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Marine Microbiology
Questions related to Marine Microbiology
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Hi everyone,
I am trying to grow marine cyanobacteria from my samples using BG11 and L1 as nutrient and agar as gelling agent. I found agarolytic bacteria thrives in my plates, liquifying my agar. So, I tried using gellan gum as an alternative to agar. In my trials, I keep getting wobbly unstable gellan gels even though I used magnesium sulphate as cations. After overnight upside-down, I got dome-shaped gellan gel surface. When I tried using cell spreader, the gelling broke. Has anyone encounter this problem?
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Kristova Yubilius Indrataruna I have two suggestions for you: 1) Use ASNIII medium for marine CB: BG-11 was elaborated for freshwater CB; 2) Use very purified agarose (like Kim Sea) instead of either agar or gellan gum because very purified agarose does not support the proliferation of heterotrophs. Good luck. Igor Brown
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you can consult this book
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Hi, everyone!
I'm looking for an untouched area or highly emerged problem that humans (probably farmers, pharmaceutical industry-related) facing recently or might be faced in the near future which can be sorted out by marine microbiological or biotechnological resources.
or please suggest to me any resource related to this matter. your valuable comments are highly appriciated.
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In the isolation of Vibrio from water sample, besides using TCBS what other selective media can be used
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Thank you @ Sambit Kisore Das. Very informative. I can't agree less.
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I want to isolate fungi from sea water. Which medium do you recommend to do so?
I was thinking about PDA modified with sea water but I am not sure if I should use either 100% sea water or a mixture distilled or deionized water/sea water? I do not know either if it is better to use natural filtered sea water or to prepare artificial sea water.
Thank you in advance
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Hi, I agree with Laith, you can use Petroleum oil-Czapek agar medium to isolate water fungi.
Best regards
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While extracting MPs from sediment samples of Peruvian sandy beaches, I found a transparent film with dark brown dots (see pictures attached). Could those be colonies of marine microorganisms? If so, what are the possible species or families? Consider this sample coming from Lima, Peru (SE Pacific).
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Hard to tell, but I doubt they are bacteria. Can you do a bacterial stain and observe under a microscope? Or culture these on agar plates?
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Dear All,
I'm interested in studying a Master Degree program on Marine Microbiology or related fields.
I wanted to know if you can recommend me a place or pogram, based on your experience.
Thank you all for your time.
Greetings,
Pamela Borja
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Mohammed Anas Chowdhury Thanks for your advice, I will search in those links. =)
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I am attempting to culture aerobic marine methanotrophs and was planning on plating them on seawater agar. I keep finding references to 'aged' seawater in media recipes and was wondering what the necessity of aging the seawater in the dark is? If the aging is necessary, should I filter sterilize or autoclave the seawater prior to aging or just before media prep?
Thanks for your input!
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First, age the seawater for few weeks (2 to 4).
After ageing, filter the seawater
Lastly, Autoclave
I found two references mentioned below related to your question,
(1) The quality of water used in media preparation is very important.
Natural seawater can be collected nearshore, but its salinity and quality is often quite variable and unpredictable, particularly in temperate and polar regions (due to anthropogenic pollution, toxic metabolites released by algal blooms in coastal waters). The quality of coastal water may be improved by ageing for a few months (allowing bacterial degradation of inhibitory substances), by autoclaving (heat may denature inhibitory substances), or by filtering through acid-washed charcoal (which absorbs toxic organic compounds). Most coastal waters contain significant quantities of inorganic and organic particulate matter, and therefore must be filtered before use (eg Whatman no. 1 filter paper).
The low biomass and continual depletion of many trace elements from the surface waters of the open ocean by biogeochemical processes makes this water much cleaner, and therefore preferable for culturing purposes
(2) "use of aged seawater (sea water kept under dark for (2 to 4 weeks) is frequently recommended, but the media prepared from recently collected and properly filtered seawater are entirely satisfactory.
Handbook of Phycological Methods: Culture Methods and Growth Measurements edited by Janet R. Stein
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The composition of the BH medium is as follows:
MgSO4 - 0.2g/L
CaCl2 - 0.02g/L
KH2PO4 - 1g/L
K2HPO4 - 1g/L
(NH4)2SO4 - 1g/L
and FeCl3 - 0.05g/L
Thank you!
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This medium lacks nitrogen source, so it depend on the nutritional requirements and N sources is one of them?
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Thanks
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Hi , I agree with Abhishek Mukherjee answer
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I am conducting a comparative study trying to isolate bacteria on different concentrations of Marine broth (MB) media + agar vs MB + gellan gum/Gelzan/Gelrite.
For the agar I have 10, 50, and 100% MB, which solidifies perfectly fine.
I have tried 10, 50, and 100% MB + gellan gum but only 10% MB solidifies. I have tried to optimise the concentrations of gellan gum from the recommended 6.5 g/L to 10 g/L. I have also tried to prepare the media with and without Mg2SO4.
Any advice would be appreciated!
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In order to gather knowledge regarding state of the art tag sequencing methods used in the field of microbial oceanography, I am looking for on going projects of marine microbial observatories/recently published studies regarding time series of marine microbial communities.
Thanks in advance!
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i am more interest to know what kind of hypothesis, question, or objective that each index usually answer. surprisingly that both indices has quite the same definition in a term of scribing the reason of using them to define diversity.  i calculate both indices and i would like to discuss the results. simply my objective is to see if there are special and temporal differences in phytoplankton diversity among 8 sampling stations.
  also i am interested to know what are the main objectives or questions of your researches that you answered using such indices. thanks
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Parallel use of species diversity indices in ecological studies is a general practice, and a typical case is the parallel application of the
Shannon–Wiener and Simpson index. However, while the Shannon–Wiener index is strongly influenced by species richness and by
rare species, the Simpson index gives more weight to evenness and common species. The effect of the sample size is generally
negligible for both of them.
The use of both diversity indices improves the output information of the dataset, which is unique for each community or sample
analyzed. Looking at the wider content—both emphasizing the richness, and the specimen distribution into the individual
species—adds to the more complex information of the diversity in ecosystems.
In this sense, H has an advantage over D because it depends more on species richness and less abundant species, so it is very
sensitive to even small diversity changes, and thus is widely used to assess the actual state of environment. On the other hand, D has
the advantage over H in counting more on dominant species and is not affected by less abundant elements; therefore, it is used to
show the trend of ecosystem diversity heading.
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Dear All,
I have been isolating Vibrio spp. from sea food using TCBS agar, but have a lot of confusion in morphological identity and biochemical tests. Most of the suspected isolates were identified as Aeromonas and Shewanella after 16S rRNA sequencing. 
I would be thankful if anyone can let me know about any other convenient method to identify Vibrio spp. and I need to know whether 16S rRNA sequencing is a reliable method to identify Vibrio.
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Dear all,
I could find this reference too for isolation and identification of Vibrio spp. and I could successfully use this method for isolation.
I think it is better to share it with you all.
Many thanks to the authors of this study (D. Ottaviani, L. Masini and S. Bacchiocchi, 2003).
Thank you,
Best regards,
Chamara.
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I am in search of protocols for culturing marine bacteria from sea sediments under anoxic or microaerophilic conditions.
My samples are from deep-sea sediments 2-5 km in depth from Antarctic waters. The sediments are within the upper 10 cm of the seabed. 
Any protocols, media suggestions, or advice would be extremely appreciated.
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Winogradsky columns might be usable...many possible ways to formulate them.
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Can you help to give some example  of high temperature tolerant marine micro algae.
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Dunaliella salina, I agree  with Dr Saber. I am warking on but to study the effect of  salinity  on the accumulation of bêta carotene. 
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Hi, I am approaching to the identification of nano and microplankton using an inverted microscope with epifluorescence. 
I would like to differentiate the organisms in 7 classes:
Autotrophic dinoflagellates, autotrophic flagellates, cryptophytes, diatoms, prymnesiophytes, heterotrophic dinoflagellates and
heterotrophic flagellates.
This type of classification has been used by Landry et al. 2014 (http://dx.doi.org/10.1016/j.dsr2.2014.02.006).
Using the epifluorescence I can distinguish heterotrophs from autotrophs, but I do not now how to identify the organisms for each class.
Do you know how to do it? 
Thank you, any help will be more than welcome,
Luca
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Hello,
I did my PhD looking at the different phytoplanktonic group and differentiate autotrophs to heterotrophs. For each samples, I was doing two observations: 1 with the traditional light to identify the species and then switching on the epifluorescence to observe if the organism is auto- or heterotrophic.   I have checked the article you mentioned, and they don't provide a lot information "Each cell was manually identified ".  I am almost sure that they should have a picture  of the organisms without fluoresecence for identification.
Today I work in a company where we do viability count for Ballast Water using epifluorescence, and identification is done by switching off the fluorescence
Bye
Aurore
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Hello,
I am analysing microbial assemblages attached to marine microplastics using 16S metagenomics, and I have a query about maximising the quantity of DNA I can extract from the substrate?
I have been using the DNeasy Kit, Qiagen (Formerly PowerSoil, MoBio) to remove the attached assemblage from the plastic (recommended by Harrison et al, 2014), but I am struggling to extract any DNA - according to PCR amplification and 0.8% agarose gel electrophoresis. I am aware that the associated biomass, ergo DNA yield, is typically very low.
One paper (Debeljak et al, 2017) suggests that I should incorporate an additional lysis step using ReadyLyse solution (Epicentre) to maximise cell lysis. However, I was wondering if there was a cheaper alternative? For example, I was thinking of creating a Lysozyme:TE Buffer solution to the desired molarity, although I'm aware that the enzymes are derived from different sources and have different efficiencies.
If anyone has any feedback/recommendations, that would be very useful!
Thanks in advance.
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Ok, sounds challenging - I also found early sampling intervals (6hrs - 2 days) more difficult to deal with. In the BMC Microbiology paper, some samples were excluded for this reason. If PCR fails, you could also consider multiple displacement amplification (MDA) or FISH (if you know which taxa you're looking for).
As a side note: This also depends on temperature... during some pilot experiments, I found that the plastic surfaces were colonised a lot quicker when incubated at RT rather than 4 degrees Celsius :-)
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Can fastidious or even unculturable marine bacteria be grown in situ? If so, what are the current methodologies for doing so?
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The main 'classic' method is just leaving a glass slide in the water and let it colonise by the bugs.
For more advanced methods I would recommend take a look on some of the work of Belinda Ferrari at UNSW (Australia).
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I am looking for standard nutrient solid media recipes or protocols used to culture marine bacteria. What are the standard types of media used to culture marine bacteria (psychrophiles)?
Also, in scenarios where these media are not able to culture these bacteria what methods are then employed?
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One commonly used media is Marine Agar 2216, which is based on ZoBell media. Works fine with my psychrophiles. :)
For those bacteria that cannot grow on this media, you might need low-nutrient media or  a special one designed for the purpose.
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Hi,
I am investigating marine bacteria for potential plastic degradation, and wanted to know if there any standardised protocols or selective media that would be worth considering during the screening process.
I have considered directly incorporating the polymer into the media (PE powder & marine agar - Lobelle and Cunliffe, 2011), Bushnell-Haas media (Harshvardhan & Jha, 2013) and the MATH assay. However, my understanding is the latter two methods typically investigate liquid hydrocarbon, whereas I am using a solid substrate.
If anyone has any suggestions, or is facing a similar issue, or has any feedback on the above mentioned methods, please comment
Thanks :)
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Hi
I work with soils and sediments from Antarctica, and as DNA is very stable under those low temperatures, I would like to use RNA extractions instead of DNA extractions from the environmental samples to do amplicon sequencing with Illumina. Is that possible? have anyone did amplicon sequencing for 16S, 18S, ITS, based on cDNA from RNA extractions?
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Dear Susana,
Let start with the basic; DNA and RNA are relative stable molecules. In nature, they are degraded mainly due to enzymatic catalysis. Of course, RNA is less stable than DNA in oxic conditions, but this is not the case here. Microbial communities in cold soils are dominated by psychrophiles which are able to have an active metabolism in those environments. Investigation of microbial communities via 16S amplicon sequencing in DNA level will show you who is present, where in RNA level will show you who is active there. This is a well known process and people doing that for long. The difficulties lying in the following:
1. Be able to stabilize your samples as soon as possible and not having community shifts during sampling and prior of extraction. Several ways to do so; Liquid nitrogen, RNA protect or Lysis buffer in situ. Since the environment is cold, and liquid nitrogen difficult to transport, I would go either with RNA protect or lysing buffer directly (and then freeze). Actually, this is how I am collecting environmental samples for RNA analysis. I used this procedure to collect permafrost RNA samples.
2. How much RNA you can extract from your samples. There is a huge discussion among experts, and dozens of protocols. Experience taught me that there is no universal protocol and if you want to evaluate a protocol you need to do it with the actual samples! This, sometimes is very difficult. In principle you need to keep in mind the following principle: harsh conditions to break the cells and release nucleic acids, not too harsh because you will co-extract several inhibitors (i.e. humic acids) and you might degrade your RNA. 
If you need more information how you can do it, limitations and amlpicon RNA seq strategies, please contact me directly.
Hope this helps
Best,
M
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Marin samples?
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My best guess is also for a squashed solitary tunicate. It almost looks as if the body has been mostly squeezed out of the tunic. 
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Hi, we are working on stool-metagenomics based on V3V4 and ITS2 amplicon sequencing. We would like to analyse data in a work station by using QIIME. Can someone help me with the best configuration (type of processor, RAM, hard disk storage) of work station that is required for analysing this NGS data (preferably for the data that is generated on an Illumina-MISeq platform; assuming for each sample nearly 2 GB of data is generated; and we need to analyse a maximum of about 120 such samples at a time).
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I have the Processor Intel® Core™ i7-6700 CPU @ 3.40GHz × 8, In this web (http://ark.intel.com/products/88196/Intel-Core-i7-6700-Processor-8M-Cache-up-to-4_00-GHz) you can check the characteristics. It have 4 cores, and 8 threads, it is good because in a lot of software you can use more than one thread, that makes it faster. And it is so important to have a 64bit operation system, because with 32bit you can't use all the RAM of the computer.
Sorry if I have not explained so well... but basically more threads is more processing powerfull and less time to finish the processing.
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I am presently carrying out research on microalgae that produces toxin in lagos Nigeria, i would like to know where i can purchase saxitoxin standard or where i can send my samples to (water and shellfishes) for quantification. Thanks in advance .
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Dear Peter
Thanks for your quick response,is noted and will be acted upon.
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This is a recurring unknown in my samples. It is about 20 um in length. The sample was preserved with Lugol's solution, and the image was taken from an inverted microscope. The sample was taken from a central Texas reservoir in June. 
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 Dear Ms.Tatiana,
I think the photomicrograph is of Hormogonium of Stigonema.( Cyanobacterium coming under  the order Nostocales).
Good luck!
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i have been given an assignment and i have to find out how to isolate marine microorganisms ... need help 
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You can use following samples to isolate the microbes.
1. Sea water
2. Marine algae
3. Corals and sponges
4. Marine fish and other organisms
First you can culture them in sea water agar plates. It can be made with bacto agar + sea water. Then if you use sea water around 100 micro liter of sample spread on the plate using sterile glass beads. Then Incubate them at 30 0C for 16 hours.
After 16 hours you can identify different shapes of colonies. But they may not have prominent colors. Then pick that single colonies and strike separately on marine agar plates. Incubate with same conditions mentioned above. Then you can see the colorful colonies. If you need further separation... separate them again with picking the single colonies until contamination will be not found. If you need to identify them, simply use the colony morphology or you can do the 16s rRNA sequencing analysis.
** When you separate the colonies, make sure your own labeling method to identify each colony. continue that labeling method until fully identify the bacteria or fungi.
Best regards.
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I want to culture the tardigrades collected from intertidal area? In the initial experiment, I tried with diatom feed(Achnanthes) but failed.
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Thank you very much for your information.
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I have isolated Botryococcus in a river, when I analysed it through LM it has colonies and mucilage threads connecting. But when I tried culturing it became unicellular but there are some visible threads, but it has a thick cup shape mucilage on the outer cell and takes 14 days to culture while optimising its culture conditions. But later on ill do some ecological studies and phylogenetic studies on that river. But my theory is that the botryococus I have isolated is not in river itself  but the source of it at top or upstream. I hope you can help me.
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Make sure you have a pure culture.  The unicellular could be anything.
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I am landlocked an unable to culture marine specimens myself. I am having trouble locating an institution which has this culture available for study.
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You could try 
Australian National Algae Culture Collection http://www.csiro.au/
or
Culture Collection of Algae and Protozoa, UK
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Found off the karachi coast
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Quddusi:
Well, your documented form could possibly be assigned to Leocrates sp. of  Polychaete worms Family Hesionidae.
Best
Syed
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I want to quantify bacteria present both in the tissue and skeleton of coral and am thinking of inoculating 96 well plates and doing serial dilutions to obtain the most probable number (MPN). I can use a plate reader to measure optical density so human error is removed when assessing turbidity of the wells. This poses the question:
1) How can I remove a standardised area, weight or volume of coral tissue and skeleton with minimal damage to the coral?
I need this to standardise the number of bacteria to i.e. number of bacteria per ml mucus or per cm2 tissue. I've tried using a scalpel to scrape or break off pieces but it is difficult to standardise this and I've tried using 25G needles to extract tissue from the polyps, though this is only successful on long-polyped morphologies. I've heard that Waterpik-ing is very crude and difficult to standardise too.
Any insight on how to quantify coral-associated bacteria would be helpful. I had also thought about qPCR but this too requires removing a standardised area of coral tissue and skeleton to begin with.
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Getting the optical density or even dry and wet weights will not be accurate for this study. The former requires liquid cultures which can be concentrated by filtering or de-concentrated by dilutions. The 2 latter methods will also give false results because you will need to harvest the bacterial cells by scrapping as you say and then weigh them out and you see you could be weighing biomass which are not bacterial cells.
I think the best way to go about this study will be to do viable plate counts on appropriate culture media as this will also aid you in doing further identifications like colony morphology, biochemical and even molecular characteristics.
 Most of the endosymbionts of coral animals have been shown to be algae (dinoflagellates) and this study will be useful in showing if bacteria are also coral endosymbionts. As for the blank used to zero the spectrophotometer you can use either bacteria-free medium or just sterile distilled water.
All the best!
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I was planning to run a qPCR assay on amoA genes (bacterial and archaeal) from my marine sediment DNA extracts. After going through literature I observed that many people were using the Arch-amoAF and Arch-amoAR to amplify archaeal amoA (Francis et al 2005) and amoAF1 and amoA2R for the bacteria (Rotthauwe et al 1997). However these are the same primers used for preparing standards for their assays. I have a pure culture N.maritimus to be used as a standard for the archaeal assay. I was wondering if I could use the above mentioned primer to amplify the amoA gene from the cell culture, purify the product and directly use it as a standard without cloning it ? My main concern was if it was fine to run the qPCR assay with primers that were used to prepare the standard itself.
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Hey thanks .. I thought so too. wanted to be sure about it.
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My lysmata seticaudata have become sterile, they are still carrying eggs as normal, and these are developing but not hatching, and are then shed with the moult. Their eyes have a growth covering them, can anyone identify this, is it likely to be related to the sterility issue? Lysmata boggessi have also become sterile, but I've moved them on before I'd noticed this eye issue. Attached are two photos, i4 is a healthy eye, i5 and i8 have the growth.
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Dear Tony, 
I could suggest you to contact Dr. R. Calado, CESAM,  University of Alveiro, Portugal who is renowned expert on marine ornamental shrimps. His group may work on these parasite issues. He may able to address your issues......
You may contact him at rjcalado@hotmail.com 
Prakash
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Does anybody know that can the Ceratium furca and Ceratium fusus produce toxic?Are they Harmful algae?THX!
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To my knowledge Ceratium species do not produce toxins, however they can be considered as "harmful algae" if they deplete nutrients under bloom conditions. The papers that Cihelio cited do not prove toxicity of the species either but only deduct it from their dominance in blooms that led to fish kills (Mijares et al, 1985 & Orella-Cepeda et al, 2004). The putative toxin that Mijares et al found could therefore derive from another phytoplankton species that was present. The publications of Licea et al (2004), and Orellana-Cepeda et al. (2004) even mentioned that Ceratium was non-toxic.
So, harmful - yes, toxic - most likely not.
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I am currently studying for my masters and my project involves studying chytrids and their association with marine diatoms.  I have a lot of papers many of which relate to previous methods and findings in freshwater environments.  I was wondering if anyone had observed possible or confirmed chytrids in conjunction with any other research into diatoms and if so did you notice any patterns or species that seemed particularly affected.  I'm not looking for definitive data just any thoughts or observations anyone may have made. 
Thanks
Lucy
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Lucy, I would be interested to follow your research on diatom parasites -  although I am not an expert on the parasite aspect of the study (only on diatoms), having published only one paper on this topic. Certainly, let me know if you find any parasitized Pseudo-nitzschia cells. You can find Pseudo-nitzschia references at the website I maintian (http://www.inter.dfo-mpo.gc.ca/Gulf/DAPR). I can also put you in touch with someone else working (occasionally) on parasites in Pseudo-nitzschia. You can contact me directly at stephen.bates@dfo-mpo.gc.ca. I wish you the best for your important research.
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Marine microbiologist
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Thank you for your answers  
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I am looking to determine the rate at which a seawater intake pipe will foul from biological material at about 40 feet water depth off southern California 
Thanks
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There is no better manner to estimate this than having previous or getting new field data - which, I am sure, you can find, since there are several universities and research institutes nearby - on the fouling development (biomass, species composition - some species are usually defined as "soft fouling", while calcified biofoulers (e.g., barnacles) are called "hard fouling" by people from more applied fields) on such surfaces. Fouling development will also depend on the composition, orientation, roughness and diameter of the pipe in question. 
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We are currently trying to create mutualistic associations between yeast and algae and our system relies on identifying a carbon source that is not utilised by Chlorella species. However, thus far it seems to be able to grow on most carbon sources that we tested.
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There are some factors that will counteract your strategy.
You have to make sure that there are conditions under which Chlorella is really dependent on an external carbon source. For it, you have to grow it under completely heterotrophic conditions in the absence of any light.
You also have to make sure that there is no dissolved inorganic carbon in the culture medium. If there is contact with air, dissolved inorganic carbon in the culture medium exists in the forms of CO2, H2CO3, HCO3- and CO32-.
Another problem is that dead organisms or even some secretions of living organisms in your setup will also provide a carbon source; this must be prevented.
With these conditions, Chlorella won’t grow solely on (polycyclic) aromatic hydrocarbons like Anthracene, Phenanthrene, Chrysene, Benzo(a)pyrene, Coronene or Ovalene.
However, these compounds also show toxicity. Moreover, the required setup conditions with missing CO2 directly influence several key enzymes in carbon metabolism, such as carbonic anhydrase, and other pathways.
Thus, I am afraid that your strategy won't be easy to put into practice.
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This species was found to cause blooming in the Mediterranean sea. This species was blooming in the Mediterranean, turning the water brown.
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Dear Mohamed, as others have stated this is indeed A.sanguinea (also known as Gymnodinium sanguineum ). It is thought to be a non-toxic dinoflagellate but it has been implicated in seabird deaths (see Jessop et al. 2009). 
Jessup DA, Miller, MA, Ryan, JP, Nevins, HM, Kerkering, HA et al. 2009. Mass stranding of marine birds caused by a surfactant-producing red tide. PLoS ONE 4: e4550. doi:10.1371/journal.pone.0004550.
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Collected on the building walls, diameter around 2 to 9 microns. both have a same size with different pigmentation and cell structure
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Dear Nur,
I doubt the first picture as that of Chroococcus limneticus  and the second one as Gloeocapsa violacea Kützing
Reference: Cyanoprokaryota ,Teil 1.Chroococcales,Komárek, J and Anagnostidis .K(1998)
Good luck!
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This question came up after reading the paper by Engelhardt et al., 2015.
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So it seems that C N and P from viruses could support benthic communities based on the work by Dell'Anno et al 2015.
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My question came up after reading the paper by Yutin et al. 2013.
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According to the International Committee on the Taxonomy of Viruses, they have not been classified: http://www.ictvonline.org/virusTaxonomy.asp.  As far as I know they have not been officially classified yet.  According to Yutin et al.'s phylogenetic analysis, these viruses seem to be more closely related to Mimiviruses (and relatives) than to the classic "Phycodnaviruses".
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Hello
I am working on macrobenthos in Iranian waters of Persian Gulf (soft bottom). I can not identify this species.
Could someone help me.
Thanks a lot
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Salam Ms. Sorour,
Please send a high quality photo. By the way, if you want to get the precise answer, maybe you can send the sample to the museum or anywhere who are expert on this animal, same as what I did for crustaceans.  Good job, and Good luck :) 
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Water samples were taken from an estuary (Salinity: 15 ppt) and brown colony formed in the agar plates after 4 weeks of plating. Photographs taken at 40 X magnification. Size of the cells: 8-15 micron 
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My dear, this seems to be a fungus.
My greetings.
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Hi everyone,
I am looking for a specific microbe and though someone here could help? For my research, I need a non-pathogenic bacterial host that is slightly halophilic. Ideally this host should be able to survive in fresh or seawater and preferably one who's genome has been sequenced in the past. 
I am currently working with E. coli NEB 10-b and it is proving to be very sensitive to changes in ionic strength. I am also working with P. putida KT2440. Though I have managed to grow this host in LB media with an ionic strength of 0.5 M, they cannot survive the change to glucose minimal media at the same ionic strength (let alone surviving in seawater). 
Does anyone have a suggestion or potential host in mind?
Thank you!
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This is great, thank you both for your help. Vibrio does look like good potential hosts that are widely available on ATCC. Plus their optimal growth rates are typically around room temperature, is a plus for me. I am also considering some Shewanella strains as a potential host. I will verify the growth rate in freshwater of some of these strains and let you know the outcome. 
Thanks again!
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That preferably don't involve the handling of radioisotopes if possible!
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To measure uptake of N you can use 15N as already said. You can use either 15N2 gas for N2-fixation, or 15NO3 for nitrate uptake or 15NH4 for ammonium uptake. Check either Montoya 1996 or Klawonn 2015  for details.
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Hei,
I have several aerobic marine bacteria strains that I would like to test for sulfide resistance. My plan would be to add Na2S to the medium (marine broth), but as far as I know the S will precipitate with the oxygen, which is not the point.
After looking online and in few books I could not find a solution.
Anybody has an idea?
Thanks.
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Dear Sven,
A couple of papers i thought helpful to you are added below:
1. Teodora Bagarinao, Sulfide as an environmental factor an toxicant: tolerance and adaptations and adaptations in aquatic organisms,Aquatic Toxicology, 1992 - Elsevier
2. JEFFREY K. KING,JOEL E. KOSTKA,MARC E. FRISCHER,AND F. MICHAEL SAUNDERS,Sulfate-Reducing Bacteria Methylate Mercury at Variable Rates in Pure Culture and in Marine Sediments,APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
0099-2240/00/$04.0010, June 2000, p. 2430–2437
Good luck!
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From a reservoir. Sample fixed wiht lugol,
Thanks in advance
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Kind of Chrysophyte call. Might be lost by Uroglena or might be Ochromonas. In case you see ingested algae (e.g. Centrales) inside, most probably Ochromonas. But it is so difficult with these creatures. We mostly count them as "other Chrysophytes" and if we are lucky we can identify (but only from a living sample)
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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 want to obtain the cyanobacterium "Synechococcus sp. MA19" to carry out experiments.
Can you provide me with a source, e.g. a culture collection? I contacted authors that describe this strain (papers from 2000-2001) and large culture collections, but so far I was not able to find/obtain a sample MA 19.
Originally, the MA 19 was isolated from a hot spring in Japan (Miyakejima).
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Here is a list of algae culture collections that might be helpful.
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I am looking for a multiparametric probe instead of a set of probes for specific nutrients, e.g. nitrate, phophates and silicates.
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Hello,
Green Eyes LLC now offers wet chemistry instruments for the measurement of nutrients in fresh and salt water.  We took over the product line from EnviroTech Instrument in Jan. 2014 and have made significant improvements since then.  If interested, please contact us at info@gescience.com..
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Thanks for help
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ex: I have isolated Streptomyces albus from marine sediments. can I name it some thing like M1 or S1. Any standard systematic codes are there to name the strain?
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The rules are working for taxa from species to class, but strains aren`t taxa, so you are free to name them any way you want
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hello
I have many phage isolated from marine environment and I want to measure their  Capsid and tail size . please if you have program for this issue  please share it to  me. 
thanks
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I am currently using the SILVA reference database and I get OTUs assigned to MALV III. I saw in Georges et al. 2014 they were observed in Southern Ocean, but find little about their relation to the rest of the MALV. Anyone has papers or hits to help me? 
Thanks !
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For MALV the reference about phylogeny is
Guillou, L., Viprey, M., Chambouvet, A., Welsh, R.M., Kirkham, A.R., Massana, R., Scanlan, D.J. & Worden, A.Z. (2008). Widespread occurrence and genetic diversity of marine parasitoids belonging to Syndiniales (Alveolata). Environmental Microbiology. 10. p.pp. 3349–3365.
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I think its Psuedo-nitzscia seriata but would like a second opinion. The sample was taking north of Shapinsay in Orkney.  
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Dear Ben:
As it was mentioned by other collegues previously it is not possible to determine diatoms with this kind of pictures.
Pseudo-nitzschia is a genus characterized by presenting cells in stepped chains united by shorter or longer overlap of valve ends, these chains are motile.
Nitzschia may be solitary or forming chain like or stellate colonies and raphe system is strongly eccentric closer to the margin and fibulate.
Your picture did not shows these elementary features, thus it is not possible to determine the material seriously.
I recommend to use the book "Identifiying marine phytoplankton" Tomas (ed). 1997. There you can see the chapter 2, Marine Diatoms by Hasle & Syvertsen with good description of generic characters and characters showing differences between species.
Kind regards.
Eugenia
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I have initiated my work in Cyanobacteria from metagenomic cyanobacterial samples, and i wish to do some taxonomy work at molecular level. I am using 16S rRNA gene based Cynano bacterial primers CY106F and CY 781 R (Nubel et al., 1997). During PCR i am getting nonspecific bands along with the primers. I have tried all things by changing annealing temperature, MgCl2, BSA . But cant able to solve the problem. My query is is there any other Cyanospecific primers available? I had seen some people were using 16S rRNA and ITS based primers for Cyano classification. I am quite optimistic with 16S rRNA gene based classification. And also how fare morphology based classification will help compared to molecular level? Comments and suggestions are welcome!!!
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Thanks for the mention of the article, George.  I uploaded it in the box above.
Venkatachalam, the above article is a great introduction to the ITS region, but we have moved forward since then.  In that article it mentions primers 1 and 2.  I have found it relatively easy to amplify this region, and it is nice because it includes a large portion of the 16S rRNA (1161 nucleotides) and all of the ITS.  Because of the problem with operons, I clone all my PCR reactions and then submit 3-4 clones with internal primers so that I get the whole read.  ITS will not sequence well without cloning.
The ITS has been used recently to separate cyanobacterial species.  The secondary structure is useful, and as we get more taxon sampling we are using this region in other ways too. I am attaching a recent paper that applies the ITS in some interesting ways.  If you contact me directly (my email is in the article), I can give you access to a lot more.
Good luck.
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I want to know if bacteria requires insoluble hydrocarbon as a sole carbon source for biosurfactant production or they can produce it during their normal growth without any insoluble hydrocarbon.
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Different microbes behave differently under different culture conditions. Some produce biosurfactants in media supplemented with water-insoluble hydrocarbon substrates, while on the other hand, others do not need any hydrophobic substrate. They can produce biosurfactants on water soluble substrates. You can find literature regarding the metabolic physiology of your isolates for the production of biosurfactants or you can check it on both type of substrates, otherwise. Another phenomenon, which has been explored is that microbes produce variety of biosurfactants under different circumstances, i.e. same microbe may produce different type of biosurfactants on water-soluble substrates than on water-insoluble substrates.  Similarly, under different cultivation conditions, microbes can also switch between the two mechanisms of gene-regulation for respective biosurfactant production. Therefore, it is of utmost importance to investigate each microbial isolate for its characteristic potential of biosurfactant production under varying environmental as well as culture-conditions, before giving any final comments.
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I will be going on a 2-3 day trip and was wondering if I could just isolate bacteria (from sediments) in the field instead of bringing the samples back to the lab. If I have to bring the samples back then I would have to shorten my trip. Are there any Dos and Don'ts for this? or anything to take heed off?
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James: you can and shd do in the field. WE made a small portable- that can be assembled in the field isolation chamber with perspex sheet. We worked in the field for about 21 days with such arrangement. While coming back from Andman islands, we were lucky to get a good captain who allowed us use an empty container as lab. It takes almost two days to return and we utilised that time also. Sterilise with spirit. Enjoy and Happy time!
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I think that is posible algae??
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I think that is gastropod
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People usually make glycerol stock and store at -80 freezer. Do you remove salts before making the glycerol stock? 
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Stab culture  with 1% agar, 20-50% glycerol stock without removing salt can use for longtime preservation it works very well for my samples.
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I'd like to get a Trichodesmium thiebautii starter culture and so far, I've only found T. erythraeum. Does anybody have an idea where/which organization I could ask? I'd be extremely grateful for any help! 
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Hi Gustaf, thank you for your response! I'm in contact with John about FedExing them here, but you're absolutely right. It's taking me a while to get the last strain I received from Bigelow back to life... It's comforting to hear that it's not my fault they're taking their time. Hope to report on a healthy T. tiebautii strain soon! By the way, I just saw you're doing some interesting research on diazotrophy and ferritin. Looking forward to reading your papers! Greetings from Kiel
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It appears that both are desmids, one is develop a spiral!!  Is this something people have seen in nature previously?
Thanks in advance!  Paul
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Hi Paul They are Closterium  and the first 2 look likely they are dying  and the upper part of 19 looks normal.. the other 1/2 looks like it is starting to die..Are these pics from preserved material or are they from live. Look like chloroplasts in dying cells. 
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I am trying to get the standards for Mycosporine like amino acids to charaterize for the same in cyanobacteria but it is not commercially available. Please suggest me in this regard.
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Maybe this article/ scientific work group could help you for the identification.
A high-resolution reverse-phase liquid chromatography method for the analysis of mycosporine-like amino acids (MAAs) in marine organisms
J. I. Carreto, M. O. Carignan, N. G. Montoya
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Adults and eggs of killifish kept in a small modular recirculation system, showed fungal disease (cotton like appearance) on the anal fin and around the egg corion.
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Use acriflavine hydrochlorid, dosage 0.45 per 100 ml in vehicle q. s. p. Treatment of adult fish: two drops of 3 liters. Eggs: diluting a water drop in a 10ml container and collected with a pipette content of the diluted and pour just a drop in the containers containing the eggs.
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I am going to investigate the growth of Celeribacter spp. ( isolated from marine environment) at different salt concentration by using Luria Bertani broth. I was wondering if anybody could tell me, what other ingredients  except trypton, yeast extract, NaCl  should be added to LB? 
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I suggest for marine bacteria isolation or culture to use more suitable medium than LB and you can use Marine Broth of Difco or add marine filtered water to prepare medium. Else, I agree with Jurgen and Karthick to try these formula
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I am looking to run survival experiment  of bacteria in a lake mesocosm and would like to count viable cell numbers over time in sediments. Ideally, I would like to be able to calculate CFU/ml and not CFU/g so I can compare to growth in lake water as well. I have tried Beat Beating with 2mm beads but my recovery is very low. Any suggestions or ways people get around the mass to volume comparisons!? 
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Hi, if you are only looking at bacteria, you can use differential centrifugation. You first spin down at 2500 rpm to pellet all the larger cells and debris, with the bacteria still in solution. After decanting the solution to a new container, spin down at 10 000 rpm to pellet any bacteria in solution.
Although this is a very basic method, it allows you to study bacteria in any environment, minus some of the background noise. You can also combine diff. centrif. with bead beating - that way you know that most of your extracted DNA will be from bacteria.
I would also suggest using DAPI to stain only viable cells.
Regards,
F
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Why I did not get good digestion for phage DNA? I need to determine the diversity of phage isolates, and the approximate genome size from the fragments generated from RFLP. My concentration of phage DNA is 338.97 ng/µl & 1.93 at 260/280 nm. I used 1µl EcoR1 (NEB), 1µl (50x) buffer, 5µl DNA and 3µl Mill Q water for 10 µl reaction. 
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Hi Nattan, your reaction mixture should contain 1uL of 10X  buffer for 10 uL total reaction mixture. If you use 1uL of  50X buffer, you should bring the total volume to 50 uL. H2O should be 43 uL in your reaction mixture, You might have to add more DNA in this case.
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I’m using specific primers to quantify Scrippsiella donghaienis (a phytoplankton species) in DNA extracts from sediments by real time PCR. When total DNA concentration is high, the melting curve shows a specific peak of Scrippsiella donghaienis at 82,5 °C. However, in sediments of low DNA concentration, I get a double peak (a very small peak of Scrippsiella and another higher peak at 88 °C). Do you have an explanation to this ? I attached 2 images of specific amplification and non specific amplification from the same test.
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Once you have fnished a rtPCR with those resutls, I would run an agarose gel of your products in order to see if you get more than one band. Under my opinion it looks that the primers are not specific enough
Good luck!
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Hello peers,
I'm looking for papers/tutorials/information on how to classify transcripts based on species. For example, if I was going to be sequencing a small anemone doing a whole-body RNA extraction I want to be able to differentiate between the anemone and the food it has eaten (bacteria, plankton, etc.).
I know that the Lehnert et al. 2014 (Extensive Differences in Gene Expression between Symbiotic and Aposymbiotic Cnidarians) designed a custom Support Vector Machine algorithm "TopSort"; however, it is not open-source. 
If anyone could provide other examples (preferably with open-source software that would require no, or very little, tweaking) It would be most appreciated. I think that gut microbiome or metagenomic research would cover this but I haven't found anything yet.
Regards,
Matthew J. Oldach
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Kraken is the closest thing I've come across, but doesn't use any fancy machine learning I think. It looks at each kmer in the sequence in question and asks whether that particular kmer has been seen before, only in a particular clade, and thus, can the sequence be classified to a particular clade.  In this way it reduces your set of input sequences to a set of diagnostic features/markers that can be used to classify each sequence to a clade.
You could build a machine learning approach around this I guess, by training on known genomes, using each kmer as a feature, and deriving a classifier model relating the predictive value of each kmer in relation to each taxon.  
Maybe SVM is only a nicety here though, and you could make use of the existing (open) kraken software.  You would probably need to make your own kraken database, enriched for Cnidarian sequences (the default kraken is bacterial as far as I know), and bear in mind that this is a job for a machine with very large RAM.
Bonus points for returning the kraken to the underwater world.
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In the culture and screening of genetically modified microalgae, which can endure against the antibiotics, cells maintained duplication but green color of microalgae slowly disappears (not perfectly). What does it mean?
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