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Microbial Isolation - Science topic

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I have 16sr DNA sequence for a bacteria which shows 86% similarity while doing BLAST search. Does this mean it represents a novel genus? In that case, is it mandatory to do a DNA hybridization test?
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You may need to perform DNA hybridization test in other to confirm your new isolate .
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Methanotrophs use methane as their sole source of C and energy. They are single biological entity, which can help in the mitigation of most potent GHG in the atmosphere. 
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How to obtain 20% methane 5%CO2 and rest atmospheric mixture in anaerobic jar?
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Microbiologists, research scholars or experts working in this feild
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As other researches told, the color of medium will change to the red, due to oxidation of Fe2+ to Fe3+ by bacteria. However, it is not a reliable way to monitor the growing of bacteria. Because Fe2+ also can be oxidize to Fe3+ due to chemical reactions. Therefore, I suggest you to use other parameters for monitoring the bacteria growth such as pH, ORP and counting under microscope.
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Ruminococcus albus
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Ruminococcus albus sp. nov. (Hungate 1957)
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I want to screen soil samples for azotobacter and azospirillium, so is there any plate or chemical assay to quickly screen samples, after that when I identify it as any one above them, then please suggest any molecular or other method to identify the strain of the same..
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Characterization of Azotobacter
Direct microscopy investigates was used for pure isolates of Azotobacter. It was found that the microbe is gram negative, large in size, oval or cocci, single or in pairs according to Bergy's Manual (1974) and Becking (1981). Identifying testes were carried out including (IMViC) tests, it was positive in indole test and negative in methyl red , voges proskauer , citrate , gelatin hydrolysis catalase , cellulase , pectinase and amylase activities .Nitrite formation and denitrification tests were carried out by the method described by Neyra et al., (1977) in addition to nitrogenase activity according to (Hardy et al., 1973) and litmus as described by Drews (1983) and Sussmulh et al. (1987)
Characterization of Azospirillum
Pure isolates being short rods of spinning motility gram negative, producing alkalinity in growth culture .The ability of Azospirilla to use glucose a sole carbon source for growth was described by Tarrand et al. (1978), positive in production of acid and gas, requirement of biotin for growth , catalase production and reduction NO3 to NO2.
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A similar  question has already been  asked by Anil Khatape, and I  had  read the answers.. 
I have enriched the archaea in liquide medium containing acetate as sole source of carbon, and have added antibiotics. However, when transferring on solid plate (with acetate), no grow is observed..  I am wondering whether someone has experience in isolating acetoclastic methanogen on solid agar culture. 
Thank you in advance
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Dear Irene Davidova
Many thanks for this suggestion.. 
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I want to isolate hyper thermophilic bacteria in general (not a specific bacterium) but i do not Know the most suitable medium; would u help me plz? 
thanks
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Halophilic medium modified from the formulation of Oren (1983) was used, comprising (per litre distilled water): 125 g NaCl ; 100 g MgCl2.6H2O ; 5 g K2SO4 ; 0.1 g CaCl2.2H2O ; 1 g yeast extract ; 1 g Casamino acids and 2 g soluble starch. The pH was adjusted to 7 and incubation was at 37oC.
Oren, A. (1983). Halobacterium sodomense sp. nov., a Dead Sea halobacterium with an extremely high magnesium requirement. Int J Syst Bacteriol 33, 381–386.
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Hi,
I would like to cultivate bacteria from fish skin in a range of different medias and isolate them afterwards. I am wondering if anyone have experience with cultivation of bacteria from skin using a medium that contains mucins? Or other medias that might be suitable for cultivating skin bacteria?
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Dear Mert, thank you so much for your response. We have now decided to first try the medium mentioned in the paper by Looft et al, that includes mucins for isolating the bacteria able to utilize mucins. Thank you for the attached paper - it was very helpful!
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Please suggest some standard pocedurte for isolation of polyethylene degrading bacteria from soil and water sample. Standard media and polythene sample is varying in different articles besides the incubation time and enrichment process. 
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How lonng the clinical isolates of S aureus can be preserved at 2-8 degre celcius?
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If you grow them up on TSA or nutrient agar slants, then seal the top of the tube so air does not get in and moisture does not get out, they will stay for several months, up to 6 months. If they are suspended in normal saline, they will probably be good for 1-2 months.
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Thanks. Please is 16S rRNA sequencing similar to 16S rDNA sequencing. Secondly can you suggest a reliable laboratory that can perform molecular characterisation of Microbial Isolates with fast turn around time to me. I need answers urgently please
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Microbial ID does A GREAT JOB.  http://www.microbialid.com
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I want to enumerate and isolate zinc mobilizing microbes from wheat rhizosphere. Any one may guide me the most appropriate media along with the zinc source please for the same?                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
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Is their any easy method by which I could separate microbial cells from saliva samples of human. It would be really helpfull if someone can share related protocol.
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Please check the pdf
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I am working on biological nitrogen fixation and I wanted to screen the microbes for nitrogen fixation attributes. Is there a method to find nitrogen fixation by microbial isolates apart from Gas chromatography method (ARA).
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The potential for biological nitrogen fixing ability can be detected initially by culturing the isolates on nitrogen free media containing bromothymol blue as an indicator. Bromothymol, a pH indicator, shows yellow colour in acidic medium, green colour in neutral medium and blue colour in basic medium. during nitrogen fixation, free atmospheric nitrogen is converted into ammonia which makes the medium basic and hence turns blue (Dobereiner, 1972).
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What is the real meaning of the iterative coryneform bacteria isolated from samples of the waning of otorrhea episodes of chronic or recurrent otitis externa ?
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The most common bacteria isolated from the middle ear in acute otitis media (AOM) are Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus. Corynebacteria also have been isolated, but are likely secondary infections of which the relative pathogenicity of these species are not yet known. See attached link for more information.
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I isolated many phages from marine water and  took a microscopic image for this phages but now I have many phages. I think this may be new phage but I'm not sure whether these are new phages or not. Also I every time I take microscopic images for phages isolated from one plaque, I see more than one phage in my sample. 
I read many articles about purification of phages from marine water. I did all that was advised but I don't get good results. Thank you for advise about all of these issues.
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thank u mr. karrar, i isolated  and conformed the phages by using the above protocols successfully.
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I was gram staining some bacteria in a microbology practical and when I look into microscope, I see handful of unknown objects that doesn't look like bacteria at all (refer to the photo attached). 
My supervisor has no idea about what it would be, but suspecting that they are some sorts of brown microscopic fungi. It is ~ 8 microns in length and ~2 microns in width and it seems to have been splitting out transparent cells that seem to contain chlorophyll inside.
Thanks everyone!
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Hi, that definitely is a fungal spore. Could be a teliospore of a rust fungus if it would be an environmental or better leafe sample or alternaria as a conatmination in the lab.
If I would run the lab I wouls spread out some open Agar Plates to test for fungal contamination in the lab. That should not get onto a bacterial sample at good laboratory praxis. Or did you drop anything?
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I am currently isolating those lactobacillus from dali products and I am using tomato juice broth with this formula:
for 400ml
Triptone 4g 
yeast extract 4g
80ml of filtrated tomato juice
I been having problems to growth them, they growth but really slow, can someone help me to see if the broth I am using is missing something? Thank you!
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Hi GYnna.
I used an MRH agar and tomate juice powder and adjust the pH
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I am looking for some methodology that decrease the time of microbiological analysis. Today we expend 48 hours to know if a sample is contaminated by bacteria and almost a week to know if a sample is contaminated by fungi. I am looking for something that could indicate this contamination in hours or, if possible, minutes.
Do you know some methodology or device that could make this happens?
Thank you so much.
Lilian
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If you are looking for general microbial growth (i.e. any type of contaminants) there are a number of companies selling products for rapid diagnostics. See for example Rapid Micro Systems. 
If you are looking for a specific contaminant or strain, then you could probably devise some phage based luminescence assay or a real time PCR assay and monitor increase in signal over short time. 
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I attached gel image with all problems on it,please analyse and give me right suggestion to optimize it.
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Hi Vivek,
I don´t think that increasing the gradient would help much. As you can see, your samples stopped somewhere around 50 - 55% and below there is an empty space you have marked. Increasing the gradient range will shrink the profiles. I would suggest the opposite, to make the gradient from 35 to 55%, then you will have a better separation of your bands. In addition, loading less amount of DNA (1/3 to 1/5 the volume you have used) will diminish the high fluorescence in the lower part of the gel, allowing to see the bands. Although, with this amounts you will lose the signal for the upper bands. Unfortunately that is always a problem with profiles that have both, very intense and very faint bands. Good luck!
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isolation criteria
suitable media
preservation during transportation
incubation temperatures
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About preservation during transportation, in my experience, I don't think you'll need any special treatment. Just take the sample as much as possible and put it on an sterile container. And you might want to inoculate the bacteria directly from your hot spring/ land fill site in the enrichment medium such as Luria Bertani or Nutrient Broth. 
Regarding the incubation temperature, I would recommend to measure the temperature in the hot spring as well as in the land fill site. Therefore you could use those temperature as a standard. However, usually you can use 37 C for soil bacteria isolation. 
For suitable media, sometimes, there're some specific compound from it's origin which is needed for bacterial growth. So, you can use the sample itself to provide those nutrients (example, water from hot spring and soil from land fill). Dissolve general medium such as LB or NB using the water from the hot spring and sterilize it using autoclave as usual. If you want to isolate bacteria from soil, you can dissolve about 10 g of soil in 100 ml of distilled water and use the water to dissolve the general medium.
I hope this would help. 
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Need Multi Locus Sequence Typing (MLST) analysis training especially for the identification of Bacillus genera upto species level by using house keeping genes like Gyrase B and rpo B.
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The php pheneplate system is a microplate based assay that allows fingerprinting the phenotype of microbial isolates (or microbial communities), basing on the cinetic of utilzation of a set of C substrates. Although it has been used for medical research, there is no publications that I know of in which this method is applied to the soil microbial community. I believe it could be used to obtain information on the functional diversity of soil microbial communities, similarly to the biolog method. In the past, my colleagues obtained encouraging results applying the assay on a very sandy loose soil. As of now, I am having troubles obtaining reproducible results, having tried 1% pirofosfate solutions as extractant on a clay soil, and dilutions up to 10-4, with and witout filtering the soil suspension. I believe the problem is not related to microbes contaminations, as I operated in a sterile environment, and my control plate does not suggest the presence of contaminant microorganisms.  Has anybody ever been able to successfully perform a php pheneplate assay on a soil microbial community? What is the best extractant to use? How much should I dilute the suspension before inoculating it into the microplates?
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Thank you for the advices!|
The pH of the pyrophosphate solution is 9.5, surely not adequate to extract lively microrganisms. Though It appears to me that the pH is a bit too high for a 1.8g/L concentration. Is the pHmeter broken? I am trying to calculate what the pH of the solution should be basing on Na4P2O7 dissociation constants, but I cannot find any pkB value. 
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isolation of environmental mycrobaterium TB
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Thanks a lot on the other hand is the PCR test enough to confirm the results or we need other tests ?and what's there names ?
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Trying to make up some different media to isolate soil actinomycetes, and also ocean actinomycetes.  I was going to go with actinomycetes isolation agar in addition to a few others, but was wondering how important the asparagine really is.
Sodium Caseinate----2g
Asparagine---0.1g
Sodium Propionate----4g
Then some trace metal salts, and sometimes addition of glycerol.  
Was also looking at Soya Flour Mannitol media, Triptic Soy Agar, and Starch Casein Agar, with perhaps addition of autoclaved ocean water when isolating from saline mud samples.  
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Thanks for the responses.  The humic acid vitamin agar seems pretty interesting.  I'm trying to follow in the footsteps of Albert Schatz and discover something great in a basement!  www.basementbiotech.org is my blog where I write about some things.
The hard part though is the structure elucidation and bio-assay guided fractionation of extracts.  Since so many things have been discovered and re-discovered, I figured looking in the ocean's soil might provide a higher chance of finding something novel.
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I'm working on mastitis organisms, even the general causative microbes are also taking a longer time for culturing so I'm waiting for good suggestions.
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Mastitis organisms were more species. So you need more cultures media (ex for Setrptoccoci use Sterptococci media count 1% sodium azide  , coliform  use Eusion methylene blue and Staphylococci use Staph No.110)
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I have isolated actinobacteria from soil, but until now I only get one actinobacteria. Most of microorganism grow there is bacteria. I used Nystatin, penicillin, and sometime use benomyl. Did I use a wrong supplement?
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You can use specific media for isolation.and also try using different antibiotics
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I have a very promising consortium, which is able to grow almost in pure oil and degrade it as well. I want to isolate cells for further analysis and sequencing, but there is a problem. When I am trying to centrifuge the medium, nothing is happening (no pellet is visible, only some scums).
Does anyone have any experience with such samples and methods of isolation?
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Just a crazy idea, but perhaps you can try some classical phase separation method: When you have your Oil with the bacteria, try to add some water, mix the two solutions and then let the water settle. Then take off the oil and spin down the water fraction and check if you got a pellet.
Best regards.
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I have learned that for the identification of microbial isolate (especially bacterium) up to species/subspecies level one have to analyse 16S rRNA/rDNA sequence, %G+C content of the bacterial genome, DNA-DNA hybridisation and FAME analysis (as reported in many papers published in IJSEM journal).
What is the role of % G+C content in identification and what it means above 50% G+C and below 50% G+C content? which is better (above 50% or below 50% G+C) to relate the evolutionary relationship between isolated bacterial cell with other already reported . {>50% G+C content means evolutionary relationship or not?}
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The G+C content is just a marker. Depending on the genus, a similar G+C content might or might not indicate a relation. For example, the next relatives of Corynebacterium glutamicum have a much higher G+C than this strain while more distant relatives have a similar one. So a certain G+C continent does not indicate relatedness.
To properly discriminate between species, you need methods with better resolution like the 16S sequence or DNA-DNA hybridization.
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I've been using bacillus subtilis to isolate thermostable alpha amylase.
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Enzyme production medium: The enzyme production
was carried out at 55°C (except temperature
optimization experiment) in a rotary shaker at 150 rpm
for 144 h in 100 mL of the basal medium of the
following composition: Soluble Starch (1%), Maltose
(1%), (NH4)2SO4 (0.2%), CaCl2 (10-4M), K2HPO4
(10-1M), MgCl2, 6H2O (0.02%), pH 7. At regular
intervals (2 h), the triplicate samples were harvested
and the cells were separated by centrifugation
(10,000´g 20 min) at 4°C in a refrigerated centrifuge.
The supernatant was used for enzyme assay and
characterization studies.
a-amylase assay: The activity of a-amylase was
assayed by incubating 0.5 mL enzyme with 0.5 mL
soluble starch (1%, w/v) prepared in 0.1 M sodium
phosphate buffer (pH 7.0). After incubation at 70°C for
60 min the reaction was stopped by the addition of 2
ML of 3-5-dinitrosalicylic acid reagent[7] and
absorbance was measured in a UV/vis
spectrophotometer (Biofuge). One unit (U) is defined as
the amount of enzyme which releases 1 mmol of
reducing end groups min-1 in 0.1 M sodium
phosphate buffer (pH 7.0) with 0.5% (w/v) soluble
starch as substrate at 55°C.
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I found a large presence of lactococcus garvieae in milk and cheese samples.
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Dear Dr. El-Baradei, we have isolated and characterized many L. garvieae strains from traditional Spanish cheeses. In some of them, they constitute majority populations during acidification. However, apparently, they are replaced by L. lactis during ripening. If present, they are easily isolated in M17 (as L. garvieae strains from dairy are lactose positive, and they behave similarly to wild L. lactis). They are already present in milk, which means that they may come from cow´s udders, as does L. lactis. You are well aware that for contributing to cheese ripening cells have to die and release the cytoplasm content, which is actually what I mentioned above. I am pretty sure that they contribute to the sensory characteristics of the cheeses in which they are dwelling. Furthermore, some strains have bee proposed and used as starters in some Italian cheeses. Hope this answers some of your questions.
Regards,
Baltasar Mayo
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We were trying to isolate some thermophilic bacteria when the idea suddenly struck us to isolate Thermus aquaticus from hot springs. But before approaching hot spring sources we wanted to try isolating some thermophiles from the soil.
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I'm planning to sample several environments looking for Staphylococci species specifically. Is there any protocol permitting to select them efficiently? For example, which medium is the most selective? What are the biochemical tests which are reliable and discriminatory, easy to perform? Even if the selection is not perfect, I am looking for reducing the number of strains to sequence (for identity confirmation).
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Ive got one easier method for you. Chromagar. BD makes selective agar plates for S. aureus and S. epidermidis - these are known as Chromagar plates. You can buy these here: http://www.bd.com/ds/productCenter/214982.asp.
Once you have done that I would do whole colony PCR using MLST primers to determine the clonal type.
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We have been trying to identify some of our bacterial isolates using the Biolog Identification system. Two of the isolates, which vary in their physiological fingerprints to an extent, have been assigned the same genus and species by the Biolog. Any clues?
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Biolog Identification is based on the oxidation of different carbon sources by bacterial strains, it is quite normal that strains of same species can have different pattern in biolog plates. So, you can't assign species name only on the basis of it, you need to confirm your results with 16S rRNA gene sequencing or FAME or MALDI-TOF (as suggested by Saban).
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We are currently handling a good collection of lactic acid bacteria and yeast isolates associated with various stages of an indigenous fermentation process for production of fermented bamboo shoot. Most of them are fairly identified by ARDRA and rRNA gene sequencing (similarity range 97-99%). However, being an untapped ecological niche which is not explored deeply, we are expecting this niche might harbour new novel species.
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Dear Wahengbam Romi,
I would like to suggest you kindly to consider folowing characters for describing new isolates as suggested by Granitsiosis. Firstly, colony properties like, colour, smoothness or roughnes, size and shape; secondly, better nutrient medium, color, shape and size of the bacterium, specific growth rate, habitat, biochemical properties like enzymatic activities, and then go for molecular analysis. It will give you better data to claim for new isolates.