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In my books on the subject of “mathematics of opinions” I have shown that even positive beginnings (must) end in complete decadence, unless there is a premature end. This is most pronounced in the area of ​​politics, which for various reasons must degenerate into a melting pot of ideologically intellectually limited underachievers.
What is fatal, as can be observed live today, is the complete disconnection from reality: the leaders not only have an ideologically limited horizon of facts, they are also surrounded by a dense cordon of followers who absorb the leaders’ secretions with sexually fervent desire (I have also shown the background to this) and do not allow anything to get to them, i.e. they also effectively shield them from attempts at rational argumentation, which of course has an even greater effect on the ideologization.
On the other hand, the brain is bound to a demand-reward loop: people face challenges that, when mastered, cause a hormonal feeling of happiness (again, more details in “Mathematics of Opinions”). The absence of happiness hormones, in turn, causes people to look for new challenges. A problem arises when there are essentially no more challenges, because any action becomes without consequences.
This is also explained live in today's politics: normally, politicians should have to answer for an action, even if the responsibility only consists in asking themselves “Will I be voted out and can no longer act like this?” Today, the political system has reached a point where this no longer applies. All politicians, starting with the last backbencher in parliament, can do whatever they want without any consequences. A health minister can break laws and even the constitution, prove himself corrupt, send hundreds of thousands of citizens to illness and death and finally declare that all of this was not necessary - and stay in office and can carry on completely arbitrarily. No action has any consequences anymore, except perhaps for the last backbencher who is eliminated in the next round of elections.
Of course, this does not only apply to politics. The people behind it who still have some intelligence, the oligarchs or multi-billionaires who at least partially play their games behind the scenes, are also affected. For someone like Bill Gates, no action has any consequences anymore and the reward of happiness hormones is missing.
Ultimately, this leads to a displacement activity according to the motto "one man's joy is another man's sorrow". What can no longer be achieved through positive experiences of one's own can be achieved through negative experiences of others, for which one is ultimately responsible. The appeal of the matter is that you don't have to feel any consequences yourself, but others do (this is again explained in more detail in the books). This starts with the reporting of critics (even the followers in the justice system are subject to such mechanisms, as they too no longer have to fear any consequences if they ignore the law) and continues all the way to compulsory vaccination. I think it is quite possible that some politician or another has experienced a kind of inner reward orgasm in their uncompromising commitment to compulsory vaccination against the fears of those affected.
Where does it end? Wild conspiracy theories report cruel rituals by the rich and powerful in which women or - as the crowning glory - children are sacrificed. Well, if you think the development described through to the end, these are not conspiracy theories, but for some of those affected it may well end there. The ultimate kick from murdering someone else because something else no longer gives them a kick.
Is that also the background to the current desire for war that prevails everywhere? In Ukraine, the machinations of these people, who preach daily that more and more powerful weapons must be sent there, have probably already cost 1 million human lives, and another million could well be saved by what the Israeli government is doing, also with the help of the local hawks. And these things are not even far away: on social and other networks you can watch videos of people dying there at any time. Does that give you a kick? Or are we all already oversaturated with the virtual reality of the media industry, which can depict blood splattering around and guts hanging from houses in much more colorful ways with the help of AI? Is there really a desire to experience that live? Or is there still so much residual reality that you would rather not experience it yourself, but at least see someone you know personally die?
There is a lot to suggest that the ideological view is becoming increasingly narrow. It seems that the invulnerability of the warmongers leads them to make invulnerability itself an axiom of their thinking, in the sense that a war can destroy everything around them, but not them and their way of life. They seem to believe that they will continue to live in a luxury villa with servants who bring them everything they ask for, when everything around them looks like something out of a Mad Max film. And unfortunately, this virus also seems to be infecting those who are just the bulk of the claqueurs.
The reality: in the EU countries, no one has enough weapons to wage a war for even a few days. No one has a war industry that could provide replacements. The population is anything but ready or willing to fight; they are no longer even willing to work. One might consider the replacement of a private as warlord by a lance corporal as progress, but it is certainly not. The warmongering has no basis in reality, but is nevertheless constantly intensifying. Even if there is no war, it will still lead to hardship and misery. And although this cannot be overlooked, there is still a majority of the population that continues to enthusiastically participate.
As a consequence of current experiences, one view must be corrected: up to now, sociological and psychological models have assumed that a proportion of 25% - 33% of realists is enough to overturn the ideology. Current developments, however, show that an absolute majority, i.e. >50%, is actually necessary for a tipping point. Or, to put it more brutally: if you want peace, you must first kill everyone who wants war.
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Well, you have addressed exactly the strategic points that we thought at least some of were developed to the point where no major accident could happen. I don't know how things are going with you, but here in Germany everything has completely failed.
Transparency and responsibility: Transparency can only be expected from management personnel with authority. Unfortunately, we are in a situation in which management personnel have positional power due to networks and cannot afford transparency for competitive reasons. Responsibility no longer exists at all, as can be seen from the many scandals that are survived unscathed.
Psychological support: There is no understanding that this is necessary. You can't even forcibly commit a leading politician to help him get back on his feet.
Public education: this requires a functioning press if it is to be even partially successful. But that no longer exists here either. And as far as education is concerned... see the PISA studies
Democratic institutions: Annalena Baerbock "I don't care what my voters want" - no consequences. The German judiciary is completely controlled by the executive, and the public prosecutor's offices have even been determined by the EU Court of Justice. Politicians do not discuss with citizens, but let the judiciary loose on them: Annalena Baerbock 500 criminal proceedings against critical citizens in the last year, Robert Habeck 800 proceedings, Agnes Strack-Zimmermann 200 per week (!)
Consultants and science: To go from being a scientist to being a recognized scientist, you have to say what politics dictates. Natural laws are not interesting.
Legal framework: well, that goes the opposite way here. Even the human rights articles in the constitution (Articles 1 - 19 GG) are no longer of concern to anyone in politics or the judiciary.
You see, the system has now been completely driven into the wall. But I think the politicians are too stupid to have accomplished everything themselves (intellectual underachievers). Regardless of who is really controlling it, they need puppets with a corresponding psychological profile to install in suitable environments. Recognizing the mechanisms can hopefully help to avoid derailment next time. How to get out of the situation at the moment is another question.
I have analyzed the development paths in my books "Mathematics of Opinions" (4 volumes). The books are in German, but you can probably handle them. I will upload them in the next few days so they can be read freely. Maybe you'll take a look when you get the chance.
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Hy, in my research I need to extract RNA from mice gut samples for further cDNA and qPCR. However, the results are really inconsistent, sometimes it's 700ng/µl, and for another replicate which has the same sample we get 350ng/µl, surprisingly it always seems that the first sample is the best and afterwards the quantity drops dramatically. We have found that it is really dificult to digest the samples since the gut samples are quite rubbery. The methods we are currently using is cutting the tissue in small pieces, adding Qiazol, trying to stamp the tissue with a micro mortar and vortex afterwards with a vortex for 30-45 min (in between vortexing we cool the samples on ice for 5 min). After phase separation with chloroform we are really carefull to only extract aqueous phase and premix it with 100% ethanol. Then we use the miRNeasy Kit for RNA isolation.
We can't think of any other reason for the inconsistency to be lying with the digestion method in the way we stamp and cut the tissue.
Also the other issue is that we always have low A260/230 ratios but really good A260/280 ratios. Perhaps this is due guanidine contamination.
Can anyone give some insight in what can be changed? Ofcoarse a tool such as a tissue ruptor with be handy, but we don't have acces to it at the moment, perhaps we can try liquid nitrogen, but this would be really time consuming when using a mortar and pestle (I need to do 85 samples).
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Here's a summary of strategies to improve RNA yield and purity from mouse gut samples:
1. **Optimizing Homogenization**:
- Use a **bead homogenizer** (if available) or **freeze samples in liquid nitrogen** and grind them with a mortar and pestle for better cell lysis. Alternatively, mince tissues finely with scissors to make them easier to lyse.
2. **Adjust RNA Extraction Protocol**:
- Extend vortexing time with QIAzol to at least 45 minutes, and consider pre-incubating the sample with QIAzol for 5–10 minutes.
- Perform 1–2 freeze-thaw cycles after initial QIAzol treatment for more effective tissue breakdown.
3. **Improve A260/230 Ratios**:
- Add an **extra 80% ethanol wash** step to reduce contaminants.
- Slightly increase chloroform during phase separation (0.25–0.3 mL per 1 mL QIAzol) for purer RNA separation.
4. **Consistency in Processing**:
- Process samples at a uniform time to avoid yield drops due to time variations.
5. **Additional Cleanup for Guanidine Contamination**:
- Increase ethanol during binding or add a post-extraction cleanup (e.g., using a lithium chloride precipitation) to remove salts.
These steps should help improve RNA consistency and purity without extra equipment.
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These are mutually exclusive either for genetic, multifactorial or unknown reasons the immune system begins to attack a body it is designed to defend or
a problem of leaky gut syndrome where a permeable digestive tract allows access to pathogenic microbes inside defenses where they cause havoc colonizing arteries and brain tissue to provoking severe immune responses damaging nerves to bone? Your comments, ideas, and remarks are welcome. Where do you stand on this question?
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CAR T quashes autoimmune diseases
"Three people with severe autoimmune diseases are in remission after being treated with bioengineered and CRISPR-modified immune cells called chimeric antigen receptor (CAR) T cells. They are the first to be treated with engineered immune cells from donors. The advance could represent the first step towards mass production of CAR T therapies for conditions such as lupus and multiple sclerosis. The ongoing success and safety of the therapy need to be demonstrated in more people before it can be considered for wider use, but it “could prove paradigm shifting”, says immunologist Daniel Baker..."
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Dear Colleagues,
I am working on histological observations of the gut of invertebrates.
My main research is on seasonal changes in the gut tissue of sea cucumbers.
This species ceases or decreases its feeding activity in summer and also decreases its feeding activity in winter.
In particular, the gut retracts or disappears in summer.
Gut retraction is recognised as a result of catabolising components in the body to conserve energy and to tolerate depleted stored nutrients.
Please advise if anyone else has researched this other reason for intestinal retraction in a professional manner.
Best regards.
Kai Tanaka
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Digestive system regression in sea cucumbers and other invertebrates can occur due to 1. Food scarcity 2. Temperature fluctuations 3. Dormancy/hibernation 4. Reproductive cycles 5. Molting 6. Desiccation 7. Disease/parasites 8. Stress/injury 9. Aging/senescence Sea cucumber-specific reasons: 1. Dietary changes 2. Burrowing behavior 3. Regeneration/autotomy Seasonal changes can trigger regression to conserve energy, reduce metabolic rate, and adapt to changing environments. Investigate: Gut morphology/histology Enzyme activity Gene expression Hormonal regulation
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I am looking to amplify a region that can be used to group the gut bacteria into higher taxa like phyla or classes. The target has to be a region that is relatively conserved within the phylum or class but variable between these groups.
I hope some here who have done studies in this area can give me recommendations on which primer sequences worked best for you. Thanks!
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Thank you Idowu Goodnews Oluwaseyi . That is very helpful.
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The role of gut microbiota and diet has become a major concern for the health of Alzheimer’s disease (AD) individuals. The literatures shows that the microbiome composition has been linked to neurodegenerative disease and plays a critical role in the gut and brain axis. Microbial fermentation may release microbial metabolites that release short chain fatty acids such as butyric acid that has a major influence on the gut brain axis with effects on brain amyloid beta levels and plaque deposition. Researchers indicate that the understanding how the gut microbes interact with drugs and regulate gut microbes may improve the therapeutic effects and clinical outcomes of drugs with relevance to neurodegenerative diseases. The use of specific drugs may have major impact on the composition and metabolic function of gut microbiota metabolites and therapeutics for AD. Research is required on various drugs to assess drug-microbiota interactions on gram negative versus gram positive bacteria to reduce microbial metabolites such as toxic bacterial lipopolysaccharide levels in the plasma and brain with relevance to Alzheimer’s disease.
RELEVANT REFERENCES:
1. Sharma, A and IJ Martins. The role of Microbiota in the pathogenesis of Alzheimer’s disease. Acta Scientific Nutritional Health. 7.7 (2023): 108-118.
2. Vich Vila, A., Collij, V., Sanna, S. et al. Impact of commonly used drugs on the composition and metabolic function of the gut microbiota. Nat Commun 11, 362 (2020).
3. Wan Y, Zuo T. Interplays between drugs and the gut microbiome. Gastroenterol Rep (Oxf). 2022 Apr 8;10:goac009.
4. Weersma RK, Zhernakova A, Fu J. Interaction between drugs and the gut microbiome. Gut 2020;69:1510-1519.
5. Pant A, Maiti TK, Mahajan D, Das B. Human Gut Microbiota and Drug Metabolism. Microb Ecol. 2023 Jul;86(1):97-111.
6. Misera A, Łoniewski I, Palma J, Kulaszyńska M, Czarnecka W, Kaczmarczyk M, Liśkiewicz P, Samochowiec J and Skonieczna-Żydecka K (2023) Clinical significance of microbiota changes under the influence of psychotropic drugs. An updated narrative review. Front. Microbiol. 14:1125022.
7. Zhao, Q., Chen, Y., Huang, W. et al. Drug-microbiota interactions: an emerging priority for precision medicine. Sig Transduct Target Ther 8, 386 (2023).
8. Wang, S.; Ju, D.; Zeng, X. Mechanisms and Clinical Implications of Human Gut Microbiota-Drug Interactions in the Precision Medicine Era. Biomedicines 2024, 12, 194.
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Impact of Drugs on Gut Microbiota
1. Antibiotics:
  • Alteration of Microbiota Composition: Antibiotics can significantly disrupt the gut microbiota, leading to reduced diversity and changes in the relative abundance of bacterial species. This can have long-term effects on the gut ecosystem.
  • Functional Impacts: These changes can impact metabolic functions such as bile acid metabolism, short-chain fatty acid production, and immune modulation.
2. Metformin:
  • Diabetes and Metabolic Health: Metformin, a common diabetes medication, has been shown to alter gut microbiota composition, increasing the abundance of beneficial bacteria such as Akkermansia muciniphila. These changes are believed to contribute to its beneficial effects on glucose metabolism.
3. Proton Pump Inhibitors (PPIs):
  • Gastrointestinal Health: PPIs, used for reducing stomach acid, can also alter gut microbiota. They have been associated with decreased microbial diversity and increased risk of Clostridium difficile infections.
4. Nonsteroidal Anti-Inflammatory Drugs (NSAIDs):
  • Inflammation and Gut Health: NSAIDs can affect gut microbiota composition and increase intestinal permeability, potentially leading to dysbiosis and gut inflammation.
Gut Microbiota and Alzheimer's Disease
1. Microbiota-Gut-Brain Axis:
  • The gut-brain axis refers to the bidirectional communication between the gut and the brain. Gut microbiota can influence brain function through various mechanisms, including modulation of the immune system, production of neuroactive compounds, and regulation of the gut barrier.
2. Inflammation and Neurodegeneration:
  • Chronic systemic inflammation, often linked to gut dysbiosis, is a known risk factor for neurodegenerative diseases like AD. Altered gut microbiota composition can lead to increased gut permeability and systemic inflammation, which might exacerbate neuroinflammation and AD pathology.
3. Specific Bacterial Strains and AD:
  • Certain bacterial strains have been implicated in the development or prevention of AD. For instance, beneficial bacteria producing short-chain fatty acids (e.g., butyrate) have neuroprotective effects, whereas some pathogenic bacteria might contribute to amyloid plaque formation and neurodegeneration.
Therapeutics for Alzheimer’s Disease
Given the influence of gut microbiota on systemic inflammation and brain health, modulating the gut microbiome is being explored as a therapeutic strategy for AD.
1. Probiotics and Prebiotics:
  • These can promote the growth of beneficial bacteria and have shown promise in improving cognitive function and reducing neuroinflammation in preclinical models of AD.
2. Fecal Microbiota Transplantation (FMT):
  • FMT involves transferring gut microbiota from a healthy donor to a recipient. Preliminary studies suggest that FMT can restore gut microbiota balance and might have beneficial effects on neuroinflammation and cognitive function in AD.
3. Dietary Interventions:
  • Diets rich in fiber and polyphenols can positively modulate gut microbiota, potentially leading to reduced inflammation and improved brain health. The Mediterranean diet, for example, has been associated with a lower risk of AD.
4. Antibiotics and Gut-Brain Therapy:
  • Specific antibiotics might be used to target pathogenic bacteria contributing to neuroinflammation. However, this approach requires careful consideration due to the potential for broad-spectrum antibiotics to disrupt beneficial microbiota.
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Hello,
my research group is rather new to the field of microbiome research. We have been collecting stool samples (stabilized in OMNIGENE gut tubes and then frozen at -80°C) and are now looking for a commercial partner for the microbiome analyses (16S rDNA amplicon sequencing).
We would very much appreciate recommendations for European companies to work with from scientists experienced in this field.
Thank you very much!
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Several European companies are known for their expertise in microbiome analysis. Here are some recommendations:
  1. CosmosID (Germany): Specializes in microbiome analysis using next-generation sequencing and bioinformatics to provide comprehensive microbial profiles.
  2. Enterome (France): Focuses on the development of innovative drugs and diagnostics based on the gut microbiome.
  3. BaseClear (Netherlands): Offers a wide range of services, including microbial genomics and metagenomics, for microbiome research.
  4. Zymo Research Europe (Germany): Provides a variety of microbiome analysis services, including DNA/RNA extraction, sequencing, and bioinformatics.
  5. Microba (Australia, with European offices): Known for its high-resolution gut microbiome analysis, offering detailed insights into microbial composition and function.
  6. Eagle Genomics (UK): Specializes in microbiome data analysis and interpretation using advanced bioinformatics and AI-driven solutions.
  7. Metabiomics (UK): Provides microbiome sequencing and analysis services with a focus on medical applications and disease biomarkers.
  8. Biomes (Germany): Offers comprehensive microbiome analysis services, including gut health reports for individuals and research services for professionals.
These companies offer a range of services, from basic sequencing to advanced bioinformatics and clinical applications, catering to both research and clinical needs in the microbiome field.
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Dear all,
This is a microplastic image from the fish gut, and I am having difficulty identifying this shiny particle. I am guessing it may be a film particle. If someone knows, please guide me.
Thank you so much in advance.
With Regards
Pragya Mehta
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Fabio Castagnino Ugolotti, Thank you so much, sir, for your response and view. What do you suggest could be possible.
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"A high-fat diet promotes cancer progression by inducing gut microbiota–mediated leucine production and PMN-MDSC differentiation"
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You may click on "Request full-text" at https://www.researchgate.net/publication/380372546. Four of the authors are ResearchGate members and will get your request. In about half of such cases I got the text from the authors this way. Or you may write an email message to the address indicated at <https://www.pnas.org/doi/10.1073/pnas.2306776121>.
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How do specific metabolic and immune factors within the gut interact with the reproductive tract, and what are the precise molecular mechanisms governing this interplay, considering the impact on reproductive health and potential implications for fertility and pregnancy outcomes?
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The relationship between gut metabolism, immunity, and reproductive health involves various molecular mechanisms. The gut microbiota composition affects hormone metabolism, nutrient absorption, and immune function, with dysbiosis potentially impacting reproductive health. Short-chain fatty acids (SCFAs) produced by gut bacteria influence immune responses, with dysregulation potentially affecting reproductive health through immune tolerance disruption. Chronic low-grade gut inflammation can impair reproductive health by disrupting ovarian function, embryo implantation, and increasing pregnancy complications risk. Gut microbiota can influence hormone metabolism, impacting menstrual irregularities, infertility, and pregnancy complications. Mucosal immunity and barrier function maintenance prevent harmful substance translocation, with dysregulation potentially impacting reproductive health. The gut-brain axis communicates bidirectionally, affecting stress responses, mood, and behavior, influencing fertility, sexual function, and pregnancy outcomes. Emerging evidence suggests gut microbiota-induced epigenetic modifications may affect immune regulation, metabolic pathways, and reproductive health, impacting disease susceptibility and offspring development. Understanding these molecular pathways is crucial for developing interventions to optimize reproductive outcomes and mitigate gut-related disorders' impact on fertility and pregnancy.
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I would like to cultivate lactobacilli in an intestinal organ-on-chip model and stain it with a suitable dye either beforehand or, if necessary, after the end of the experiment with a suitable antibody for immunofluorescence microscopy.
Briefly, I would like to check the Lactobacillus attachment/localization to/in the intestinal tissue.
Is there anyone with experience in this area and could explain possible procedures?
Thank you very much in advance!
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May consider using Permai fluorescence dye.
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I'm working in a project where I need to test the enzyme activities of an insect's gut fluid. I've already tested it and found some good activity. Now I want to know whether the enzyme, showing high activity, is endogenous to the insect, or just produced by gut microbiota. There is no genome sequence or any other information available about that insect. How can I test it? How can I prepare enzyme solution, containing only endogenous enzyme of the insect?
I've read about the antibiotic feeding process. Anything else?
I found a paper where they incubated the insects for 24 hours, dissected, collected the gut fluid and then filtrated the gut fluid through 0.22 micrometer syringe filter, then demanded that this filtered solution contain no bacteria, neither their enzymes. But how can they demand there is no bacterial enzyme? If it is for starvation, then isn't it obvious for the insect's enzyme to reduce too? Can anyone provide any reference on support of this method, or concept? Please let me know.
I'm struggling in this issue and can't find any solution. I can't use the antibiotic method for a reason. Please help me out. Thank you.
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If you can't kill off the bacteria in the insect gut by the use of antibiotics, can you somehow raise the insects in such a way that they do not have any gut bacteria, the way it is done with mice? This would involve first eliminating the gut bacteria of some parental insects with a combination of antibiotics, maintaining them in a sterile environment with sterile food, then breeding those insects to produce offspring that could be studied without antibiotic treatment. I don't know if anyone has ever done this.
Even if you had the entire genome sequence of the aseptic insect, it would not necessarily help you with your experiment if you are measuring the enzyme activity level with an enzyme assay, since that does not necessarily uniquely identify the enzyme(s) responsible for the activity. You would have to do a proteomics study to see what proteins were present in the gut fluid. You might be able to identify the enzymes that way by comparison with genomic sequences of related insects. You might also consider obtaining the full genome (or at least the full exome) sequence of your insect to inform your proteomics work. It's not that expensive nowadays.
You should also consider that there might be an interaction between the insect's gut lining cells and the gut microbiome that could affect the insect's secretion of enzymes. In other words, the insect and its microbiome may not be independent, so you would get a different amount and/or set of enzymes secreted by aseptic insects compared to insets with a normal microbiome.
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I am working with a marine non-model organism that has a planktrotrophic larval type and I found out that the larvae are not very selective while feeding. I am planning to do RNAi by feeding them bacteria (E.coli) expressing dsRNA. As a preliminary experiment, I am planning to feed them with bacteria for three hours. I would like to know whether or not they are ingested the bacteria. Is there a method to stain ingested bacteria? The larval type of my organism is transparent and the gut can be visualized using an optical microscope.
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As someone mentioned above, maybe some type of GFP or other color (red/far red) engineered strain of your bacteria. If that is not possible, can you perform sectioning and staining, you could use primary antibodies that specifically target bacterial antigens.
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Introducing a Super Unified Theory of SU(11) of 10-dimensional Space-Time of the physical universe instead of four dimensional Einsten's Space-Time of GUT SU(5) of SM, which explained the forces of Strong by SU(3), Weak by SU(2), Electromagnetic by U(1)and a new fifth force called latent force by the new particles of SU(6). The strength of SU(6) are so large that it can changes the exotic matter fluid into ordinary matter then everything.
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In the past, repeatedly discussed about the new energy group SU(6) in perspective of Standard Model of Physics, such for Higgs Field, for the discussion of Majorana particles, Fermions etc. But in the present, my introduced SU(6) discussed on the basis of new energy bosons beyond SM which unified with old energy group SU(3), SU(2) & U(1) of the SM by a Super Unified Energy Group SUT of SU(11) instead of GUT of SU(5).
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Hi everyone,
im using this established protocol:
I already optimized everything, written under "troubleshooting" in this protocol. I directly obtain the samples from the endoscopy, its less than 20 minutes between extraction and starting work in the lab. During this time the samples are on ice and in the media recommended in the protocol.
So far i was only able to generate organoids from childrens biopsies, but not from older patients. It seems, as if they needed a stronger Wnt-activation?
I use recombinant human Wnt3a at the correct concentrations. Would you recommend switching to Wnt-surrogate or Wnt-conditioned media?
The protocol should also work with samples from older patients.
Is maybe the tissue piece from the endoscopy too small and i need larger samples from the surgery?
Any other ideas, what i could try?
Thank you,
Marco
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Not relevant to to this discussion but somewhat related indirecly. When I was isolating mesenchymal stem cells from patients bone marrow taken from their femur undergoing knee surgery. There was variation among patients. Some patients' bone marrow stem cells did not grew further or very little grew. Whereas some patients bone marrow stem cells provided me millions of cells after 2 passages. I wanted to to go back to history of the patients what medicines and what diseases they had.
May be you should try to relate to patients medical history besides age group
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Dear Joanna,
I just saw your nice video contribution about your study about acupuncture as treatment in diabetic neuropathy - great: I wish you a great success! Ich hoffe, Dir geht es gut: Berlin macht sicherlich weiterhin viel Spaß inmitten Deiner Familie und an der Charité. Bei mir sieht es ähnlich gut aus, es gibt so viele Dinge hier in Dresden und Leipzig - wunnebar:-)
Wünsch Dir eine tolle Zeit in 2021, bis denne und liebe Grüße,
MIchael
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here is an article that creates link between trigeminal neuropathz and trigeminal dysphoria
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I presently use the powersoil pro kit for the extraction but I am not getting my desired result maybe cos the starting material (Daphnia magna gut) is too small. i'd like to know what methods has been used that yields a good result.
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I use the MasterPure Complete DNA and RNA purification kit from Biosearch technology. To carry out my DNA extraction, I use 10 adult daphnia which I crush with our small homemade device which vibrates in the lysis buffer of the kit. I advise you to read the following paper, we based ourselves on it. Optimization of DNA extraction from the crustacean Daphnia by Athanasio published in 2016 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867708/)
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I'm currently working on probiotic adherence to human's gut and, while writing the manuscript, received a question from one of assesors whether there is a minimum adherence inhibition? Maybe in 30% 50%?
As far as I read papers, none has ever mentioned.
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Could you be little more clear on the question ?
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I want to isolate Bacillus subtilis from animal gut digesta and was wondering what is the best medium to grow it effectively. is it TSA? also what are the conditions of the growth, aerobically or anaerobically?
Thanks
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Hi! Hope you are doing marvellously well
In my case i do not isolate, i just take them from a pure culture, and they grow in LB or LB-Agar.
Liquid culture conditions are 37ºC but we also tested for 28ºC. between 140-200RPM works fine. I have a protocol were i escalate in small scale just for the sake of some things we are working on, "1 colony" from LB-agar plate put on 4mL LB to grow overnight (14hs) and then that culture put on an Erlenmeyer with 200mL LB to grow again for aroundv 20Hs, at that point you will reach around 3-3.5OD600nm.
Hope this gives you an idea how it work on Luria Bertani media
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Dear Friends,
Please, join a discussion on Theory of Universality in Physics, a comprehensive GUT. The discussion is live in Academia now.
My Theory is proved to be experimentally correct on 4 aspects, namely :
1. The increase calculations for Sun (both radius and mass).
2. The increase calculations for Earth(radius and volume)
3. Dark matter speed, u
4. Gravitational constant, G.
Kasibhatla Surya Narayana
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Everything in space of universe is weightless, thus, mass of object such as moons, planets and suns are weightless. In addition any calculation of mass of object is just wasting time. Also, everything of nature is changing constantly through temperature and pressure and interaction of chemical elements. Read some of my articles, they all are unprecedented and are not last century ideology.
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I need to purify DNA and RNA from frozen GUT tissue (rectal biopsy tissues from monkeys mainly). The tissues are frozen at -80°C with or without RNAlater. I am thinking to use TissueLyser, since it will be safer when using infected tissues. According to the protocols, if the tissue are stabilized with RNAlater, I can thaw it at room temperature and proceed with next steps for disruption and homogenization. What about frozen tissues without RNAlater? How to take and weight a tissue, if it is frozen in the medium at -80°C and should not be thawed (according to the manuals)? What size of stainless steel beads would you recommend? (I am thinking to use 5 mm beads). I am also not sure what TissueLyzer would be better to use...
I will appreciate any suggestions related to the use of TissueLyser for rectal tissue samples.
Thank you everyone in advance!!!
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Which are the relatively more successful refutations so far of the foundations of quantum physics? How do they compare with Relativity when it comes to efforts to build up a GUT on bases that may apply also to many other sciences?
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Raphael Neelamkavil I'm unsure if that was directed at me, but if it was, thank you a million times over. My past work with philosophy and reading of Shulgin has greatly influenced my writing and presentation style. Absolutely wonderful compliment to wake up to. I hope you know your questions and conversations are equally competent and enlightening. I appreciate your time following any of my work and will continue to follow your thought provoking discussions. Sorry if this is non-sequitir due to me being late to the party. I was unprepared because physicists don't usually get invited to parties.
I am typing an answer on QM but am basically a half-asleep zombie. Will engage in QM conversation soon.
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I have already processed my fish gut samples for microplastics analysis from extraction to filtration and drying. The extracted microplastic particles were stored in dried glass fiber membranes. Is it valid and possible to detect PAHs traces by using the membranes alone and directly analyzing it with gas chromatograph or HPLC? Will the retained PAHs can be detected if I carefully pick the particles and testing it directly with the gas chromatograph or HPLC? Thank you in advance.
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Hi, I'm searching for studies on the diet of the mayfly Ephemera vulgata (Linnaeus 1758)? From a European database (freshwaterecology.info), its feeding type preferences is given as 20% gatherer (FPOM sediment) and 80% active filter feeder (FPOM, CPOM and micro prey from the water column). The reference for this information is given as the AQEM expert consortium. I would be really interested to see the original study providing this information, or any other studies that explores food item preferences (e.g. gut content) of E. vulgata. If you know about any papers, thesis, reports etc. with such information, even anectodical, I would be grateful if you could share this information with me.
Tor Erik
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Crisis and emergency alert http://youtu.be/Ng1-KJueYiU Time for the people to stand together to bypass, help us build the bypass. We have the foundation's know
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In bee pathogen diagnostics, we often use different sets of primers to identify pathogens in bee guts.
One of the primer sets we have been using a while in PCR, we are now using to quantify pathogen loads via qPCR.
However, we discovered that a frequently used primer set, published years ago and used frequently in different papers, is showing a strange curve. It amplifies a fragment of the 18S rRNA gene, of Microsporidian pathogens belonging to the genus Nosema.
We tried different conditions and concentrations, but the curves stay the same.
In addition is a BioRad data file with the standard curves, and a picture for those who cannot open these types of files.
Efficiency is always low when using these primers, while R² is high, so we think it is due to the weird curves.
Does anyone know what could be the reason for the amplification curves?
Or the reason of the low efficiency?
Thanks!
More info:
Primer info:
Forward = Nosema universal, edited by Fernandez 2012, originally from Higes 2006 (TATGCCGACGATGTGATATG)
Reverse = Nosema universal, from Higes 2006 (CACAGCATCCATTGAAAACG)
qPCR Program:
2 min at 95°C, 39 times: 15 sec at 95°C, 30 sec at 56°C, 45 sec at 72°C
Fernández, J. M. et al. (2012) ‘Asymptomatic presence of Nosema spp. in Spanish commercial apiaries’, Journal of invertebrate pathology. J Invertebr Pathol, 111(2), pp. 106–110. doi: 10.1016/J.JIP.2012.06.008.
Higes, M., Martín, R. and Meana, A. (2006) ‘Nosema ceranae, a new microsporidian parasite in honeybees in Europe’, Journal of Invertebrate Pathology. Academic Press, 92(2), pp. 93–95. doi: 10.1016/J.JIP.2006.02.005.
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Hi,
The strange amplification curve you are observing in your qPCR assay could be caused by a few different factors. Some possibilities include:
  • Non-specific amplification: The primer set you are using may be amplifying non-target sequences in addition to the 18S rRNA gene of the Microsporidian pathogens. This could be due to the primers binding to regions of high homology with other genes or sequences in the sample.
  • Primer-dimer formation: The primers you are using may be forming primer-dimers, which are non-specific amplification products formed between the forward and reverse primers. This can result in a smeared amplification curve.
  • Presence of mutations: The primer set you used is for a specific Nosema genus, but your sample may contain a different strain of the pathogen with different genetic variations, which can affect the specificity of the primer set.
  • Low primer specificity: The primer set you are using may not be specific enough for the target sequence and is amplifying other regions of the genome.
The low efficiency of the PCR reaction can be caused by one or more of the above factors.
To troubleshoot this issue, you can try the following:
  • Verify the primer sequences by comparing them to the published sequences and check for any potential errors or mutations.
  • Check the specificity of the primer set by performing a melting curve analysis and see if there are any non-specific products.
  • Try optimizing the PCR conditions, such as the annealing temperature, primer concentration, MgCl2 concentration, etc.
  • You can check the specificity by performing a BLAST search and see if the primer set matches the expected target sequence.
  • Try using another primer set for the same target to confirm whether the problem is with the primer set or with the sample.
It's worth noting that a high R² value in the standard curve is usually an indication of good correlation between the Cq and the log of the input template, but it does not necessarily mean that the primer set is suitable for quantifying the pathogen load in your samples.
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Dear colleagues! We plan to isolate mitochondria from freshwater amphipods, but didn't find any methods in literature - the closest found was the method of isolation from whiteleg shrimp Litopenaeus vannamei.
The problem is - the amphipods are quite small - around 1 cm long, so it's hard to isolate the gut before mitochondria isolation.
Will it work if we use just the sample of 10 g (or is that too much?) of amphipods and blender to homogenize it in isolation medium? Or it is crucial to select only some parts - for example only the amphipods legs and antennas?
P.S.: we do not have chitinase, nor the chance to get it in time.
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Isolating mitochondria from freshwater amphipods involves several steps, including homogenizing the tissue, centrifuging the homogenate to pellet the mitochondria, and separating the mitochondria from other cellular components. Here is a general protocol for isolating mitochondria from freshwater amphipods:
  1. Collect and prepare the tissue: Freshwater amphipods should be collected from their natural habitat and kept on ice until they can be processed. To isolate the mitochondria, you will need to use a small amount of the amphipod's tissue, such as the muscle or gill tissue.
  2. Homogenize the tissue: To break open the cells and release the mitochondria, the tissue should be homogenized using a glass homogenizer or a tissue grinder. The tissue should be homogenized in a buffer that is appropriate for the specific goals of the experiment, such as a hypotonic buffer for enzyme assays or a detergent-based buffer for protein isolation.
  3. Centrifuge the homogenate: After homogenizing the tissue, the homogenate should be centrifuged at low speed (e.g., 1,500 x g) to pellet the mitochondria. This will separate the mitochondria from other cellular components such as the nuclei, cytosol, and plasma membrane.
  4. Separate the mitochondria from the other cellular components: To separate the mitochondria from the other cellular components, the pellet should be resuspended in a buffer and centrifuged at high speed (e.g., 10,000 x g). The resulting supernatant should contain the mitochondria, while the pellet will contain the other cellular components. The mitochondria can be further purified by centrifuging the supernatant at an even higher speed (e.g., 100,000 x g) to pellet the mitochondria.
It is important to note that the specific details of the protocol may vary depending on the specific goals of the experiment and the resources available. It is also important to follow good laboratory practices and to handle the samples and chemicals carefully to avoid contamination and to ensure the integrity of the results.
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Hi,
I need to compare the alpha diversity of three communities (the gut bacteria; the gastro-intestinal helminth community ; the bacteria community found in the spleen of rodents)
For the gut bacteria, I have abundance data
For the two other communities, I hace presence/absence data
Alpha diversity : I was thinking of using richess and Shannon index, for all communities (even the two with presence/absence data only). I know that Shannon index is made for abundance data, but is there any statistical problem in using it for 0/1 data ? It would be a shame to analyse gut bacteria using only presnece/absence data and richness index for all communities...
Beta diversity : I was thinking of using Bray-Curtis distances for all communities. I know that Bray-Curtis is made for abundance data, but is there any statistical problem in using it for 0/1 data ? It would be a shame to analyse gut bacteria using only presence/absence data and Jaccard distance for all communities...
Thanks,
Nathalie
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Hi, Nathalie. Try to use iNEXT R package (check on google for the easier version online - http://chao.stat.nthu.edu.tw/wordpress/software_download/inext-online/)... you have options with binary and abundance data for interpolation and extrapolation. The output figures are great. Also, you can save the results in excel to check the confidence levels for any interception if the charts are not clear.
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probiotics can be used for controlling pathogenic microorganism in the gut of poultry, but what else these probiotics can be used in poultry
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i mean what are the main uses of probiotics for poultry and livestock health
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The research topic is for final year graduation from bachelor's degree. I had a topic in mind such as Leaky gut syndrome: The imbalance between microbiota and
harmful bacteria, but due to unavailable equipment in our labs, I need other topics. Your help is appreciated.
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What are the available resources and facilities in your lab?
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If I’m looking at the association between cardiovascular disease (CVD) risk and gut microbial beta diversity, I understand that this would show the association between levels CVD risk and inter-individual differences in gut microbiome composition. However, I’m finding understanding that association in a biological context a bit tricky. For example, if it is a positive significant association, is that suggesting that harbouring a more distinct gut microbial community compared to somebody else , may be linked with a CVD risk (i.e there is no “core” microbiome signature associated with CVD risk per se that would be common across individuals, more likely to be different compositions when comparing).
I'm more familiar with comparing beta diversity between disease vs control, rather than association with an endpoint, so any explanations would be appreciated - thank you.
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Thank you very much for all of your help! I've selected the answer in line with the initial question.
Thanks again
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I stained dissected gut of Drosophila larvae using DCF-DA for visualisation of ROS. Gut tissue was fixed with 4% PFA overnight at 4°C. Under fluorescence microscope, bright red fluorescence was noticed insteed of green fluorescence. Is it normal ?
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Assuming you had the correct stain:
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Hi,
I am trying to study the gut microbiota of honeybees. Is it necessary to separate the host tissue (gut particles) after we homogenize the gut? Our lab is using DNA amplification of 16S rRNA and the culture method for the study. I am curious if the host tissue has an impact on the bacterial DNA extraction process and/or in qPCR analysis.
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I think these points will help:
1- Primers are well designed with no cross matching with Honey bee genome
2- Your amplification conditions are OK and tested to be sensitive and specific against the chosen genes. You can try it on bacterial samples before starting work on your GUT samples.
3- The extraction process exclude inhibitors.
4-The DNA yield is good
5- qPCR better to use probe based assay.
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As we have known, the bacteria such as E. coli and P. gingivalis can secret LPS, EVs to induce the immunogenic response of the host. So, I wonder if there are other components that can be secret into the gut by bacteria? Thanks very much!
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Visit also the following useful RG link:
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This would include tools for assembly, binning, and annotation. If it can be of any help, it's for gut samples.
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I'd vote for MetaWRAP pipline (Metaspades for the assembly)
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I am running RNA extractions on whole gut samples for downstream RNAseq. For one individual I realized there was a length of gut tissue still in the original collection tube that I didn't add to the homogenization solution. I'm not sure what region of the gut it actually is or proportionally how large it is relative tissue that was homogenized (it is smaller), but I'm worried that if there are regional differences in RNA expression profiles that will bias the RNAseq data towards the already-processed portion of the gut.
Is this sample salvageable? If I extract RNA from the leftover tissue, could I just combine the total RNA sample volumes from both prior to sending in for sequencing? Alternatively, if we sequence both separately could we normalize and combine reads somehow? Are there any other strategies that would be more robust to prevent bias? Thanks in advance.
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Was the gut that didn't get homogenised effectively protected from RNases? If the leftover tissue was: stored in a reagent like RNAlater the whole time, OR frozen the whole time and never thawed, then I think it would be fine to extract the RNA from it now and add that RNA to the existing RNA from the same sample. But if the leftover tissue sat around in the collection tube at ambient temperatures for even a minute longer than the other samples - it's gone, let it go. Substantial differences in RNA quality between samples can be a major source of bias in RNAseq experiments, so deliberately adding RNA which is likely to be degraded is a bad idea. (If you are in doubt at all, do you have access to a BioAnalyzer or TapeStation or other way to check RNA integrity? You could extract the RNA from the leftover tissue, compare the RIN of the new extraction to the previous extraction, and mix them only if they are comparable.)
I don't think preparing a second library from the RNA made from the leftover tissue and then sequencing that would be worth the effort and expense. I don't think it would be possible to neatly integrate this data into your experiment.
Whatever you end up doing, this sample will be different in some way to the other samples in your experiment. So you should run an outlier analysis at the bioinformatic level, and seriously consider excluding this sample entirely if it looks different from the other samples. Building in some extra n into RNA-seq experiments to account for errors like this (they happen to us all!) is a very good idea when feasible.
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I have metagenomic data of gut bacteria of tribal population of Himachal Pradesh, can anyone help me regarding analysis of same for writing a research article. we will share authorship for the same.
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Many statistical analyses can be done on metagenomic data. Adding some details to your question will help narrow down the type of analysis you can do based on your research question.
  • What is your research question?
  • What type of samples did your study use? I guess stool samples?
  • What sequencing technology was used, and what was the library prep. protocol?
You can send a private message if it's easier to chat.
Best,
Hamza
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Can anybody help me with the protein extraction from the gut of adults and Larvae of Zebrafish?
I have a problem: I can't get a good result in Western Blot with adult zebrafish gut or with zebrafish larvae (5 dpf). When I extract the proteins, the bands on SDS-page appear not very concentrated and after the transfer, when I paint the membrane with ponceau red, the result is not clear and there aren't defined bands , but everything is very blurred as if the loaded samples were dirty. The protocol we use provides for the homogenization of the sample with lysis buffer
(SDS 1%, EDTA pH 8.0 10 mM, Tris-HCl pH 8.1 50 mM, PMSF 1 mM, protease inibitor) or Ripa buffer (Sodium chloride 150 mM, Tris-HCl pH 8.0 50 mM, Nonidet P-40 1%, Sodium deoxycholate 0.5%, SDS 0.1%, protease inhibitor).  Then I heat the samples for 5 minutes and centrifuge them at 12,000g for 15 minutes at 4°C.  After, I load from 20 to 50 ug of protein in each well in a 10% or 15% gel.  I do either dry or wet transfer, but either way it doesn't work well. What could I do to improve my homogenization of samples and transfer?
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Make a fresh loading buffer and ensure that the protein samples are quickly mixed in loading buffer and the vials are kept in boiling water for 7-10 min. And reduce the total protein/lane. Hope this will resolve your issue.
Best
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We have procured metronidazole powder ( active ingredient) for an aquaculture experiment. The purpose is to study the effect of metronidazole on gut flagellates. But we have a problem with dissolving metronidazole in pure water.
Could someone suggest how it could be done?
Regards
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It is only slightly soluble in pure water. However, it is soluble in acidic solutions (at pH 1.2). Read the article below
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With regard to patients suffering form spontaneous bacterial peritonitis, it seems counterintuitive that those patients tend to NEVER have anerobes as the causative agents despite the common leakage theory that should allow anerobes (that fill the gut) to leak whenever the opportunity is present? Is the leakage theory a sloppy theory? Do we need to tinker it a little bit? Should we adapt a new theory?! Or, should we go playing more in our labs hoping to uncover the real etiology and decipher this anerobes enigma?
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The main bacteria causing spontaneous bacterial peritonitis (SBP) are Escherichia coli and Streptococcus viridans) and not Pneumococci
Usually, 75% of spontaneous bacterial peritonitis is caused by infections by aerobic gram-negative organisms (50% of these being Escherichia coli). The remainder has been due to aerobic gram-positive organisms (mainly Viridans group streptococci).
Anaerobic organisms are rare because of the high oxygen tension of the ascitic fluid. A single organism is noted in 92% of cases, and 8% of cases are polymicrobial.
For details, please have a look at this Medscape article link under Pathophysiology and Etiology:
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Hi, I'm trying to take picture after immunolocalization with insect guts. After I did all process, I have a problem when I mount the guts. I used pipette and pipette tip (I cut the tip) to do this. But, most of the guts stuck to the tips.. Do you have any idea to solve this problem?
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June-Pyo, I don't know any specific names or suppliers, but you can get some from people who works with glass or metal. For example, glass blowers know how to make simple tools like that in a few minutes by melting and pulling the glass. You may also be able to make one yourself by heating and pulling a tall piece of glass (eg, pipette), though you should be careful as it is potentially dangerous. It is better to find someone who knows what they are doing.
Refik
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I have assessed the effect of trypsin and pepsin on phages. Thus, I want to compare the normal concentration range with the concentration where phages remain viable.
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Trypsin : 50-800ug/ml.
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Hello everybody
I am facing an issue in fish gut digestion using 30% H2O2. After the digestion process, I am getting a fat oil layer that might trap plastic particles. Kindly help me out with this to digest the oil layer or separate particles from it.
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Thank you sir for your response C.A. (Kees) Kan
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i need help regarding designing oral feeding bioassay for adult beetle (scotinae, coleoptera). my study involves profiling the effect of a toxin on midgut of the insect through transcriptomics. i have tried some semi artificial diet based protocol. but it did not work and i am unable to find much literature on it. please suggest any other way to feed the insect so as the chemical reaches the gut.
i have been working on adult beetles. can i also use lower developmental stages (larva or pupa) for such a study.
thank you :)
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Dear Aisha,
Good job. This seems really an interesting topic for me to work on and therefore, I got inspired to have a quick survey in Google scholar about it. Interestingly I came across bunch of papers describing the transcriptome analysis profiling the effect of toxins like Bacillus thuringiensis based biopesticides on mainly lepidopterans. Just few of them worked on Coleopterans. So, your work may be a great job to find new achievements in this case. Hereby, I send you two related papers published recently one on lepidopterans and one on coleopterans. First read the method part and then check the references you may have the chance to find new approaches in the case of your research. Good luck.
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I want to study the sorption and desorpotion of an antibiotic by microplastics in the gut of a marine fish species. I'm wondering if I can prepare a solution that is extremely similar to the intestinal environment (simialr pH, compotiosion of ions, disgest enzymes and etc), and then do the kinetics test in that solution? Has anyone done a similar study of this? Could you kindly share the protocol to prepare the "intestinal environment-like solution"?
(I speculated that the intestinal contents in fish are more like liquid than a solid? Am I wrong?)
Thanks in advance!
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I cannot answer this, exactly. But if I want to test it, I prepare antiobiotic coated microplastic. Then I mix it with the fish feed using a suitable binder. Then I offer the feed to the fish and monitor serum antiniotic concentration within a time frame
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I want to extract the midgut of Callosobruchus maculatus larvae to isolate their proteases. This will be my first time dissecting a small larva (2nd/3rd instar). I read some publications about how they dissect and prepare the gut homogenate. However, some research protocols were different from the others. Sometimes, the insects were dissected by only using cold distilled water, but others by using buffers (citrate-phosphate or sodium-acetate) or NaCl solution.
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Actively feeding final instars were dissected for isolation of midgut in ice-cold MET buffer (330mM mannitol, 5mM EGTA, 17mM Tris-HCl with pH maintained at 7.5). The midgut contents were isolated and washed thoroughly in the dissection buffer which transferred to 1.5ml eppendorf tubes containing 50µl MET buffer for homogenization. After addition of 50µl of 24mM MgCl2homogenate was centrifuged at 4500rpm at 4°C for 30min. The supernatant was collected and stored at -20°C for alkaline phosphatase characterization
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I am following Karam et el (2017) method on digestion at 40C instead of 60C as most publications reported disruption in the plastic components. KOH had the most optimum digestion as well compared to other bases and acids by having the least damaging effects on the components. Im following 1:3 ratio (organ:solution). Most of my samples are cloudy. How to increase the digestion of my samples? Thank you.
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Alia Nordin Maybe at 60ºC it will work better. There are some studies that also add shaking tables stages, with 85-100 or plus rpm.
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I want to isolate the microbiom from the gut of the larvae but I have problems with the dissection. I tried to use needles and a scalpel from a dissection set for biologists but I had problems to open the larvae and to find the gut. Is there any trick?
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Larvae of Lepidoptera they have well developed head capsule, 3 pairs of thoracic leg and 3-5 abdominal leg; Duster headless and legless; Coleopyeta well develped.head capsule and only three pairs of thoracic leg; Hymenopteta well developed head capsule. Three pairs of thoracic legs and greater than five pairs of abdominal leg.
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For example: The intestine/gut of zebrafish as model organism.
I would like to study metabolic profiling by using GC/MS. Could you please suggest suitable protocol for determining metabolites in tissues.
Thanks in advance.
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In this work, a proper extraction methodology is described. Please see section 2.6.
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I'm looking at gut bacteria in heat stressed birds but I'm not certain after how long can I sample faeces after episode of heat stress. I don't want to sample early before the changes shows in faeces or too late.
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@Vineet Singh thank you. The broiler article is very interesting and informative
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Dear Colleagues,
I have faced a challenge in the process of primer design for Real time PCR.
Two main bacterial populations exist in our gut, firmicutes and bacteroidetes. I am wondering how to design a specific primer for such wide populations? what genes should I target for primer design?
I appreciate your help and guidance because I really need that
Thanks
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Thank you Jetty Ramadevi so much.
I will study and search more and will ask for your help
thanks for your time
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Hi, we found some fish parasites in the gut of Serranus cabrilla, and in the gut and on the skin of Thalassoma pavo? Does anybody have a clue to which groups this parasites might belong to?
Thank you very much in advance
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Please find attached an illustrated chapter for some parasites in fish.
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Hi all,
My experiment is to find the differences of micro bacteria in gut of marron Cherax caini. there were 2 groups of marron , one fed every day, and one fasted for 4 weeks. At the end of trial, marron gut were applied RNA gene sequences.
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What do you want to ask?
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Our lab performed some 16s rRNA sequencing works recently, we acquired more OTU/ASV for oral samples than feces samples. We wonder if this result reliable or not, but we don't find there are any papers indicated the gut has more microbiota than the oral cavity. I will be very appreciative if you can give some valuable suggestions.
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Oral microbiota may be more than faecal microbiota depending on many factors. Not all the microorganisms that enter the mouth that can survive through the gut. Some are killed by HCl in the stomach. The glycoproteinous membranes and cell walls of others are damaged by some enzymes.
If you repeat the experiment using new samples, the results may likely change. This is because oral microbial loads and diversities vary and depend on activity: recently brushed, recent consumption of some food types, oral sex etc.
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Earthworms were stored at minus 80 degreed freezer. I need to extract the gut from earthworms for microbial analysis. However, i am having problem to extract gut because the earthworm became wet from inside after defrosting and its really hard to pull out the clear guts.
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I have incubation several pots of earthworms and I want to find out my treatment effects on earthworm gut microbiota. But I need to go back to China next month and I can not extract the DNA of earthworm gut right after I finish my incubation due to the condition limited. So I need to freeze dry the gut sample and take them back to China for DNA extraction and analysis. Could that be possible?
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To understand the gut microbiome-brain axis
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It seems yes. I read a study just last week that found that microbiota were associated with depression. Unfortunately, I deleted the source so I can't give you the reference :-( Sorry!
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I need to compare the human gut microbes with human to identify functional genome similarity. Is there any online tool for comparing the genome between different species?
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Do you mixing two similar but different things, i.e., functional genomics and comparative genomics.
If you want to compare genomes, it will be comparative genomics which would require genome (nucleotide) data.
For functional analysis and comparison, several other datasets would be required depending on the experimental design.
For comparative genomics too, you cannot use whole microbiome to compare against human genome. Such comparison itself is illogical. Why would one need to compare genome of a microbe to that of human.
  • There are several tools which can be used for the comparative analysis, but that can be discussed once the basics has been understood.
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I did the hemolytic test, The result showing after 24 hours indicated that the bacteria have gamma-hemolytic (non-hemolytic), but I kept the plate in the incubator for 48 hours, then the clear halo zone showed around the colony (Beta-hemolytic). So which result should I use? I read some papers and normally they did incubation for around 16 -24 hours.
Another found was that 75-90% of my isolates from the fish gut have Alpha or Beta hemolytic. and almost 100% of my isolates with antibacterial activity are hemolytic strains. So I would like to ask two more questions:
1. Is hemolytic really a necessary safety test for probiotic due to many bacteria with hemolytic is available in the environment and we still can scope with them?
2. hemolytic activity and antibacterial activity are in correlated or not?
Thank you very much for your suggestion and comments!
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If Your bacteria is showing zone on the plates in between the incubation of 24 hours then there is no need to provide the more incubation.
Thank you
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Good day. I am working on collecting larvae samples of a saturnid moth (Imbrasia belina), of which i would like to use for DNA barcoding and SNP genotyping. How do i prepare the specimens to avoid contamination from the gut contents? do i have to degut the larvae prior to preserving in absolute ethanol? so as to extract good grade DNA?
Many thanks in advance..
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Thanks very much Sven Thatje ..that is so helpful. the larvae is about 5cm length, on average....so i believe i will get enough muscle tirrue from it.
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I have dissected 2-3cm intestinal segments, flattened and stretched on a platform, fixed in 4% PFA for 24 hours, and dehydrated in 0.03% sodium azide for a maximum of 4 days (is this too long?). After this, what are the best solution and temperature conditions to store this tissue if needed for immunohistochemistry?
Also - what is the best method by which the myenteric plexus and submucosal layers of the gut can be isolated from these whole mounts for histological staining? Thank you!
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Since its emergence in 2019 the SARS-CoV-2 remains a challenge for scientists, clinicians and the overall population to solve. Besides its rapid spread and harsh respiratory distress it did induce, further secondary outocomes are observed. In particular, recent works highlifhted a preferential accumulation of its spike protein in skin tissues, specifically sweet glands. It appears that skin epithelial cells could also be a secondary host-cell of the viron.
this instigates several questions including:
i. did the viron targets all epithelial cells, such as skin and gut epithelia?
ii. if so, does it really rely on angiotensin converting ezyme receptor abundance in these cells? would these cells act as reservor for the virus ARN?
iii. could spike protein modify the skin physico-chemical properties?
etc.
I really wish to have a deep discussion of this fact
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the expression of SARS-CoV-2 spike protein in skin cells might be a probe for the virus replication into different cell types, in particular epithelial ones. if so, these cells, as spared from clinical investigations, might be the origin of recurrent auto infection and probably explain its transmission way from animals (eg. bats) to humans. furthermore the usage of the ARN-vaccine could amplify the spike protein presence in cutaneous tissues and change their physico-chemical properties and function.
it is thought that evaluating the abundance of the spike protein in the skin of a large group of patients (notably at different severity grades) is required to testify such hypothesis and better manage the disease.
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Hello everyone. I have collected intestinal biopsies from stem cell transplanted patients in RNAlater and wish to quantify neutrophils only via qPCR. As we know that the mucosa of gut is filled with several immune/epithelial cells, I want to target just one cell population. Is there any singular magic marker for neutrophils? Please let me know. Thank you in advance.
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Thank you everyone for you invaluable input. After intense discussion in my lab, I finally got an opportunity to run micro-array on my patient samples. I hope to find some answers soon.
Best,
Sakhila
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I am comparing the gut microbial diversity of 2 groups of individuals. I found that the alpha diversity is significantly different but the beta diversity is not significantly different. Can I still assume that the groups are different in their microbial population?
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Alpha diversity indices are most influenced by the abundance of local communities. When looking for beta diversities, the index you are using is crucial to understand if these abundances are taken into account or not. Bray-Curtis and Sorensen, for example, are based on the same equation. The only difference is the assumption of abundance-based (Bray-Curtis) or presence/absence (Sorensen). You can normalize alpha diversity matrices before running beta diversity tests by using Raupp-Crick correction, for example. It decreases the bias that different abundances would cause in beta diversity inferences. I would rather use beta Bray balanced.
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I want to evaluate if some groups of humans have endotoxemia or are at risk for. So, I have this huge doubt whether I must choose serum LPS ou serum LBP or even other marker/protein, because the research papers do not discuss what is the bettter marker for diagnosis endotoxemia, or the better marker (from gut) for activating serum monocytes.
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Hi Aedrian, I appreciate your suggestion. In fact, I have thought about this design but I will not be able to perform a propesctive study at this moment. For this, I have study several technical papers regarding endotoxemia markers to choose the best marker for my research question. It seems that LBP is better than LPS due to lipoproteins and other proteins present in blood that may interfere on LPS detection. And LAL test may underestimate the LPS detection. On the other hand, LBP at high concentrations not necessarily activates TLR4 pathway in monocytes. I have read about endotoxemia tolerance, which is also confusing in a clinical matter.
Anyway, thanks for comment here!
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Anyone who has a protocol to study microbiota? How to maintain the guts, etc?
I want to begin studying microbiota from aged rats and I really know nothing about how to do that.
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Maria Jose Bellini .-. To begin with find some of the great reviews (I will try to find some to post later), but you can even begin with the basic information at https://en.wikipedia.org/wiki/Gut_flora . If you intend on culture methods, you need to find a good microbiological laboratory in your institution or a university that can process the samples. From the way you wrote the question I am assuming you might be more interested in a genomics approach (or metabolomics). For this you will need to find a lab that can process your samples (this would be too hard to set up for your own lab), and there are even commercial companies which can process your samples. I have used https://www.mrdnalab.com/ and there website will give you insight into a variety of methods.
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Fecal microbiota transplantation (FMT) is the administration of a solution of fecal matter from a donor into the intestinal tract of a recipient in order to directly change the recipient’s gut microbial composition and confer a health benefit.
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Dimitri this is an interesting question that I do not believe anyone has researched (in part for ethical and legal reasons). A lot of questions would need to be answered first - what is the diversity of the elderly person and the potential younger person - is the elderly person a risk from any of the hundreds of organisms in the donor - Would it really have any great effect - is there a specific pathological need in the older person - is there any research showing the loss of any part of the microbiota having a pathological affect on causing disease or aging? Many more questions need to be raised, and a lot of research to do to understand whether any benefit could actually occur. Or could there be harm to the older recipient?
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I heard about the beneficial using of bone broth that is rich in collagen for healing leaky tight junction in gut; but I did not find scientific evidence about it, may you help me to find scientific evidence and studies ?
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There are no peer reviewed articles on your topic in Pubmed, but there is a Master’s thesis that considers bone broth and gut microbiota in relation to stress.https://bearworks.missouristate.edu/cgi/viewcontent.cgi?article=4308&context=theses
and a number of perr-reviewed papers on peptides and gut epithelial integrity: https://pubmed.ncbi.nlm.nih.gov/28174772/
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Recently I have been interested in microbial communities from gut & intestine of the same fish species popular in Indonesia marine aquaculture, Grouper (Epinephelus sp.), but are raised in different farm.
For example E1: monococcus 36%, streptococcus 29%, ... bacteria 70% gram negative ... etc
compared to E2: monococcus 31%, streptococcus 35%, ... bacteria 63% gram negative ... etc
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To conclude your study you can identify the sources of specific microbiota on a specific population with their feeding habit. The ranges of microbiota vary with feeding zone and water quality.
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I am working on the gut bacterial diversity of thrips and I sequence 16S V3-V4 regions of one species of thrips with the larvae pupae and adult life stages.To my surprise, I have 99-100 % bacterial genus Arsenophonus in all my samples. What may be the possible reasons for this. Do this bacteria replace the other bacteria and what experiment does I need to do to confirm this?
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is Arsenophonus found in all samples collected from temperate and tropical locality? Being symbiotic may play a role in plant disease transmission if your thrips is plant disease vector. Presence or absence of Arsenophonus may decide vector status of a population of that species of thrips in that area.
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Please share your preferred host DNA depletion method for microbial shotgun metagenomic sequencing. Please explain why. If your preferred method is to sequence everything and remove host reads through bioinformatics, share that too.
Some context: I will be sequencing degraded formalin-fixed ethanol-preserved DNA collected from amphibian gut swabs. Debating if I try one of the chemical removal methods or simply deal with this computationally. Most of the methods I have found are setup for removing mammal host DNA, so not sure of things like removing differentially methylated DNA, etc. I am open to trying new and developing methods for this.
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Great, thank you for the feedback Jacob and Vadim. I will Be opting for no host reduction for this experiment. I may try adding a few samples that have had host DNA removed as a sort of side experiment/comparison. As this is a NovaSeq experiment, I unfortunately don’t want to add many more samples and another lane/run is over budget.
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Good morning,
for our project we will dissect bumble bee trying to obtain and separate the external with the internal part (gut),
do you have any suggestion or tip do it?
we tried just after took the insect from the -80 °C and dissect on ice, but the terminal part was tough to remove and consequently the gut was contaminated with the external portion, we will try other times, if you have any suggestion, we would appreciate a lot!
thanks
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I would have taken the same approach. This delicate work might require a lot of training and may be it is more of an art than a skill, that everyone can acquire! Check around your staff (if possible) to find the most succesfull one in doing this. Depending on the purpose of your experiment, you might also try more harsh methods to fix the animal eg parafin embedding.
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I read some articles on mollusc and fish diet analysis. The formula for percentage frequency of occurrence stated above:
%F0 = Nei/Ne x 100
and percentage numerical abundance :
%NI : Ni/N x 100
especially in mollusc. I want to know how is the calculation for Nei, Ni and N take place, is it an individual organisms count under microscope slide or do we need to used specific device or equipments such as haemocytometer or sedgewick rafter to count the number of the cell per area before we can fill in the Nei, Ni and N in the formula?
Thank you.
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If you are counting food items, then both formulas will give you exactly the same result. Now, the real catch is what we mean when we say abundance. Is it the amount of events of finding a given food item? Is it how much biomass of a given food item a mollusc is ingesting? Is it a standardized methodology of counting? This is something that you have to think about.
Every time you want to find out how much of something is ingested we need to think about this: A count is meaningless in its nature. The count is only meaningful if you scale it to the representation of the total. Here is where the percentage comes into play. It will always capture the proportional amount of something. Therefore, we will always do it as: part/total X 100.
Now for your methods, I don't know much about molluscs but perhaps you could put the contents of the crop into a slide. The slide is now your sample. The Total is the total count of food items. The parts are the frequency of a given item. The same logic apply if you decide to quantify abundance using biomass (which is perhaps better but way more difficult to do), but the counts will be replaced with weights.
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the gut related diseases like White feaces, white muscle, white gut are doing much damage to shrimp by retarding growth and increasing FCR and eventually heavy losses. is there any medicine or cure available now apart from preventive measures?