Anaerobic Culture - Science topic
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Questions related to Anaerobic Culture
How to decrease an Anaerobic Chamber from 22 ppm to 0 ppm. A few things come to mind (have not tried) :
1. Decrease the water evaporation rate (Needed to remove H2S)
2. Increase the temperature to 37C (Currently 30)
3. Spread the zeolites on larger surfaces
Do you have any other input? Thanks in advance.
I have come across 'Anaerobic atmosphere generation bags' from Sigma. Has anyone used them?
I have a sample of fat and I want to reduce sulfate in it without breaking the fats, what are the best SRB species to use? I have found Desulfovibrio vulgaris to be the model microorganism in many studies, but I am not sure which organism to choose.
I've recently started working in an environmental analysis laboratory and wondered why is it that, in order to detect Legionella spp. from water samples, the incubation is done in a closed bag with a candle. I understand this is done to create an atmosphere enriched in CO2 but I'm guessing it shouldn't be anaerobic?
I have been doing some research but I can't seem to find the reason why it's done this way, other than because of this paper:
- Feeley, J. C., Gorman, G. W., Weaver, R. E., Mackel, D. C., & Smith, H. W. (1978). Primary isolation media for Legionnaires disease bacterium. Journal of clinical microbiology, 8(3), 320–325.
However I am looking for an explanation, if Legionella spp. are mainly said to be aerobic in literature why are they incubated in such conditions?
My lab needs to grow several strictly anaerobic bacteria that are of Biosafety Level 2. We need to use the Hungate technique (example: https://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/Kultivierungshinweise_neu_CD/engl_Anaerob_update.pdf) to keep the culture tubes under anaerobic conditions, because we do not have access to a proper anaerobic chamber. We also prefer using a biosafety cabinet to prevent handler from contacting infectious aerosols. Since the Hungate technique requires using nitrogen gas and flame to create sterile and anaerobic environment, does anyone know how to implement that inside a biosafety cabinet? Or is there any alternative to suit the purpose?
Anaerobic: Gardnerella, Blood agar.
- Incubated in anaerobic condition for 24-48 hrs for growth. (Done)
After the growth, we are intended to take single colony in TSB,
-> then to shaking incubator.
-> After that, glycerol stocking.
But do the anaerobes single colony would survive, after taken to shaking incubator?
Would they survive in such aerobic condition?
I am planing to work with Geobacter sulfurreducens bacteria which need purging by nitrogen and carbon dioxide for growth. I cant bring gases in the lab where I am working with.
Is gas-pak enough for such bacterial growth under anoxic conditions in anaerobic jar? If not, could you recommend other ideas?
I have been preparing anaerobic media in the traditional way (and as mostly outlined in DSMZ recommendations) and adding additions to the vials post autoclaving. This has worked well in that I've never had issues with contamination and my cultures always grow. However, this means that the vials are not technically replicates and require individual vial pH amendment at this point. It also is more labour intensive. I have the equipment for the Widdel flask method, however I am a little concerned with how best to avoid contamination and also when additions are added to the main media (post autoclaving and during sparging) do I then pH adjust the media at this point?
Any advice or help is greatly appreciated.
I am constantly dealing with Clostridium sporogenes and botulinum contaminations in anaerobic cultures, both suspended and plated. Does anyone have experience with this and insights on how to deal with it? Thank you!
I plan to do some research on antibiotics in anaerobic cultures. Given that these cultures are likely to become resistant to the antibiotics, I was wondering if there are any additional safety precautions that should be taken when working with these cultures? Much of the work will be done in anaerobic chamber, but some preparation for analysis is done on a work bench.
I am getting a white crystalline things in my anaerobic culture bottles. I have used same medium from a year and this never happened before. Can anyone suggest me, what this could be?
Just for information: I incubated these cultures last month and this white thing wasn't there in last two weeks.
SRB: Desulfovibrio vulgaris ATCC 29579
Medium: Postgates Medium B
Conditions: 15 mL Falcon Tube sealed with parafilm, Aerobic incubator 37C
The entire tube was black at some point. The tubes was opened once inside an anaerobic chamber to extract 1mL of the growing culture. The tube maintained its black color until all of a sudden it stratified into layers. The bottom is still black, followed by a yellow layer and the majority is now clear. Mixing does not help.
Is the culture still alive? Is the color change due to pH change brought upon by the production of H2S?
Does anyone have any suggestions as to 96 well plate seal that is not permeable and can be used in a plate reader?
I am running a few experiments that require me to isolate live cells from fresh stool samples. I've found Nycodenz to be perfect for my needs as it gives me large quantities of clean cells. I am now trying to improve my processing methods to prevent cell death from oxygen during the collection and centrifuge steps. I have a vinyl anaerobic chamber to work in, but I have to take the samples out to centrifuge.
Up till now I have been using regular 50ml and 15ml conical tubes, but I read that polypropylene allows oxygen diffusion at a fairly high rate. I ordered glass tubes with butyl rubber screw cap stoppers, but they will be more difficult to work with because of the smaller opening and insert plugs that will likely get covered in stool (not fun). Is oxygen diffusion through polypropylene something to really be concerned about during a 40 min spin?
I have been advised that adding L-cystine to all of my solutions could be used to neutralize oxygen. I've had trouble understanding exactly what it does besides lowering the reduction potential of media for bacterial growth. At the bottom I have a link to a paper that says it has a "redox buffering" effect when exposed to oxygen, but does this actually protect the cells from oxygen atoms, or just the redox potential of the medium? Could adding L-cysteine to the PBST that I collect the stool into help protect the sample during transport into the anaerobic chamber?
Another side note, should I be doing all of this at room temp or chilled? I've been told that some organisms like vibrio cholerae wont tolerate refrigeration, but the nycodenz seems to work better at 4c.
We have some sludge from a running SBR reactor and want to culture it under anaerobic conditions for some time prior to inoculation for our research. Unfortunately, our laboratory has no anaerobic chamber or jar, does anyone has an idea how to simply but efficiently create favourable anaerobic conditions to culture this sludge.
I am suffering of high air percentage under ongoing anaerobic bioreactor. I tried with CO2 flush for couple of hour but it did not reduce effectively. Higher air level is killing my consortium. I ensure air leakage at all the ports and tubings junctions manytimes but did not work. Please help if somebody has an idea over it.
Currently I am growing Lactobacillus crispatus, Prevotella disiens, and Prevotella bivia in purely anaerobic conditions. I was wondering if anyone knew if Prevotella species are viable in microaerobic conditions? Microaerobic conditions means they would be growing in an environment with increased oxygen levels compared anaerobic conditions, however still a reduced amount of oxygen compared to true atmospheric conditions.
References to literate would be very helpful, thank you.
I working on a project to prevent biofilm formation and bacterial proliferation in drinking water. In my quest for a suitable assay, I have been looking for a good media that represents drinking water conditions.. I can easily find M9 salt medium recipes online, I can not find recipes for M8, I assume that there are similar...
A male 17 yrs of age attended local hospital with complain of two small swellings on the abdominal wall just below umbilicus with slight tenderness but no fever. On palpation swellings were soft and slight fluctuating. They were drained with pus and bloody discharge. Cefurixime was given for 7 days but they remained unhealed. After two weeks patient was given anti tubercular drugs and continued for next 1 and half month without any progress. He was admitted in Rangpur Medical College Hospital. In the mean time another two swellings appeared on the upper part of the abdominal wall. On investigation blood sugar,CBC were normal. Tuberculine test negative. Microscopy showed gram positive bacilli, gram positive cocci and gram negative bacilli. Culture showed pseudomonas aeroginosa only sensitive to gentamycin. Anaerobic culture not done for non availability. Thinking it as necrotizing fasciitis, Injectable metronidazole plus, amoxicillin and cefurixime started. Two weeks passed without any response. Anti tubercular drug not stopped. Four days ago a palpable inguinal lympnode was removed -biopsy done reported nonspecific inflammatory cells, good number plasma cell seen. What is the possible diagnosis. Patient is poor in a poor country Bangladesh.
Dear friends a group of microbiologists has recently condemned our sterility tests (for aerrobic, microaerobic and anaearobic bacteria) on some vaccine on the grounds that instead of using screw capped tubes or flasks we used 100 ml cotton plugged tubes?
For anaerobic culture and microaerobic cultures Thioglycollate medium was used.
Yours replies are important as it will be lesson to us to modify the techniques established and used in bacteriological culture techniques.
I am trying to reactivate an anaerobic culture unsuccessfully, I am using liquid media in Balch tubes. The only difference between the medium recipe and what I am doing is the CO2 content in the anoxic gas. I am using 100%N2 and not 80/20 N2/CO2. I asked the distributor for the reason to use 20% CO2, their answer was that 20%CO2 buffers the medium and CO2 and may be also required as additional electron sink or carbon source. I am not convinced with the answer since the media already contains 30mM of sodium bicarbonate that should be enough to play these rolls.
I operate a high rate anaerobic/aerobic digester (HRAAD) that treats various types of wastewater. The system produces biogas, R2, and R1 water. The treated effluent is used to grow food/fuel/ornamental crops in an advanced hydroponic system. I am looking for a type of grass/sedge that would be suited for riparian zones that can be gasified. My system does have a height constraint of about 3ft. However, I have grown crops that exceed this height constraint by altering lighting regimes and producing rapid flowering/fruiting.
I am trying to design a lab scale Down Flow Hanging Sponge Reactor to treat municipal wastewater. However, I plan to use only a maximum of about 10 lts as the volume of the reactor. Flow controlled feed water let dripper/sprinkers etc are possible. Are there any specific constraints to be kept in mind?
My organism is an obligate anearobe but aerotolerant, Hence a certain degree of anaerobic conditions has to be maintained.
I am well aware of the medium requirements of my organism and the basic necessity of a reducing medium.
But I want an insight into a practical suggestion with flask preparation, maintenance, subculturing, sampling, etc.
I am working on microbially triggered drug delivery system so I want to do dissolution studies for tablet.
I need to cultivate C.Saccharolyticus which is a thermophilic (70° C), strictly anaerobic asporogenous bacterium. We dont have the facility of anaerobic chamber. Can we culture it using a Thioglycollate media by inoculate the ampoule culture (by inoculating 200microliters of media after cut opening the vial) onto the media.
There is a patient with infective discharge, with high rate and non stop, we examined by aerobic and anaerobic culture and fungy sabror culture, no growth. then we examined 16srRNA presence, but again negative and in direct smear there is nothings other than WBC. So for diagnosis what we can do further??
I isolated the DNA of yeast strains obtained from the Sauerkraut brine and subsequently performed the PCR-RAPD for the strains. Before sitting down to PCR-RFLP, I would like to get busy with PCR-RAPD for the bacteria (hopefully mostly LAB) and here is the thing - what ways of ensuring anaerobic conditions can you suggest, besides using airless chamber or wrapping test tubes with foil?
I haven't experienced big difficulties in cultivating so far, yet I would appreciate it if you could provide me with some solutions in case I'd have to modify something. Thanks a million in advance!
I just want to know what are the media are using for themophilic biopolymer producing anaerobic bacteria during the isolation.
We tried several times to make petri dishes cultures isolation techniques, flat and inclined agar methods, and still appears 2 strains in 16s pcr. We want to contract a service of strain isolation fast and reliable to deposit as soon as possible.
Any suggestions?. Thanks
I am using RCM and ATCC 2107 media for culturing C. chauvoei but sevaral times I get mixed culture even while reviving from stock pure culture. for ATCC 2107 i am using anaerobic gas pack also. Suggest me the protocol for culturing this organism without any contamination.
The requirement for methanogens are 20% CO2 and 80% N2. How do we obtain this mixture?
we use an anaerobic chamber that allow to use 2 gasses but we cannot control the amounts of gases.
Currently I'm having an internship in an alcohol plant. The plant uses seed fermenter (aerob, 2.2 m3), pre-fermenter (aerob, 44 m3) and main fermenter (anaerob, 830 m3). I am placed on the main fermenter area. The substrate used was molasses (contains glucose and sucrose), and some data which given are Brix and pH in 44 hours. In one batch , the fermentation product contains 8.9% v/v of alcohol with %Sugar (analysed by Somogy method, only for monosaccharides) 1.735% w/v. I've processed all the data I have and found that the yield was too high. I even have substract the alcohol in the final product of main fermenter with the alcohol from pre-fermenter. Based on theory, the yield affect the stoichiometric coefficient of alcohol. The maximum stoichiometric coefficient is 2, but I got 2.9. What should I do?
Briefly : based on the data I have, the alcohol is more than should be, or the sugar consumed (based on approximation using Brix data) is lower than it should be.
I think it is not possible for the alcohol to be produced in the molasses tank since the production will also produce CO2 and damage the tank. I;ve made a relation between Brix and %TS (based on disaccharide analysis data), and also between Brix and %TS (based on Somogy analysis data). It seems that the combination of both data give the best result, assuming that Brix and %TS related linearly. I think its not valid, but I have nothing to do anyway, the company did not allow me to get another data.
Your help would be meaningful, thank you :)
For growing anaerobic bacteria on solid agar plates, I am using an "anaerobic jar" with "gas pack" that consumes all the oxygen inside the chamber after it is sealed. We usually seal the petriplates containing solidified agar medium after inoculation, to avoid contamination (which works well with aerobic cultures). Should we do the same for growing anaerobic bacteria in anaerobic chamber?
I doubt that if we seal the plates, the oxygen inside the plates will be trapped, while the gas-pack removes the oxygen from the chamber. And therefore making it difficult to maintain anaerobic condition inside the plates. But if we do not seal, how to avoid contamination?
I'm currently growing Bifidobacteria in BSM broth for my experiments but the broth is reacting with a particle that I am exposing the bacteria to and causing it to agglomerate, having an effect on the results. When I expose the same particle to lactobacillus growing in MRS, this agglomeration doesn't happen. Is there any modifications I can make to the MRS broth to allow the Bifidobacteria to grow in it too?
MRS media: probiotic bacterial and yeast strains being used, some strains are anaerobic while some are microaerophilic.
I'm going to culture the anaerobe (ie.Bifidobacteria spp and Clostridium perfringens) from fece (or cecum content), but the preparation of the anaerobic solution isn't going well. According to the description, it must be colorless after autoclaving and pink means it can't be used. But every time I tried, it wasn't pink or colorless but faint orange.
The attached file is the composition of the anaerobic solution.
Is there any reason why people don't use abundant marine sediment as inoculum? Marine sediment contains high sulfate reducing bacteria (SRB) which will compete with methanogenic archae so it will lower the methane production. Is that one of the reasons?
One of my colleges is working with a anaerobic chamber to culture Clostridium, but for several weeks he had problems to obtain growth of. He suspects that there is a problem with the chamber and he asked me if I know any oxygen indicator, a colorimetric or another kind or chemical indicator that he can put inside the chamber to detect any oxygen. Can anyone help him?