Mycobacterium Tuberculosis - Science topic

I am working on a cytoplasmic protein(85 KDa) of Mycobacterium tuberculosis which is N and C-terminal His-tagged protein and expressing in it E.coli strain i.e BL21(DE3)Gold. By Ni-NTA column chromatography, I am getting two non-specific bands, one is around 120KDa and one is around 60KDa other than my desired protein. What should i do to remove this?

Can you recommend an antibody for detection of M. tuberculosis H37Rv by fluorescence microscopy? I've tried a couple of them with no success; the background is so high. I'm looking for intra-macrophage bacteria. .

I am doing a resazurin assay to determine the MIC of plant extracts against Mycobacterium tuberculosis. The problem is the MIC is coming at a very high value. I read in one paper that even if there are 80 cells the color changes from blue to pink. I am diluting the inoculum also (1:20 dilution). How to solve this problem. Kindly give suggestions. Should I dilute the inoculum more?

Hi

I am working with Mycobacterium tuberculosis variant bovis BCG strain TMC 1011 which requires 12/16 days to grow as visible colonies. I had inoculated my culture on 7H10 slant but before my organism grows the slants and the plate cracks or dehydrates. I have made slants having small surface area and larger butt to prevent dehydration. I have also tried sealing both my slants and plate with parafilm. However with prolonged incubation at 37 C the parafilm turns sticky and it becomes difficult to open plates and plug off slants.

Please suggest some ideas to avoid dehydration of my media.

Regards,

Arti Sharma.

I would like to work on a project on Mycobacterium tuberculosis, but I am worried about the safe conditions for extracting DNA from Mycobacterium tuberculosis culture.

Does anyone have a complete protocol to successfully knockout genes in M. tuberculosis and complement back the genes? Please kindly assist.

It is believed that between a quarter and a third of the world's population is latently infected with Mycobacterium tuberculosis. The importance of latency is reflected in a huge drive by research funding organizations to study the biology and epidemiology of latent tuberculosis infection and to create medications that particularly treat latent infection, with the goal of eradicating tuberculosis globally. Mycobacterium tuberculosis' incubation period lasts around 2-3 weeks following the first infection. The objective is to reduce the incubation period so that the patient may be diagnosed and treated as soon as possible.

Dear colleagues,

If anybody knows about the existence of special database of nucleotide sequencings for Mycobacterium tuberculosis?

We would be very grateful to get the information about assembled genomes or nucleotide sequencings of genes of drug resistant MTBc.

what are the potentials of RHA 1 and Is there is any functional similarity between RHA 1 and Mtb ?

Hi.

I extracted total RNA from Mycobacterium tuberculosis (MTB) isolates. The RIN for some of the samples is >7 but rRNA ratio is below 1. Tapestation RNA screen tape was used. I am attaching the electrograms for reference. Should one consider the rRNA ratio..isnt this factored in RIN calculation?

Will these samples be good for downstream RNA seq application?

Thanks!

I have planned to infect THP-1-derived macrophages with Mycobacterium tuberculosis. After infection, cells would be lysed to quantify intracellular viable bacteria. How should I prepare 0.2% Saponin for cell lysis?

Hi

I am working on Mycobacterium Tuberculosis and recently i started line probe assay for the 1st and 2nd line drug. For the 1st line drug there are no problem i faced because the band comes properly after hybridization. For 2nd line drug i utilized same DNA extracted sample .But the band is not clear in that time.Moreover the band are invisible in some cases. I cannot identified why this problem is occurring?? Because the same samples gives well result for first line drug.

Anyone one how knows about that ,please kindly share your valuable thought.

Thanks

Hi, I am a research student, In order to find a new enzyme targets to treat tuberculosis, could anyone help me to find out new enzyme targets which should be used only in Mycobacterium tuberculosis and why that enzymes are used as a target for anti-TB.

How to simulate the antimicrobial activity between a microorganism (mainly Mycobacterium tuberculosis) and an antimicrobial peptide? Bioinformatics!

Anyone interested un partnership?

I have important question about my master’s thesis. I want To choose my topic . Can you help my about new topics about Mycobacterium Tuberculosis?

How can we extract good quality DNA from MGIT tubes manually for Mycobacterium tuberculosis?

kindly suggest!

Hello, I am infecting THP-1s with mycobacterium tuberculosis bacteria. I am looking for a mycobacteria dye which can enable me to see infected cells through flow cytometry. Do you know of any which work? Many thanks for your time.

Currently we are using the hybridization stripes from Hain Lifesciences but I am curious if there is also a (good) Real-Time-PCR to differentiate between the members of the TBC complex.

I would like to extract Mycobacterium tuberculosis RNA from infected host cells. Is there a method that I can use. Aim is to study bacterial gene expression using RT-qPCR.

I will really appreciate your assistance

I would like to extract Mycobacterium tuberculosis RNA from infected host cells. Is there a method that I can use. Aim is to study bacterial gene expression using RT-qPCR.

I will really appreciate your assistance

We have not grown this bacteria before. Any suggestions related to media, growth conditions, how long to grow it, etc would be greatly appreciated.

We are looking to test an antimicrobial substance against Mycobacterium tuberculosis strain H37Ra. We want to use a standard zone of inhibition assay on an agar plate. Based on previous suggestions, we would likely use Middlebrook 7H10 agar. Any suggestions on how best to conduct this experiment? I assume we could swab the bacteria on the plate as 'normal', but how long would we likely have to incubate to see bacterial growth? Any suggestions on incubation temp and conditions? How do we keep the plates from drying out during incubation?

I preserved my MTB strains directly in MGIT tubes and stored at -20. According to some papers we can preserve our strains in MGIT without adding glycerol. Now the issue is, after one year when i am trying to recover my strains, i am unable to get any growth. I have tried both solid and liquid media for re-growth. Has anyone experienced this before and if someone can suggest me what should i do to recover these strains? Would be grateful for all the suggestions.

Hellow my fellows

Iam working on Real time PCR technique for detection the gene who is responsiable on Tuberculosis as well as the AFB stain so i hope that any one have worked on the same project that can assist me by sending articles or researches that might be useful to complete it .

Thanks

Hammad Shihab

Assistant lecturer

College of education for girls

Mosul University

I am running an experiment in which I need to calculate the CFU/ml of a Mycobacterium tuberculosis growing in 7H9 broth. Is there a reliable method to calculate cfu/ml for MTB since a culture of MTB clumps unlike E coli culture.

Mycobacterium tuberculosis grew in liquid medium（7H9+OADC，no Tween80） to OD₆₀₀~0.7-0.8. Phage D29 was then used for infection（MOI~1）.But I did not observe the bacterial fluid to become clear.What is the possible reason？

Respected researchers,

Please enlist here some troubleshooting tips and practical issues regarding Mycobacterium tuberculosis, M.leprae, NTM and other acid-fast organisms handling, detection and difficulties faced while dealing with them.

Dear researchers,

I'm planning on quantifying ROS production using DHE superoxide indicator in human macrophages challenged with Mycobacterium tuberculosis. Due to bio-safety issues, samples need to be fixated with PFA 2% overnight prior to flow cytometry analysis. Therefore, I would like to know if anyone had experience using DHE with a fixation step and how it affects the staining.

Also, which other methods could replace PFA fixation and guarantee bacteria killing plus effective DHE staning.

Kindly, 

Murilo Delgobo

We are planning to diagnose Extra-pulmonary tuberculosis in our lab and we are unable to find the right RT-PCR kit to differentiate typical and atypical Mycobacterium Tuberculosis. If there are any company please suggest us.

Thank you

I have glycerol stocks of M.tb in -80°C freezer. But it is not reviving properly; shall I rapidly thaw it? or Shall I keep in -30°C for one hour , 4°C for one hour and in room temperature (25 °C ) for one hour and use for inoculation?

Which one is correct and what is optimum percentage of glycerol should be used for preparation of glycerol stock for Mycobacterium tuberculosis i.e. final concentration

Thanks in advance

Dear all great researchers,

I intend to measure the level of iron concentration in monocytes before I infect them with mycobacterium tuberculosis. The idea is, I want to assess the role of intracellular iron metabolism in TB pathogenesis.

I will be profoundly grateful if you can share a laboratory method to measure iron in monocytes with me.

my email is ^{ebdanso@mrc.gm} or ^{lshed6@lshtm.ac.uk} .

Hoping to get your usual help with this.

Ebrima.

Does alternative medicine effective against Mycobacterium tuberculosis?

Hi

what are the most country around the world have tuberculosis that many cases can recorded daily ?

I was trying to culture Mtb clinical strains in Sauton’s media ( Potassium phosphate, monobasic 0.5 g, L-asparagine monohydrate 4.0 g

Citric acid monohydrate 2.0 g, Ferric ammonium citrate 0.05 g 1% Zinc sulfate monohydrate 0.1 ml, Magnesium sulfate heptahydrate 0.5 g 100% Glycerol 60 ml, Water to 1 L, 20% Tween 80 2.5 ml). But the OD600 start declining after 3 weeks of culture, before reaching log phase. I want to avoid protein contaminants from OADC. So, do you think that using Middlebrook 7H9 Broth Base with out OADC/ADC will solve the peoblem?

If one have been vaccinated with BCG vaccine for Mycobacterium tuberculosis, will they test positive for TB skin test even though they are not infected?

Human tuberculosis (TB), a devastating disease caused by the gram-positive, acid-fast eubacterium Mycobacterium tuberculosis, was classified as a global health emergency by the World Health Organization in 1993. TB remains one of the deadliest infectious diseases with an estimated 1.8 million deaths occurring per year, mainly in the developing world (World Health

I am currently facing difficulty in the DNA extraction because of the viscosity of the sputum samples. I am using the Chelex based extraction method which does yield in DNA but it is inefficient with the sputum samples treated with NaOH- NALC. I have come across some solutions such as cetylpyridinium chloride (CPC) and Dithiothreitol (DTT). Please guide me.

Many Thanks.

Hello,

I am interested in checking stained mycobacterium tuberculosis by flow cytometer. I started with significant amount of cells, then passed through a 5 µm filter to get rid of cell clusters. At this step, more than 50% cells were lost. Then I added my dye. After staining, I resuspended bacteria in 10% formalin for 30 min, and then washed cells with PBS. After washing, most of cells were lost. Does any one have a solution for this?

Thanks,

Jun

I really got confused in different protocols

how can I use: Guinea Pig Spinal Cord (GPSC) and

Complete Freund’s Adjuvant (CFA) and

Mycobacterium tuberculosis

what is the real material?

how can I prepare them?

can i use Mycobacterium Tuberculosis H37 Ra, Complete Adjuvant in Oil ( DIFCO 231131)?

Hi everyone,

I wanted to preserve Klebsiella pneumoniae, Rhodococcus equi and Mycobacterium tuberculosis. What is the best technique to preserve these bacteria for a longer period of time? Also, I wanted to know how long we can preserve them. Please specify preservation conditions as well if possible.

Thanks Thanks

Dear researchers,

I have to extract total RNA from infected lung tissue samples to mycobacterium tuberculosis in good quality, high concentration and high integrity for producing RNA-seq data using Illumina Hiseq platform. would you please guide me in this matter?

Thank you for your help

I try to used the line probe assay detect Mycobacterium tuberculosis (MTB) complex and to differentiate it from Non Mycobacterium tuberculosis But, the way I work is unclear

I have a gene of 1.7 kb size from mycobacterium tuberculosis, my forward and reverse primer have Tm 72 and 76 degrees respectively.

I have tried PCR with many different annealing temperatures but my gene is not being amplified.

PCR reactions already done are:

T=95 for 5 min

T=95 for 1 min

T=(68, 65, 62, 59, 55) for 1min

T=72 for 1 min 30 sec

Repeat 32 cycles

T=10 min

this article was in fact withdrawn upon my request to a Biolife's journal. Is it Biolife's responsability or the first and corresponding author's responsability to ask for withdrawal off all on line databases such as Research Gate and/or Pubmed.

Apparently the article's abstract is still on line and the first author wants to resubmit an improved version and fears plagiarism.

Here is his e-mail : sajidjan@live.com

Mycobacterium Tuberculosis moved from P3 lab after confirmed Inactivation with Heat Inactivation.

How to extract dna from mycobacterium tuberculosis by CTAB. is there specific extraction kit? i want to really know the technique before using it

I had already extracted gDNA from Mycobacterium tuberculosis, then measuring the DNA concentration (ng/ul).So, I want to know how I will convert the DNA quantitation into the CFU/ml of microorganisms.

Thanks in advance.

# Tuberculosis continues to be the major health concern for India and has the high burden status in the world.

#India is committed to stop TB under its National Strategic Plan for TB Elimination by 2025.

Therefore, to achieve these objectives, one of the important contribution is from the scientific community to discover a potent vaccine and fast acting drugs to control the ongoing infections. But still nothing substantial research has come out in the form of the final products.

I think T cell based vaccine has the potential to induce a robust immune response and therefore we need to concentrate on antigens which can activate T cells with a highest population coverage.

What are the different strategies we can adopt to achieve complete eradication of tuberculosis?

I need to isolate a tiny fraction of macrophages that could successfully kill the internalized bacteria (for example, M. tuberculosis). I think the best way is FACS, but I need fluorescent dye(s) that deferentially stain these two populations, while keeping their RNA content intact so that I can perform RNA-Seq.

How can Get good yield from DNA of Mycobacterium tuberculosis in sputum with good quality..?

hello all,

A research group and I are finding out the antimicrobial activity of A. fumigatus over susceptible and resistant strains of M. tuberculosis.

We are proceeding the MOPS method using the Aspergillus' extract in DMSO 5%. However it is not working well, so we read about a PRE-TREATMENT (an still unknown reagent for us) to be applied with the liquid medium in MOPS test.

So, this REAGENT can disolve the lipidic bilayer of Mycobacterium and the extract can diffuse and perform much better.

Besides, this reagent mustnt have antimicrobial activity by itself; in order to avoid a bias.

I look forward for your help please!

Thanks in advance

What is the preferred technique for studying methylation in Mycobacterium tuberculosis complex?! other than NGS and SMRT

I came across the two contradicting views that ROS levels must be low under low oxygen (microaerophillic) or in absence of oxygen (hypoxia).

Other researcher shows that ROS levels are high due to inhibition of ETC. Generation of superoxide anions by NADH dehydrogenase shown to play role.

I wish to knew what happens under physiological conditions in tuberculosis inside granuloma or macrophage where conditions are hypoxic or similar ?

Kindly support your answers with appropriate references.

Any suggestions study have been done about mtb in Malaysia.

Will heating at 95 °C for 30 min in a heat block successfully kill the Mycobacterium?

Has anyone got any long term (minimum 1 year) culturesof Mycobacterium Tuberculosis?

In low resource countries, it is difficult to carryout genotyping studies wheere facilities are not available. i am wondering anyone could give me ideas how Mycobacterium Tuberculosis sputum samples from MDRTB patients be kept in storage container, transported by flight into another country to carryout the genetic analysis where facility is available? any suggestions or ideas as I am preparing my research proposal and will be interested to get samples from my country and do the analysis in other country.

why the culture medium viz. LJ media is regarded as gold strandard in TB diagnosis?

i need to know the new enzyme targets of Mycobacterium Tuberculosis and the compounds and/or drugs acting on them 

We discovered new antimicrobials with a rather large spectrum of activity (first publication as poster at ASM Microbe 2018, in June). We just found activity also on M. smegmatis, so chances are that there is also activity on M. tuberculosis. I know, this is not certain, only 30~50% according to Altaf et al (2010), but still worth a try I think. We are BSL2 and have no experience with Mt strains. Important, these antimicrobials are based on extracts of synergistic mixes of edible botanicals, and we managed to be likely eligible to enter directly into phase II (according to FDA guidelines to industry for botanical drugs). Please contact me if interested for testing them in vitro (even in animal!) under MTA, results to be published.

For culturing Mycobacterium tuberculosis strains, we use 7H9 media supplemented with 10%OADC. Once prepared separately, how long can these be stored before use? Also, what might be the proper storage conditions? Is it advisable to mix 7H9 and OADC (without any antibiotics) to make a stock, and aliquot from it as and when needed ?

can somebody tell if there are any other static drugs for mtb besides emb and at what concentration are they static

I took a sample from a Mtb culture that is growing in Middlebrook 7h9 enriched with OADC(oleic acid,albumin, dextrose and catalase). The bacteria were harvested by centrifugation, and the supernatant was frozen at -70°c. How can i get rid of all that albumin from the medium. Unfortunately i don't have acess to a synthetic medium like sauton's broth to inoculate the bacteria after MB7h9. Maybe TCA precipitation method? Should i start with a new medium?

We plan perform Mycobacterium tuberculosis whole genome sequencing of about 1,000 samples, now we're assessing different DNA extraction ways. The first results we got (picture joined) have destabilized me.

* electrophoresis gel shows smears (right image, degraded DNA) with Omega kit than CTAB (center view);

* we got higher DNA amount with CTAB but the average coverage is very low (we used CLC Workbench software (Qiagen), this is not the case with Omega kit where the trend is inverted. We do not plan to analyze our NGS data with that software in our study.

--> What's wrong? Why those unexpected results?

[Could someone recommend to me a working protocol that they are using or they used?] -- NGS

I am currently culturing an Erdman strain of Mycobacterium tuberculosis. After culturing we need a way to disrupt the bacterial clumps at a microscopic level. We have tried homogenization with a pestel and a homoge tube and sonicatoin up to 8min in a water bath but have had little success. What a is a good method that people have found that has worked for their lab? Thanks in advance!

I have extracted DNA in 100ul TE buffer from Mycobacterium tuberculosis culture using CTAB method. Now I want to remove the RNA contamination by using RNase A having concentration of 10mg/ml.

What is the best concentration and quantity of RNase A should be used to remove the RNA contamination.

Need your valuable suggestion and output

Usually it is hematogenous spread from another primary focus. Is there any evidence to prove if there is particular tissue element with affinity to Mycobacterium tuberculosis like Staphylococcus aureus osteomyelitis? Is anyone working on this field?

Trying to find an effect alternative lysis buffer not that different to GITC that can ensure effective lysis of infected macrophages with Mycobacterium tuberculosis. If planning to extract RNA from intracellular bacteria lysate. The reason why GITC is used is because it helps to prevent activity of RNase enzymes not to damage the extract, but I worry that the lysis with GITC doesn’t seem to be effective enough and end up less intracellular bacteria material to extract RNA from.

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?

I digested insert (in pJET) and pNIP40b (mycobacteriophage MS6-derived) vector with XbaI; then I transformed the ligation (ratio 1:1 and 1:3) and the linearized vector (control) in Stbl2. After incubation, I observed huge growing in both plates.

How to solve this problem?

Molecular cloning. Complementation strain. Tuberculosis.

Please can someone suggest a commercial laboratory anywhere around the globe that can assist with testing drug conjugates for antitubercular activity against Mycobacterium tuberculosis (Mtb) H37Rv strain, cytotoxicity and ex vivo intracellular killing activity or macrophages Intracellular Mtb killing ?

Does anyone have a protocol to share? Thank you!

I have to evaluate insilico / computational studies for pncA(Pyrazinamidase) in Mycobacterium tuberculosis and Pyrazinamide resistance.

I am working on characterization of Mannose-1-Phosphate Guanylyltransferase enzyme (M-1-P GT) from Mycobacterium tuberculosis, by colorimetric method. For this i uses the following reagents in reaction,

Tris-Hcl (pH 7.5)

Mannose-1phosphate 2mM (Substrate of enzyme)

GTP 10mM (Substrate of enzyme)

MgCl2 4mM

pyrophophatase (PPase)0.004U/ul

M-1-P GT (enzyme)

H2O (final vol 50 ul)

As in the presence of enzyme (M-1-P GT) the above mentioned substrates converted into products (GDP-mannose and PPi). These PPi in the presence of PPase converted into 2Pi which can be detected by malachite green dye (malachite green is yellow color dye which turned green upon reaction with Pi) and reading could be taken by spectrometer.

I run the Test (which includes all of the above reagents) and Control (which includes all of the above reagent except M-1-P GT enzyme). so theoretically in the absence of enzyme no substrates could convert into product and malachite green couldn't turned into green color. therefor control should have less reading on spectrometer as compare to control. but i am experiencing the intensive color change in even control (without enzyme) which is false reading.

so kindly suggest me what could be the possible reason of this false reading in control?

and what should i do to solve this problem?

Thank you

I used innuprep blood DNA minikit based on binding nucleid acids onto the surface of a spin filter membrane. It is not designated for sputum and pus sample so I conduct prior treatment before get into the kit procedure. The prior treatments are decontamination with NaOH for 30 min RT, cell lysis with modification buffer lysis (contained Tris HCl, EDta,Triton X and Tween 20) and incubation in 55C for 1 hour. I usually obtain very clear solution then used it for next procedure by adding Binding Solution from the kit and this is where the issue happens. Sometimes addition Binding Solution make the solution cloudy. When I apply it to the spin filter and centrifuge, debris restrained onto the filter and really disrupt the washing process (the washing solution has not completely passed through even at higher speed or longer centrifugation time). The kit do not mention about Binding Solution composition so I am not sure where the DNA remain (whether it is precipitated or in the supernatant) if I do centrifuge before apply the cloudy solution to the spin filter. I go search about general Binding Solution composition and still have no clear idea. Please help me to figure out this issue. Thank you 🙂

Do we need both TST and pulmonary x ray to work with nonhuman primates?

Is it possible that negative in pulmonary x ray and positive in extrapulmonary TB will be contagious?

Any references or guidelines?

Is there any report that shows the presence of biofilm made by Mycobacterium tuberculosis in vivo/ inside granuloma?

P-gp inhibitors enhances the drug level inside cells by inhibiting the efflux. 

My specific question is whether a P-gp inhibitor can be used to enhance the level of rifmapicin in lungs and especially macrophages, if the inhibitor (P-gp) lacks the mycobacterial efflux inhibitory and self antimicrobial potential. As some P-gp inhibitors are developed as bioavailability enhancer of anticancer agents.

There is lack of this type of literature. 

firstly i used pET16b expression vector and BL21 strain but i could'nt got protein in soluble fraction. So i changed the vector to pCOLDII with the same expression strain BL21  and i used 0.2, 0.5 and 1mM conc. of IPGT but still i could'nt get protein over expression. i found that the protein conc. was same in sample with or without inducer. Kindly suggest me what i need to do to get over express protein in soluble fraction??

Thank you

For Cloning EspD of Mycobacterium tuberculosis H37rV