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I tried making MRS broth but it is hard as a brick. Is there any practical tip to make it or it is what it is?
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MRS Broth is a medium for the cultivation and enumeration of Lactobacillus spp. This product has the same formulation as LAB093 MRS Agar with the omission of agar. You can see the Composition in Technical Data. Or you can Buy the ready MRS medium from the Chemical company.
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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.
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Is the chamber working with any sort of metal catalyst? If so, I suggest you replace it. Sometimes, depending on the anaerobic chamber usage, it may loose it's efficiency in removing oxygen traces.
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we want an alternative method instead of using Methylene blue strips
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I can understand your situation. Blood agar shoes red color and could not show color of indicator in the medium. It's just suggestion; prepare extra medium just agar only but including resazurin. It can be work as indicator.
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I have come across 'Anaerobic atmosphere generation bags' from Sigma. Has anyone used them?
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About pyrogallic acid + NaOH as oxygen scavenger ― check my post at: https://www.researchgate.net/post/Can-somebody-recommend-a-method-for-making-48-96-well-plate-anaerobic
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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.
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Vulgaris, as you mention. I also used desulfuricans a lot, and I isolated my own strains. But this was a long time ago. The best reference at the time for fatty acid profiles was Postgate’s book. I’m not sure who is best now, but Schlegel and his coworkers became world leaders on SRB.
None were fast, but by far the best way is an anaerobic glove box to minimise exposure to oxygen.
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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?
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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?
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I use cultivation media such as thioglycollate,
or media coated with sterile liquid paraffin.
Anerobiosis jars with bags can be used to create the
oxygen-free atmosphere (GENBOX) from Biomeriux
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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?
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I think it depends on the material that you are using.. Quantity of glycerol need for sticking may differ
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Dear all,
I am applying a budget of around 5000 USD. Which combo of the equipment for anaerobic culture is good for me? Thanks in advance for your help!
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Dear Anh Huu Dang, can you provide further information on what equipment you have already. In my experience for anaerobic culturing you require an anaerobic glovebox system (Coy or Plaslabs) - gloveboxes are quite expensive we paid £10,000 inc vat for one recently, for liquid culture - hungate vials, bungs and seals, syringes, needles, metal cannula, cotton wool and gas mixtures for sparging (BOC). This review by Prof. Caroline Plugge has a great description of what is needed and other pieces of kit like the Widdel flask . Best wishes, Ciara
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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?
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Maurice Ekpenyong Good answer
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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.
Ciara
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I have extensive experience of Widdel Flask and used this system for 4-years without any contamination. I generally add all the heat sensitive component like vitamins , amino acids post autoclave and used filter sterilized gas for flushing the vials and head space. I never experienced any contamination in any Hungate tube or serium vials. Just take precaution and follow aseptic techniques.In case of bicarbonate buffer sufficient saturation of medium with N2: CO2 require and it takes some time
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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!
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Dear Sarah,
Greetings!
Before answering your question, I have some queries.
1. Which anaerobe do you want to culture?
2. Where you are culturing? If it is in anaerobic chamber you have to sterilized/ wiped with Benzalkonium chloride. (You can use this solution because, other chemicals such as ethanol or isopropanol are flammable, since you are using hydrogen inside the anaerobic chamber)
If organism of interest grow on specific substrate, you just increase the particular substrate concentration, to avoid the contamination. For example, for sulfate reducing bacteria, you have to increase the sulfate concentration to hinder other anaerobic contamination.
Thank you !
Best
VELRAJ
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If I cannot use the commercially available ones? Thanks!
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Also, you can use any antifungal agent with MRS media to prevent growth of candida
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Does anyone know the range of pressure that 120ml anaerobic serum bottles can tolerate (10-100 psi)?
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Stoppered serum bottles are relatively convenient and inexpensive way to culture strictly anaerobic bacteria. Media can be prepared in large batches filling serum bottles which can then be stored at room temperature for several months ready for inoculation.Turn valve to the vacuum position and watch for the pressure to equilibrate to around -20 in Hg ( about 3 - 5min. For more information consult https://openwetware.com
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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.
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I don't think you just have to think about anaerobic workbench or laminar AF bench. If your lab is not bio safety level-2 approved, you should not do it. Apart from working, the waste disposal should also be according to protocol.
Similar discussion earlier on here might help you further
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I prepared the medium according to DSMZ, for M. barkeri. I accidentally added absolute methanol instead of 50% of methanol (v/v). Is there any way to resolve this?
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Hi Jiayi! I can not help you because it's not my subject field
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Do bacteria grow in soil that remains under water all the time? Or, are bacteria growing under water all anaerobes? If not, where from do they get O2 if there is not much agitation for O2 to be dissolved?
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Agreed !
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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.
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Hi Pryanka,
without knowing the chemistry of your solution and coal powder it is hard to give a definite answer. My guess would be that some minerals have leached out from the coal causing a change in pH and association with other dissolved matter resulting in precipitation. The best would be for you to extract the white flocs from the bottle using a syringe or pipettor and analyse under SEM-EDX for morphology and elemental composition.
Hope this helps?
Kind regards, Rob.
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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?
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Hello, if you grown SRB, than you will have dark sulphides in media bulk water, then they fall down to the bottom,. If you open the falcon you need to look how many oxygen in it now.
and look for bottom of the flask sediments if it still dark you will have a hope for gaining some living cell, if it sediment rustic red color there is no hope.
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Thanks in advance for your replies.
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Dear Shivalkar
I think this paper is good for you:
Diaz PI, Zilm PS, Rogers AH. Fusobacterium nucleatum supports the growth of Porphyromonas gingivalis in oxygenated and carbon-dioxide-depleted environments. Microbiology. 2002 Feb 1;148(2):467-72.
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How to make anaerobic FeCl2 solution? we tried to make the solution and blow N2 and CO2 gas into the serum bottle then autoclave it didn't work. Is there anyway to make the solution anaerobic?
thank you!
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I work with anaerobes and this is my way of preparing anoxic solution.
1) Place your solution in a serum bottle, seal it with butyl rubber septum and aluminum cap.
2) Use a 21ga needle to pierce through the butyl rubber septum (this serves as a gas outlet).
3) Connect your gas supply to another 21ga needle and pierce the needle through the butyl rubber septum. Ensure that the needle is inside the liquid phase. Turn on the gas (I use 500mL/min of gas at 40psi).
4) After 10 mins, remove both needles simultaneously.
5) Autoclave your sample.
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Does anyone have any suggestions as to 96 well plate seal that is not permeable and can be used in a plate reader?
Thank you,
Elizabeth
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I've successfully used these ThermaSeal PCR sealing films (Excel Scientific) to provide an anaerobic environment for the culture of green sulfur bacteria, however the growth on the outer wells is unreliable because of the texturing on the edge of my 24/96 well plates makes them difficult to seal. Stick to the inner wells (Rows B-G, columns 2-11) if you want to have more consistent results.
The plastic on these ThermaSeal tubes is polypropylene, which is OK as an oxygen barrier for some anaerobic strains such, as green sulfur bacteria, which can scavenge oxygen, allowing them to survive in its presence, but not grow. Other bacteria may flat out die. If that's the case, you might want to try to explore a plastic film material that's designed to prevent gas exchange, such as Ethylene Vinyl Alcohol (EVOH). This plastic is used widely in the food industry to keep oxygen out when packaging (according to Wikipedia), so it may be very useful to grow cells with However, I don't think anyone sells this film in a form that makes them convenient for use with 96-well plates. Perhaps with a heat sealer you can make an anaerobic seal around the plate.
In my old lab, I would carefully melt birthday candles (a cheap source of paraffin that's conveniently shaped) to make glass-glass seals for the anaerobic growth of photosynthetic bacteria between a microscope slide and a cover slip. It may be feasible to just seal a 96-well plate with paraffin (or a combination of paraffin with petroleum jelly to make the paraffin more maleable). Nail enamel also works really well, but it can be toxic to your bacteria if it comes in contact with the growing medium and is generally more "runny" than paraffin, so it may not work well in sealing larger gaps.
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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. 
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Thankyou for the thorough answers. I'm still waiting on some materials before I can start this experiment, but this help me a lot for planning. Thanks!
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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. 
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I may add some more info to the message of Yifan. A serum bottle + rubber stopper + flash gas phase with nitrogen gas via syringe needle = perfect possibility to get anoxic conditions with activa sludge.
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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.
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Dear all,
I have a similar problem in my anaerobic fermentations (glucose fermentation by C.beijerinckii). I flush the fermenter with N2 (0.5 l/min) entire time and stir.
I prepare my anaerobic media accordingly so that there is no dissolved oxygen in the begining of the fermentation. However, during exponential growth phase, dissolved oxygen level increases drastically, up to 60%. The problem is not the probe nor the equipment. I observed the same pattern in different setups (Aplikon fermenters and microreactor fermentations).
Does anyone experience a similar thing?
Regards,
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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.
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i would start here
and i would follow the links to the original papers that describe the species of the genus Prevotella . . .
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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...
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In drinking water microbiology R2A medium is a very good alternative for total counts, sometimes you need some long incubation times, however you also can recover interesting micro-organism
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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.
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My suggestion  is leishmaniosis or pyoderma gangrenosum
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Trying to grow anaerobes.
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i may help you if you provide refrence of  the paper youvwant to follow. i have many papers of tassel and wilkins so if you give full refrence i will help you out.
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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.
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Cotton plugs do increase the risk for contamination quite a bit.  How do you keep them sterile when doing transfers?  The screw cap would be preferable when using thioglycollate media as tightening the tubes helps increase the anaerobic conditions.  Loose screw tops work well for aerobic cultures, but the culture tubes with loose caps are ideal as they are easy to handle if doing transfers, and they remain sterile.
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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.
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20% CO2 seems a little high, but the function of the COis providing the acid for the buffer H2CO3/HCO3 -.  For 30mM bicarb, 20% CO2 seems extremely high and would render the culture media acidic.  25mM bicarbonate is used with 5% CO2 to render a pH ~ 7.4
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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.
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Reed and water hyacinths has also been used in the Philippines and in NASA. But the most practical approach is to select locally thriving grasses with massive root systems. This way, the supply  source and environmental setting are no longer and issues. Then test them in the effluents to get their survival and thresholds.
For metal uptake, NASA has experiments on those since the 1980s. Maybe you can ask them.
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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?
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Dear Abhishek
High rate anaerobic treatment processes, like up flow anaerobic sludge blanket (UASB) reactor have been intensively used for the treatment domestic wastewater because of their low operation costs, smaller space requirements, high organic removal efficiency, low sludge production, and net energy benefit through the production of biogas .
The DHS module column consists of four identical segments connected vertically, each segment will be equipped with 25 L of polyurethane foam (PF) warped with plastic material randomly distributed in the whole reactor. The DHS system will be made of PVC, with a capacity of 0.3 m3 and has an internal diameter of 0.16 m. The height of the reactor is 0.88 m. The reactor will be filled with PF which represents 34% of the total liquid reactor volume. The characteristics of the PF (sponge) are surface area 256 m2/m3, density 30 kg/m3, void ratio 0.9, and pore size of 0.63 mm. The total volume of the PF will be 100 L. The dimensions of the used sponge (PF) (cylindrical shape) will be 27 mm height ×4 mm diameter. The AR effluent will be flowed by gravity to the distributor which will be located on the top of the DHS module and will be rotated at 15 rpm.
Please find attached herewith related article,
Regards,
Prem Baboo
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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.
Thank you
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Many obligate anaerobes can tolerate some exposure to oxygen but would be stressed. I would think that depending on your research goals etc, you would be best to grow them in serum bottles under a nitrogen atmosphere with septum caps. If you want to agitate them you can still do it of course. Sampling is done with sterile needle and syringe. 
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I am working on microbially triggered drug delivery system so I want to do dissolution studies for tablet.
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commercial jars are better
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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. 
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I have transferred my  lyophilized  C. acetobutylicum strain without the use of anaerobic chamber in a laminar air flow. Well, it is not the correct and recommended method, but since I didnt had any other options, I had to do it in an improper way. But it worked in my case.
        In my case i had to add a bit of milk media in my lyophilized culture and then inoculate in to milk media. So i prepared few serum/anaerobic bottles of milk media anaerobically using gassing manifold and a few empty bottles just purged with nitrogen at normal atmospheric pressure. Then i autoclaved all the bottles and used it in a clean laminar air flow. I then used a disposable syringe and needle, which was flushed 5-6 times in the autoclaved nitrogen purged bottle before use. Now you need one more person to help you in laminar air flow, for breaking lyophilized vial. Using this purged needle, i quickly add the few ml of milk media in lyophilized vial, mix 2-3 times and quickly inoculated  into another bottle of milk media  with the help of same syringe. The remaining lyophilized culture were inoculated into another bottle. This were my master cultures and then immediately i sub cultured in to another sterilized milk media bottles.  With that I had a total of 6-7 bottles containing my lyophilized culture.
         The thing to be noted is to maintain aseptic conditions and speed. You can add a bit cysteine-HCl as a reducing agent after inoculation, but don't add in large amount as it has been proven to inhibit growth of some anaerobic bacteria. Dont use Na2S. There are many more tricks that can be used depending on the facilities available.
Best of luck
            
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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??
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I'm surprised that the 16s PCR is negative, as discharges are seldom sterile. However, such samples may be highly inhibitory. I would advise reanalysing the sample with a small amount (ca. 2 ng) of bacterial DNA added. If it still comes up negative, the sample is inhibitory (try further purification, or just dilute it). If it comes up positive, your sample is probably free of bacteria. 
Note however, that some bacteria may amplify poorly with the 16s primers. 
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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!
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There are many options, depending on your resources and anaerobic requirements. I work with strict anaerobes, but the concepts apply to facultative anaerobes.
-A rigorous method is given here: Hungate, R. E., & Macy, J. (1973). The roll-tube method for cultivation of strict anaerobes. Bulletins of the Ecological Research Committee, 123-126. 
-A more recent paper for liquid media prep: Wolfe, R. S., & Metcalf, W. W. (2010). A vacuum-vortex technique for preparation of anoxic solutions or liquid culture media in small volumes for cultivating methanogens or other strict anaerobes. Anaerobe, 16(3), 216-219.
Sounds like you want simple, however, proper anaerobic culturing requires some work. These papers should give you a start and provide background on what to look out for. Good luck!
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I just want to know what are the media are using for themophilic biopolymer producing anaerobic bacteria during the isolation.
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Thermophilic bacteria are sensitive for temperature so kindly maintain the desire optimum temperature requirement of the organisms. For the production of biopolymer provide high concentration of simple carbon source and nitrogen source in the minimum amount, this kind of medium will enhance the production of biopolymer. 
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Reagents such as Resazurin, Cys-HCl are available with us.
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What is an anaerobic workstation ? Is it an anaerobic chamber or a station providing various anaerobic gases?
If it is an anaerobic chamber you have to provide a system in which you insert anaerobic gas into your medium or devices, which all have to be gas tight. With anaerobic gases (most likely nitrogen) you can flush out oxygen from the system. With some indicators (resazurin) or measuring devices (optode) you can check the oxygen content in liquid medium for example.
Hope that helps!
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Do you know companies interested in trace metals dosing in anaerobic digestors?
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I think TERI www.teriin.org can be contacted as they are active in anaerobic digestion of different types of wastes. 
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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
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Antonio,
What is the originating source of the anaerobic strain?  You may be faced with a codependency problem where one microbe is dependent upon the other for survival.
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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. 
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Hi,
In the past I have had some issues with ATCC stocks being slightly contaminated, or even the media that comes along with them. Recently, I have had problems with contamination while growing Clostridium species in the same way as you- on RCM and in anaerobic jars with gas packs. What I did was prepare my own RCM, not what ATCC gave me, streaked my culture into 3-4 quadrants, and selected the predominate species on the plate. The previous freezer stocks were not grossly contaminated, so it was easy to find the predominate species. Simple to do, and I also made several pure stocks of my own.  
I am sure you already know this, but you might also check the O-ring around the jar to make sure it still is tight. In addition, replace the gas packs as suggested on the packs you are using. I hope this helps
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What is the survival time of anerobic bacteria like porphyromonas gingivalis, actinobacillus, prevotella intermedia, fusobacterium nucleatum in aerobic environment? How they survive? These are periodontal pathogens which are of significance.
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Hi.
You might find these two papers of interest in terms of survival of P. gingivalis.
Diaz and Rogers 2004
Regards,
John Smalley
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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. 
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You CANNOT do this in a chamber! It's DANGEROUS! Very Explosive!
You can do it only in a tube, serum bottle or Oxoid anaerobic jar!!!
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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 :)
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It is not about the question itself. It is about the trust of pre-grade students to the people here and not in other web sites.
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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?
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Petri plates are designed to provide sterile environment for the growth of microbes on solid media. There is no need to seal the petri plates as all aseptic operations are performed strictly under qualified Laminar air flow (LAF) cabinet. Microbes won’t enter the petri plates in any way when they are closed and handled properly. To be extra precautious some microbiologists will follow this practice but it not an absolute requirement. So, you need not to worry that unsealing of plates may lead to contamination.
I create anaerobic conditions in a very simple cost-effective manner by using glass fish tanks as candle jars. Position the plates (inverted) in the tank, light candle, place aluminum foil over the top of the tank, and seal foil tightly with the tape. Tanks should not be filled to more than 50% capacity to ensure consistent and adequate growth.
I handled lot of bacteria under anaerobic condition by following the above simple technique without using gas-pak system.
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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?
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As they said, you can grow them in MRS supplemented with 0.05% Cys (0.5g/L) in anaerobic conditions. In addition, I would suggest you add mupirocin which makes the media more selective for Bif:
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MRS media: probiotic bacterial and yeast strains being used, some strains are anaerobic while some are microaerophilic.
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It all comes down to finding an O2 concentration where both are at least a bit happy. In my experience the best way to try is in an anaerobic cabinet (like the ones Don Whitley Scientific supplies). You can use those completely anaerobic, but with a bit of imagination you can play with the atmosphere composition in a controlled way. (You have to remove the O2 absorbent for the chamber).
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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.
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How are you preparing the anaerobic solution? The presence of color, leads me to believe you might not be reducing it, or doing it the wrong way. If you can add information on how you do it, I might be able to check where the possible problem is.
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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?
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Marine sediment can be used as inoculum for the anaerobic treatment of saline wastewater. SRB will compete with methanogens only if sulfate is present, which is the case in sea water.
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Looking for a method for isolation of anammox bacteria from estuarine and coastal ecosystems
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Using Nitrogen gas and monitoring by Resazurin is not working well, since it remains red instead of yellow.
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You may consider adding reducing substances such as cystein-HCl and/or sodium sulfide (e.g. 0.5 g / L each). It works very well for culturing methanogens.
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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?
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Oxoid sell anaerobic indicator strips which are impregnated with resazurin, a redox indicator, which change colour to indicate partially (pink) and fully (colourless) anaerobic conditions when opened inside the chamber (http://goo.gl/YqKaD). However in my experience the best way is to install an O2 and H2 analyser (e.g. http://goo.gl/pjQ6J) which will indicate in ppm the exact level of O2 (and H2 in % if using gas mix for catalysts). A cheap alternative to both is to add a few drops of resazurin reagent (final conc. 1mg/L) to anaerobic medium and leave the top off. If only partial anoxia exists inside the chamber, the medium will be pink; if fully anoxic it will turn colourless. Note that a transition back to pink from colourless is possible only once in the event that conditions become less anoxic. Hope this helps.