, lab mat work space, syringe, gauze, alcohol, glucose strips, glucose meters and freshly made 20% glucose solution.
Make sure that glucose meter and strips are the same code before starting.
Stick one strip into the glucose meter all the way.
Obtain the Control Solution; check that date of use is before the discard date. If not, discard and acquire a new control. Write the discard date on the control bottle three months from the date opened.
Shake the control bottle before using and put one drop of the control on the glucose strip in the glucose meter. Make sure control solution fills up the strip and touches the base.
Once reading appears compare control reading to the control range on the test strip bottle. Press the right arrow button to log the reading into the glucometer, L1 will appear on the top of the screen, and then press the power button on the side of the meter.
Record the control reading for internal purposes.
Weighing and separating mice
Start weighing the mice in numerical order so that the mice weighed last have some time to calm down before getting a baseline reading.
Grab a mouse out of the cage with deliberate movements trying to reduce any chasing.
Have the cage close enough to the scale that mouse is not suspended by its tail for more than 3 seconds.
Place the mouse on the scale and record its weight. Place the mouse in a duplex by side by itself. The cage should be clean or the clean cage from fasting making sure the mouse has access to water. The mice should be separated so that they are in numerical order. No mouse should be in a duplex with a mouse of the opposite sex.
Once weighing is complete move cages to IPGTT room nicely on a cart. Place cages in numerical order nicely, avoiding any shaking or bumping of the cage.
Procedure
Calculate and record the volume of 20% glucose solution required (2g of glucose/kg body mass) for IP injection as follows: volume of IP glucose injection (µl) = 10 x body weight (g).
Set the test strip in the glucose meter, part of the way. If left in all the way, the meter tends to turn off before blood is administered.
Recap the strip container every time after obtaining a strip. Do not use strips that have been left open to air for extended periods of time.
Clean tail with gauze soaked with 70% alcohol, then dry tail with dry gauze. Make sure tail is dry.
Score the tip of the tail using a fresh or sterilized scalpel blade, only a millimeter or two is needed.
Push test strip all the way into the glucose meter, drop indicator will show up.
Milk the tail and discard the first small drop of blood by dabbing on lab mat.
A small drop of blood (<5µl) from the tail is placed on the glucose test strip in the glucose meter. Make sure blood fills up the strip and touches the base before the 5 second count down ends to get an accurate reading.
Record this baseline glucose fasting level.
Inject the mouse intraperitoneally with the appropriate amount of glucose solution determined earlier by weight and note the time-point of injection on the record sheet.
The blood glucose levels are measured at 15, 30, 60 and 120 minutes after glucose injection, by placing a small drop of blood on a new test strip and recording the measurements. Start the bleeding again by removing the clot from the first incision; massage the tail if blood flow is inadequate from base to tip. Record results on the record sheet.
Ensure that further blood loss from the incision is minimal by briefly applying pressure to the incision after each measurement. At the end of the experiment add food and recovery gel to the cage and make sure water is available to the animals.
Monitor the animals carefully to observe any abnormal behavior(s).
 
Clean-up:
Wipe down countertops, glucose meters, and other materials with Clorox wipes.
Change your gloves between each cage. To save time, double glove for every two cages.
Discard used lab coat due to possible blood contamination.
 
Notes:
Calibrate the glucose meter routinely as outlined in the manual.
Record equipment name, manufacturer, model and serial number each time.
Record if mouse was restrained or not and type of strip used (whole blood glucose).
 
Reference List:
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The anode compartment contained Shewanella oneidensis, along with Sodium pyruvate, Casien hydrolysate and MSM, I have a basic understanding of how the system works and I require someone to explain what the role of the bacteria is in degrading the dye as well as what the dye breaks down to.
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Thank you Dr Alan and Dr Mohamed I appreciate it.
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We are about to genome sequence a probiotic strain of Bacillus. For trials, we are using a spontaneous rif mutant (to enable enumeration). Would anyone recommend sequencing both the mutant and original strain to determine what the difference is between the two? For some context - this Bacillus is being prepared for commercialisation, we would be aiming for ~170X coverage, and prob won't close the genome. Any advice would be much appreciated.
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Hi Alan.
We did the same strategy for Streptococcus pneumoniae (for B lactams and quinolones, see our publications ), a new meachnisms were described (ABC, and efflux pumps).
And we did the same for M. tuberculosis vs Rifampin.
In M. tuberculosis, all induced resistant mutants harboured mutations in the small region determining resistance to rifampin. and some mutations are known to be associated with high level resistance. it is also important to see if your mutant are also resistant to other rifamycin drugs.
It will be worthy to seq both strains of Bacillus and see if there is other rearrangements, mutations in efflux pumps or ABC transporters...etc
If you want to close the genome the PacBio is better but more expensive, but for this kind of analysis Illumina Miseq is sufficient to analyse.
To achieve 170X coverage or more, keep in mind that plasmids are also sequenced, and you may need to know how many are present and how much copies ? in some instance rif resistance is also mediated by plasmids.
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I would like to develop a simple method to distinguish antibiofilm surface but with a loss cost and a reduced incubation time.
The surfaces I hace to study are differents, some times are PP, PE and other times are metal surfaces.
i read several standart method like:
-E2196 − 12
Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with Medium Shear and Continuous Flow Using Rotating Disk Reactor1
-E2647 − 13
Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow1
But in all methods, I have to spend many money and time to build a reactor.
Finally I have decided to do a stactic simple reactor, I am going to use a Falcon (50 ml) with TSB and some micronutrients with a inoculum ( e coli 10^4 UFC/ml) and after I introduce my sample  but i an not sure if I  have to change the medium every day or how many time i have to incubate the samples?
I would to like to have a homogeneus surface in 72 hours.
thanks
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Dear Lydia, we previously tested biofilm formation ability of our biofilm forming S. epidermidis strain on different ion implanted metal alloy surfaces. We used 24-well plate. The size of our coupons fit these wells. After incubation with bacteria we washed samples and removed bacteria from surfaces and count them. By this way we can compare different surface treatments. 
Another way to use optic microscope after  staining bacteria to determine biofilm formation on metal surfaces, however, to compare the results a little bit difficult.
Best regards.
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First of all let me state that i am not a microbiologist but i have to interprete some microbiological results in water. In our laboratory we use the m-endo les mebrane filter method for the detection of total coliforms and the tbx method for the detection of e. coli. IIs it possible to have more colony count for the e coli than the colony count for total coliforms? eg Total coliforms 10, e coli 28. Thank you very much in advance for your replies!
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Hello,
E. coli is a Coliform. So it seems unusual to have more E. coli than Coliforms.
But it is possible by using different media.
TBX is optimized to the growth of E. coli. If you make a dilution and you add the same inoculum to both media you will have more E. coli on TBX than on ENDO
If you want to do more water tests, I would recommend the Chromogenic Coliform Agar according to the ISO 9308-1. There you get both coliforms and E. coli on one Plate in different colure and you will not have these difficulties by interpreting the results.
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Could anyone please tell me where I can get the following strains?
AHL degrading strain Pseudomonas fluorescens (P3/pME6863)
AHL non-degrading strain Pseudomonas fluorescens (P3/pME6000)
Agrobacterium tumefaciens AT 136 (pZLR4)
Maybe someone could provide me with a sample?
Thanks :)
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my dear, 
i sorry, I suggest you to contact the people who worked with these bacteria.
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I study pattern recognition receptors (PRRs) protein in shrimp like this (http://www.sciencedirect.com/science/article/pii/S0145305X1400010X) and (http://www.sciencedirect.com/science/article/pii/S105046481400148X).  I suffered a problem in the part of bacteria binding assay and agglutination assay. My recombinant protein can bind to all bacterial (gram-negative, E.coli and Vibrio harveyi, gram-positive, Bacillus megaterium and Staphylococcus haemolyticus and Yeast, Saccharomyces cerevisiae) but cannot agglutinate V. harveyi, S. haemolyticus and S. cerevisiae. I presuppose that binding of my recombinant protein to V. harveyi, S. haemolyticus and S. cerevisiae might activate phagocytosis or encapsulation, agglutinated by  my recombinant protein to E.coli and B. megaterium might activate nodule formation.
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Chapter 1: The immune response to infection
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Extraction and purification of antibiotics
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Dear Ali
A four step solvent transfer method was used for the extraction of penicillin from different shake flask cultivation systems. Briefly, first penicillin produced in shake flask culture was extracted in to amyl acetate and then transferred from amyl acetate into phosphate buffer. In third step extraction was made from buffer solution in to chloroform and finally transferred from chloroform in to water. The extracted material was soaked in to sterilized filter paper discs for quantitative analysis.
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I need to spike blood with a homogeneous culture of A. brasiliensis to ensure equal quantities of inoculum are present in the PC and the test sample when I aliquot from an in vitro spiked stock.
I have tried various techniques such as increasing the agitation (250rpm), decreasing pH in SD and PD broths (down to pH 2.04), changing temperature conditions (between 28 and 37 C) etc.
I recently tried using the culture to plate and make spores for spore preps in order to increase the level of inoculum...
Has anyone managed to do this or does anyone have any suggestions for what else I could try to either (1) create a homogeneous culture (2) ensure equal inoculum present in PC and test samples?
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hi, how do you do
you can find some thing useful in your project in www.atcc.org
good luck