Science method

# 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.
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Hi there,
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)
Equation A:
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 .
Equation B:
BE (%) = (Initial diesel residue - final diesel residue)/Initial diesel weight x 100
can anyone tell me why there are 2 different equations? thanks!
Diesel per se is a complex mixture. "% biodegradation" can be misleading as some fractions may be readily metabolized and some not.
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I am to start working on a hydrocarbon(long chains) biosynthesis project and look for methods for the purification of alkanes from bacterial culture.
8/5/22
Dear Wubishet,
What is the nature of the hydrocarbon(s) you wish to purify? Are they completely immiscible in water? Are they charged or neutral molecules?
You may be able to purify the HC's using solvent extraction, e.g., hexane, iso-octane, etc. You will first need to remove the cells from the culture (e.g., centrifugation). If the HC's are charged (e.g., contain carboxyl groups), you will need to eliminate the charge or the HC's will be hydrophilic. E.g., in the case of carboxylate residues, acidification will protonate the carboxylate to produce neutra l-COOH groups, making the HC's hydrophobic.
I hope this information helps you.
Bill Colonna
Iowa State University, Ames, Iowa, USA wcolonna@iastate.edu
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Hi everyone!
I would like to measure the zinc content of a bacterial culture supernatant using the spectrophotometer. Any suggestions?
Thanks!
Anna
Thanks Jorgaq, but I was looking for something cheaper than a kit.
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My lactic acid bacterial cultures that were growing a month or two back isn't growing now. I am unable to subculture it from glycerol stock stored at -20°C. How can i revive it?
How old is the media you're subculturing it to? Some people in our lab thought our Enterococcus faecalis freezer stocks were going bad but we eventually figured out that E. faecalis stops being able to grow in BHI once it's been left at room temperature for about a month. That stopped happening when we started putting prepared BHI in the refrigerator for long term storage. It might be worth remaking your media if you haven't already - lactic acid bacteria tend to be fussy about their nutrient requirements.
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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?
In other, most bacteria can be measure with microscope
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Hello,
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.
No, if you use expire sheep blood then you can’t get your desire results. Thus my recommendation to collect fresh blood.
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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.
Tap water also has pharmaceutical residues and metabolites. I don't know what is the status in your country but in Kuwait, we do have very reliable data that we have often at trace levels.
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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!
Tey one control without inoculum, so that you can find whether it is because of inoculum or agar,
I too faced this kind of issue I found some white color spots after, Instead of troubleshooting I opened a new agar jar and compared the presence and absence of the spots and processed with the new one,
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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?
Hi!!!!
Please do standard for determining protease activity with a substrate and compare your bacterial culture's protease activity
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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.
My question is 'why bother'?
M9 is not necessarily cheaper than LB and alike. The main benefit is it is a define media so you have a more consistent growth. If you want to better understand the issues with LB see this post by Hiroshi Nakaido. A text I will recomend any microbiologist/biochemist
After you read this decide what are you trying to do - overexpress protein? produce single cell protein (and from what)? study metabolism? demonstrate bacteria growth for high-schoolers? DIY molecular biology? depending on what you are trying to do you will need to the correct media and the decision need to take much more into account then "does it have Leucin".
The following paper has media recipes:
Also many webpages have such - simply google "E. coli media recipes"
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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?
viable count
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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?
Thank you soo much Sir@Prem Baboo
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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?
In general you will get better aeration in flasks than you will in tubes, which may or may not make a difference for your experiment. This of course will depend upon the volume of culture broth relative to vessel size and the rotation speed. But growth in tubes is fine for many purposes.
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I'm trying to extract plasmid DNA of e.coli and I'm growing a pre-culture in LB agitated medium at 37ºC. I should have an OD(600 nm) 2,6 to start another culture but it only reaches OD 1,16 and then starts to decrease. What should I do?
Hi Carolina,
In addition to Benedik's answer, after the stationary phase the bacterial growth enters the death phase. So the OD600 reaches until 1,5-1,8 and then declines. Please measure OD every 30 min to get a growth curve and then indicate the culture time for highest OD. Actually, you don't need to arrive OD 2,6 to start another culture. An overnight culture (about 16 hours) is enough to reculture for extracting plasmid DNA. An important point is that what is the inoculation amount to be added into the LB medium. For example, I add 10 microliter thawed E. coli culture to the 100ml LB medium and incubate it for overnight.
Have a good study.
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Many biochemical tests of novel bacteria characterization, will use tween as one of the carbon sources (tween degradation).
Why is tween significant in bacteria biochemical characterization?
Tween (mostly Tween 80, an anionic detergent/surfactant) is added to the bacterial culture media (in this case the novel bacteria) because of many reasons:
1. Check the carbon sources utilized by the bacteria
2. If checking for hydrocarbon-degrading bacteria, it can be used as one characterization.
3. Tween 80 provides bacterial cells with exogenous fatty acid (oleic acid) to protect against adverse environmental conditions.
4. During xenobiotic compound degradation Tween increases the solubility of compounds in the aqueous phase which are otherwise less soluble when used without a surfactant.
Depending on the background and hypothesis of the studies you can use the characterization accordingly.
Hope this helps.
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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.
Kailash Srivastava and Shuraik Kader Thank you so much.
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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.
If it is not a protein , why is there a difference between the untreated and treated sample?
In what cases does proteinase K not affect proteins?
Abdelahhad Barbour Thanks for the idea !
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I am working on bacterial secondary metabolites so I need to know about their isolation method from liquid culture.
Hi,
After you grow the bacteria in liquid media, centrifuge them and save the cell pellet and supernatant. Find a solvent in which your secondary metabolite is soluble, but is not miscible in water (Butanol, ethyl acetate, chloroform, or dichloromethane are good places to start). Then:
1. Add the solvent to the liquid culture at a 1:1 ratio and shake at room temperature for 1 hr.
2. Allow the mixture to separate into phases (you can centrifuge the sample to speed this up)
3. Collect the solvent and evaporate to a workable volume.
4. Analyse your sample by LC/MS or thin layer chromatography.
You will likely get a large range of metabolites in the crude extract, which you can separate with a column if necessary. You can also do an extraction on the cell pellet using the same method, though you can use methanol or another miscible solvent because you will not have a large amount of water in the cell pellet.
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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?
In case you wish to raise a secondary culture (in a minimal medium) from a primary culture (grown in a nutrient-rich medium, eg: Nutrient broth, Luria broth, etc.), you should wash your cells before inoculating them. This is done to remove the spent (used up) media from the primary cells or any metabolites or nutrients that would contaminate your minimal media.
Whenever we shift down the culture (transferring cells from a nutrient-rich to a minimal or nutritionally-low media), it is recommended that we wash the cells before inoculating them to raise the secondary culture.
Washing will not have any impact on the growth pattern. But, instead change/shift in media will cause a definite and notable change in the growth pattern. Shifting down would certainly extend the lag phase of your culture. If you wish to perform any growth-related experiments, using a common media for raising primary and secondary cultures is advisable. This would not extend your lag phase and your cells will begin to grow quickly.
Good luck!
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Dear All,
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?
Thank you.
Ellen
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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?
12/23/21
Dear Sayak,
I agree w/ Phil Geis' answer. Filtration will sterilize the oil. However, most oils are thick and viscous, so running them through a sterile filter will be time consuming. Autoclaving is a better way to go.
I hope this information helps you.
Bill Colonna Ames, Iowa, USA williamjcolonna6@gmail.com
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I wanted to study the functional groups attached on bacterial cell via FT-IR. I am unsure of the procedure regarding the sample preparation for it. How do I make kbr pellet from bacterial culture?
Mixed culture estimation -
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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?
Haoyang Zhou I suspected oxygen, too. I tried double agar overlay and it seemed to have solved it partially. However, not good enough. I think there is more to it than just oxygen. My bacteria was bloodborne pathogen so I am also thinking the environmenta pH may also play a role. In TSA, the microenvironment of even several bacteria may be changing enough to inhibit growth. However, in TSB buffering capacity is larger thanks to diffusion. I even considered of using blood and the buffer that are used to cultivate Bordetella pertussis, but it is a bit expensive for me currently.
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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
Read above mention two papers, hope it will be helpful to conclude the importance of bacterial growth factors.
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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
Aerosols generated e.g. by pipetting and (shared) centrifuges might pose a problem. Use designated sets of labware and establish a protocol to minimize contamination risks, e.g. by doing routine tissue culture before bacterial work, cleaning surfaces of the bench and frequently touched objects, as e.g. incubator doors) changing gloves, etc. Make sure to educate all people properly, esp. those who are new to the matter.
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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.
@ Michael, I think Ascorbic acid may be alternative for Antimony Potassium Tartrate.
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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?
There are many tests, have you tried a simple means comparison test?
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Hi,
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.
Thanks,
Cleaning the cells means centrifuging the cells and washing them with a simple buffer system (e.g., phosphate-buffered saline). The less components in the wash buffer the better. The problem with this, however, is that the metabolic composition of the cell changes pretty quickly and you are effectively stressing the cells by doing this. You also want to avoid heat or cold shocks so the buffer should be at the same temp as the growth media. Snap freezing the pellet in liquid N2 or dry ice after the wash helps preserve the metabolites.
The issue with the derivatization will be what in the buffer system also gets derivatized. Simplifying the wash buffer helps with this, but you should be aware if there is any carryover derivatives in the wash buffer. The concept of a control is more related to comparing the metabolome between growth conditions. LC/MS is not quantitative because of ionization differences between individual molecules. Therefore, when you are comparing LC/MS results between two culture conditions, all you get is a ratiometric measurement of what went up and what went down. You don't get an absolute concentration. You can only get to an absolute concentration by spiking samples, preferably with stable isotope versions of the metabolites that you want to track. see:
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• 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.
- 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.
Hi Christian,
I suggest you to take just half of the volume per well of gel. In addition always run your gel between 70-80V range. One more thing i wish to add is monitor the buffer temperature, above 40 degree C may hamper the integration of gel subsequently resolution may affect.
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Hello,
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?
The precise dilution factor does not matter. It does seem slow. Something is probably suboptimal. It can still work fine. Try them out.
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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 !
It May be a reagent problem. You can troubleshoot the purification by taking an aliquot of every binding and wash eluate from the column. Precipitate a sample of each using iso propanol and you should see which step is not behaving as it should and you can investigate the reagent concerned
It is possible that you have a small and invisible precipitate so dissolve the residue that you have and measure its OD260. Perhaps you simply do not have much plasmid
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(The glycerol stock = ATCC Salmonella Typhimurium and is basically aged 3 years)
Hello
Usually bacterial glycerol stock is stored at - 80 degree C for long-term storage (many years). In this case, the probability of bacteria reviving is less because the glycerol stock has been stored at - 20 degree C for 3 years.
Nevertheless, you can revive bacteria from the glycerol stock by placing the frozen tube on ice, and with the help of sterile loop, pipette tip or toothpick scrape some of the frozen bacteria from the top and streak the bacteria onto LB agar plate. Grow the bacteria overnight at the appropriate temperature. You can then isolate single colony and inoculate in broth medium. This is done to remove any contaminants that could be present in the glycerol stock.
Care must be taken to see that the glycerol stock does not thaw.
I hope this helps.
Best,
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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?
The World Health Organisation recommends the use of disinfectants on hard surfaces and hand sanitizers. The formulations that offer the best results are alcohol-based disinfectants. We use Ethanol and Isopropanol (IPA) in our hand sanitizer formulations. These two alcohols are equally effective when it comes to killing bacteria, but to understand the differences between them kindly check:
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I need suggestions that is it necessary to to place frozen culture into water bath prior to streak that culture into agar plates??
Although I did not freeze the bacterial culture to such a degree, I think the principle is the same...First, the thawing should be gradual so that it is not left at room temperature, but first in the freezer of the refrigerator for 1 hour and then transferred to the refrigerator 4 ° C overnight, with the preparation of fresh media, preferably the same media that was used in isolation, and if the media that was used previously was a selective media, do not add the selective supplement, because the bacteria are stressed and then after thewing transferred from the broth, a 2 loup full, streaked on agar and incubated with the same previous conditions ...And then if you want to freeze again, it is preferable to prepare double-strength broth to preserve these bacteria for a longer period, and medical glycerol is added to it by 20%. I have used this method many times with Campylobacter and it was very satisfactory although Campylo is a very sensitive and fastidious microorganism... As for putting culture in the water bath, I do not advise that because you may expose it to shock due to the great difference in temperature...I wish you good luck
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I need to know the specific column and HPLC conditions required to standardize the protocol.
Here are some articles for your reference:
Removal of B. cereus cereulide toxin from monoclonal antibody bioprocess feed via two-step Protein A affinity and multimodal chromatography
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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?
I don't know about ATCC 27853 specifically but it's pretty common for a lot of Pseudomonas aeruginosa strains to spontaneously form iridescent plaque-looking areas. A lot of older papers talk about it, ex:
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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
Hi Miriam. I am currently working on protein purification and I use centriprep centrifugal devices, which you can buy here (https://www.sigmaaldrich.com/US/en/substance/centriprepcentrifugalfilterunit1234598765?gclid=CjwKCAjwlYCHBhAQEiwA4K21m7zoy6w8Qsiub-mIiySPQKaDbYAx-0oV1bSY1ERlRHlPU3wKD9GTrBoCW20QAvD_BwE).
Any proteins smaller than the molecular weight cutoff are collected as flow through, or the solution that passes through the membrane and is consequently not concentrated by the centriprep device. You could then concentrate this flow through using a SpeedVac or anion exchange.
Hope this helps!
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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??
When transporting glassware from one place to another, I do not think that you can avoid contamination 100%, but as much as possible it can be reduced by wrapping these utensils with sterile cellophane and transporting them with the dishes in a sterilized container, knowing that it is better to transfer bacteria through transport media. In general, if you use special selective culture media, you do not need to worry, because such media will prevent the growth of any species other than the one you are investigating.
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Hello
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!
Hello
Using 400g for 5 minutes in tubes of 15 and 50mL I have been able to form pellets of cell lines and tissue cells in suspension without problems. To pellet 96 well plates, I have used 900g for 2 minutes.
Best.
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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!
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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?
Have a look at attached TEM image of bacteria sampled indifferent media. The image is from the following publication:
You can consult authors of the manuscript for details of methodology as their paper does not reveal much.
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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!
Hello Leanne Cleaver, we had used the Bioanalyser for checking the RNA quality of DNA chips (Affymetrix). I never did these analyses for my own, but have also seen degraded RNA. Then the two peaks are lower or not visible within a lot of other peaks. I dont know the exact definition of the peak at 25-30s, but be aware, that you can see all RNA here.
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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?
Not totally familiar with this kit but since most of Maxiprep kits work on the same principle I can try to point out one possible mistake.
After filtrating your Lysate/Precipitate on the syringe you have to add a binding buffer to your filtrate before to apply it on the DNA binding column. Did you do this step ? Omitting it results in very poor binding of the plasmid DNA to the silica column and ends up with super low amount of purified DNA
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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.
Did you think about centrifuging your rumen fluid at 4 C at about 5000 rpm for about 30 minutes? .Then the supernatant can be taken and you can filter it again later if you want using a syringe filter
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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.
I agree with Dr. Kien T Nguyen 's answer. As your isolate is growing well in your broth culture you can first store it at -80C. For that, you can add 500 microliters of your culture broth and 500 microliters of 10-15% glycerol into cryovials and store. Then you can try changing the growth parameters of your solid medium. Try to use proper incubation temperature, specific growth requirements. As Dr Kien said it is likely that your culture has specific growth requirements to grow in solid media. Moreover, when you grow isolates in broths you shake it for aeration which usually enhances the growth. That might be another reason, its aeration needs. By the way, it is much better to identify your isolate after growing in broth. When it is identified, you can provide the specific growth requirements on a solid medium.
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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.
Thank you so much for the answer Michael J. Benedik . I shall take note and apply to my research.
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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)?
Biological systems can be filtered in all size ranges, with predictable molecular sizes and weights retained or passed through each. The manufacturer of filters can give advice.
You might consider centrifuge to remove the particles, if there is a small density difference.
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Hi everybody,
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!
The problem primarily arises if clones (or phages) have unequal growth rates then you will amplify any bias you have. When you go through the single colony phase although there may be size differences with an overnight growth on plates there is ample opportunity for the slower growers to somewhat catchup with the faster growers. But this is much less true in liquid. But if everything is growing at the same rate then it might not matter so much.
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Hello,
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...).
Thank you
As Sandhye said for your situation candle-jar is the best solution (like the uploaded photo) but still for solid media like agar plates you can use a thin layer of sterilized liquid paraffin or glycerol on top of ur culture media to generate an anaerobic condition.
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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
kindly check
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Hi everybody
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 for the info!
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I am going to store fecal samples for culture dependent and culture independent bacterial diversity analysis.
Please use filter paper for LONG TERM STORAGE
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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.
Liannie,
The protocol of Friesenegger et al. (1991), which depends on repeated glycerol washes, has been used by other groups with success.
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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?
Since you are not overexpressing the protein, the only reason why it would be found in the low-speed pellet is that you did not adequately disrupt the cells. After a really thorough disruption, such as can be obtained by 2 careful passes through a French press at 18,000 psi, there is hardly any low-speed pellet. The membranes can then be pelleted by high-speed centrifugation.
When resuspending the cell pellet in buffer prior to disruption, you should include lysozyme, DNase and Mg2+. The lysozyme breaks down the peptidoglycan cell wall. The DNase + Mg2+ breaks down the DNA, reducing the viscosity of the lysate. You can also include RNase, optionally.
This method of purifying the cell membranes by centrifugation before dissolving them provides a substantial first step of membrane protein purification by removing all of the cytoplasmic protein.
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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?
Dear Natalia,
1. Pick up a single fresh colony grown in freshly prepared agar medium.
2. Suspend it in 2-3 ml of suitable broth and incubate it at 37 °C for 2-3 hours with vigorous shaking.
3. After incubation, centrifuge at maximum speed at for 5-10 minutes and discard the supernatant.
4. Re-suspend cell pellet in 50 μl of 10 mM EDTA and heated at 95-100 °C for 5 minutes.
5. After heating, the lysate is vortex and briefly centrifuged.
6. 1-2 μl of the lysate is taken and used as template for PCR in 50 microleter PCR reaction.
Good Luck
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What would be the perfect protocol to perform this test signifying the positive and negative results?
Gelatin Hydrolysis Test – Principle, Procedure, Uses and Interpretation
Gelatin is a protein derived from the connective tissues of vertebrates, that is, collagen. It is produced when collagen is boiled in water. Gelatin hydrolysis detects the presence of gelatinases. Gelatinases are proteases secreted extracellularly by some bacteria which hydrolyze or digest gelatin. The production of gelatinases is used as a presumptive test for the identification of various organisms, including Staphylococcus sp., Enterobacteriaceae, and some gram-positive bacilli.
Principle
This test is used to determine the ability of an organism to produce extracellular proteolytic enzymes (gelatinases) that liquefy gelatin, a component of vertebrate connective tissue.
The reaction occurs in two sequential steps: in first reaction gelatinases hydrolyze gelatin into polypeptides and then polypeptides are further converted into amino acids. The amino acid is taken up by the cell and used for metabolic purposes.
The presence of gelatinases is detected using a nutrient gelatin medium. When an organism produces gelatinase, the enzyme liquefies the growth medium by hydrolyzing gelatin present in the medium.
Media:
Nutrient gelatin medium
Enzymatic digest of gelatin (5 g), beef extract (3 g), gelatin (120 g), per 1000 mL, pH 6.8.
Method
There are several methods for determining gelatinase production, all of which make use of gelatin as the substrate. The standard and most commonly employed method is the nutrient gelatin stab method.
Inoculate the gelatin deep with 4 to 5 drops of a 24-hour broth culture.
Incubate at 35°-37°C in ambient air for up to 14 days.
Note: Incubate the medium at 25°C if the organism grows better at 25°C than at 35°C.
Alternatively, inoculate the gelatin deep from a 24-hour-old colony by stabbing four or five times, 0.5 inch into the medium.
Remove the gelatin tube daily from the incubator and place at 4°C to check for liquefaction.
Note: Do not invert or tip the tube, because sometimes the only discernible liquefaction occurs at the top of the deep where inoculation occurred.
Refrigerate an un-inoculated control along with the inoculated tube. Liquefaction is determined only after the control has hardened (gelled).
Nutrient gelatin plate method
Stab-inoculate a heavy inoculum of an 18- to 24-hour-old test bacteria onto culture plates prefilled with nutrient gelatin (23 g/liter nutrient agar, 8 g/liter gelatin).
Incubate inoculated nutrient gelatin plates at 35oC for 24 hours.
Note:
In some cases, plates are flooded with saturated ammonium sulfate to precipitate unhydrolyzed gelatin, making the clear zones easier to see. Results are often observed within 5 to 10 minutes after flooding with saturated ammonium sulfate.
Expected Results
Positive: Partial or total liquefaction of the inoculated tube (the control tube must be completely solidified) at 4°C within 14 days. On plates, gelatin hydrolysis is indicated by clear zones around gelatinase-positive colonies.
Negative: Complete solidification of the tube at 4°C. On plates, no clear zones around colonies are observed.
Gelatin Hydrolysis Test Tubes
Gelatin hydrolysis. A, Positive; note liquefaction at top of tube. B, Uninoculated tube.
Gelatin Hydrolysis Test Results
Gelatin hydrolysis. A, Positive B, Negative
Uses
This test is used to determine the ability of an organism that produce gelatinases.
This test is helpful in identifying and differentiating species of Serratia, Proteus, Bacillus, Clostridium, Pseudomonas and Flavobacterium.
This test differentiates pathogenic Staphylococcus aureus which is gelatinase-positive from non-pathogenic epidermidis which is gelatinase negative.
This test can be used to differentiate Serratia and Proteus species which are gelatin positive from other members of Enterobacteriaceae family.
Bacillus anthracis, B. cereus and several other members of the genus are gelatinase-positive, as are Clostridium tetani and perfringens.
Limitations
Some organisms may grow poorly or not at all in this medium.
Gelatin is liquid above 20°C; therefore determination of results must be completed following refrigeration.
Gelatinase usually acts at the surface of the medium. Shaking the tube while it is warm may result in false-negative interpretation.
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Hey,
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?
Reduction of CFU may or may not have any correlation with biofilm reduction, it all depends upon the protocol you are using as to whether you are really reducing biofilm or merely reducing planktonic cells.
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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.
Hope this can be useful.
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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.
Hi Nidhi
From your question it is not clear that which bacteria you are trying to culture on nutrient agar medium. As you know certain bacteria could not give a good response on NA. To get the growth of those bacteria you need to modify the media composition.
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I want to extract plasmid dna from bacterial culture guide me what will be the most appropriate method.
Save money and time and go with the commercially avaliable kits. In addition , as Balamurali Krishna Vasamsetti said , the quality of isolated plasmid DNA is high and therfore it is suitable for various downstream applications such as sequecning, transfection and restriction digest.
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Zone of Inhibition
Prasun Kumar .-. I am not sure that I understand why you are saying that there will be greater diffusion from a larger disc. Could you please explain this. It seems to me the issue of diffusion is related to the amount of antibiotic on the disc, as diffusion moves from high concentration to low. The same amount of antibiotic on a larger disc would thus be more 'diluted' on the disc and thus it seems that there should be a lower ratio, and the distance from the center of the disc to the outside edge would reduce the amount of antibiotic diffusing out from the edge. Please let me know of any research related to this. Thanks!
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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?
Ammonium chloride
Ammonium sulfate
Di-potassium hydrogen phosphate
sodium dihydrogen phosphate dihydrate
sodium sulfate anhydrous
Di-ammonium hydrogen citrate
Glucose
Magnesium sulfate hepta-hydrate
Thiamine Hydrochloride
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
DH5alpha does not require arginine to be added. The argF gene, which is deleted, is a duplicate gene so is not essential. Assuming you have the right concentrations for your chemicals and are following a recipe for a standard medium, then check the glucose concentration you are adding and also the pH. Both of those could be problems. I would also suggest you might try using a different minimal medium just to see if it grows better.
Note that BL21 is RecA+ and DH5a is RecA- so will not grow at the same rate or to the same density as BL21.
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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?
If you want precisely 20oC then you need to either use an incubator in a cold room or a refrigerated incubator. If you don't need a precise temperature, you might be ok with just using ambient temperature in your lab. Alternately maybe you can opt to compare 25C vs 37C instead of 20, that way you can control with the incubators you have.
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Microbiologist’s opinion needed
Hello everyone,
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?
please feel free to message me if you want to discuss more.
best wishes,
Gloria
Mrs Dana Ulanova , Mr. Ciamak Ghazaei and Mr. Andrew Macrae thank you so much for answering! Your answers help us aim for the right research approach and certainly raise a lot more questions. We don't have specific microbiological equipment you're mentioning, but some of the equipment that we use in electrochemistry measurements might do the job since the methods are sensitive and require to be controlled. Need to research more on that. Also, there's a clinical microbiology lab at University Clinical Hospital, we'll make sure to know if they are available or can contribute.
Best wishes
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How long is it possible to store the bacterial culture at 4C?
It all depends upon what you intend to do with them. If you are just storing them and then plan to streak them out again later, strains should last for months at 4C. However if you want to actually do experiments on the cells you are storing, then probably 24 hours at most before you start having physiological changes.
Note that with E. coli strains if the strain is a recA- strain (like many cloning strains), then their lifespan is MUCH shorter and you will start getting significant cell death in a week or two at 4C.
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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 agree with dr Andrew Jenkins
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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?
You should inoculate the bacteria in eneichment broth medium, like lauria medium, brain heart infusion broth with double- concentration, then transfer growth on suitable agar medium and conferming its identification.
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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.
Hello, I recommend to consult Berlyn MK (1998) Linkage map of Escherichia coli K-12, edition 10: the traditional map. Microbiol Mol Biol Rev 62(3): 814-984 (see attachment). The Instructions to Authors of the Journal of Bacteriology as a great resource as well. They explain genetic nomenclature for bacteria.
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Infections and colonization or contamination criteria?
If you know a paper to review, I was wonder if you can introduce me.
Contamination: contamination is the presence of non-proliferating organisms on the surface of a wound
Colonization: presence of multiplying micro-organisms on the surface of a wound, but with no immune response from the host
Infection: presence of multiplying organisms with associated immune response and clinical signs of inflammation
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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.)
Be sure that you are using sterile medium as your blank for your OD measurements. Having an OD600 of 2 is pretty normal for a dense culture of E. coli, so if you are getting an OD of 2 then it should be fine. Normal overnight cultures are 1.5-2 OD.
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Except lyophilization what is the best why for bacterial culture storage?
25% glycerol in TSB ,Store at -20 °C for 4 month & at -80 °C for up to 1 year.
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Hello
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?
I have solutions of your problem, follow it then you will get the plaques
1.Do spot assay
2.Next day scrap that clear area and mix with host bacteria and broth
3.Next day centrifuge and filter this preparation
4.You will get plaques without any problem.
5.If still you are not getting it..let me know I have many solutions for this problem.
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how to keep the p.acnes samples grow anaerobically? is the anaerobic jar helpful ?
Thank you very much :)
Propionibacterium acnes is the most commonly isolated anaerobic gram-positive non-spore forming bacilli usually isolated from blood cultures bottles that may act as a contaminant.
All blood cultures were processed by the hospital microbiology laboratory using a standard blood culture system (Bactec 730 or Bactec 9240; Becton Dickinson, Sparks, MD).
P. acnes clinical isolates 889 (type IA), 6609 (type IB) and ATCC 11828 (type II) strains were cultured on pre-reduced Columbia agar base supplemented with 5% (v/v) bovine blood, vitamin K1 and haemin (Oxoid, UK).
Bacteria were grown under anaerobic conditions (anaerobic chamber; Bactron Sheldon Man, Oregon, USA) at 37°C.
A single colony forming unit (CFU) was inoculated in brain heart infusion (BHI, pH 7.4; Oxoid) broth cultures, and the samples were incubated at 37°C for 48 hours.
All the best,
Steph
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Hi everyone,
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?