Science topic
Yeasts - Science topic
Yeasts are eukaryotic micro-organisms which belong to Ascomycota and the Basidiomycota phylogenic groups. 1,500 species have been currently described and are estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding.
Questions related to Yeasts
Hello all. I am repeatedly streaking one yeast strain (MATa MAL2-8C, SUC2) in 5-FOA plates aiming to disrupt the URA3 gene. After spreading thrice, the yeast is still able to grow in SD-URA plates. Am I doing something wrong? Do you guys have any tips?
It is my first time trying this widespread protocol to select URA3- cells. PS: I solubilized 5-FOA in DMSO, filtered and added to autoclaved medium (w/ YNB, SD w/aa, glucose 20%, agar 2%) to final concentration of 100mg/mL. Unfortunately I cannot try to disrupt URA3 by using CRISPR in the moment.
Thank you all.
Does yeast grow in MRS agar medium? If so, what might be the reason?

Could anyone recommend a kit for total DNA (genomic and plasmid DNA) isolation from yeast cells? Thanks
I am using the PCR based methods to tag Saccharomyces Cerevisiae strains with antibody and fluorescent epitopes derived from plasmids. This method takes advantage of Yeast's homologous recombination system to integrate DNA fragments into the yeast genome.
Each plasmid region with the epitope also contains a selectable marker for transformant screenings (i.e. URA3/KANMX6). However, when amplifying tagging cassettes from these plasmids using primers with gene specific sequences homologous to the C-terminal regions of my desired genes (confirmed by gel electrophoresis), which then undergo DNA precipitation to be used as raw DNA for Lithium Acetate yeast transformations, only approximately 1/20 screened potential transformant colonies grown on selective media would have the desired edit in the gene I am working with after genomic DNA extraction, diagnostic PCR and agarose gel electrophoresis confirmation.
I have tried to extend the gene specific primer regions to theoretically improve homologous recombination efficiency for efficient transformations, yet my yield of success has been very low.
I am working with a mutant that has been shown to have Homologous recombination hindered to prefer Non Homologous End Joining. However, even in my wild-type strain, it is very difficult to have efficient and successful gene editing in my experience. Also, my negative controls (competent yeast cells lacking the tagging cassete with the selectable marker) also do not have any colony growth as expected so I know my transformations are at least visibly successful.
Has anyone who has used PCR mediated tagging methods found ways to improve transformation efficiency?
Here are the Papers I am using to aid in protocol:
Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., & Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast (Chichester, England), 14(10), 953–961. https://doi.org/10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U
Lee, S., Lim, W. A., & Thorn, K. S. (2013). Improved blue, green, and red fluorescent protein tagging vectors for S. cerevisiae. PloS one, 8(7), e67902. https://doi.org/10.1371/journal.pone.0067902
Please let me know any tips for improvement!
Thank you!
How do you guys count your bacterial or yeast colonies? My fungus grows like yeast with distinct colony dots. I usually rely on the Fiji app(ImageJ) to manually tag and count them. What’s your go to method? 🧫 #colonycount #fungi
I am working on yeast surface display library. After I work with yeasts for a while, I obviously see them at very bad conditions.
If I let the media stand for 1 min, I see cells aggregate very fast, form weird turbid media, and precipitate to the bottom super fast.
I assume it might be because the freezing with DMSO was harmful? But I honestly followed every step from a published protocol, so what could be the reason and what should I do?
We have been performing transformation associated recombination (TAR) in yeast. We believe that some of our yeast colonies (derived from a single cell) are maintaining two different TAR plasmids, one containing a large piece of DNA, around 35KB, the result of a successful recombination event into the plasmid. The other plasmid is empty, because it did not successfully receive a recombination event. It should be noted that the TAR plasmid contains the CEN/ARS origin of replication for maintenance in the yeast cell. The plasmid has a trp1 gene for selection on dropout medium.
Hi all, I plant to creat a yeast mutant with two gene knockout. I want to use homologous recombination method. I found lots people will knock out one gene first, then continue with the second one. Could I knock out these two genes with kanMX and natMX at same time, and screen the positive colonies in media containg G418 and nourseothricin?Thanks.
I've tried DMSO before, but it doesn't work with the high concentrations of beta glucan
if you have any suggestions, I'll be happy to know it
I am conducting an experiment on comparative gene expression in yeasts using the real-time qPCR method. However, I am experiencing amplification in all my negative controls.
I use MilliQ water, have changed my plate and adhesive setup, work with micropipettes dedicated exclusively to qPCR, wear gloves, and perform my work in a cabinet where I turn on UV light for a few minutes before starting.
Does anyone have any tips on where this contamination might be coming from or how to resolve it?
Hello, colleagues.
I am currently working on the production and scaling of biosurfactant produced by Ustilago maydis. The methodology we currently use involves production in YEPS medium (1% yeast extract, 2% casein peptone, and 2% sucrose). We perform the extraction of MELs with a 1:1 ethyl acetate solution in the cell-free medium, and we recover the supernatant through evaporation. Additionally, we have searched the literature for other extraction and purification methods without much success.
Is this toolkit only an extension for YTK (MoClo Yeast toolkit), does it assume that I have this kit and is based on YTK level 0 fragments?
Are there any regular YTK promoters or only the newly added inducible promoters?
I've been working on fungal projects and recently, my petri dish has been constantly contaminated by 'this' thing. I'm not 100 percent sure if it is bacteria or yeast. Someone please tell me what that is and how to prevent it. Thank you so much!

My cells can not survive with these bright spots (20x and 40x)!! I thought it was the yeast but now I don’t think so…. Please help me!





This is a primary glial culture (taken from pups @PD2). Picture taken at 10DIV. Astrocyte layer is completely confluent. We are actually after the microglial layer underneath the astrocytes (Will use Saura et al. mild trypsinization procedure at DIV15). I'm thinking that the smaller cells might be yeast, except some of them have small projections. Thoughts? We are very strict about our cell culture protocols - all culture work is done in a BSC that is rigourously sanitized with 70%ETOH. Anything brought into the hood is sprayed/wiped with 70% ETOH, so not sure how yeast would get into this culture? The only thing I can think of is that during initial harvest, we don't have a downdraft table - it is done in the cell culture room on a counter that is wiped down with 70% ETOH and all instruments are soaked/dried prior to use. Could it be coming in via initial harvest?

why am i getting to see growth of yeast colonies after performing dilution spotting on the respective dropout media when only the empty prey and bait vectors were co-transformed? the yeast strain used was AH109 and the vectors used are PGADC1 and PGBDUC1
Hey, i am working on oral probiotics, using MRS agar for lactic acid bacteria isolation, but now getting yeast contamination with bacterial colonies. how to prevent yeast growth on MRS agar without affecting bacteria?
I have prepared a liquid broth containing Yeast (S. Cerevisiae) that i need to add from it to fermentation media of lignocellulosic hydrolysate.
I need to know based on what parameters do we add milliliters of yeast to fermentation broth?
Good day to everyone,
I have little experience with culturing yeast, so I was wondering if someone could help with an issue I've encountered. I haven't seen many defined colonies from my plates of baker's yeast grown on agar, but I have been seeing these cloudy and spreading colonies of growth, and I am questioning if these were still yeast. Non of my control plates seem to have this issue, only certain treatement plates (the pink-tinted plate is also agar, just with some coloring).
Could this be contamination, perhaps from bacteria or some other fungi?
I appreciate any help, as I would like to avoid this in the future!
Best regards.


I need to perform yeast cell transformation after Golden Gate assembly in a 96 well plate. The cassette will be excised from the vector by RE digestion before transformation and usually, we clean the DNA by precipitation before transferring it into the cells. However, we are trying to avoid this "in between" step and I'd like to know more about alternative and faster purification/enzyme deactivation methods. Or else, what are the chance of success in case I jump straight to the transformation step?
Any suggestion, tips or comment are very welcome.
Thank you :)
I am working on yeast transformation of a calcium channel. I performed twice transformation and no colony was observed after 3 days. The transformation protocol works well before. Is this gene toxic to yeast for the calcium channel activity? If so, How to achieve the transformation?
Hello Everyone,
I've been working with THP-1 cells and recently noticed some blackish clusters in the differentiated cells after 48 hours of differentiation (I've attached images of these differentiated THPs). To differentiate the cells, I use PMA at 100 ng/ml for 24 hours, followed by a 24-hour rest period in PMA-free media. Initially, I suspected a possible yeast infection, but the clusters didn't grow, and surprisingly, they even reduced after 72 hours post-differentiation. To be cautious, I discarded that batch of cells and started fresh.
However, in the new vial I revived from frozen stock, I observed something similar—reddish-black clusters(images of the undifferentiated THPs taken on the day of revival are also attached).
Has anyone else encountered something similar with their THP-1 cells? Any insights or advice would be greatly appreciated.
Thank you!



+1
I want to make some yeast extractions with a protocol that uses Isopropanol and NaOH.
So in the end i will have an azeotrope of Isopropanol and Water that comes from the cell and NaOH.
Can you recommend a protocol to recover the isopropanol - so it can be reused in later extractions again?
The plasmid that was transformed originally is correct (sequenced). I am expressing different gene targets from the yeast genome to optimize my protein. When I sequence the incorrect size, it matches one of my gene targets (also from the yeast genome) , not the one I transformed and it happened with many targets (they all match with one of the gene targets from the genome so this is not an operational mistake. My colony PCR Primers are on the promoter and the terminator and I check all the targets with the same primers. I got many right sizes too (targets from the yeast genome) in the same batch of transformation.
I have change primers and the Tms too, but the gel is either empty or the sequence matches with another target not the one I transformed.
hello everyone, do you have any advice on advice for Agar disk-diffusion yeast method?
like, medium agar to use and how many microlitres to use to wet the disk-diffusion?
thank you all for your help
Image attached is at 20X. Cultured media for checking presence of bacteria and yeast and both tests were negative.

Hello, community,
Could you please clarify whether current legislation permits the reuse of algae biomass after it has been used to treat non-hazardous decontaminated laboratory organic waste?
Specifically, I want to understand any regulatory constraints or guidelines that might apply to this process, even if they are not directly concerned with using algae but other biological means (bacteria, yeast).
Additionally, are there particular conditions under which this reuse would be allowed or prohibited?
Cheers,
Gabriele
I'm planning to perform BSA degradation using YCB and agar, but I need a protocol. Does anyone have one they'd be willing to share?
Hi,
I've recently come across the colony of Geothrichum silvicola (based on sequencing result) isolated from soil with slimy morphology, unlike the colony morphology I've found on the journal below, which is moldy-like. Does anyone have experience with the difference morphology of yeast? and is there any chance the colony morphology of the yeast can change under certain circumstances?
I hope I have been able to convey what I want to ask.
Thank you for your answers.
Journal:
Hi all,
We performed Yeast-Cell-Glucose-Uptake assay to estimate the antidiabetic potential of the test compound.
The Metformin treated yeast cells, resulted in taking up more glucse than normal cells, however, the yeast cells treated with the test compound was observed to contain more glucose than normal yeast cells ?
Has anyone faced this similar problem ? Does it happen that the yeast cells will release glucose into the system than absorbing it.
the reaction was for 1hr in room temperature.
I am already using BL21(DE3)pLysS cells but I am still getting leaky expression in pre-expression samples before IPTG is added. LB broth is tryptone, salt and yeast.
Thanks :)
Is the instant dry yeast, or Brewer's yeast, Saccharomyces cerevisiae, had the ability to provide calcium and nitrogen minerals in addition to ethahnol ? Please support answers with published research.
The yeast K. marxianus is well known for its ability to ferment de disacharide lactose. It is also able to ferment the disaccharide maltose or not? If you can share citations with respect to this issue I will be grateful.
Please ask if you need additional information.
I am new to Nanoformulations. I have done the synthesis of NLC for an antifungal compound using the protocol published in At the end of the process, I got a mixture of milky white solution with solid coagulants. Is this a correct form of how NLC looks? I have also doubts about %w/w calculations. Kindly help me with the calculation
Just revived a sw480 cells from a 2019 batch.
Was normal on the first 2 days. After 5 day, the culture media became cloudy, and lot of suspicious white dots.
Is this yeast contamination, as I have never encountered this before.

I'm currently trying to capture a biosynthetic gene cluster using Transformation-Associated Recombination (TAR) in yeast. After identifying positive yeast clones, I extract, via an alkaline lysis method, the plasmid from yeast and electroporate it into E. coli DH10B.
However, I have not been able to find any positive hits upon cPCR on Eci clones despite testing about 100 colonies using the same diagnostic primers I used to identify the yeast positive clones. Then, on some of the E coli that I pick and miniprep, I consistently only see my original capture vector (with no gene cluster inserted).
The issue may lie in the yeast plasmid extraction, the transformation, or the plasmid isolation prep from E coli. I know that yeast plasmid extractions are hardly ever clean and tend to be "dirty," contaminated with yeast gDNA and other DNA it has inside due to the IPA precipitation required to perform the method. That tends to lead to poor transformation efficiency into E coli. But I feel like I'm stuck going in circles trying to bring the plasmid into E coli and isolating it. if anyone has ever worked with TAR and has experienced any troubleshooting at this stage of the process, I would appreciate insight. Or any advice on things I can try to better improve yeast plasmid extraction or transformation/isolation in E coli. Many thanks!
I tried glycine buffer but the yeast cells acidify the buffer so the pH goes down to 7-6 overnight. I need something that will stay around 9 and that isn't toxic to the cells.
Hello!
When constructing the plasmid, I used the same promoter. However, the direction of each expression cassette promoter in the obtained plasmid is the same, and the plasmid with the opposite direction of promoter cannot be obtained. In this case, I am worried that homologous recombination within the plasmid may occur after integration into yeast, resulting in the loss of some expression cassettes. How should I do toget the plasmid with the opposite promoter direction?
Thanks in advance!
I would like to make solid plates without the metals iron, copper or zinc for growth studies in yeast. I'd like to test the effects of the absence of each of these metals individually while leaving the other two constant. Does anyone have a method for doing this? Normal YPD contains trace amounts of all these metals. I'd like to make something similar to what is found here, but in solid media.
Good morning,
I used TCA/aceton protocol to precipitate proteins. I incubated vesicles from yeast with TCA. After centrifugation 14000xg, 10min at 4°C I saw black pellets in each samples... What could happen? Is there any option to purification such protein pellets?
I'm currently confused on the centrifuge speed needed for separate yeast cells (Saccharomyces cerevisiae) from its aqueous medium
I need the yeast cells to still alive to ferment herbal extracts
Most of the GFP-ATG8 studies use yeast-derived plasmids rather than integrating into the genome. For the ones that integrate GFP-ATG8 into the genome, they are using a commercially available URA3 marker for the URA3 locus, which is not available in BY4741. Is there any other way than making a new plasmid? Thank you!
I'm running RNA extracted from Saccharomyces cerevisiae in a Tapestation 4200. The RINe value is excellent, but the sizes of the 18S/28S bands are lower than expected: ~1000 and ~1800 instead of ~2000 and ~4000, respectively. The internal control (lower marker, 25nt) in each sample ran as expected. Yeast don't seem to have a hidden break in the rRNA.
Has anyone experienced a similar problem?

Hello everyone. I am working on antimicrobial properties of yeast isolated from kombucha. So I chose 2 (out of 4) yeast strains with the highest antimicrobial properties, ran a PCR test and then send my samples in order to be sequenced. The BLAST results showed me Candida parapsilosis. I wonder, is this a relevant finding? I searched and read a few articles about this yeast strain's role in fermented foods, it's potential as a probiotic (somehow?) and it's technological properties in fermentation, especially in fermenting glucose and even findings of Candida spp. in kombucha. I am currently writing my thesis, yet as a Master's student, I am somehow worried and not sure to even report it. I am quite sure of my PCR test, as I saw sharp bonds on my electrophoresis gel and used proper primers (ITS and NL-4). Is this data considered relevant or am I being overly-nervous?
Thanks a lot in advance!
Iam trying to optimize the Comet assay protocol for Yeast cells in my lab but iam unable to lyse the yeast cells with the regular protocol and hence those cells are seen intact even after the treatment. I looked upon some protocols which suggested the use of Zymolyase/ Lyticase for the Yeast cell wall lysis, but my lab doesnt have either of these enzymes and hence can someone suggest any other enzymes apart frombthe above mentioned that I can use for the same? Also can I use Lysozyme for digesting the Yeast cell wall?
Yeast from a small scale fermentation was heat inactivated and stored in a refrigerator after centrifugation - about 60-70% water content.
The Yeast was planned for lipid/sterol extraction. Sadly i got sick for about a month before i could do the extraction.
Now the refrigerated sample is moldy.
Do you know what influence mold can have on such a sample - if it may be still usable after removing the mold, what the mold may have used/altered as food source to grow?
As to get to this point was quite time consuming I would prefer not to need to repeat the experiment to this point - and maybe find a way to save the sample.
I found a paper investigating the Effect of bug damage and mold contamination on fatty acids and sterols of hazelnut oil (DOI: 10.1007/s00217-016-2778-x)- where the lipids/sterol decreased. Not sure if this is similar in a liquid solution with yeast settled at the bottom.
Do you have any experience and/or advice for such a situation?
Any tip and help is much appreciated
What solvent should I use for the yeast cells? Precautions to take when preparing the solution (pH...)? concentration range for non-toxic efficacy?
Hi everyone!
I would like to start researching the response of human mast cells to yeast. Which commercial human mast cell line do you think is the best? I have read that LAD-2 is difficult to passaging and ect. What do you think about HMC1.2 and LUVA?
Hello.
I have a question about these two behaviors, the Warburg and Crabtree effects. I am a master's student and my teacher said to me that Warburg occurs in cancer cells and Crabtree in yeast, basically that the Warburg effect on cancer is analogous to the Crabtree effect on yeast. However, I see some scientific articles that say that cancer cells have both. In yeast, I understand that high concentrations of glucose inhibit oxidative respiration. I am confused. Can someone explain this to me? Cancer cells only exhibit the Warburg effect or also exhibit the crabtree effect?
Thanks
I am relatively new to conducting Western blots. I employed the alkaline lysis method to extract whole proteins from yeast. Subsequently, I introduced the Opti Protein Marker (ABM, CAT log NO: G252) and completed the gel electrophoresis. The proteins were then transferred to a nitrocellulose membrane using the wet transfer method. Following the transfer, I performed a Ponceau staining, during which the protein markers were clearly visible.
Moving forward, I proceeded to block the membrane for 1 hour in 5% BSA in TBST (Initially, I attempted blocking with 5% non-fat skimmed milk, but encountered high background signals). Subsequent to blocking, I washed the membrane with TBST (3 x 7 mins) and TBS (1 x 5 min). Following this, I incubated the membrane overnight at 4 degrees Celsius with a primary antibody (Beta-tubulin, Rabbit IgG Polyclonal), 1:1000 dilution in 5% BSA in TBST). Afterward, I repeated the washing steps with TBST (3 x 7 mins) and TBS (1 x 5 min).
For the next step, I incubated the membrane with a secondary antibody (Rabbit anti-Goat IgG (H+L), HRP, Polyclonal, 1:10,000 diluted with 5% non-fat skimmed milk in TBST) for 3 hours. Subsequently, I performed additional washes with TBST (3 x 7 mins) and TBS (1 x 5 min). Finally, I carried out chemiluminescence detection with an exposure time of 30 seconds.
However, my results were unsatisfactory as I observed multiple nonspecific bands, and the protein marker disappeared. I seek assistance from experienced researchers, especially those familiar with Western blotting of yeast proteins. I would appreciate any insights or suggestions to identify and resolve the issues.
Additionally, please refrain from suggesting the use of monoclonal antibodies, as it is currently beyond my budget constraints.
Thank you in advance for your help.

LAPT broth: 1.5% Bacto Peptone, 1% Bacto Tryptone, 1% yeast extract, 1% glucose and 0.1% Tween 80
I have a query about carabcillin antibiotics is good option to put it in ypd agar for growing yeast or is it possible to grow them without antibiotic?
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)
I've been trying to isolate a low copy (CEN/ARS) plasmid from yeast that has selective markers for both yeast and bacteria (Trp and Amp). However, when I try to recover the plasmid I can't get any positive bacterial clones when transforming the DNA. Running the DNA on a gel it looks like I mostly isolate genomic DNA.
Does anyone have experience with or can provide references to studies that have employed the RNA Plant Micro Kit for RNA extraction from yeast cells?
Looking for a straightforward approach to isolate nuclei that does not require the use of an ultracentrifuge... we generally work with mitochondria, however, we are interested in examining some nuclear proteins. Our crude nuclear pellets from our mitochondrial isolations appear to be contaminated with broken mitochondria. Any suggestions would be greatly appreciated!
I was just thinking could it be possible just because 2- 5 hr storage of my yeast cells before soptting could switch to anaerobic or post diauxic shift
I am studying the phagocytic activity of frog blood cells. After incubating frog whole blood with yeast cells, I had this microscopic image; Could be possible for frog erythrocytes to engulf yeast cell?

I have disrupted yeast gene in genome now I want to verify it without delaying it for overnight culture and then genome isolation so can I directly check it (gene disruption )from transformed yeast colony plate by colony pcr?
Kind and warm greetings to all
I'm working on Antifreeze proteins(AFP) that is expressed by Yeast So which method \ assay is more reliable and effective to detect AFP by western blot or ELISA with references ? and why? Kindly if anyone working now or previously on Antifreeze protein can you guide me.
Protocol used for transformation (TAKARRA)
Upon linearization of plasmid DNA for electroporation mediated transformation in yeast, is it necessary to precipitate out the DNA from the restriction reaction via alcohol (With 75% ethanol). Isn't it possible to just deactivate the restriction enzyme via heat treatment and proceed towards transformation using the same restriction mixture?
I did yeast two hybrid transformation a few days ago and no yeast colonies grow in the DDO+ 225 AbA plates after 3-5 days. OD600, carrier DNA, competent cell, as well as the amount of AD and BD plasmid were correct according to TAKARA manual. Can anyone know the reason why no colonies could grow in the plate?
Hello. Hopefully, everyone is doing well. I usually use the Pichia protein expression system to express my recombinant protein. Every time working with this system was so easy for me and had no problem at all but for the last two months whenever I try to express the same proteins in this system, after purification i notice lots of DNA contamination that cannot be removed from my protein sample. I want to know if anyone faced the same problem before as I do not understand what has been changed in my procedures which make me face this huge problem. If anything you can mention which may help me is really appreciated. (I also tried so many wayed to get rid of this contaminant but was not useful) THANKS

Hello,
I'm working on an optimization experiment related to yeast growth and the best conditions for growth. Optical density is a measure of yeast growth, and from the literature, this yeast strain grows best at 30 degrees Celcius. Suprisingly the results have shown that optical density is higher at a lower temperature, around 20 degrees. What statistical test should I use if the null hypothesis is that the highest optical density is achieved at a temperature of 30 degrees Celcius?
Please be kind. I have minimal background in stats; I'm just a lab tech. Thank you
So far I have seen that organic acids are used to kill off spent yeast from the brewing industry before being fed to animals such as pigs. The amount of acid needed to achieve a satisfactory kill off seems to be quite cost prohibitive so I was wondering why I have never seen formaldehyde containing products being used for the control of yeast in animal feed in regions where formaldehyde is allowed? Thanks
We are working with Pichia pastori and must take our yeast to another country. What are the considerations for the transportation? I mean, besides the air company considerations.
Dear ResearchGate Community
Which type of yeast does exist in this field?
Is there still a possibility of yeast contamination despite the continuous use of 70% ethanol and Bleach 10 % ?
What can be done if such contamination is observed?
Thank you in advance for your time and consideration.

I'm working with a Saccharomyces cerevisiae strain that tends to form big cell aggregates. In order to take this strain to a flow cytometer, I need to get single cells, but although I've used up to 250 mM of EDTA and strong mechanical forces, I've been unable to disaggregate the yeast's chains. Does anyone know a way to make cells separate?
