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Clinical Microbiology - Science topic

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Questions related to Clinical Microbiology
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Recently , clinical microbiology and immunology techniques used were mostly automated, and the conventional methods are almost left behind . Do think these new technology produces a well-trained and understood graduates that really understand what are microorganisms and immune components and their activities and mechanisms ?
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Why should they be mutually exclusive. The automated methods are generally based on classic methods, so understanding the old methods is important, but knowing how to apply modern technology is equally important.
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Despite being a gram positive bacteria, Clostridium tetani can produce a greenish fluorescence color on MacConkey medium containing neutral red indicator, why is that?
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I support the views raised by Prof.Phil Geis.
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I am learning about predatory bacteria.
Could you give me a recommendation for researcher & research group that focuses on predatory bacteria? Especially the predatory bacteria from animal samples, not just environmental samples. Hopefully, this can be a list for someone interested in this topic.
Thank you in advance.
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If you were referring to predatory as in bacteria that prey on other bacteria and you dont get answers here then I’d recommend pubmed and going through recent publications or reviews on predatory bacteria eg
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I need to sequence an entire bacterial gene (6.1 kb). Is there a way to do it other than amplifying several smaller overlapping fragments for Sanger sequencing? Can I Sanger sequence the whole amplified gene using "walking primers", and if so, how is it techincally performed?
Is there any other method to sequence a single gene?
Thanks
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You can ,as you say, pcr amplify overlapping 800 base amplimers ( you get no useful sequence under the sequencing primer or for the next 30 bases). If you can amplify the whole 6KB in one amplimer then you just need overlapping sequencing primers at 800 base intervals along the whole sequence but just the one template dna. If you have a friend with a minion sequencer or NGS then new sequencing technology is good and quick but not cheap or easy to analyse the results
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I am having bacterial strains in the agar plate. I want to extract these strains and store them in glycerol for long-term use. Is there any stepwise procedure for this process?
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This is the procedure I use for E. coli stocks:
- Take a freshly saturated culture in rich medium (2YT or LB) and spin 2 ml in a 2-ml microfuge tube for 2 min in a microcentrifuge.
- Discard 1 ml of the supernatant and use the remaining 1 ml to resuspend the pellet
- To the concentrated suspension, add 0.5 ml 60% (v/v) sterile glycerol and mix thoroughly by pipetting up and down.
-Transfer the mixture to a cryotube and store at -80 °C
I have kept such viable stocks for decades. When needed, scratch the surface of the frozen stock with a toothipck and use the scratched material to inoculate a fresh culture, without thawing the rest of the stock, which should be returned immediately to the -80°C freezer.
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Hi.
We have some carbon nanoparticles and want to check antibacterial activity against drug-resistant bacteria. Before working on drug-tolerant bacteria, Are there any protocols or parameters for checking nanoparticles whether have the antibacterial capability on drug-resistant bacteria?.
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Follow
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Hi.
I have prepared hetero-atom-doped carbon dots (Nitrogen with sulfur, boron, phosphorous) for the eradication of biofilm of drug-resistant bacteria. But, I did not see any bacterial death at low concentrations when I am using doped carbon dots. Should I change any parameters or something else?
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Dear Saravanan Arumugam thanks for sharing this very interesting technical question with the RG community. Personally I'm not an exert in this field of reserach (we are inorganic / organometallic chemists) and I was not even aware of the fact that hetero-atom-doped carbon dots have antibacterial properties. However, there are various reports in the literature in which this effect has been demonstrated. For example, please have a look at the following potentially useful articles which migth help you in your analysis:
Applications of N-Doped Carbon Dots as Antimicrobial Agents, Antibiotic Carriers, and Selective Fluorescent Probes for Nitro Explosives
and
Antibacterial Activity of Nitrogen-Doped Carbon Dots Enhanced by
Atomic Dispersion of Copper
The first article is freely available as public full text, while the second has been published Open Access (please see attached pdf file). Apparently it might be worth a try using the hetero-atom-doped carbon dots in combination with copper or silver nanoparticles for enhanced antibacterial activity.
Good luck with your work!
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Hi to all,
We successfully created in our lab a new RT-PCR system, designed for amplification of the S1 segment of Infectious bronchitis virus (IBV). Yet, we obtained the end point PCR product only by using RNA from enriched allantois fluid, derived by inoculating SPF embryonated eggs. What is the best one-step RT-PCR kit/enzyme that we can use to enhance the segment using RNA directly extracted from tracheal/cloacal swabs? according to what I read, the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase is the best option, due to the increased sensitivity and the broad temperature range of the cDNA synthesis (We currently use the Verso one-step RT-PCR kit).
Will appreciate any insight,
Ido
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The best way of end-point amplification of target region using RNA directly extracted from tracheal/cloacal swabs is making complementary DNA (Step 1) and amplify target region from complementary DNA (Step 2).
One-step RT-PCR system can lead to lower yields or efficiency, more complicated to troubleshoot, and does not provide a stock of cDNA for future use. Since RNAs are unstable so, you can reverse transcribe them immediately and perform PCR later (Two-step PCR System).
Still if you want to use One-Step RT-PCR system, you may use following kits;
2. SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (you mentioned).
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In all labs I saw, I found that ethanol is used for sterilization of hands etc. in experiments with microorganisms. Why is isopropyl alcohol not used while it is relatively cheaper? Is it harmful for skin upon frequent use?
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The World Health Organisation recommends the use of disinfectants on hard surfaces and hand sanitizers. The formulations that offer the best results are alcohol-based disinfectants. We use Ethanol and Isopropanol (IPA) in our hand sanitizer formulations. These two alcohols are equally effective when it comes to killing bacteria, but to understand the differences between them kindly check:
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I have some isolates of salmonella typhi that is oxidase positive and on API they show that it is salmonella typhi. 
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I have run into a similar identification problem with a gram-negative bacillus that results as Salmonella typhi, S. gallinarum or sometimes as Shigella spp. in biochemical assay (usually using Remel RapID ONE in my case), but consistently produces an oxidase positive reaction (from growth on SBA, MH and others). In addition to this contradiction, the isolate has yet to be H2S positive on any medium (without significant acid production from sugar fermentation/utilization that could hide H2S positivity - good times for any of you out there that run into lac+ Salmonella isolates), and presents significantly enhanced growth under increased CO2 with difficulty at first isolation in aerobic subculture from blood culture vials or thioglycolate broth.
To complicate the identification further, MALDI-TOF protein biotyping always results the isolate with low confidence as Salmonella - oxidase positive genera have never broken the top 10 matched patterns by rank of score value. We test a lot of isolates which cannot be matched in currently available MALDI-TOF spectra libraries so I have worked under the assumption that these low confidence ID's were "overly confident." The isolate obviously needs to be sequenced at this point, but I unfortunately will not have the opportunity to do this and am unsure anyone will pick this work up behind me. As such, I am left only with the contribution of a comment to this thread in case we are encountering the same problem and someone here can solve it.
After repeated testing following countless passages in subculture across various media, my best guesses are as follows:
1. Aggregatibacter actinomycetemcomitans
2. non-motile, L-form Salmonella typhi or S. gallinarum with unknown interference mechanism resulting in false-positive oxidase test result for cytochrome C oxidase activity (thinking along the lines of something like manganese oxide interfering with oxidase test reagent)
Best of luck solving your mystery should you not be able to sequence your isolate...
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We are examining tissue from a cluster of human and canine patients with a similar pattern of systemic illness of unknown cause. All members of the cluster have evidence of motile zoospore-like objects in their blood and other tissue aspirates. Control wet-mount preps from healthy relatives of these patients do not show the presence of such motile objects.
Despite the tiny size of these motile objects, the “swimming” motion seems more consistent with the “falling leaf” forward motility pattern associated with a eukaryotic flagella than with bacterial motility patterns. Also, the staining patterns and SEM appearance of these objects appears more consistent with a eukaryote.
Preliminary sequencing studies have suggested sequence homology with stramnopile-type organisms. We are attempting to sequence cultured colonies of the organism but are having extremely low DNA yields despite robust growth of the organism in culture.
We would appreciate the opinion of those familiar with the morphology and zoospore motility of oomycete and related type organisms about the similarities and differences seen in these movies of motility in unstained, aseptically collected adipose tissue nodules from a patient in this cluster, suspected to be infected with a novel or emerging type of eukaryotic pathogen.
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sure, I'd like to see the pictures - philageis@aol.com
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I'm looking for the validated method or a standard.
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Hi Hachemi,
There is more simple method instead of the DNAse agar. You may test it too:
Good luck,
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As per recent diagnostic criteria for the diagnosis of NTM, there is a chance of overdiagnosis of NTM, thereby enhancing the potential toxicities of the drugs. There is also a chance of undertreatment. How do you defend this column? What do you think?
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Hi, good day! Nontuberculous mycobacteria are a diverse group of mycobacterial species that causes different clinical infections in children and adults. According to Ingen (2013), 25 species have been strongly associated with NTM out of the 140 known NTM species in the literature/studies. Isolation of M. kansasii and M. malmoense from pulmonary specimens usually indicates disease, whereas Mycobacterium gordonae , M. simiae, or M. chelonae are said to be contaminants rather than causative agents of true disease. Mycobacterium avium complex (MAC), M. xenopi, and M. abscessus form an intermediate category between these two extremes (Ingen,2013). NTM species differ in their clinical relevance therefore, the need for correct species identification is very important. As per your question, to prevent the chance of overdiagnosis and undertreatment of NTM, it is crucial to identify the particular causative agent that caused the condition/illness as well as its clinical relevance. to make a firm diagnosis, a culture of the clinical specimens, and a histological examination of tissue biopsy specimens are needed. Diagnosing NTM disease is complex therefore a good communication between clinicians, radiologists, and microbiologists may help in optimizing the culture conditions in line with the particularities of the patients.
if you want to know more about the diagnosis of NMT, this article might help you:
Ingen, J. (2013). Diagnosis of Nontuberculous Mycobacterial Infections. Diagnosis of Nontuberculous Mycobacterial Infections. DOI:10.1055/s-0033-1333569
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Is it better to use kanamycin aesculin azide agar or bile aesculin agar when selecting for enterococci? Are there advantages of one media over the other? Thanks
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The Bile Esculin Azide Agar is a good and recommended selective medium for the isolation and identification of Enterococci. The presence of the bile salt in this agar acts by inhibiting the growth of mostly gram-positive organisms while esculin is hydrolyzed by Enterococci providing the means for selective isolation and presumptively identification of the Group D Streptococi and Enterococci like the Enterococcus faecalis and Enterococcus faecium.
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Burkholderia pseudomallei as we know is a non-fermentor of lactose. However, it appears as pink-colored colonies in MacConkey agar media following 48hours of incubation. I have cultured strains myself and found them as pink wrinkled colonies on MacConkey agar following 48-72 hours incubation. What is the reason for this pink colored appearance?
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It was nice to see this discussion both because there was a clear question, but even more importantly the answers were knowledgeable with helpful information. When possible references should always be given, and several of the answers would have been more helpful if there was a citation that could be checked out.
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We have designed an in house LAMP assay for the detection of a pathogen. We have used ThermoPol buffer, betaine, MgSO4, Bst polymerase and dNTPs in the reaction. I wanted to know why MgSO4 is used instead of MgCl2 (like PCR) in the LAMP assay?
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I do not think that it makes any difference whether you use chloride or sulphate. The important part is the magnesium ions
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A 70 year female admitted to the ICU with UTI. 
Report: Isolate- Serratia marcescens 
Susceptibility report: 
Aztreonam- resistant
Cefazolin- resistant
Cefotaxime- resistant 
Ceftriaxone- resistant
Cefepime- MIC 4 microgm/ml
Cefotetan- resistant 
Piperacillin/tazobactam- resistant
Imipenem- MIC less than 1 microgm/ml
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i agree with Jay Siddharth
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Does Proteus mirabillis capsule have any role in urinary stone formation? if yes please what is the mechanism?
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Please take a look at this link.
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Hi dears
Anybody know the percentage of MRSA and MSSA of S.aureus in the differen population? or totally in the world?
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Article Methicillin Resistant Staphylococcus aureus (MRSA) Nasal Car...
Article Comparison of chromogenic agar medium and diffusion disk tes...
Article Detection of methicillin resistant Staphylococcus aureus (MR...
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What is the most suitable biochemical test for differentiation of Klebsiella pneumoniae (isolated from clinical samples) from other Klebsiella spp?
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the API system is a good one, Klebsiella sp, the (probably) easiest way would be by the Indol test. K. pneumoniae is indol-negative, whereas K. oxytoca and K. ornitolytica are indol positive. But be aware that e.g. Enterobacter spp are also indol-negative.sugar fermentation
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I used falcon tubes instead of cryovials for the preservation of microbial cultures.
As cryovials were expensive for me to buy So, instead I used falcon tubes.
But I'm looking for the scientific reason or say difference between cryovials and falcon tube preservation materials.
What are pros and cons of cryovials and flacon tubes?
What If someone asked me about these preserving materials I used especially falcon tubes,
How should I justify that I used cryovials or falcon tubes because of blah blah.....?
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You can simply use regular 1.5ml/2ml Eppendorf tubes. The only downside, less writing space on the side.
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Microbial biofilm is the formation of sticky adhesion layer attached to the surfaces of animate or inanimate objects
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Establishing the relationship of Double J stent Catheters with the episodes of CAUTI and CRBSI and subsequently link its prolonged exposure leading to bacterial translocation as a risk factor for the development of the sepsis.
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In recent years, the most of researchers and interesting ones in the field of microbial biofilm documented that nano particles like silver, metal ions play a significant role in biofilm approach.
Here i would like to concentrate a light on the modern technique in biofilm and the role of nanotechnology in combating medical resistant microbial infection.
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For developing good portable sterilizer for field use (maybe for forest, desert) which source can work with smaller battery source, with less energy, in a smaller box without producing hazards to the personnel?
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I know this reply is really belated but I was wondering if portable UV-C devices abundantly available on line will do the job. Here is an example of what can be found on ebay: https://www.ebay.com/itm/Nail-Sterilizer-Box-Disinfection-UV-LED-Pedicure-Brush-Cleaner-Tool-HL/123814321847?hash=item1cd3e89eb7:g:2t0AAOSw4Vpb0ofU
I am really curious if anybody used them in research. So far could not find any reviews of such products by scientists at Amazon :)
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Mycobacterium Tuberculosis moved from P3 lab after confirmed Inactivation with Heat Inactivation.
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Thanks for all the tips! This was extremely helpful
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As we know from the Bruker's instruction or CLSI, the recommended bacterial load for identification by MALDI-TOF MS is around 10^5 CFU. Moreover, it is also clear that species identification would be wrong when the bacterial load is low in Bruker's MALDI-TOF MS. On this basis, however, I am curious why we fail in getting reliable identification when the bacterial load is low. Is it the problem attributed only to low peak intensity or bad S/N ratio? What would the MS spectra look like? Does anyone has such the experience? Do we have opportunity to improve the identification under the condition by more advanced bioinformatics?
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Shortly, a multicenter study on S.maltophilia biofilm formation starts.
Clinical strains will be needed.
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Yes, I am interested @Giovanni
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I'm performing assays on a number of bacterial strains, some of which are capable of forming biofilms.
What effects would a "tissue culture treated" plate and a "non-tissue culture treated" plate have on bacteria suspensions??
Would a TC treated plate cause lower amounts of planktonic cells and/or promote biofilm formation as compared to non-TC treated plates?
Thanks!
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Hello Kevin. My team compared the biofilms on different brands of 96-wellplates (TC-treated, non-TC-treated), and found that some non-TC-treated plates (super cheap) had the lowest biofilm of the same bacteria. We then concluded that non-biofilm experiments can be used on certain non-TC-treated plates, while biofilm experiments should be used on TC-treated plates.
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Mannitol Salt Agar is the most common technique used to differentiate CoPS from CoNS. But I am curious to know any other techniques or media which can be used to differentiate CoPS from CoNS on an agar plate. Kindly suggest.
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You can not. The only available test for this purpose is coagulase test.
Other tests or agar media are unreliable.
Regards
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I know that vancomycin is effective only against gram positive bugs but it seems that there is a bacterium that vancomycin covers.
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It's well-known that most Gram-negative bacteria are intrinsically resistant to vancomycin because their outer membranes are impermeable to large glycopeptide molecules (with the exception of some non-gonococcal Neisseria species).
Regards
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What are New techniques in diagnostics clinical microbiology -identification and biodiversity?
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Following
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why always we use  0.5 McFarland standard specifically in determine susceptibility testing of anti microbial agent?
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I also use 0.5 Mcfarland to determine number of bacteria cell after incubation but in most articles they use Uv/visible spectrophotometer at 600 nm to determine cell concentration. Whic way is more reliable?
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9 peoples including a hospital nurse died due to the Nipah viral outbreak; plz mentioned how to manage this contagious entity.
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At least we would be very happy to test the antiviral activity of ZnO tetrapods, let me know if someone is interested, I am hopeful that it will work.
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American Society for Microbiology, "Culture of Responsibility Workshop", at Lovely Professional University on 5-7 May 2018, Registration Link: http://www.lpu.in/hrd/ (seats are filling fast)
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It is so interesting, unfortunately I will not be able to be there.
My best wishes
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Morphological culture colonies is very important for preliminary laboratory diagnosis of bacterial genus followed by confirmatory tests.
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Can you provide additional information e.g. Gram stain, colony morphology in other culture medium?
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Need to upgrade our laboratory. To provide a timely diagnosis of various bacterial, viral, fungal and mycobacterial infections and do epidemiological typing of various nosocomial pathogens.
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Next to molecular biology ( and perhaps even more promising) you should focus on mass spec techniques.
Read as an introduction:
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The oncogenic potential of the high risk HPV types lies in the oncoproteins early (E6 and E7) which can bind to and modulate a number of different gene products, in particular, the tumor suppressors proteins (p53 and pRb).
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If I understood your question right, you want to know if other HPV proteins (different from E6/E7) can induce cervical cancer. Besides E6 and E7, E5 has been showed to be an oncoprotein as well. One well explored mechanism of E5 is regarding the evasion of immune system. The high‐risk E5 protein interferes with classical MHC class 1 processing, but also has many other functions related to proliferation.
E5 as an oncogene has been largely investigated in bovine papillomavirus BPV:
Check the review from Dr. Doorbar below:
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Hydrolysis of β-lactam antibiotics by β-lactamases is the most common mechanism of resistance for this class of antibacterial agents in clinically important Gram-negative bacteria. Because penicillins, cephalosporins, and carbapenems are included in the preferred treatment regimens for many infectious diseases, the presence and characteristics of these enzymes play a critical role in the selection of appropriate therapy.
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Hey, for a real time update on classification of beta-lactamases in general, you got to always check this online database
From the website you have all the known abd emerging beta-lactamases list and classifications
Cheers
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The role of the infection with Brucella on the abortion of infected animals and no such occur in human being  
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Hi,
It is believed that brucellosis causes fewer spontaneous
abortions in humans than it does in animals because of
the absence of erythritol in thehumanplacentaandfetus
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Hi everybody,
All is in the title : What are the uses of the Galleria mellonella model other than an infection model ? We ask me that question and I don't find other uses except study bacterial diseases by pathogenicity for exemple or efficiency of antibiotics / phage therapy.
Thank you
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You can use Galleria for compound toxicity testing, virulence testing, efficacy of novel compounds, evaluation of pathogenicity between strains of bacteria/fungi. In addition, you can now purchase research grade Galleria from https://biosystemstechnology.com/ which are grown free of any hormones or antibiotics and are genome sequenced
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Fisher is out of Mueller Hinton with 5% Sheep blood, will using Chocolate Mueller Hinton be acceptable?
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Chocolate Mueller Hinton Agar has been developed for fastidious organism susceptibility testing , originally for Haemophilus species and A. pleuropneumoniae and H. somni (both veterinary pathogens).
Currently the CLSI suggests the use of Haemophilus test medium for H. influenza and H. parainfluenzae species.
If you are testing streptococci I would suggest that it is not appropriate as it has X and V factors which were added to this media specifically to allow the organisms above to grow.
CLSI quality control ranges and interpretive criteria for Streptococci, Listeria and N. meningitidis were developed using 5% sheep blood Mueller Hinton agar . So I would not recommend the use of chocolate Mueller Hinton for these species.
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I want to separate E.coli dependent diarrheas from antother type of diarrheas. I wonder to is there any way to separe them via clinical symptom or laboratory test.
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I think it is very difficult to differeniate clinically E.coli diarrhea from that caused by other agents.However,in the lab after isolation of E coli from stool there are many Known laboratory methods confirm that the causal is E.coli (strain associated with diarrhea) of those
1- demonstration of toxin e.g Biken test to detect Labile toxin(L.T)
2-Animal pathogencity
3- serology serotypes . certain serotypes Known to cause diarrhea
4- virulence factor may be detected by molecular methods
Best Regards
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Dear all,
I am involved in a small project to investigate the way scientists communicate their work and are engaged in social media. We have created a small survey, which you can find following the link below.
The survey is anonymous, and we calculated that you would need maximum 5 minutes to complete it. I know you are all very busy, but I would be extremely grateful if you could find few minutes to complete the questionnaire.
Thank you very much for your time and your help!
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I would love to get updated with the results of the survey... How can we stay in touch?
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When the clone, the bacterium fluid grows, but the bacterium fluid PCR amplification does not come out, is how to return a responsibility?
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What is the ARMS Plus?
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What is the role of extracellular DNA in bacterial antibiotic resistance and how can it be detected in the lab?
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Understanding the mechanisms by which biofilm bacteria develop resistance to antibiotics is paramount to expanding the treatment options available to patients with chronic biofilm infections. ... Extracellular DNA, a known component of biofilms, was found to induce antibiotic resistance
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Can somebody tell me the most common microorganism isolated from ICU?
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Dear Manar, this article will help you:
Semin Respir Crit Care Med. 2003 Feb;24(1):3-22.
Epidemiology, prevalence, and sites of infections in intensive care units.
Richards M1, Thursky K, Buising K.
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Hello everyone. I'm getting confused about my work. please help me. I put a few Antimicrobial disks including Imipenem, Amoxicillin/tazobactam,
piperacillin/ tazobactam and cefuroxime for measuring the Susceptibility (S,R,I) for Staphylococcus aureus. The problem is here, because the CLSI 2013 and older version  haven't any guidelines. I don't know what I am suppose to do? Thanks.
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You will find what you want in the updated version of CLSI 2016 document (attached file).
Regards
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Dear Everyone,
I'm looking for the fulltext for NCCLS M22-A2 and A3 procedure. May you kindly help if you have? 
Thanks!
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Thank you very much for all your help. I really appreciate that.
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Antibiotic sensitivity testing of colistin was done by Kirby Bauer method.I have isolated DNA by boiling method.
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Becouse of other mechanisms of resistance 
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Hello, would like to ask about agar medium. Mueller- Hinton (MH) is usually used for antibiotic susceptibility testing due to the ability to absorb toxin. Is MH suitable for bacteriocin screening as well? If no, what agar is suitable for this kind of work? I'm working with Burkholderia spp. Thanks.
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Pseudomonas aeruginosa Have low percent Of biofilm Formation Although It's Clinical Isolated From sputum And Pus .
What Is The Explaination Of This Low Percent ?
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Pa is known to form biofilm and responsible for some of the horrifying nosocomial infection. As pathogen it will grow very slowly outside the host body in absence of body fluids.
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Is it possible to get a high count of E. coli than faecal coliforms when using the  m-LGA (Membrane Lactose Glucuronide Agar) which is used for the differentiation and enumeration of Escherichia coli and other coliforms through the membrane filtration technique?
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Thanks Marion! That answers my question.
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I test colistin MIC values of some clinical A.baumannii and K.pneumoniae isolates with microdilution method. In last experiment, I mistakenly prepared standard Mueller-Hinton broth instead of its cation-adjusted version and I obtained like the values of 128, 64 or 32; whereas I expected that they had to be between 2 and 4 (based on the information from hospital). The previously given values can also be wrong, so can it be possible that usage of standard MH broth could affect the results that much?
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I use the CLSI guidelines but that was just a mistake the using standard MHB. For the information, I repeated the procedure with CAMHB and the results didnot change significantly, just for a few samples as only one value of MIC. 
Thanks a lot for your comments, 
All the best
Ezgi
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I am working on the prevalence of asymptomatic carriers of group A streptococci (GAS)among school children of 5-15 years. Culture yield was very low for GAS, but with the use of qPCR (spy1258 gene) i could pick up more positives. what should be the actual bacterial load in throat swabs to confirm as carriers?If the ct value is between 36 and 40 can we take as positives?
We serially diluted the sample of known DNA concentration. ct value of 24.45 corresponded to DNA concentration of 37.6ng/µl (apprximately1.5 x 10^8 cfu/ml).  ct value of 38.56 corresponded to DNA concentration of 37.6fg/µl (apprximately1.5 x 10^4 cfu/ml). 
What concentration of DNA/ bacteria should be there in throat swabs to be interpreted as asymptomatic colonization?
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Thank you Dirk Schmidt.
For patients the ct values ranged between 20 and 30.
Is there any guidelines about detection GAS asymptomatic carriers (especially about the bacterial /DNA load)
If the qPCR flags positive at 36 to 40 is it really carrier state positive, since group A streptococcus won't be present as normal flora of upper respiratory tract.
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 I am looking for a specific visual method if any to check contamination while incubation of Mtb in Dubos medium(Selective medium for Mtb)?
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This pdf It may help
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Purple urine in the bag is a known entity.
Blood and urine examinations normal
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The pathogenesis is controversial. According to the most popular hypothesis , dietary tryptophan is converted to indole by gut bacteria, which is further metabolized in the liver to indoxyl sulphate and then excreted in the urine. Constipation favors conversion of tryptophan to indole by gut bacteria.
Once excreted, indoxyl sulphate can be processed by bacteria colonizing the urinary catheter to indoxyl, which is further converted to indigo (blue) and indirubin (red). ( Initially had red tinged urine which became orange as urine flowed)
The most commonly involved
bacteria are Providencia stuartii, Providencia rettgeri, Escherichia coli, Klebsiella pneumoniae,( like our patient) Proteusmirabilis, Morganella morganii, Pseudomonas aeruginosa, and Enterococcus species [4]. These bacteria produce indoxyl phosphatase and sulphatase enzymes.
so it solves our puzzle!!
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I am working on antimicrobial resistance in bacteria isolated from non anthropogenic environment. Majority of the isolates are non pathogenic, however resistance are there? So is there any guideline like (CLSI guidelines for clinical isolates) to deal with such resistance?
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This is hardly surprising and was shown multiple times in the past. Keep in mind that most antibiotics were originally produced by natural microflora... Take a look at these papers:
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is there any correlation between biofilm formation and antibiotic resistance in clinical bacterial isolates, if yes what is the mechanisms? 
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Yes indeed there is. Well established bacteria have the ability to resist better the effects of agents they are exposed to including antibiotics.  
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Hi,im currently in the stage of publishing my research work on evaluation of several methods of detecting Burkholderia pseudomallei culture, may i know the best journal platform to publish?. Im looking foward to the journal which has open access and if possible free fees, so that i can share the findings to others.
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People interested in an update on predatory journals, following the cessation of Beall's List updates and it's removal, might like to refer to an article in today's Times Higher Education (6 April 2017, pp. 42 & 43), reporting work by Larissa Shamseer and David Moher (link below but access may be restricted to subscribers).
Their study resulted in a list of 13 warning signs:
1. The scope of interest includes non-biomedical subjects alongside biomedical topics, or multiple, wide-ranging and unrelated fields of study are combined
2. The website contains spelling and grammar errors
3, Images are distorted/fuzzy, intended to look like something they are not, or are unauthorised
4.The home page language targets authors rather than readers
5, The Index Copernicus Value is promoted on the website
6.  Description of the manuscript handling process is lacking
7.   Manuscripts are requested to be submitted via email
8.   Rapid publication is promised
9.   There is no retraction policy
10.   Information on whether and how journal content will be digitally preserved Is absent
11. The article processing/publication charge is very low (eg, <$150)
12. Journals claiming to be open access either retain copyright of published research or fail to mention copyright
13.  The contact email address is non-professional and non-journal affiliated (eg, @gmail.com or @yahoo.com)
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identification of S.aureus by morfological and microscopical picture and by PCR.
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Couple of thoughts:
  • There are several commercial specialty agar media products available to differentiate MRSA from MSSA. They typically inhibit growth of MSSA and vary in the color of MRSA colonies.
  • You can make your own MRSA selective agar by supplementing standard Mueller-Hinton agar with 6 mg/L oxacillin and 4% NaCl. This inhibits the growth of MSSA (oxacillin) and supports growth of heteroresistant MRSA (NaCl)
  • Verify that you incubate at 33-35°C, not 37°C
  • Like all staph, it will show up as gram-positive (purple) cocci in clusters on standard gram stain. This will not differentiate MSSA from MRSA
  • Colony color is variable from almost pure white to a deep golden color and is not useful to differentiate MSSA from MRSA
  • PCR amplification of the mecA element is the current rapid-diagnostic standard, although it does not detect the (fairly rare) cases of borderline resistance or resistance due to mecC elements
  • The "real" way to do it is to use a cefoxitin disk screen test or broth microdilution per EUCAST/CLSI standard. The latex agglutination test is another alternative.
Lots of options depending on how many samples you will be processing, how much you're willing to pay and what turnaround time you want. Good luck!
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The surgeon sent all tissues for pathology in formaline for TB .Can we perform any microbiological studies or PCR on such specimens with formalin .?
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As far as microbiological methods are concern; isolation is not possible from formalized tissue. Acid fast staining, histopathology and immunohistochemistry can be performed. Pcr can be uesd to detect the presence of bacterial DNA.
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there is no information about florfenicol in CLSI
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I'm not aware of defined inhibition zone diameter breakpoints for E. coli and Florfenicol.
In a paper of O Ahmed (2010) [https://ann-clinmicrob.biomedcentral.com/articles/10.1186/1476-0711-9-12] the breakpoint was set to ≤18 mm. Miller et al (2003) [http://jcm.asm.org/content/41/9/4318.full] found E. coli ATCC 25922 (susceptible to florfenicol) showing inhibition zones of 18-33 mm (grown at 28 C!!!) while NCCLS (at that time) approved 20 - 30 mm for that reference strain.
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Anyone who shares the supplemental file M27-S4 used as a reference for antifungal susceptibility testing of yeasts?
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??
You are confused. This question is not for you. This is a general question available for anyone who is a Researchgate member.
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Kindly help me by suggesting the mechanism of different antibiotics and supplements used during culturing of H. pylori.
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We use selective media with amphotericin, vancomycin, cefsulodin and trimethoprim for H. pylori culture and it works very well.
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i am planning to run a multplex pcr for IMP, VIM, KPC, NDM genes and i dont have control strains for VIM
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 If one of the species of the Enterobacteriaceae family  that is resistant to ceftazidime and cefotaxime,  also ceftazidime-clauvunic and cefotaxime -clauvunic and the difference of zone diameter between CAZ and CAZ-CV and also for CTX are less than 5 mm(0 mm).it is ESBL positive or not?
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One causes of failure of Disk Diffusion confirmatory test of ESBL production in detecting ESBL-producing enteric bacteria is the over-expression of ESBL and AmpC genes.  Sometimes a bacteria has ESBL and AmpC genes and AmpC enzymes were over-expressed. When this situation occurs, AmpC enzymes is suppress the ESBL confirmatory test. You should check presence of  the bacteria for AmpC genes.
You have to check the susceptibility of your isolate to cefoxitin. 
The attached table may guide you to the suitable answer.
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Hi . I have moraxella   isolates in tryptose soya agar slant for period one month but i do subculture in  brain heart and tryptose soy broth  but no growth . what can i do for maintain for some months?
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Brain heart infusion broth with 2% glycerol. Then store in -20 °C. This will maintain your culture for 4-6 months.
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What is the significance and implications of asymptomatic throat carriers of beta hemolytic streptococci?
Significance Group A streptococcal isolation from throat swab of healthy children and the treatment is still a dilemma! What can be done if the child is found to be a carrier of GAS? Is it recommended to detect the asymptomatic carriers in community other than outbreak investigations?
In our study among school children of 5 to 15 years, the asymptomatic carriage rates of Group F and Group C streptococci was very high when compared to that of Group A. Is there any significance for this and how can we explain.
Do we need to treat the children?  If yes what is recommended?
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asymptomatic throat carriers of beta hemolytic streptococci & healthy children should not be treated with antibiotics. 
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I want to know why occurs D shape between some antibiotic in some gram negative bacteria ? like that erythromycin and clindamycin in staph and strep? How we can find the reason?
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The classic example of inducible resistance, as you provide, is the D-shaped disk diffusion patterns in gram positives (staph and strep) when you add both erythromycin (ERM) and clindamycin (CLI) disks on the same plate. A strain with inducible clindamycin resistance would appear susceptible if only a CLI disk is used.
This is because clindamycin does not release attenuation of the ermC or similar MLSB mRNA transcript (so it is never expressed, thus no resistance). However, treatment of infection caused by such an organism with clindamycin monotherapy has a high likelihood of treatment failure due to small subpopulations with mutated ermC sequences able to constitutively express  high-level MLSB clindamycin resistance. While spontaneous generation of these mutated ermC sequences that are not attenuated is rare, the number of microbes in an average infection is often sufficient to have some of these resistant subpopulations already present. In contrast, even small amounts of erythromycin bind to the ermC or similar MLSB mRNA transcript, release attenuation and the transcript is expressed resulting in high-level ERY/CLI resistance in the entire population given the presence of erythromycin.
Thus, there will be a blunting of the CLI zone of inhibition where ERY has diffused into the media resulting in a "D" shape because ERY "turns on" expression of ERY/CLI resistance in all members of the population, not just the vanishingly small subpopulation with a mutated (read: constitutively expressed) ermC-like transcript.
Similar inducible resistances can occur in gram negative organisms (e.g. with tetracycline, etc). Hope this helps!
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Is there any method to distinguish between pathogenic and non pathogenic E coli strains grown on a plate using their cultural characters?
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Pathogenicity and detection of virulence genes are necessary for completing the identification criteria for an organism. So PCR test for on of the virulence genes or pathogenicity using experimental animals are two main cases enable us to know wheather the organism is pathogenic or no.
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To direct diagnosis meningitis in children as the quick result and depended on as a result  of treatments and compared with other tests as culturing and  molecular  techniques PCR 
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No ...since there are a lot of commensial niesseria which may add to confusion on gram stain. so culture or molecular dtetection is necessary
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I have a strain of enterococcus sensitive to vancomycin and resistant to teicoplanin, Is that correct ?
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Thanks for your suggestion
I will check the CLSI, I have 26th edition one, Is it the last version do you have?
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This is a no-brainer. Bacteria culture, especially with drug resistant strains like MRSA, should be conducted in a separate area than that in which cell/tissue culture takes place; however, I am having a very hard time tracking down something that says this in writing from NIH/CDC or WHO.
For perspective: Currently, the lab in question cultures are variety of tissue (BSL-2) as well as cells (MSCs), and a student is trying to introduce a MRSA project for biofilm applications. Can someone please provide me documentation outlining why this is dangerous for the biosecurity of the cell/tissue culture work currently ongoing in the culture room? 
Any documentation outlining why bacteria and cell/tissue culture MUST be conducted in separate research spaces would be greatly appreciated. (Also, there is one biosafety cabinet, so this bacteria would be introduced into the singular culture room/hood and incubator system). 
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Under my personal criterion, it is better to separate both activities, in our laboratory Vero cell cultures are maintained in an area and laminar flow hood separated from the room where we infect those cells with intracellular parasites mainly, the laminar flow hood is also different . In your case one would think Staphylococcus aureus strains are extracellular, but that vision has changed. Attach a reference that might help you.
Curr Protoc Microbiol. 2007 Feb;Chapter 9:Unit 9C.4. doi: 10.1002/9780471729259.mc09c04s4.
Tissue culture assays used to analyze invasion by Staphylococcus aureus.
Cheung AL1, Bayles KW.
Author information
 
Abstract
Many successful pathogens have developed the ability to adhere to and invade animal tissues. Recent experimental evidence suggests that S. aureus, generally perceived as an extracellular pathogen, can also invade and, in some cases, multiply within host cells. As a proxy to infections in animal hosts, the study of S. aureus interactions with tissue culture cells has become an important research tool in many aspects of bacterial pathogenesis. In this unit, we describe two cell culture models, including bovine mammary epithelial cells and human umbilical vein endothelial cells, that investigators have used to study the interactions of S. aureus with host cells.
PMID:
18770623
DOI:
10.1002/9780471729259.mc09c04s4
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I'm not able to conclude my result. I am using Acinetobacter species strain to perform Serum bactericidal Assay.
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Thank you so much for your reply sir,
I'm already used as control E.coli as well as Klebsiella pneumoniae as control of serum sensitive but all the ways getting serum resistance. 
I want to measure complement factor in serum could inhibit bacterial growth.
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Hi everyone
i want determine antibiotic susceptibility of biofilm-state bacteria to compare with planktonic-state bacteria. i searched some papers and met some description like MBEC, "MBEC" is the same meaning with "MİC of biofilm cells". and is there any method for Escherichia coli.
Best wishes
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MBEC is biofilm eradication concentration in which impact of compounds is assessed on pregrown biofilm. MBIC is similar to MIC.
Generally crystal voilet assay is done for quantifying, resazurin assay is also used to assess the viablility.
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many research used water instead of DMSO to make serial dilution from fluconazole while other antifungal agent diluted with DMSO like Terbinafine or Griseofulvin
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