Marine Microbiology - Science topic

To determine whether epiphytic bacteria on seaweed have a positive effect on seaweed growth or nitrogen fixation, you can perform a variety of experiments and physicochemical tests. Some potential approaches you could consider include:

I hope this helps! Let me know if you have any additional questions.

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

Check https://www.frontiersin.org/articles/10.3389/fpls.2017.00049/full

Thank you @ Sambit Kisore Das. Very informative. I can't agree less.

Hi, I agree with Laith, you can use Petroleum oil-Czapek agar medium to isolate water fungi.

Best regards

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?

Mohammed Anas Chowdhury Thanks for your advice, I will search in those links. =)

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

http://ina.tmsoc.org/CODENET/culturenotes.htm

(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

I believe this issue has been discussed before https://www.researchgate.net/post/Why_3g_L_of_phytagel_could_not_set_in_SM_media

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.

Winogradsky columns might be usable...many possible ways to formulate them.

Dunaliella salina, I agree  with Dr Saber. I am warking on but to study the effect of  salinity  on the accumulation of bêta carotene. 

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

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 :-)

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).

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.

 Zobell Marine Agar is also good medium ti isolate marine bacterium. but you have to modify it for isolation for plastic degradation.

The media you mentioned in your statement are also good only concentrate on salinity ,temperature  and pH of your sample and environment of your sampling place 

http://aem.asm.org/content/66/5/1834.full.pdf+html 

http://download.springer.com/static/pdf/406/art%253A10.1007%252FBF01458031.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2FBF01458031&token2=exp=1487234528~acl=%2Fstatic%2Fpdf%2F406%2Fart%25253A10.1007%25252FBF01458031.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252FBF01458031*~hmac=2079d5b87d4c7bc24260cc90832134851d3b5a0866a69f19d25fbd4e4591d015 

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

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. 

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.

Dear Peter

Thanks for your quick response,is noted and will be acted upon.

 Dear Ms.Tatiana,

I think the photomicrograph is of Hormogonium of Stigonema.( Cyanobacterium coming under  the order Nostocales).

Good luck!

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.

Thank you very much for your information.

Make sure you have a pure culture.  The unicellular could be anything.

You could try 

Australian National Algae Culture Collection http://www.csiro.au/

or

Culture Collection of Algae and Protozoa, UK

Quddusi:

Well, your documented form could possibly be assigned to Leocrates sp. of  Polychaete worms Family Hesionidae.

Best

Syed

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!

Hey thanks .. I thought so too. wanted to be sure about it.

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

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.

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.

Thank you for your answers  

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. 

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 CO₃^2-.

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.

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.

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!

So it seems that C N and P from viruses could support benthic communities based on the work by Dell'Anno et al 2015.

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".

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 :) 

My dear, this seems to be a fungus.

My greetings.

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!

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.

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!

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)

thank u mr. karrar, i isolated  and conformed the phages by using the above protocols successfully.

Here is a list of algae culture collections that might be helpful.

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..

Dear Trabelsi,

I hope 2 links below are useful to answer your question.

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

Maybe you will be interested in this:

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

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.

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.

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!

I think that is gastropod

Stab culture  with 1% agar, 20-50% glycerol stock without removing salt can use for longtime preservation it works very well for my samples.

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

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. 

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

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.

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

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.

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!

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.

Obviously, chlorophyll biosynthesis is suppressed somehow and/or its degradation is promoted. Possible reasons include (but not limited to) direct antibiotic action, or increased light sensitivity (mediated e.g. by ROS accumulation and/ photosynthetic appaartus inhibition).

Water treatment plants vary greatly in their processes, especially relating to the source water.  Plants treating surface water are going to have low numbers of bacteria survive the treatment processes, especially Bacillus species and other endospore forming bacteria, because many of the endospores survive even chlorine.  Well system will have fewer Bacillus spp., but might have other bacteria persist because the water isn't treated as vigorously.  You also will have the development of biofilms in the system, especially in the plumbing of houses and businesses. 

Hello, Adeyemi,

Four years ago, I taught an optional Marine Biology class at the University of Cyprus that was very well attended. I wanted to introduce the junior and senior Biology students to the incredible abundance of microorganisms, and sought supporting documentation for exactly such statements, as the one you are asking about.

The source I ended up using was a 2007 paper by Larry Pomeroy and colleagues in the Oceanography magazine. The article citation is:

Pomeroy, L.R., P.J. leB. Williams, F. Azam, and J.E. Hobbie. 2007. The microbial loop. Oceanography 20(2):28–33, http://dx.doi.org/10.5670/oceanog.2007.45.

And here is a link to the article on The Oceanography Society web-site, where you can download the paper:

Make sure you check out Figures 1 and 2 on page 30. I think you will like them.

Best regards,

Angelos

Zobell marine broth 2216 contains ferric citrate and sodium sulfate. In the presence of sulfur reducing bacteria in the marine sediments, concurrent reduction of ferric and sulfate to ferrous sulfide.

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.

I agree with the above comments. It really depends on what you are interested in. Are you interested in overall changes in the coral microbial community, or do you want to know the composition of the microbial community within each niche in the coral (e.g. skeleton, tissues, mucus)?

It is always important to take samples from adjacent water, or other potential sources of influence as a control. This way you may control whether the microbes that you find within the coral microbiome are coral-specific or also found within the surrounding environment.

You often find papers working on the coral microbiota with microbial community profiles from both the coral and the surrounding water (see examples in links attached).

I hope this helped.

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