Microorganism Identification - Science topic

8/20/22

Dear Hooi,

Do you see this in every smear that you examine w/ the microscope? Or is this the only one you've seen? At first, I thought it was a small length of hyphal (fungal) filament, but it looks more like a tiny stand of cotton fiber than a microorganism.

Bill Colonna Iowa State University, Ames, Iowa, USA wcolonna@iastate.edu

It may be Actinomycetes. It needs microscopy test. So the picture is not clear enough to identify it.

1) By adding organic matter humus can form phosphohumic complexes which is easily assimilated by plants, humate ion can replace phosphate ions and humus can form coating around the Fe and Al ions so that coating prevent P from fixation....

2) There are P - solubilizing organisms a) Bacteria - Pseudomonas and Bacillus

b) Fungi - Aspergillus and Pencillium

3) VAM Fungi can increase the absorption of P from soil by extension of root system

4) Placement (Band placement) of fertilizers (Placing the fertilizer below the seed can reduce the fixation of P by reducing the contact between the soil and fertilizer)

5) Liming of acid soils also release fixed P from Fe and Al compounds

Hard to tell, but I doubt they are bacteria. Can you do a bacterial stain and observe under a microscope? Or culture these on agar plates?

If possible micro-organisms, a traditional gram stain might be revealing. If they stain by gram, you will be able to see their morphology (and gram reaction). If they do not stain by gram, it is very likely that they are not micro-organisms. I realise that this is a simple approach, but it is quick and might be useful.

SAI of Manchester UK make 3 MALDI ToF products

with Bacterion software for microbiological ID www.saiman.co.uk

As an example of what you are looking for:

Sp1 (a transcription factor) interacts with the gene of HIF-1alfa (another transcription factor) that controls more than 100 genes.

Folia Microbiol (Praha). 2013 Jan;58(1):61-8. doi: 10.1007/s12223-012-0179-5. Epub 2012 Jul 12.

Abstract : Soil microbial populations play crucial role in soil properties and influence below-ground ecosystem processes. Microbial composition and functioning changes the soil quality through decomposition of organic matter, recycling of nutrients, and biological control of parasites of plants. Moreover, the discovery that soil microbes may translate into benefits for biotechnology, management of agricultural, forest, and natural ecosystems, biodegradation of pollutants, and waste treatment systems maximized the need of scientists for the isolation and their characterization. Operations such as the production of antibiotics and enzymic activities from microorganisms of soil constitute objectives of industry in her effort to cope with the increase of population of earth and disturbance of environment and may ameliorate the effects of global climate change. In the past decades, new biochemical and molecular techniques have been developed in our effort to identify and classify soil bacteria. The goal of measuring the soil microbial diversity is difficult because of the limited knowledge about bacteria species and classification through families and orders. Molecular techniques extend our knowledge about microbial diversity and help the taxonomy of species. Measuring and monitoring soil microbial communities can lead us to better understanding of their composition and function in many ecosystem processes.

Some PDFs enclosed

Samples from freshwater, fixed with  formalin, 100X magnification

Dear Vipin 

The picture likes E. coli or one specie of Enteriobacteracae family. You can use Sodium Azid with MRS for growth inhibition of   E. coli

It's a good paper for the collothecidae, you have to see the lobes form

http://zookeys.pensoft.net/articles.php?id=3330 

Dear Rebati, if you get colourless transparent colonies in Ashby's Mannitol Agar plates, you could assume them to be Azotobacter colonies. However, for confirmation you should have to go for morphological, biochemical and molecular characterization of the isolates. 

Regards,

Sosanka

172

Hi Kenfackvoukeng

I agree with Dr.Hugo

156

Hi,

I think that it was some kind of yeast to hyphal phase.

good luck

I have used Bergey's Manual of Bacteriology for the identification of bacteria, it was very useful

Cannot be Rotifer. I see some kind of annulation on the last Photo. Is cuticula hard or soft? And size?

Hello; 

Could you send me please your email? To sending you some articles about DNA barcoding in Collembola group. You can also follow David Porco. 

Best regards. 

Malik. 

Dear Gang Dai,

As it was already mentioned by colleagues answered to you, microscopic observation is necessary for preliminary identification. Mycelial / non-mycelial organization help to narrow a group of possible organisms and other structures revealed can help to choose a relevant method for further more precise identification.

Please, provide  microphotos to get helpful answers,

best wishes,

Elena

Thank you Indrayanto for your suggestion  

Not an expert in "morphology of frozen samples" but in general, cell-walled organisms conserve quite well the morphology, at least from the optical microscopy point of view.

For electron microscopy, your samples might not be so suitable though. But even so, I think it would be too laborious. I guess you didn't snap-freeze your samples and even if you do cryo-SEM or similar, I wouldn't be very confident due to the slow freezing process.

Easy way, extract DNA, do amplicon sequencing and try to correlate the taxa in the results with known filamentous bugs. Off course, after confirming they aren't fungi.

hi 

I am sure it's Staurastrum arachne

The methods in the papers below may be able to help.

Hi jose, I agree with Christine and I would say that they are some kind of crystal. I occasionally see similar patterns when some liquid comes out from the culture dish, and it dries under the plastic layer. Maybe you can check if the crystals stay in a focus different from the one of the cells. Hope it helps!

Just go straight into high throughput sequencing.

You can also use FISH, but it can be a real pain to count so many bugs and having appropriate probes for all the taxa you want to evaluate.

As pointed out by previous experts it is certainly not from the genus Actinotaenium. It also seems to be similar to C. tumescens Turner 1892, which Turner suggests comparing with C. maculatum in the same paper. Based on the morphology from the picture provided, this specimen is also most likely a tychoplankter.

Regards,

Alex.

Dear Andrés,

I am not an expert in filament identification but these images look like Type 021N to me. It is normally >200µm long, 1.5 to 2.5µm in diameter with obvious indented septa and no sheath. However, its morphology may change depending on whether it is being favoured by a nutrient deficiency or septic conditions (sulphur granules in slides 2 and 3 ??) and it can therefore be mistaken for other filaments. An F/M ratio > 0.25 can eliminate Type 0041 and staining may rule out Nostocoida species (Type 021N stains Gram and Neisser negative in the absence of sulphur granules). S natans and Type 1701 will be unlikely if the dissolved oxygen is high.

Dear Chenna, As suggested by Subir consult G.P. Sinha or Dr. Upreti, NBRI.

In my opinion is not a parasitic worm, it looks like an invertebrate larva which was eaten by fish. Probably it remains in the mouth or gill cavity because the fish was catch immediately after feeding. It should be a oligochaetan or insect larva, Chironomus sp. i.e. 

Thermo Fisher Scientific offers a wide range of biochemical identification kits to clinical microbiologists, providing reliable results in simple and convenient formats. The range, which includes the rapid and easy-to-use Remel RapID™ microbial identification systems and the Oxoid Microbact™ biochemical identification kits, requires no capital investment, so it allows even small laboratories to confirm the identification of clinically significant bacteria, yeasts and fungi. The RapID range is the fastest and most convenient identification system of its kind. With its simple, one-step inoculation procedure, it allows accurate identification of a comprehensive range of micro-organisms in just four hours. The RapID method detects pre-formed bacterial enzymes and, therefore, is not dependent on the growth of the organism. This allows the panels to be incubated aerobically without the need for oil overlays, which saves time and resources. Each RapID kit has the same simple procedure, allowing it to be adopted easily in laboratory workflows, and the biochemical reactions are clearly visible as distinct colour changes. These are interpreted quickly and conveniently by theElectronic RapID Compendium (ERIC™), a user-friendly, Windows®-based software package, to minimise misidentifications and to ensure accurate and reliable results.

I hope this will help

This won't be the specific answer you are looking for, however, since you are working with halophiles I wanted to make you aware of the Halohandbook.  Anyone who works with halophiles should have a copy of this invaluable collection of protocols put together by Dr. Mike Dyall-Smith.  You can find the Halohandbook here (it is free to download): http://www.haloarchaea.com/resources/halohandbook/Halohandbook_2008_v7.pdf

Dear Eyasu. Species identification using an key is complicated. Tipically, more powerful tools for ultrastructure determination are necesary. A good strategy: Identify to genera level and later use phycological specie level description from research articles.

Regards. 

It is a characteristic for hypersaline waters ciliate Fabrea salina

Hi,

this is difficult without some fungal structures like spores or conidia. Do you have the fungus on agar plates?

Hi Hamudul!

The first one is the green alga Pediastrum duplex var. reticulatum

and the second one is the green alga Pediastrum tetras,

both green algae belongs to the family: Hydrodictyaceae, order: Chlorococcales, class: Chlorophyceae, division: CHLOROPHYTA

best wishes

Bohuslav

Literature: KOMAREK, Jiri & FOTT, Bohuslav (1983): Chlorophyceae (Gruenalgen) Ordnung: Chlorococcales. In: Huber-Pestalozzi, Gottfried (Ed.), Das Phytoplankton des Suesswassers, Systematik und Biologie, E. Schweizerbart´sche Verlagsbuchhandlung (Naegele u. Obermiller), Stuttgart, p. 283-308.

I could not make a better picture, I am sorry. I have done that yesterday, without a camera.

Dear  Isable

did you have any picture from scolex and mature proglottids, these are essential for correct diagnosis

If you have access to DNA sequencing facilities you may amplify the standrd region between ribosomal 18 and 23 S and send it out for sequencing; the data you will obtain provide you with the species name, or nearest hit.

Mass Spec is a quick and easy way to get a size/mass based result

I probably found the answer to my question.

On the website:

anamorphy image contains Peziza succos. Is analogy, this is what I found could be anamorph Gyromitra fastigiata?

I think this is a Chlorella-like microalga. Try to make a wet mount and observe under the light microscope. If it is what I think, it will be quite difficult to remove from the culture; The same had happened to me when dealing with micropropagation. One of the solutions may pass through patience - as soon you have your tissues growing, take them out from the initial plate and plant them in new fresh media. Or try again :-)

Dear Tarun

It is necessary to note that radiolarians capture and digest a large variety of small prays (e.g. silicoflagellates, ciliates, tintinnids, diatoms, crustacean larvae, copepods and bacteria). This is done by use of their “sticky” pseudopodia (in particular the axopodia and/or rhizopodia). Several different feeding strategies have been reported for radiolarians (Anderson 1983; Matsuoka 2007). The captured food is enclosed in a digestive vacuole and carried into the ectoplasm where the digestion occurs. Although many radiolarian species are assumed to be omnivores, some feeding preferences do exist. There are, for instance, species preferring algal- before animal prey and vise versa (Anderson 1983). Some radiolarians are not actively hunting their food, but live primarily of nutrients obtained from their symbiotic photosynthesizing algae.

some link that useful for your question

This is difficult to say, simply from these characteristics. If you have the possibility, prepare the 16S-RNA and sequence that. This is a highly conserved molecule, which will tell the taxonomy of your strain. Otherwise, go more systematically through a microbial identification protocol, which can be found in Bergeys Manual and other source.

I don't know the magnification of your pictures but it looks like yeast. My supervisor brings down the hammer on people that get this type of contamination, because it's always of human origin. Ethanol doesn't always put down fungal contamination, neither does bleach or UV, unless the exposure is prolonged. I doubt your surfaces are to blame, it is almost always originating from lack of proper prep and sterilization of ones self. To avoid this I have often gone "over the top" with my clean-up before tissue culture work, spraying my arms up past the elbows with 70% ethanol, spraying both the inside and outside of my gloves as well as my lab coat sleeves, and spraying compulsively throughout any procedure (once ever 2-3 minutes). Not light mists either but enough to get things quite wet. And since I work without the face shield typical of most biohazard flow-hoods, I also wash my face, neck and chest with harsh soap, with an additional spray of ethanol to the lab coat neckline. This is the only way I know to avoid yeast contamination. You can try antifungals like cefotaxime or econazole, if even scrupulous cleaning does not solve the problem. Also does your lab share media, or do you use personal supplies?

Thank you all for your response.

We also provide identification biochemical tests, which are based largely on the use of sugars and therefore do not work for identification, as Edoardo writes. We have identified strains Maldi TOF as A.citreus or A. cummimsii and all were isolated from swab ear during otitis media. We consider also as commensals.16S rRNA method do not perform. Thank you for the simple methods of differentiation resulting from years of experience microbiologist. Such methods usually work well even without molecular genetic practices.

Thank you Stefan and Edoardo for an answers and experiences.

                                                                                                 Petr

it sure looks like a pennate diatom sp. potentially Navicula sp.

1% cetrimide is apparently the disclosing agent (http://www.sciencedirect.com/science/article/pii/S0944501306000966).

Cetrimide agar apparently doesn't serve that function - it has less cetrimide (0,3%) and that lesser amount is spatially limited by agar.  suggest you follow the published procedure with 1% solution.

Nice work,

Watching your biochemical test results, on the first side I thought of some Serotype of Salmonella ?????????????

Best

Vesna

identified. This foraminifera is undoubtedly unattached, so identified by the appropriate symbol (1).

Hi Randa,

You want to isolate Jatropha leaf degrading microbes from soil. It is very simple, bury leaf litter in soil at 4-5 different places at similar depth with proper moisture content. Take samples from soil at different time intervals and isolate using serial dilution methods. You also can study the diversity of microorganisms using metagenomics approches.

http://ac.els-cdn.com/S0734975008000839/1-s2.0-S0734975008000839-main.pdf?_tid=9973abea-8807-11e4-b3aa-00000aab0f26&acdnat=1419052874_5bed79598e0200bfa94b0fd35b14fab5 

Look at Bergey's Manuals

Dear Manikandan Subramanian,

I think that is a Cladocora sp. (Madreporaria)

Best wishes,

I agree with the above answers, but one should also test the lower tolerance for sodium chloride as well as the upper tolerance. If it doesn't live at low salinity, that's a pretty good indication that it is not terrigenous .

As many of my colleagues suggested , best way is to discard the cells and start your work with new vial of cells.

Follow the good laboratory practices.(A must)

Fumigate the hood and also the whole room periodically.

If you are using the waterbath for warming your media then put copper sulfate in it.It will avoid the carry over contamination.

Test for all possible contamination every monthly (bacteria, fungi, mycoplasma).

Bacterial and fungal contamination can be easily identify whereas mycoplasma contamination can be difficult. However, mycoplasma testing kit is available. I am using from the TAKARA company.This kit is a mycoplasma PCR detection kit. It is very simple if you are familiar with PCR technique. Mycoplasma can also be detected by hoiest staining.

If your cell is contaminated with mycoplasma and you don't have other option then treat your cells with ciprofoxin for 14 days. Check the cells for mycoplasma and reuse it. It is not recommended but if you don't have the alternative, it can be followed.

Use mask because a common source of mycoplasma is saliva.

All the Best.

I would prepare liquid media (e.g. Alkaline Peptone Water) with different concentrations of sodium ions and determine the turbidity after a defined time interval.

Thank you Dr. Hendricks for your helpful comment and suggestion. The reference will be used. I hope someday I could make a good network with you and make a join research. Have a nice weekend and thanks

To identify a species, you need the number of striae in 10 µm and lineolae in 10 µm.

N. spartinetensis : average 15 striae in 10 µm and 25 lineolae in 10 µm (see Witkowski, A., Sullivan, M. J., Bogaczewicz-Adamczak, B., Bąk, M., Rhiel, E., Ribeiro, L., & Richard, P. (2012). Morphology and distribution of a little known but widespread diatom (Bacillariophyceae), Navicula spartinetensis Sullivan et Reimer. Diatom Research, 27(1), 43-51.)

N. flanatica : 10 striae in 10 µm and 20 lineolae in 10 µm (see Witkowski, A. (2000). Diatom flora of marine coasts I. Iconographia diatomologica, 7, 1-925.)

It could be also N. phyllepta, very commun in mud (as N. spartinentensis). For this species, shorter than N. spartinetensis and N. flanatica, striae are also 15 in 10 µm but with 40 lineolae in 10 µm (see Witkowski, A. (2000). Diatom flora of marine coasts I. Iconographia diatomologica, 7, 1-925.)

I hope this help.

Regards,

Vona

Hi,

It looks like a rotifera (as per Ana's suggestion), but must to say, rotifera are multicellular animals with a complex structure (internal). So this maybe to small for that. It maybe interesting to see how many of these organisms are present in a sample.

For a nice web resource about rotifera:

rosalba

Dear Patel

MALDI TOF identificattion of microorganism is getting more usual these days, but continuos without being so common. In my opion is a really fast method.

I hope it can be interesting for you.

MALDI-TOF MS analysis

The strains were analysed using an Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Leipzig, Germany) equipped with a 200-Hz smartbeam laser. The sample preparation and the MALDI-TOF MS performing was carried out as was previously published [http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0020223] using a matrix of saturated solution of a-HCCA (Bruker Daltonics, Germany) in 50% acetonitrile and 2.5% trifluoracetic acid. As indicate the mannufacturer we used amounts of biomass between 5 and 100 mg to obtain the spectra and the calibration mass were the Bruker Bacterial Test Standards (BTS) which were as follows (masses as averages): RL36, 4365.3 Da; RS22, 5096.8 Da; RL34, 5381.4 Da; RL33meth, 6255.4 Da; RL29, 7274.5 Da; RS19, 10,300.1 Da; RNase A, 13,683.2 Da and myoglobin, 16,952.3 Da. MALDI-TOF MS identifications were classified using the score values proposed by the manufacturer: a score value between 2.3 and 3.00 indicated species identification; a score value between 2.0 and 2.299 indicated genus identification and possible species identification, a score value between 1.7 and 1.999 indicated genus identification, and a score value <1.7 indicated no identification. Cluster analysis was performed based on comparison of strain-specific main spectra created as described above. The dendrogram was constructed by the statistical toolbox of Matlab 7.1 (Math- Works Inc., USA) integrated in the MALDI Biotyper 2.0 software. The parameter settings were: ‘Distance Measure = Euclidian’ and ‘Linkage = complete’. The linkage function is normalized according to the distance between 0 (perfect match) and 1000 (no match).

Thank you very much for all your inputs. Due to limited resources, research students in our institution needed to prioritize tests for proper identification of isolates from mangroves. I guess for initial data, we can use Vitek 2 since its available. But for publication, genomic analysis will be a necessity.

Dear Michael:

Other way is to use yeast selective media for different species in order to detect which one control your media, this technique is easy to reproduce at any laboratory without too much stuff. Also sometimes by microscope observation you may distinguish among some genus such as Saccharomycodes, Schizosaccharomyces, Kloeckera, some Candida…

Best regards.

Ive got one easier method for you. Chromagar. BD makes selective agar plates for S. aureus and S. epidermidis - these are known as Chromagar plates. You can buy these here: http://www.bd.com/ds/productCenter/214982.asp.

Once you have done that I would do whole colony PCR using MLST primers to determine the clonal type.

I think you can used PCR method to detect P. fluorescens treated but firstly you should prepare the right primer.

Have a look at this article http://www.nature.com/nrmicro/journal/v6/n6/execsumm/nrmicro1872.html

Biolog Identification is based on the oxidation of different carbon sources by bacterial strains, it is quite normal that strains of same species can have different pattern in biolog plates. So, you can't assign species name only on the basis of it, you need to confirm your results with 16S rRNA gene sequencing or FAME or MALDI-TOF (as suggested by Saban).

Dear Wahengbam Romi,

I would like to suggest you kindly to consider folowing characters for describing new isolates as suggested by Granitsiosis. Firstly, colony properties like, colour, smoothness or roughnes, size and shape; secondly, better nutrient medium, color, shape and size of the bacterium, specific growth rate, habitat, biochemical properties like enzymatic activities, and then go for molecular analysis. It will give you better data to claim for new isolates.