Bacterial Cell Culture - Science method
Bacterial cell culture is the complex process by which bacterial cells are grown under controlled conditions, generally outside of their natural environment.
Questions related to Bacterial Cell Culture
i have a simple question here. I notice that the diesel biodegradation community has 2 ways of calculating the biodegradation % of a microbial/bacterial culture.
some uses the abiotic control (equation A) in the equation while others use a straightforward equation (equation B)
BE (%) = 100 - (Wr x 100/Wi)
Where, BE (%) is the percentage of diesel biodegradation efficiency calculated in terms of gravimetric difference between the residual mass of diesel in sample, Wr and the residual mass of diesel in negative control, Wi .
BE (%) = (Initial diesel residue - final diesel residue)/Initial diesel weight x 100
can anyone tell me why there are 2 different equations? thanks!
I am to start working on a hydrocarbon(long chains) biosynthesis project and look for methods for the purification of alkanes from bacterial culture.
I would like to measure the zinc content of a bacterial culture supernatant using the spectrophotometer. Any suggestions?
Hello. I am working on a project about wound infection. I want to establish a wound infection mice model. The way I do this is by subcutaneously injecting an bacteria inoculum on the mice dorsal skin. However, recently I found that my experiments were not consistent in terms of skin lesion formation. I did observe skin lesion 1 day post inoculation for the first several attempts, but the lesion formation can not be reproduced in the later experiments.
The bacterial strain I used is E.Coli RP437. The inoculum was prepared as followed: overnight grown bacterial culture in LB medium was sub-cultured by 1：5 and allowed to grow to an mid-log phase (OD600=0.7 in 200uL LB medium). The bacterial suspension was centrifuged and pellet was harvested, then washed with sterile PBS. After centrifugation, the pellet was resuspended with desired volume of sterile PBS to make desired cell density.
A guess: for the first few successful try, I didn't sub-cultured the overnight grown bacterial culture. I guess will this sub-culture be the reason for the inconsistency?
I need to revive the anaerobic bacterial culture of Corynebacterium durum. The protocol recommends using Tryptic Soy Broth and Sheep's Blood. I have a bottle of Sheep's Blood but that has expired in December 2019. I was wondering whether I can still use it?
Thank you very much.
Can we use tap water for bacterial culture? Tap water contains impurities such as calcium and magnesium and their ion traces. So if I use tap water, will it contaminate the bacterial culture or not? Is there any alternative water? Please kindly give an appropriate answer. Thank you.
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?
Hi! I added 10 ml of bacterial culture to my sample and let it ferment and I measured the protease activity for 7 days. How can I know how much is the protease activity in 10 ml?
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
Could anyone kindly share the protocol for determining cytochrome c concentration in bacterial cultures? I am planning to use UV VIS spectroscopy at 410 nm.
Do the cultures need any pretreatment?
How to prepare ammonium sulphate standard for ammonia production test given by plant growth promoting rhizospheric as well as endophytic bacterial cultures.
It should be made in miligram/ml or in micromol/ml?
I usually prepare liquid cultures of S. aureus in sterile Erlenmeyer flasks sealed with aluminum foil, flask volume 4x culture volume (25mL culture in 100mL flask overnight, then diluted to 12.5mL subculture in 50mL flask the next day).
The volume that I require for my experiment is much less (<1 mL), so I was wondering if making the cultures in sterile bacterial tubes (15mL with dual-position cap) instead of glass flasks would affect bacterial growth, in terms of the shape and material of the container?
Please guide on how to determine the changing dissolved oxygen (DO) level of wastewater which has been inoculated with a desired bacterial culture intended for water bioremediation? Should I filter and centrifuge the wastewater sample before autoclaving it and then add the desired culture? How much should be the culture and wastewater ratio? Also for how many days should I take the readings? Any paper or research ideas regarding the same are welcome.
Testing for proteic nature of a putative bacteriocin:
I used 1mg/ml Proteinase K to digest the putative bacteriocine in a cell free supernatent (with unknown concentration, but the supernatant was harvested from bacterial culture with McF3) , then incubated for 3 hours at 37°C , then deactivated it at 100°C for 10min.
I then performed a deep well diffusion agar assay and find out that the putative bacteriocine treated with proteinase K was partially digested with proteinase K: In comparison with the untreated putative bacteriocine , the halo is norrower but still have an inhibition zone.
Can you please help me understand why is the inhibiting activity remaining after proteinase K treatement?
If it is not a protein , why is there a difference between the untreated and treated sample?
any suggestions please?
In what cases does proteinase K not affect proteins?
Thanks in advance.
1) Using a secondary culture for inoculation in a minimal medium.
2) Washing the overnight culture cells and then using them for inoculation in a minimal medium.
3) Using directly the overnight culture for inoculation in a minimal medium.
Question.1) Does washing a culture preferred over using a non-washing of a bacterial culture?
Question 2) Will washing a bacterial culture affect growth pattern?
I am trying to grow M.bovis bcg for electroporation on 7H9 liquid medium supplement with tween80, ADC, sodium pyruvate and casitone or 7H10 solid agar supplement with ADC, sodium pyruvate and casitone. Both medium were autoclaved at 120oC 10min. However, I found that my culture keep on being contaminated. Do you guys know if there is any way to prevent contamination like adding bacterial inhibitors eg. malachite green?
For production of bacterial biosurfactants, it is recommended to add sterile oil to the culture broth (MSM or LB). While the medium can simply be autoclaved, how can the oil be sterilised prior to adding into the broth? Is filter sterilisation the way to go? Or is UV-sterilisation enough?
We have grown some plant endophytic bacteria in TSB medium. Now we are trying to grow them in liquid medium (TSA) for genomic DNA isolation ,some are growing and some are not ,what can be the reason?
While preparing a bacterial culture, if one can provide only one of either controlled temperature or continuos shaking, which one yields good growth of bacteria
I used to work in a separate tissue culture room before, but now have to set the BSC and TC incubators in an isolated (but not closed) area of the lab. It is working well, but now we are going to start doing bacterial work for molecular biology and preparing GST-fusion proteins in the lab (outside the area where the TC facilities are, but still within the same room). Wonder anyone has encountered contamination problem for the cell culture because of growing bacterial culture in the same room? Thanks
I'm determining the phosphate stabilization potential of some bacterial isolate.
Bacterial cultures were grown in NBRIP medium and after centrifugation, I need to extract the P in the solution before proceeding with the test for available P.
Can you kindly suggest the best statistical test to compare the yield of a protein from a bacterial culture carried out in different pH? Is one-way ANOVA a suitable method?
If you are an expert in GC-MS metabolomics I need some help here!
I am doing a metabolomic analysis of a pure bacterial culture using derivatization with MTSFA using Fiehn´s protocol.
I am not very clear about how to prepare the cells for extraction and derivatization. I have a nutritive broth with a high cell count, then I have to somehow "clean it" so I can make the metabolite chemical extraction? or how do you account for the contents of the media on the sample?
In the protocol, it says I need to have a blank with only the media both treated as the samples. But is it not better to clean the sample first somehow?. I am confused.
- The experiment we conducted involved digesting bacterial plasmids from two cell cultures (Bacterial cultures of NFκB2-Ex3-sgRNA CRISPR/Cas9 and pSpCas9n(BB)-2A-GFP)
- Materials used: Plasmid miniprep kit component, FastDigest BbsI, FastDigest AgeI FastDigest Green Buffer (10X), Nuclease-free water and Plasmid DNA, 1 μg
- Incubation: 37 °C in a heat block for 30 min
- Gel electrophoresis: pre-cast 1% DNA agarose gels that contain FluoroSAFE dye, 120 Volts for 40 min.
- Loading amount: 15 μl of digested DNA fragments
- This gel was shared between two groups (we're university students). The first and fifth lanes were 1kB DNA ladder: NEB N3232S) and the streaking pattern was observed in both group's markers and lanes.
- We believe it may be an issue with the electrophoresis machine itself or the pre-cast gel but would like to get a second opinion. Our professor couldn't make heads or tails of it.
I was making DH5a competent cells but I was confused with the condition of bacterial culture in the protocol I followed.
The second step of the protocol is to "Dilute 20 μl of overnight culture into 10 ml of LB broth. Grow at 37◦C with shaking to early log phase (OD600 = 0.3)", which means the dilution ratio of the culture is 1:500. I'm wondering how long should it take to grow a bacterial culture with 1:500 dilution ratio to early log phase? I followed the protocol and it took me almost 5 hours to grow the culture. Is it normal to take really long time like this?
Is the 1:500 dilution an error in the protocol?(maybe it should be diluting 200μl of overnight culture instead of 20μl?) Or this is for particular purpose and I should follow the protocol?
Thanks for answering!
Hi everyone. I know it sounds weird but I was doing a midi (qiagen) prep yesterday and just after the last centrifugation, all isopropanol-eluted DNA just dissapeared. I saw the cloudy phase forming before the centrifugation step and when it finished I literally saw no pellet at all. My best guess Is that pellet was formed but is really llittle and I just didn't see it.Any opinions? I kept the tube at -20°c for trying again on Monday. Thanks again if anyone can share their theory about this !
(The glycerol stock = ATCC Salmonella Typhimurium and is basically aged 3 years)
In all labs I saw, I found that ethanol is used for sterilization of hands etc. in experiments with microorganisms. Why is isopropyl alcohol not used while it is relatively cheaper? Is it harmful for skin upon frequent use?
I need suggestions that is it necessary to to place frozen culture into water bath prior to streak that culture into agar plates??
I need to know the specific column and HPLC conditions required to standardize the protocol.
I have observed lysis plaques on ATCC P. aeruginosa 27853 bacterial lawn prepared from overnight bacterial cultures.
The bacterial strain is reported to contain prophage sequences in its genome.
ATCC website also mentions that bacteriophage may be present in the culture.
Could it be prophage induction (maybe due to stress conditions from high cell densities?) or phage contamination?
Does anyone have this problem before?
Hi, I want to isloate low molecular weight protein with ultracentrifugation, but I'm not sure what is the best way to do. I have a supernatant from bacterial culture and only want to keep low molecular weight proteins (below 15KDa). what is de best protocol to do that?. Thank you
If I want to transport the sterilized laboratory glassware and bacterial culture in petridshes from one lab to another lab of a short distance then what will be the complete best strategy to avoid the contamination??
Today i want to ask about whether there is optimal condition of centrifugation
(speed, time, and temperature) to obtain cell pellet.
I have already searched many protocols, but i only can find those mentioning about bacterial culture.
What i want to ask is about the optimal condition of centrifugation to get pellet of HUVECs. For me, i collect HUVECs to 1.5ml tubes with 1X PBS and I spin down the samples with small centrifuge for about just 30 seconds.
Then i can see the pellet at the bottom.
But i learned that there are numerous conditions of pelleting down cells
so i want to ask whether my protocol described above is ok
Thank you very much!
During screening of microbial extracts for antibacterial activity I keep seeing an unusual effect on the growth of P. aeruginosa (PA), which I hope you can see in the images attached. Basically I am doing disk diffusion assays, so plates are inoculated with bacterial solution matching 0.5 McFarland standard. Whenever I do this with PA I see what looks like viral plaques. I have tried preparing the inoculum using both the overnight growth and direct suspension methods, but still see the same effect. The same thing happens with two different strains of PA, and on two types of media (MHA and BHA). Interestingly, the technician in the lab has tried to replicate this several times without success.
Also, there seems to be a strange effect where there is a halo of these plaque looking things around some of the disks, yet the bacteria are growing within the zone.
If anyone has come across this before, or has any idea, I would love to hear from you!
Hello everyone, my question is how to prepare a bacterial culture for TEM or rather, how to including a sample in epoxy resin?, How can bacteria be connected to each other?
Hi there - I've been trying to optimise total RNA extraction from bacterial cultures, but I keep getting RIN = N/A on the Agilent Bioanalyser and a very sharp peak on the trace (about 25-30s). I still have 2 very distinct 16S and 23S bands. Is this degradation or is this a small RNA peak? Any advice would be greatly appreciated. Thanks in advance!
I recently did a maxiprep using an Invitrogen HiPure MaxiPrep kit with the syringe precipitators and upon completion and spectrophotometer analysis I got yields of 0.9 and 89 ng/uL. I've gone over the protocol several times and examined the expiration dates of the components and found nothing of concern other than the columns and filter cartridges being ~3 months expired.
I can't imagine these components would completely lose functionality just 3 months after the expiry so I was wondering if there are any other possible explanations?
For context I used ~300mL bacterial culture grown in LB + Ampicillin which yielded two large pellets. I don't believe I missed any steps in the protocol. Suggestions?
Hi all. One of my media recipes calls for "clarified rumen fluid." From what I found, that means through cheesecloth. The problem is that cheesecloth is very porous and I am trying to filter out more than that. I have gotten it as far as 3um but cannot get it past that. My project manager would like it to get to at least 1.0um or smaller pore size. I cannot get it to this point. Has anyone worked with this or something similar and been successful? I have tried a syringe and also tried a vacuum. Nothing is working. Any tips would be greatly appreciated.
Hello everyone, I am facing this problem where a few of my bacterial culture are not giving abundant growth as seen previously by the strain (still not identified). I tried growing in the nutrient broth it is growing there but when subcultured on nutrient agar slants the bacteria has very tiny nonvisible colonies. On subsequent subculture, it is nonexistent. I don't want to loose the strains. Please help me revive it.
For the moment, I am using LB broth to enrich the E. coli to get a concentrated stock of phage but I would like to know if ever there are other media that are more apt in yielding a high phage stock.
We need to filtrate bacterial culture media (prior to culture) to remove small particles which are problematic in electron microscopy. We have previously used 0.22 um filters but it would be useful to perform the filtration at 0.1 um. Does anyone have experience in filtering media like MRS broth or GAM broth using a mycoplasma filter etc (0.1 um pore size)?
I am building up some highly diverse phagemid libraries. In order to not make my lab work eternal, I was thinking that instead of plating endless amounts of plates to reach the desired diversity, I could just inoculate a large ON liquid culture for doing afterwards a giga-prep.
My idea would be then to compare the difference in diversity via NGS to see if there is any bias. I know plating is essential for most cloning cases but in my case I am not entirely sure... Does anybody have any ideas and/or experience in this?
Thank you very much!
is it possible to generate anaerobic conditions for growing bacteria without buying those ready-made bags sold by, for instance, ThermoFisher? That is, instead of buying the bags can I use the individual reagents (chances are that they are already in the store...).
Sudan black B stain is used to isolate the PHB producing bacteria from bacterial culture. Kindly give the procedure for the preparation of sudan black B stain
Does anyone could please let me know if there is any special procedure for using cottonseed flour in culture media? It is non-soluble, I don't know if it is used such like that or should I do something before adding it to the culture media.
Thanks a lot in advance
I am going to store fecal samples for culture dependent and culture independent bacterial diversity analysis.
Does anyone have experience in making v583 electrocompetent cells? I'm having trouble making them, if I follow the protocol by Gilmore, the OD has to reach between 0.3 to 0.7 but I can only achieve 0.1 as the highest. How to increase the efficiency? Besides using glycine, I tried lysozyme but it is ineffective. Please help. When I get more than 0.3 after electroporation the bacteria culture are contaminated and I don't know why. Supposingly gram positive cocci, but I've got gram negative for my positive colonies that grow on selective agar plates.
I plan to purify endogenous membrane proteins from bacterial culture for cryo-EM studies, and I am a bit confused with the first centrifugation step described in many articles.
This centrigugation is run generally at 10kg for 10', just after cell lysis. I suppose its purpose is to remove potential cell debrits as we keep on with the supernatent. However I have lots of my protein in the pellet as assessed by WB... Does it mean that my protocol for cell lysis is not efficient?
Thanks for your advices
Is it an effective method to isolate DNA from Gram-negative bacteria with only the use of a boil and freeze-thaw protocol, without any chemical purification? Also, is there a media to maintain the bacterial cultures before DNA extraction, that doesn't affect PCR products?
I want to check my phages against the bacterial biofilm. I have read papers in which they have added phages in a specific MOI (0.6, 1 etc) either in pretreatment or post treatment of phage infection.
I have a question if cfu/ml of bacteria is1×10^12, it means 1ml of bacterial culture contains 10^12 viable cells. MOI will be calculated accordingly. I don't know the no. of bacterial cells in 100 or 150ul.
How much volume of bacterial culture do you pipette in 96 well microtiter plate? I'm a bit confused as 200ul is the total volume you can pour in 1 well of 96 microtiter plate. How to maintain a specific MOI in each well of microtiter plate?
How can i get nonsporulating bacterial bacillus subtillis strain Δ spoIIE without antibiotic growth conditions ?
This ΔspoIIE::erm strain is showing growth conditions with antibiotic erythromycin , but I need wildtype Δ spoIIE without antibiotic growth conditions. Can I order only Δ spoIIE strains if yes then please suggest.
I am facing a problem. I have isolated few bacteria from soil and preserved them in glycerol stock (50 % glycerol v/v). Now when I am inoculating these cultures in nutrient broth and incubating overnight at 30 degree I am getting the growth but when I am streaking the NA plates either using the glycerol stock or the overnight grown bacterial culture I am not getting any growth. Kindly suggest the solution.
My culture medium consists of elements mentioned below, but still the OD doesn't exceed 0.3-0.5!!(on LB the OD reaches 5!)what am I missing?
Di-potassium hydrogen phosphate
sodium dihydrogen phosphate dihydrate
sodium sulfate anhydrous
Di-ammonium hydrogen citrate
Magnesium sulfate hepta-hydrate
Ammonium Heptamolybdate tetrahydrate
Calcium chloride dihydrate
Copper(II) sulfate pentahydrate
Disodium tetraborate decahydrate
Hydrochloric Acid 37%
Iron (II) sulfate
Manganese (II) sulfate monohydrate
Zinc sulfate heptahydrate
For my thesis I am trying to perform experiments with E. coli at 20°C, but I keep running into technical issues and would be very grateful for any advice/experience reports on the matter.
Most of our equipment can only be set to ambient (or higher) temperature, meaning 22°C or more, depending on how many people are in the lab/how much equipment is currently running/etc. Until recently, I grew my bacterial cultures in a shaking water bath set up in our 4°C room and thus able to maintain 20°C. However, the shaking water bath was only borrowed from a neighboring lab group and I had to return it.
Besides this, I would also like to record growth curves of my strains to compare the generation time at 37°C and 20°C. Curves at 37°C were measured in a plate reader, but again this device can only do ambient temperature or higher and belongs to another lab group.
I am a bit at the end of my rope here and would appreciate any new ideas. How do you grow bacterial cultures at 20°C?
Microbiologist’s opinion needed
Short version: We want to study microbiologically influenced corrosion in simulated seawater. How do we cultivate marine bacteria in the laboratory?
my mentor and I have been studying electrochemical behavior of metals and alloys in simulated acid rain. Since we came across alloys (steel and bronze) used in the shipbuilding industry we thought about changing the media for seawater. If we do so, we are obliged to take into consideration microorganisms living in the seawater and participating in direct corrosion processes through the formation of complex bioflms.
We were wondering, is there any “easy” way, or to say beginner cultures to start with? We’re aware of a hustle it may be to get the experiment and the conditions right or take care not to contaminate the working station. What interests us the most are bacteria or algae from Central or Southern Adriatic. We may also order a MIC culture from abroad (sulfate-reducing bacteria is the most popular one atm). Either way, we don’t have confidence in growing them in the laboratory or the right knowledge to set up conditions for the experiment. Thought someone would share some insights and help us plan ahead. Should we try getting pure culture from natural seawater sample or just make an online purchase?
We’re only interested in examining corrosion parameters on the metal surface, but if anyone finds it interesting and would like to contribute with their knowledge more, we’d be more than happy to broaden our horizons and collaborate. I have a Master’s degree in Biology and Chemistry and would love to explore the dynamics of processes and interactions between microorganisms and metal surfaces more.
Lastly, is bacterial culturing even worth trying if we don’t have a Microbiology expert by our side?
Looking forward to the answers,
please feel free to message me if you want to discuss more.
Thank you for your time,
I streaked e.coli from the glycerol stock on an agar plate, and after 48 hrs, I only got three single colonies on the plate. The colonies look fine. However, since the only thing that grew on the plate is these three colonies, I'm a little worried about contamination.
This is my only stock, and I've been trying to revive the same bacteria and prepare some new glycerol stocks for a couple of weeks. But this is the only time that I managed to get results.
Do you think it's a good idea to use these colonies to make new stocks? Or should I continue trying until I get proper colonies on my plate?
I've been trying to recover some bacterial cells by streaking them on agar plates, without success.
The stock has been kept in good conditions. But, there is a white pellet at the end of the glycerol tube, which I'm not actually sure whether it's the bacterial cells or not.
Do bacterial cells form pellets at the end of the glycerol stock tube?
Is there any way to recover them from the glycerol stock?
Could anyone give valuable inputs of this strain nomenclature F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, ΔpotD783::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514?. Actually I need only pot D mutant bacteria having kanamycin resistant!. This strain is mentioned on CGSC Strain as JW1109-1on yale stock culture center.
I can see with bare eyes that my defined medium for bacterial culture has a pretty high opacity, but the OD doesn't exceed 2 at 600nm.I checked the sterility of my glassware and medium, the medium is pretty transparent in the beginning. What else could cause this problem?(I have high glucose concentration and low leucine concentration in my media.)
I am doing phage propagation using s. aureus for the first time but the phage titer is very low! then when I did the EOP no plaques were observed, I use 200 μl of both phage lysate and bacterial culture. Any suggestion for increasing the phage titer?
how to keep the p.acnes samples grow anaerobically? is the anaerobic jar helpful ?
Please state the sources :)
Thank you very much :)
I have a question regarding diluting overnight bacterial culture to measure OD. I diluted my overnight culture with PBS. When I measured OD using spectrophotometer I used plain PBS as blank or PBS with media (same dilution as measured soloution) but I got either negative or zero readings. Is using PBS to dilute overnight culture a good choice, or should I use media for dilution?
Hi. I'm currently working on my final year project, which involves microbiological and protein works.
I got a little confusion on the method to measure OD for my bacterial culture in order to construct a microbial growth curve. I got 2 methods here:
Method #1: Take a sample of 1 mL from the suspension culture and transfer into a cuvette. Then, measure the OD600 against the sterile broth as the blank.
Method #2: Take a sample of 1 mL from the suspension culture and transfer into a 1.5 mL centrifuge tube. Centrifuge the sample and remove the supernatant. Then, wash and resuspend the pellet with 1 mL saline and transfer into a cuvette. Measure the OD600 against distilled water as the blank.
Can someone explain to me regarding this matter? Thank you.