Bacteria - Science topic

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

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..

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!!!

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

tldr; we're having massive contamination in our bacterial agar plates, and cannot figure out where it's coming from, even with testing. It doesn't appear to be coming from our autoclave, the Petri dishes, the agar media, the room, etc. I need to pour 1500 more plates by the end of the month but can't keep having 50-100% contamination when I pour.

I work as the lab manager for the biology teaching labs at my university, and pour over 10,000 agar plates every year (almost 20 different kinds) for the students. We didn't have any issues until November of 2021, when we started seeing massive contamination issues on our bacterial plates, and we've yet to figure out where it's coming from. I would love to hear some perspective from others to see if there's anything obvious I'm missing or something we should try differently.

Our plate pouring background:

We pour all of our plates by hand. Typically, we'll make 3 x 3L of media at a time, autoclave the media (45 minutes), let them cool to ~55-65C, and then pour. We do our pouring in a UV room, where we disinfect the bench top with lysol and then ethanol, and then leave the UV light on for at least 15 minutes. I often will come back after the 15 minutes, lay ~50 Petri dishes, and then turn the UV light back on for another 15 minutes. To pour, we use a Bunsen burner to thoroughly flame the neck of the 6L erlenmeyer of media, then we pour some of it into a sterile 500 mL erlenmeyer (which was also flamed). We again flame the neck of the 500 mL erlenmeyer, and then pour. If any media drips down the side, we wipe with a paper towel, flame again, and continue. After we fill all of the plates on the bench, we'll put a second layer of plates down and continue. 9L of media gets us 275-325 plates. After about 30 minutes, we flip them. We normally would let them sit out for 1-3 days, then put them back in their sleeves, and store in our cold room until needed. Most of the plates are used within six months, but we can sometimes use them a year or 18 months later with no issues. Normally we see less than 10% of plates becoming contaminated. This is how things have been done for years (decades) by many people before me.

The problem:

Last fall, we pulled out some bacterial plates (LB, lambda, and TKC) to use for the students, and found that they were contaminated. In November, I decided to pour more to make sure that we had enough. 100% of these were contaminated. We tried pouring again. More than two-thirds were contaminated. And we tried again. Same result. We've poured over 40 batches of plates since then (over 6000 plates), and our results are all over the place. We'll go through periods where 100% of the plates are contaminated, and then we'll get a couple batches that are okay. There's no rhyme or reason that we can find.

In the beginning of May 2022, we poured 12 batches (close to 1000 plates), and most of them had negligible amounts of contamination. But then halfway through May, we started seeing contamination again. We've now started seeing contamination again out of the blue. We're at a complete loss for where it's coming from.

The contamination itself is these tiny white/yellow/pinkish specks floating in the agar (not just on top). It looks like snow from a snowglobe, scattered throughout the plate. It takes about 3-5 days at room temperature for us to see it start growing. And it smells terrible if we leave it too long. With our bacterial plates, we now leave them out for 5 days before packaging, just so that if there is contamination, we'll be able to see it and discard those plates.

Since we typically do three large flasks at a time, we try to keep track of which plates were from which flask. Sometimes, an entire flask-worth of plates will be contaminated. Other times, it's random plates in the batch, with non-contaminated plates in between contaminated ones.

Oh, an an important thing - we do not see any contamination whenever ampicillin or kanamycin are added to the agar media for the plates. So whatever it is, it's killed off by those antibiotics.

Things we've tested:

The autoclave:

- First thing to note is that none of our liquid media has ever been contaminated during this whole ordeal. We make a bunch of liquid media and keep it for a while (months), and we have had zero bottles of media show contamination.

- There was a period of three or so months where our autoclave was the only operable one in the building, and I had about 20 other people using it. No one else ever had contamination or sterilization issues while using our autoclave. (Our contamination issues started a couple months before this)

- Our autoclave is serviced every 3 months. It passes their inspection every time.

- Originally, we used foam stoppers in the necks of the erlenmeyer under the foil. We tried getting rid of the foam stoppers and just using foil, but aren't seeing a difference.

The agar media:

- As stated above, the liquid media has had no contamination.

- I have tried autoclaving the agar media and then just leaving it in the flasks to see if anything grew, but we didn't get any contamination.

- We have tried reserving small amounts of agar media in the flasks when we're done pouring, so that we can see if contamination grew in the flasks if we saw it in the plates. However, whenever we've done this are of course the times that we don't see contamination in the plates, so it's not super helpful information. It's hard for us to do this all the time, because we need to use the flasks and not let them sit around for five days while we wait.

- We already use Millipore DI water to make our agar, but we decided to change out the tubing on the end of the system and also we autoclaved our water carboys in case that could be a cause of contamination (although it all gets autoclaved again with the media, so it shouldn't matter, but we are desperate and will try anything).

Pouring method:

- We've had four different people pouring, all of which seem to have the same issues.

- We decided to try using a pump to pour for the first time last week. The test batch (2L) looked good. The second batch (3 x 3L) has issues - at least a third of the plates were contaminated, and we're waiting to see if any more have issues. What's extra confusing is that the contaminated plates were from the flask that was poured first, and my coworker did not change the pump's outlet tubing, so we can't figure out how the second and third flasks of plates don't have contamination since they were using the same output tubing as the contaminated flask!

- We tried flaming the top of the plates after pouring (which we do to get rid of bubbles, but we started doing it to all the plates in case it helped). This did not have an effect. The contamination is in the agar anyways, so I didn't expect it to help.

- We've also been using plates from all different manufacturers due to supply chain issues (Fisher, VWR, Corning, etc), and there's no difference, so the contamination isn't from the Petri dishes themselves.

Location:

- When this started happening, I disinfected EVERYTHING in our pouring room - the walls, the ceiling, the floor, everything. I used disinfectant spray and ethanol. I changed the UV bulbs to new ones. We left the UV light on for a couple hours. This did not help.

- I tried leaving some bacterial plates open in our pouring room. 30 minutes of being open did not show contamination (I closed them then let them sit out). When I left them open for 24 hours, I did see some contamination. But when we pour plates, the Petri dishes are open for just seconds, so I don't know how the 24 hour window would correlate.

- We started pouring in different places. We've poured in three other lab spaces, and the contamination seems just as random. We've tried pouring in UV hoods. Still no difference.

- One thing I will note is that we had some summer programs, and some students swabbed the bench top in our plate-pouring room, grew out the bacteria, and sent it for sequencing. It came back as Staphylococcus hominis. Now I will say that we had not done our normal sterilizing procedures (disinfectant, ethanol, then UV light) before they swabbed, and if that is indeed the contamination, I'm not sure why the disinfection methods don't kill it. Also, I'm not sure how that bacteria would get from the tabletop into the plates, and be spread so thoroughly throughout the agar. We constantly ethanol our gloves (and we always wear our gloves when pouring), so it seems unlikely that we're transferring it. And why would it happen now, after years and years of not being an issue?

I need to pour 1500 more lambda plates before the end of the month for students, but I can't keep pouring batches of 300 plates where I throw out 250 of them! If you have any ideas of what I can try or where the problem might be coming from, I'd greatly appreciate any insight!

Hi.

We are working on tree growth/health enhancements and were wondering what sort of bacteria or fungi (or a consortium thereof) biostimulants or biofertilizers (plant growth/health promoting bacteria or fungi) are available for purchase in Europe (i.e. comply with EU standards)? Can anyone help?

Thinking of better way to obtain restriction enzymes.

I tried making MRS broth but it is hard as a brick. Is there any practical tip to make it or it is what it is?

Hi!

Bit of a strange question.

This Amycolatopsis culture has been growing for 7 days, it usually grows white colonies. I have added 100ug/mL hygromycin to check for natural resistance and the culture is turning yellow?

Why would this happen?

Thanks!

I am new to the microbiological field. I understand by using the Colony - Forming Unit (CFU) assay I will get the total number cells in the original bacterial sample. As CFU includes making several dilutions (ex. 10-fold dilution) and plating each dilution. The colonies will grow within 12-24 hrs. Then, I will be able to count the number of colonies. The acceptable plate should have between 30-300 colonies. Then, by using the following formula to calculate the number of cells which is the concentration of the original sample:

CFU/ml = (No.of colonies x dilution factor) / volume of culture plate.

I am planning to test an antimicrobial agent on E. coli MG 1655 (planktonic and biofilm forms). So, I will conduct the CFU before and after the exposure to the antimicrobial agent. So, I have to find the initial concentration to start my experiment with.

So, I could not understand a certain point about the Colony-Forming Unit (CFU) assay, which is many authors in the literature review use cfu/ml unit for the initial bacterial concentration instead of cells/ml which is can be achieved by measuring the OD (very quick method).

I understand letting the colonies to grow will take 12-24 hrs. to let bacteria grow and figure out the correlated appropriate dilution to use it as the initial concentration to start their experiments with.

My question is: How can Scientists use a certain dilution to start their experiments without waiting for at least 12 hrs. to let the colonies grow? For example, in one publication: they mentioned that they measure “the optical density on the day of the experiment was measured and adjusted to 0.15 using fresh LB media which corresponded to approx. 8 Log10 CFU/ ml”.

If crRNA sequences are ~20 nucleotides, the genome of each virus offers a multitude of potential crRNA sequences, yet natural CRISPR systems work remarkably well.

How do bacteria know which 17-23 nucleotide sequence to extract as crRNA?

Other questions: are crRNA sequences often off-target (i.e., incorrectly target non-viruses) or are they usually precise and specific to a given virus? Do bacteria extract multiple nucleotide sequences for the same viruses? Is there a pattern to how bacteria choose crRNA sequences?

Hi all,

Thanks for your help.

Mehrdad

Hello,

I'm going to be attempting to perform a bacteria killing assay using E. coli ATCC #8739. I have performed a bacteria killing assay using this bacteria before, however the previous protocol I used it for started with E. coli in a dehydrated powder and I can't seem to find them in that form anymore (in Australia). However, I can find E. coli ATCC #8739 in capsules where the bacteria is stored on the lid and just needs to be rehydrated (https://www.thermofisher.com/order/catalog/product/R4717050?SID=srch-srp-R4717050).

Having not used this product before I'm curious if anyone else has used them, and could explain in a bit more detail how they work.

For my previous protocol, from what I remember (was 5 years ago), I rehydrated the powder form of E. coli ATCC #8739, plated it on agar overnight, and then used an inoculating loop to take a sample and make a stock solution of desired concentration. Does anyone know in what way these capsules work differently? or do they essentially work the same?

Any further information would be appreciated,

Thank you,

Elliott

Hi! I work with Bacillus sp commercial protease in liquid, but in the bottle is written 16 U/g and I want to know how units contain 1ml of the protease?

Hey guys, super newbie here, I just started micro and "reconstituted" my E. coli in the nutrient broth instead of the tryptic soy broth as instructed in the lab manual (the video just stated nutrient broth and I was following along) I noticed my mistake when I reread the instructions. Will my bacteria still grow in the NB? Thanks in advance!

Hi!

I left an amycolatopsis strain to grow in double autoclaved SFM agar for three days and the agar has turned black?

Anyone know why this might be?

Thanks!

Hi! Our research partner lab sent a strain of coli (that carries a valuable plasmid) to a gene synthesis company that will modify it. That company is in China because... costs.

Then another round of lockdowns happened in China. Are those stabs still good?

(I guess we need to sequence it, if anything is alive in there).

Presumably, they have been stored at room temperature for 2 months.

How long, really, do stabs last? Google says both "up to two weeks at room temperature" , "several weeks if stored cold" , "two months" , and alledgedly, agrobacterium can even survive up to two years in stabs.

I would appreciate hearing your experiences. And I will post if the plasmid is still the same after sequencing.

Our ultimate aim is to grow bacteria and fungi in the same medium (both liquid and solid). We have already tried with czepek-dox medium and czepek-dox medium with trace metal and vitamin solution from the M9 medium. However, only fungi grew well, whereas bacterial growth is poor in both the media. Kindly provide some suggestions with literature (if possible).

Thanks!

Hello everyone

These days, I've been doing some research on how to grow successfully an E. coli DH10B strain by using "common and inexpensive" materials.

The fact, as you may be concerned about, is that DH10B strain is leucine auxotroph.

It's important to say that I'm using M9 media, and therefore this mixture won't provide the previously mentioned amino acid.

My question is, taking into consideration the fact that my bullet isn't enough to get lab grade l-leucine, can I use the leucine which is intended to be used as a dietary supplement?

And one thing more. Is there a big difference if I use l-leucine or d-leucine?

I'd be very grateful to get your help, and even getting some "tips" in order to succeed in this homemade culture.

THIS IS THE LINK TO THE DIETARY SUPPLEMENT I'M THINKING ABOUT

A number of samples were analyzed for isolated colonies of bacteria. However, in more than one sample, Total score was identical between several strains.

How can I choose the right strain?