Bacteria - Science topic

For WGS, we need to obtain pellets of Avibacterium paragallinarum (Pasteurellacea-like baceria). What are the preffered centrifuge conditions for this bacterial family? Thanks

I do not have -80 C refrigerator for storage of my bacterial cultures. Can they be stored in -20C refrigerators in 50% glycerol slants.

I would like to do genome assembly for the bacteria isolate from the environment. Can any provide me the information or any tutorial? It would be helpful!

thanks!

I'm isolating bacterial from soil on Nutrient Agar media but after 4-5 days I see fungal growth start in the plates. I read about use of Nystatin to prevent fungal growth. How much quantity or concentration of Nystatin should I use for 250 ml of culture media.

Hello,

My E. coli cells express both green fluorescent protein as well as mCherry. So I need a fluorescent stain of color other than green and red fluorescence to enumerate their viability. Please suggest. Thanks in advance.

I am expressing dsRNA using E.coli as vector. I would like to check that my bacteria culture is expressing dsRNA. I am planning to extract RNA from my bacteria culture and then load the total RNA into an agarose gel. Is there any good protocol for loading dsRNA into a gel? Do I need to add formamide to my sample in addition to the loading buffer?

I use 1ul SYTO9 and 1 ul PI per ml of water. The bacteria are attached to a surface and I cover them with 20 ul of this mixture for 10 mins (in the dark), before removing the dye and imaging. The dyes are mixed just before use. At 10 mins, MRSA on steel surfaces are staining both green(live) and red(dead), but I know by culture that they can survive on steel for hours if not days at a time. The filters do not allow cross-fluorescence. The culture is an O/N growth of MRSA in LB broth, and I centrifuge and re-suspend in PBS before use. I have reduced the amount of PI, I have washed the cells to try and remove extracellular DNA/media debris. I can't think of what else to do. Thanks in advance for any suggestions from people who also use this staining kit.

I would like to cultivate lactobacilli in an intestinal organ-on-chip model and stain it with a suitable dye either beforehand or, if necessary, after the end of the experiment with a suitable antibody for immunofluorescence microscopy.

Briefly, I would like to check the Lactobacillus attachment/localization to/in the intestinal tissue.

Is there anyone with experience in this area and could explain possible procedures?

Thank you very much in advance!

Dear researchers, what could be the formation mechanism of the structure that is tiny in size at the end of the first day in the electroless nickel plating waste solution, grows like a mushroom day by day, and has the following appearance after about 2 weeks? What is this structure? I think this is the field of biochemistry and I am not knowledgeable as a materials researcher researcher and I could not find an answer to this in the literature. Maybe because this is not within my field of study, I could not do a proper literature search, I don't know.

What species of pathogenic bacteria can infect soybeans plant ? and what are the characteristics of soybeans plant (leaves, stems, etc) that are infected by these bacteria?

What genus of bacteria can grow in YDCA medium and What color is the colony of these bacteria when grown on YDCA medium ?

Dear scientists,

I have encountered a problem where my bacteria (Staphylococcus Epidermidis) grow perfectly fine in liquid media (Tryptic soy broth) but not on agar plates (freshly prepared TSB plates). For the TSB plates that did grow bacteria, often only one small corner grew but not other parts although I streaked all over the plate using a glycerol stock (image1). I also plated the diluted bacteria solution from a liquid culture (OD was about 0.06) on the plates yet nothing grew (image 2). When I used the bacteria that did grow on the plates to streak another agar plate (TSB), they did grow but the middle of the plate didn’t grow anything (image 5). However, when I used a 6 month old LB agar plate for streaking, the bacteria grew perfectly fine (image 3). In addition, I also used an E. Coli liquid culture to streak a TSB plate, and they grew perfectly fine (image 4). I don't understand why my bacteria have problem growing on TSB agar plates but can grow in liquid TSB media. TSB media is recommended by ATCC for the growth of S. Epidermidis. This problem has halted all of my CFU experiments as the bacteria don't grow on agar plates. Would you be able to give me any suggestions why this is happening? Your time and help are strongly appreciated!

Bacteria isolated from soybean leaves have characteristic spots. The bacteria are in the form of bacilli, gram negative, positive catalase test, positive oxidase test, and the color of the colony is as shown in the picture. Bacterial colonies are shiny, slimy, convex and raised.

Bacteria isolated on NA medium showed a yellow and slightly orange color. This bacteria was also isolated in YDCA medium and showed a cream color.

I'm looking for pathogenic bacteria that cause disease in soybean plants. Please, help me. Thank you.

Im planning to do some BAC maxi preps, a total of 8, and due to the restrictions in terms of equipment, i would only be able to do 2 at a time since only two 1000mL Erlenmeyers fit in the shaking incubator.

I was wondering if i could do all of the starter cultures together and then leave some at 4ºC to then do the maxi cultures the following days (so the maximum time a culture would be at 4ºC would be around 3 days).

My concern is that by putting them at 4ºC ill be losing the inherent efficiency of a starter culture, i.e., to have actively dividing bacteria, in the logarithmic phase.

I need to know to which taxonomic levels (from phylum to species) the following approaches allow prokaryote identification:

a) morphology-based approaches

b) chemotaxonomy

c) GC content

d) DNA-DNA hybridization

d) phylogeny

For instance, can morphology-based approaches be used to identify species?

Basically, I would like to quantitatively detect total bacteria in mice feces. How can I obtain a standard curve to reveal total bacteria quantitatively?

By the way, I have one bacterial species that I grew in a suitable medium, and I obtained a standard curve by making serial dilutions, and I found that bacteria in the DNA whose amount I did not know by substituting it in the Ct equation (obtained from the standard curve). But I don't know how to quantify total bacteria. I would be glad if you help.

Hello, can you help me ?

I'm working with E.coli BL21 transformed with a plasmid that codes for a 13.271 kDa His-tagged periplasmic protein.

I've already done the induction part with IPTG, but I'd like to verify the correct expression using a Western blot. The problem is that I'm finding a lot of contradictory information and in the end, I don't know which parameters would work best in my case.

Washing: Place samples on ice and resuspend the pellets in PBS (previously cooled to 4°C) by vortexing. Centrifuge for 1 minute at 4°C and 4150g and discard the supernatant.

Resuspend the pellets in cold lysis buffer (5 ml per gram of cells).

PMSF 100 mM

Ethanol 100% qsp 10 ml

MgCl2 1 mM

Lysozyme 10 mg/ml

PMSF 1 mM

DNAse 20 mg/ml

PBS pH 7,4 qsp 40 ml

Add lysozyme and PMSF (= anti-protease) just before the experiment. Add DNAse after sonication.

Lysozyme has an Mw of 15 kDa. If the protein being studied has a similar Mw, it may be better to use a different lysis buffer. Do not add EDTA if the protein of interest has a histidine tag.

Make a stock solution of PMSF as it is not very miscible in water.

Still on ice, sonicate the samples for 8 minutes at a rate of 30-second cycles every 50 seconds (6 cycles) at a frequency of 23 kHz and an amplitude of 10 microns. The probe must be completely immersed in the sample, without touching the tube.

Centrifuge for 1h30 at 4°C and 4150g. Separate the supernatant from the pellet (a pause of a few days is possible at this stage if the samples are stored at -80°C in glycerol). If the protein of interest is membrane-bound, keep the pellet; if it is cytoplasmic or periplasmic, keep the supernatant.

After 1h30 centrifugation, depending on the protein of interest, add 1X Laemmli buffer to the pellet (how many ml?) or 2X Laemmli buffer to the supernatant.

Tris pH 6.8 200 mM

SDS 8 % (m/v)

Glycerol 40%

Bromophenol Blue 0,4 % (m/v)

DTT 400 mM

H2O qsp 40 ml

Store the loading buffer without DTT at room temperature. Add DTT just before using the buffer. 200 mM β-mercaptoethanol can be used instead of DTT.

Heat the samples for 5 minutes at 95°C. Then cool for 5 minutes on ice.

Keep samples on ice for use (or freeze at -80°C in glycerol for a few days for later use).

Hello there.

I've been having a really hard time with my cultures of human immortalized podocytes. Everytime I put them under differentiation conditions, they die because of contamination.

For more context, this culture is expanded in DMEM F12 10% FBS at 33°C until confluence. Then, for differentiation, it's subcultered into some plastic previously coated with laminin/fibronectin and maintained with RPMI 1% FBS 1X ITS at 37°C, with media changes every other day.

When the cultures are expanding they're fine, but when I start the differentiation they die before a week, that's to say, about the second media change. Cells look detached, media looks cloudy and slightly basic, and I've seen small dark dots, so I'm guessing it's bacterial contamination.

No other culture at 37°C gets contaminated, we've prepared new media, new PBS, I've thawed several vials frozen at different times and everytime I get the same results.

So, do you think it's bacterial contamination? Is it possible that the source of contamination is the laminin/fibronectin solution or the ITS? Obviously the problem starts when that is used (I have some other podocyte cultures that are not contaminated when expanding, even for weeks) and those are the only reagents that haven't been changed, could bacteria resist there?

Than you in advance

My question is self explanatory.

For cytometry purposes, I am using Accumax for tissue digestion but I need to also evaluate bacteria from the digested tissue.

HELP!

Our lab has recently discovered these dancing blobs in our TC cultures - across cell lines, primary cells and organoids. So far they are not behaving like any bacteria/fungi/yeast we have ever come across (not responding to antibiotics/antifungals and no turbid media). They seem to be amorphous and both extra/intracellular..

Someone has suggested they may be protozoa? If anyone has seen something similar or is an expert microbiologist please help us identify them!!

(Picture included for attention but really need to watch the videos to distinguish from cells/debri)

Hi, I'm looking for a product recommendation. I want to seal microplates containing bacteria for an overnight growth curve at 30-37 degrees C. The job of the seal is to keep moisture from escaping but allow oxygen in. It has to do this and also be transparent enough for absorbance readings through the seal, be sterile to not contaminate cultures, be sticky on the bottom (i.e. not for a heat sealer but rather peel and stick), and not be sticky on the top (i.e. not gum up the works inside our plate reader). In the past, we have used "Breathe Easy" seals but they're not so transparent and they're too sticky on top. What do you use for these situations? It seems a common enough methodology that surely there's a solution out there. Thanks!!!

C2925H, JM110,SCS110, HST04

I have a doubt regarding the plant compound that is partially soluble in water, which I need to test for anti-bacterial activity.

I'm dissolving it in 50% ethanol and using it for anti-bacterial activity tests. Is that fine to use like that?

Can anyone please suggest me some better options?

Thank you in advance

Hi all,

I am currently planning an experiment that involves viewing E. coli cells tagged with gold-conjugated secondary antibodies using a scanning electron microscope, and I am running into the issue of cost for primary antibodies. I might have the option of using primary antibodies previously purchased for Western blots, but I am unsure if these antibodies can also be used for SEM imaging. I do not yet know enough about the chemistry and reactivity of antibodies to answer this question, thus I find myself here!

On a related note, if anyone has any recommendations of good websites to purchase primary antibodies for E. coli that work with SEM, I would love some! I have found a few websites, but each of them only has 2 or 3 antibodies for this purpose.

Thanks,

Joel

Hello everyone,

I am encountering bacterial growth in my diluted western primary antibodies (in TBS, without any milk/bsa, with 0.01% NaZ). We keep the antibodies in +4 C since we use them frequently (Our incubations are also o/n at +4 C). Almost every 2-3 weeks I observe the contamination. I filter the antibodies with 0.4um filter every 1-2 months.

I am wondering why there is that much of bacterial growth even with NaZ. Also, is there a better way of decontaminating antibodies? Can I keep the antibodies in -20 C (how many times I can freeze/thaw them?)

Hi-

I have analysed some PI stained samples through flow cytometry.

The results show a much lower concentration of cells in those samples which have been treated than those which haven't (samples were adjusted to same CFU/ml then treated with antimicrobial- then washed- then stained- then washed again)

Am I seeing a lower concentration due to complete lysis and washing away the DNA as it is no longer intracellular? so now PI does not have much DNA to stain other than those which just have damaged membranes?

Any suggestions/advice I would be grateful!

Thank you

Storage and maintenance of pathogens is a costly and time-consuming affair, the recent study indicated that most of the pathogenic bacteria can be stored for several months at room temperature in sterile tap water without any hustle.

Ref DOI: 10.13140/RG.2.2.34672.84480

Is there any sensitive plates to understand the movement of bacteria?

I carried out a microdilution test for extract and fraction samples of 80 mg samples with DMSO 300 µl and distilled water 700 µl. For the negative control I used DMSO 300 µl and distilled water 700 µl without sample. First I put 50 µl of media into each well, then 50 µl of the sample on the 1st well, and the sample was resuspended. Finally I put 50 µl of bacterial suspension on each well. After 18 hours of incubation, I did some resazurin staining.

[based on my calculation I think the percentage of DMSO in the 1st well is 75 µl/1000 µl=0,075 so it is 7,5% (v/v)] and the literature said that the E. coli can tolerate 10% DMSO

300 µl/ml x 50 µl = C1 x 100 µl (50 µl media + 50 µl sample)

C1 = 150 µl/ml

150 µl/ml x 50 µl (after resuspension) = C2 x 100 µl (+ 50 µl bacteria suspension)

C2 = 75 µl/ml =75 µl/1000 µl = 7,5%

On microdilution with S aureus ATCC 25923 bacteria, the negative control did not inhibit the bacteria. However, microdilution with the E coli ATCC 25922 was inhibited in well 1 and 2 in the negative control (it's on the photo). I used the same negative control on both bacteria.

I don't know why this is happening, is there any explanation?

The smaller white colonies are Rhodococcus, however the yellow ones I am not sure! I collected some of the isolated yellow looking colonies? and streaked them on a separate agar plate and what I got looked like Rhodococcus. Could it be mutant colonies or just bacterial lysis? Thank you!!

Hello everybody, I'm a master degree student. I'm working with 16S data on some environmental samples. After all the cleaning, denoising ecc... now I have an object that stores my sequences, their taxonomic classification, and a table of counts of ASV per sample linked to their taxonomic classification.

The question is, what should I do with the counts for assessing Diversity metrics? Should I transform them prior to the calculation of indexes, or i should transform them according to the index/distance i want to assess? Where can I find some resources linked to these problems and related other for study that out?

I know that these questions may be very simple ones, but I'm lost.

As far as I know there is no consensus on the statistical operation of transforming the data, but i cannot leave raw because of the compositionality of the datum.

Please help

biofilm and quorum sensing genes of E.coli, not used drug but chemical material (Thiophene)

I am hoping to be proven wrong.

Kindly go through the attachment and provide me with your thoughts on this

I have performed a colony PCR with two unknown bacteria (in triplo). Lanes 6, 7 and 8 shows one bacteria with a nice outcome. The band that I expect is 1465 bp, because during the colony PCR, I use a 27 forward primer and a 1492 reverse primer. Lanes 3, 4 and 5 shows the other bacteria with also the expected outcome of 1465 bp, but there is also a band around 250 bp.

I've asked this question before and the conclusion was to change the hybridization temperature. In the last week I have tested 10 different hybridization temperatures, and all the outcomes have the same 250 bp band on the gel.

Why is this band and what can I change in the PCR settings?

I've performed a colony PCR with two unknown bacteria (in triplo). Lanes 6, 7 and 8 shows one bacteria with a nice outcome. The band that I expect is 1465 bp, because during the colony PCR, I use a 27 forward primer and a 1492 reverse primer. Lanes 3, 4 and 5 shows the other bacteria with also the expected outcome of 1465 bp, but there is also a band around 250 bp. Why is this band and what can I change in the PCR settings?

I have been regularly amplifying bacteria with my plasmid of interest and performing midipreps yielding 400-600 ng/uL. As of late, my yields have been very low tanking to almost 80 ng/uL. I initially thought this may be due to antibiotic degradation so both fresh LB and ampicillin were made (working concentration of 50ug/uL). This actually decreased the yields.

I have also noticed a decrease in my transfection efficiency. May this be due to the diluted plasmid yields increasing the volume added to the wells? Any tips on how to resolve this?

Best

The thawing of permafrost, which has been present for thousands and millions of years in areas near the Arctic Circle, mainly in the Arctic, caused by the accelerating process of global warming, will result in the release into the atmosphere of thousands and possibly millions of tonnes of hitherto frozen methane, a gas that is many times more greenhouse-generating than CO2, which will result in a significant acceleration of the already rapid process of global warming. However, this is not the only very dangerous effect for human civilisation and for the state of the planet's biosphere of the progressing process of global warming, a process which has been taking place since the first industrial revolution, i.e. since the 18th century. Among the significant negative consequences of the increasingly rapid global warming process triggered by the industrial revolution based on the dirty energy of burning fossil fuels is the increase in the risk of a future pandemic caused by viruses emerging from the thawing of the permafrost in areas near the planet's Arctic Circle. These viruses emerged and were frozen many thousands and perhaps millions of years ago, i.e. when there was not yet a modern species of homo sapiens on planet Earth. Therefore, humans may not be immune at all to these strains of different types of viruses that functioned on the planet many thousands of years ago. In addition, the existence of many species of both wild animals and farmed livestock may also be threatened if thawing viruses from many thousands of years ago prove to be completely unfamiliar to the immune systems of said animals. According to CNN media reports, there are virological research laboratories currently working on revived viruses taken from thawing permafrost. These revived viruses are referred to in the media as "zombie viruses". In addition, high summer temperatures have thawed the corpses of people who died and were buried in cemeteries many years ago, as well as animals, from whose thawing bodies pathogenic strains of viruses and bacteria have emerged. The thawing of the permafrost in recent years, for example, has been identified as a major source factor in the occurrence of the anthrax epidemic in Siberia, because the high temperatures experienced in Siberia for the first time in many thousands of years allow viruses and bacteria to be released from human cemeteries and animal corpses, i.e. micro-organisms that functioned thousands of years ago and which may be particularly dangerous to humans and animals living on the planet today.

In view of the above, I address the following question to the esteemed community of scientists and researchers:

What is your opinion on this subject?

Please respond,

I invite you all to discuss,

Thank you very much,

Best regards,

Dariusz Prokopowicz

We are doing a research on Biofilm formation of Bacteria, for knowing each isolate strong, moderate or weak, by calculations tables of Standard deviation, Variance and Cutoff (Ct) etc… and with the help of Microsoft Excel but the results in the program differ from the hand written and don’t know the best way to calculate and compare the results, any help will be very appreciated, Thank you

Ali

Hi, I'm currently working with PAST 4.01 and I have my data organized on a table of 4 columns (treatments) and 45 rows (different bacteria genders) with the amount of genders found in every treatment and they are numbers like these: 6,7E+04 ; 5,2E+05 and 0.

Last week I got diversity indices, diversity t test, diversity permutation test and everything went great. But I had to change just ONE VALUE that wasn't 0 and since then, every time I try to get the rarefaction the program doesn't respond or if I try to get diversity t test and diversity permutation test the values are wrong (it's 0 and trust me, when I did it the first time I got numbers like 0,005 but not just 0). Funny thing is that when I try to get the diversity indices and beta diversity with the same data, results are the same from the first time, it works with those options.

Please if someone knows what am I doing wrong or if this time I'm missing something... I'd really appreciate the help!

Hello,

I made a cell lysate with a gram-positive bacterium using lysozyme and sonication. I need to inactivate the lysozyme because we are using the lysate in cell-stimulation assays and do not want the lysozyme to influence the cellular response. Inactivation by heat seems to be the way to go, however, we need to maintain the integrity of bacterial lipids. Has anyone had experience with inactivating lysozyme? What temperature and how long was necessary? Will heating at this temp destroy lipids?

Thank you!

I understand the extreme pH can kill the normal skin microflora bacteria. But how. can someone explain the process?

My topic is related to antimicrobials, and after testing intracellular ATP levels I found that intracellular ATP is increased at antimicrobial concentrations. In most studies, intracellular ATP levels are decreased after drug treatment and may be related to cell membrane disruption and drug-induced apoptosis. However, I did not find any explanation for the increase of intracellular ATP and the antibacterial mechanism. Can anyone answer my question?

My colleague and I are planning to do a culture-independent study on identifying specific bacteria in a river system. We just have some questions before we undertake this study.

1. If we happen to sample pathogenic bacteria, do we need to work in a BSL-2 laboratory?

2. What is the general procedure for trying to identify specific bacteria? Do we need to perform DNA extraction, cultivation, etc.? We are planning to perform 16S rRNA metagenomic analysis and are scouting sequencing centers around our country.

We have a Flask that contains broth, and we want to inoculate it with Bacteria inoculum, Can we simply take a touch by the loop or by micropipette?

Hi, I'm trying to build a dataset of Acr and Cas protein interactions and I had a couple of questions. First, most of the literature includes which Acrs interacts with what Cas proteins and they don't mention negative examples. So, I was thinking If I know for example that AcrF1 interacts with Cas7, can I assume it doesn't interact with all other Cas proteins?

Second, some research papers mention that a certain Acr protein inhibits the CRISPR system in a certain bacteria and they don't mention anything about what Cas proteins are affected. Can I assume that For all sequences in one Acr family, they all affect the same Cas protein? e.g. if one AcrF9 inhibits Cas8 and Cas7, all AcrF9 sequences will interact with the same Cas prote ins?

I'd appreciate it if you explain these to me, and if you have any useful material please do share it with me. Thank you.

is there a specific ratio to follow during the addition?

Hi there,

I am looking for a protocol to isolate RNA using low total CFU of bacteria (S. Aureus). We already have a good isolation protocol for mammalian cells (cell culture and tissue), but now we also want to isolate RNA from bacteria. We already found several protocols, but these protocols are all based on high CFU numbers. Like a 50 ml culture of a 1x10^8 CFU/ml. For our experiments, we want to isolate RNA from only 1 ml culture with a concentration ranging from 1x10^6 - 1x10^8 CFU/ml. We have tested several things, but our yields are just too low to use for downstream applications. Hopefully, you one you has an answer for us that works.

Thanks in advance..

Hello all.

I isolated some bacterial isolates from the environment. I have been trying to culture several of these isolates in liquid minimal media (with various concentrations of the gaseous C-source used to isolate them) but i never seem to get any growth. However, they grow well on minimal media plates when i use a similar amounts of the C-source.

Can anyone help explain this and how to circumvent it?

Thank you in anticipation.

This is my first time culturing and working with bacteria.

I am culturing Bifidobacteria in the anaerobic gas chamber. According to the references I am using BL broth media supplemented with 5% defibrinated blood.

Previously while culturing other bacteria such as lactobacillus, I simply centrifuged the tube and add fresh media, dissolve the bacteria pellet and proceeded with further experiment.

In this case after 24-48hr incubation while centrifuging blood particles also collected at the bottom. Is it normal or I should follow some sp[ecial technique?

I am attaching the media details and references here for better understanding. Any suggestion is highly appreciated.

There are many complicated and detailed steps, devices like cold centrifuge, O.D measurement, silica gel, columns, HPLC, If possible please i need a short recap of the procedure and advices with Many Thanks to you

Ali

Dear all,

What could these things be in a clean mammalian cell media? Yeast? Mycoplasma colonies? Media components sedimenting?

They are as big or a little bigger than lymphocyte cells. No organelles are visible, these oval things are very smooth and of different size. I am hesitant to use this media because it much more orange than the other vial of same media I made on the same day. It has been filtered through double 0.2 um filter.

I made a cell media and set up a mock plate with all medias I made and cells to monitor whether there is any contamination.

After 5 days, this media is not turbid, cells are growing well in it.

Not sure if these things are dividing.

I will submit it for mycoplasma testing.

What could it be though? Any ideas?

Thanks,

Maria

Hello, could you suggest experiments and bioinfo tools to analyze the binding/interaction of peptides with bacterial surface & EPS (planktonic cells and biofilms).

Thanks!

Hi, I need to check microbial growth on wastes rich in proteins that tends to ferment pretty quickly. To get an idea of what and how much it's growing, I thought about using Plate Count agar, YS or YM for screening for yeasts, maybe Violet Red Bile for gram negative. What can I use to check protease activity? Since it's a mixed colture, I cannot use super selective media.

Could this course of action work for a totally uknown mixed colture? The goal is to identify the microrganisms responsible of spoilage and fermentation.

Thanks in advance for the help.

I was able to transform bacteria sucessfully with small inserts (+-500bp and 1500bp) using infusion technic. However, when it comes to larger inserts (5500bp and 6000bp), it doesnt work. We already follow the troubleshooting guide descript on the protocol, and tried differentes approaches (concentration, proportion, longer incubation).

Our primers were designed following Takara instruction with 15bp of homology and were already checked.

Our linnearized plasmid was diggested by Xho1 and Sal1 and it its 5004bp long. The final concentration of the linnearized plasmid is 195ng/uL. Our insert is 5542bp (larger than the vector) and its final concentration after purification is 27ng/uL. I'm using competent E. coli Stbl3. We use the concentration around 50ng/uL up to 150ng/uL in the infusion solution.

We tried to transform bacteria by using different proportions between the vector and the insert (1:1; 1:2 and 1:3 each). We also incubated the infusion solution for 1 hour at 50ºc (even knowing that the protocol says longer is no better). I already checked the reagents by using the positive control.

We use the heat-shock protocol, by defreezing bacteria for 30 minutes in ice; adding the infusion solution (3uL) on bacteria and leting it incubate for 30 minutes in ice; then we heat shock the bacteria for 45s at 42ºc and quickly put them into the ice again. Final step, we plate it in a petri dish with agar LB and streptomycin and let it incubate for 16-20h.

The thing is that we dont have any colony and when it appears, it doesnt have our interested insert. I dont know what else i can do.

I am looking for an efficient tool for gene knockout in a gram positive bacterium..

I have an extract that is more effective at lower concentrations against tested strains of bacteria. I have done some research and some says it might be to do thickness of the extract that doesn't allow it to diffuse into the plate equally. but to the naked eye it doest seem that way.

Any suggestions would be greatly appreciate it.

Hello,

I need to convert the e coli bacteria measured in pmol to cfu. How can i do this?

The cells are kept at RT in a desiccator (vacuum).

I have done a blue-white screening with E.coli and pUC19 with lambda DNA. Then, I selected some white colonies (and blue colonies as a control) and gel electrophoresed them next to pure pUC19. All lanes have a band similar to pure pUC19. I am stuck on why this is the case. Any help is appreciated!

Hello,

I recently found out a contamination in my cell culture.

However, I would like to ask, what could it be, exactly???

Contamination initially does not change the color and pH of the media, this happens after about 5 days.

According to the microbiological agar smear, it does not seam to be mold.

Could it be yeast contamination?

Thank you for the advice.

Have a nice day.

Our lab recently started S.aureus culture. We inoculated it on agar plate (LB) and everything went well at fisrt (attached file).

However, there was some contanimation (blurred, big colony cover the s.aureus colony) and colony with irregular shape appeared.

We already did the following experiment to find the problem causing contamination:

1. Placed a clean agar plate into incubator, no colony was shown. The incubation should be clean.

2. We conducted experiment under laminar flow hood, by placing an open agar plate in laminar flow hood. There was no colony.

3. By streaking method , we first confirm our bacteria stock was clean. Then picked one colony for further cultivation.

4. The agar plates with contamination (blurred, big colony cover) have strong odor.

The contamination already last for a month. After cleaning the incubator and laminar flow hood, the contamination keep appearing.

Any advice would be very much appreciated.

Thank you,

Ian

For example, E. Coli? Is it known how much LPS different bacterial species have?

Hi! While I was incubating some unknown bacterial strains isolated from soil samples, with mineral media and polyolefins powder, I found what I assume is a contamination in some plates. The growth was bright pink, I isolated it and incubated on LB agar plates, and started some basic tests. On gram staining, under microscope, it appears to be very small gram negative cocci, it's catalase and oxydase positive, and grew vigorously on MacConkey agar (so I'm pretty sure it's gram negative and it's not an error in the staining). The colonies are bright fire-red, that tends to become more dark red as time passes. What could it be? I thought about Acinetobacter, but the morphology is different and it's oxydase negative.

I will add some pictures

Hello Scholars,

I am an undergraduate at the University of Cross River State, Nigeria currently pursuing a microbiology program. For familiarity and enhanced understanding of the course, I wish to seek recommendations on the virtual/simulation laboratory software that would be very helpful to me and my colleagues. With my interest in research too, I will be pleased if a research simulator is recommended to help widen my understanding of Microbiological research.

Your recommendations would go a long way to significantly contribute to my academic career as well as my colleagues.

Thank you

I want to separate the bacterial/fungal cells from the growth medium loaded with ointment/cream. Please, anyone, share the techniques to separate cells alone after treatment with drug

I need to know the best growing conditions for MRSA and MDR P. aeruginosa.

The incubation time and temperature and the best medium.

Hi everyone!

A little backstory: we have these samples of Staphylococcus aureus that are representative of multiple colonies (we call these pooled samples) and we want to test their susceptibility in the Sensititre GPALL3F assay. We are looking to see if these pooled samples have more AMR genes than single colonies picked off the same plate.

To do this, we figured that growing these up in broth rather than picking the 3-5 colonies would represent the diversity more, as it'll give all of the colonies a chance to grow, not just the random 3-5 I would have picked off if following the protocol for these Sensititre plates.

I was told I would have to do a dilution scheme from this broth, and I wasn't sure where to start because I am subpar at math (pls don't judge)!!!!

Basically, my plan so far is to grow up these samples from a frozen stock to a blood agar plate and passage them twice to ensure optimal growth, then put them into 5 mL of TSB broth and allow them to grow overnight. Here is where I'm not sure about the next steps.

To create the 0.5 McFarland standard, how much will I have to dilute my 5mL sample? Should I do a 1:10 or 1:100 dilution of the staph TSB juice and then put that diluted broth into the Sensititre water? Or will I dilute the 5mL TSB by just putting the stock 5mLs into the water and not diluting it in TSB first? Has anyone tried doing a 0.5 McFarland from the broth before, and if you did, how much TSB did you put in to achieve the proper turbidity?

Thank you so much everyone! I hope this makes sense.

Hi,

I'm wondering if anyone has experienced this.

I'm working with a species of actinomycetes, and after a certain time the media colour changes to an almost red/maroon?

Is this melanin production? I'm unsure as to what it could be?

Thank you in advanced

Hello everyone!

Inducing diarrhea in BALB/c mice is a challenge for me. I gave the mice 2x10^8 CFU/mL of E. coli orally, and I monitored the mice for diarrhea. However, there are no cases of diarrhea. When I primed the mice with antibiotics for three days in a row before infecting them with E. coli on the fourth day, again none of the mice had diarrhea.

Could someone advise me on how to create a model for experimental diarrhea caused by E. coli?

Thank you in advance!!!!

Stay safe! Keep smiling!! Be positive!!!

I am doing live cell imaging of bacteria, and I am wondering if anyone has had luck washing and reusing a chambered coverglass for this application? I have been using an 8-welled version (ibidi #80807), but after 24-hour imaging there is usually some cross-contamination of the unused wells. Just washing with PBS doesn't seem to remove the adhered cells, so I think I need a better method for washing out those slides.

Here's a method I've tried:

1. 3x wash with DI water

2. 3x wash with 70% ethanol, let air dry

3. Store in parafilm until ready to use

4. 3x wash with PBS before using, to remove any lingering ethanol.

It does seem to remove the cells that had adhered to the glass, but I'm worried that trace ethanol could be interfering with cell growth. Does that seem like it would be an issue? Has anyone else tried similar or different methods to wash and reuse these slides?

Tetracycline, Streptomycin, or any other available options...?

Hello,

My colleague carried out an experiment in which she grew epithelial cells in a well plate followed by infection with Pseudomonas and treatment with phages. She wanted to determine the effect of the phages on cfu over 2, 4 and 24hrs. At each time point she removed the supernatant from a well and diluted this ten fold from 10-1 to 10-8. These dilutions were plated onto a plain Columbia agar plate and incubated.

The results in my opinion are opposite to what would be expected e.g. the most bacteria appears in the most diluted sample and no bacteria is seen in the 10-1 dilution. My colleague thinks that it's possible that there were many active phages in the first dilution that continued to kill the bacteria and that in the lowest dilution the number of active phages is lower so the bacteria grew the most.

I cannot understand how the bacteria wouldn't be diluted out as well as the phage and wondered whether it's more likely to be a error in dilution or plating.

Thank you for your help!

Rosemary