I find unknown organism which have these features: 1.Blood agar medium (small grey colonies and beta hemolytic colonies). 2.Egg yolk medium (red pigmented colonies with lecithinase positive). 3.MacConkey agar medium (non lactose fermenting colonies). 4.Gram positive bacilli non spore forming 5.catalase positive 6.Oxidase positve 7.Glucose fermenting without gas production 8.Maltose and sucrose fermenting I need some help to know this organism please help me...!!!
A quick check of Bergey's Manual of Determinative Bacteriology (9th Edition) indicates that for "Regular, Nonsporing Gram-Positive Rods" (Group 19) that the only Genus that are both catalase positive and lactate negative are Kurthia and Renibacterium. Kurthia spp. are motile (with numerous flagella) and arise from animal feces and meat products (non-pathogenic). Renibacterium spp. are nonmotile and usually originate from salmonid fish (pathogenic). If the bacterium exhibits "Irregular, Nonsporing Gram-Positive Rods" (Group 20) it will be a bit more difficult to identify. Aside from a 16S ribosomal RNA identification, perhaps the flowchart from this link might help: http://www.uiweb.uidaho.edu/micro_biology/250/IDFlowcharts.pdf.
Good luck!
PS - In response to other comments I don't think it's Listeria since it is oxidase positive & I don't think it's Clostridium as it doesn't form spores.
PPS - If it really is Gram Positive then it couldn't be Serratia (which is Gram negative). The only species of Serratia that exhibit red pigments are S. marcescens and S. plymuthica, but these ferment glucose WITH gas.
A lovely picture.
Very few Gram-positive bacteria exhibit an oxidase positive reaction. Bacillus species is the only one I can remember off-hand. The other comment is which MacConkey agar did you use as the one with crystal violet should inhibit Gram-positive bacteria? Bacillus species usually produces spores.
Therefore, I suggest the following:
Check the Gram's staining reaction. A simple test is to emulsify a few colonies in 3% potassium hydroxide solution, a gel formation within 60 seconds indicates a Gram-negative bacterium, whereas lack of gel formation as Gram-positive.
Just to follow on the red-pigment is of interest. is that due to the presence of a sugar in the medium with a fermentation indicator or is it a pigmented bacterium?
Red-pigmented Gram-positive bacteria include Rhodococcus and Staphylococcus roseus - rest of description does not fit; Gram-negative bacteria: Serratia marcescens, some pseudomonads.
There are also a number of soil and water bacteria that exhibit this characteristic i.e. red species of cyanobacteria and halophillic archaebacteria. Some Bacillus cereus strains produce red-pigment on some media, but not others.
@Khalid. the details you provide doesnt help solve the puzzle? what is the source of this isolate- clinical(human or animal) ((including site if clinical)) or food or environmental? this could help in narrowing down the identity of bug! furthermore you may need to do further biochemical profiling (if you dont have access to commercial methods (API) or molecular characterization!). Also share pics of the isolate on the other media you have put esp a non-selective media and also gram stain picture.
I agree with Jackie Picard. In clinical bacteriology, we do identify a bacterium from clinical (human) samples and use Mc Conkey agar II that inhibit gram positive bacteria.
Certainly partial DNA r16S sequencing and comparing to published bacterial sequences will help a lot. However, it is really good to at least have an idea of what you are dealing with - family or genus. A few additional low tech. and cheap tests will get you there and will assist you in determining if it is worth pursuing the matter further.
The post peculiar feature of Your isolate is the red pigment. In Your apt description I only need motility and the origin of the culture?
If You are fortunate to have access to Versalovic J et al. (Ed.): Manual of Clinical Microbiology, ASM, Washington, 2011 (10th ed.), You may consider the "rare, gram+ rods" incl. Actinomyces on photographs p. 448.
If not, You should inquire whether Rhodococcus equi is the answer? Especially if it is some sort of clinical sample.
Please, if u would provide more details about source of isolation than it would be more easy to identify it,otherwise we have 16s analysis option.
Without knowing the source of the culture makes this dificult. I would first rule out bacillus species, the fact it is growing on the MAC is confusing. Gram stain the growth from the MAC plate to make sure it is a gram pos rod
if it is gram positive should not grow on MAc plates if you are adding crystal violet, and you should do biolog and 16srRNA for further analysis because the combination you are describing is bit confusing..........................and please check the incubation time because Rhodococcus equi take at least more than 8 days for growth and not able to grow in presence of CV
Thank you guys all for your kind replays but i would like to say its a Gram positive bacilli for sure because i did the gram stain so many times by my self and other persons and we had the same result (gram positive bacilli) and also it grow well in MacConkey agar medium as non lactose colonies and i cant do 16s rDNA analysis and phylogenetic analysis or any other molecular test because i haven't here in my country and also i would like to say the growth after over night incubation.
Also i would like to say the source of the sample from semi sold deep preservation medium and i found this organism as contamination on the medium.
Khalid
try this ones
http://www.tgw1916.net/bacteria_logare.html
this a online soft ware where u can identify the bacteria and my suggestion is go for sequencing more then 1500bps..
there are more advance methods found now a days like FAME identification by GC,biochemical identification by DADE BEHRING,BIOMERICS.
Good Luck
Dear friend,
I think this organism related to "Arcanobatherium and genus of phcea". However, you schould exam other tests for conformation . You schould exam indol, manitol, sim, imvic and other differentail tests taht written in microbiology text book. So, I asked you if you found this name of this isolate send the name for me to my e-mail: [email protected].
I hope succed in your work.
could you expalain your culture condition (aerobic or ......) and I need source of this organism??
Determine the Gram Type Using the Reaction Between Polymyxin B
and Lippolysaccharides of the Outer Cell Wall of Whole Bacteria. check
http://mic.sgmjournals.org/content/128/10/2261.full.pdf
16s analysis is the best method
Great article Karthika
Repetition of the same test indicates test repeatability, which is good to pick up test variability, other errors such as reagent failure are detected by the use of controls. However, in order to improve specificity, bearing in mind that certain bacteria will not stain according to their classification, one has to resort to the use of other tests.
Could you furnish the following test results?
Motility test. I use thiogel (2 ml thioglycollate broth with 5% gelatin in a tube). It allows one to detect motility, aerobic or anaerobic growth and gelatin breakdown.
3% KOH gel test
Antibiotic susceptibilities
MacConkey agar used. No 1 is pink and No 2 has a purple tinge.
A picture of the Gram's stain and growth on blood agar.
Many contaminants are environmental bacteria and therefore often difficult to identify as commercial biochemical tests usually target human pathogens. Many contaminants are oxidative in their use of sugars. The fermentative reaction rules many of these out.
If you want to identify the unknown organism,First you can go to the referral laboratories of Microbiology, Not to social networks.If its very strange they can study Molecular analysis.
Well Said Mehvish, one has to draw a line here!. RG should continue to be a platform for everyone with passion for scientific research to share and learn from each other. with reference to this particular topic, one should understand that though molecular tools have revolutionized biomedical research including diagnostics but still conventional techniques are a mainstay in laboratory practices not just in resourse poor settings but also in so called referral or reference centers. Even labs with several sequencers dont go about sequencing right from the word go. there are established guidelines and algorithms for microbial identification. Someone who is rather new to the field of microbiology should try and learn the basics of culture and identification and then proceed to molecular typing. Not only does it make one proficient in the subject but also helps saving money (in the long run!). Well yes in clinical settings, turnaround time is the key for patient management so there one wouldnt want to do elaborate time consuming analysis whereas in research facilities one would want to do things elaborately with no time constraints. So one has to see the situation and setup available and plan out course of action.
I thank all of you who have contributed to this discussion and am extremely happy to be a part of this.
according to Bergey's Manual of Determinative Bacteriology (9th Edition)...its may be Listeria or Corynebacterium but I'm not sure..
Thank you Guys for your support and Srikanth Aluru i think you are so rude to say something like this but i dont about what you said..!! for the other i will do all the biochemical tests and i will follow all your suggestion you mentioned later and i will tell you what i find.
Pls verifly the gram nature and oxidase test as the pigment would had interfered ..its likely to be Serratia sps. try growing on XLD and TSA agar..if you get pink colonies on XLD and red pigment on tsa its most likely Serratia.
Dear Abdullah, perform r 16s analysis, i also suggest to contact biolog.com
Hello guys i would like to say i did some biochemical tests and these is the result:
1.Indole test : Negative
2.Urease test : Positive
3.Citrate test : Positive
KIA test : Red slope and yellow butt without gas and H2S production.
and also i chick Bergey's Manual of Determinative Bacteriology (9th Edition) as you suggest for me guys but i didnt find any match...!!!
Many thanks Khalid
Did you do the motility and KOH gel tests? Antimicrobial susceptibility also gives you a very good idea of what group it belongs to.
The results that you gave do give me an idea that it is a member of the Enterobacteriaceae with a strong bias towards Serratia/Enterobacter/Klebsiella. The red pigment indicates Serratia marscens. Serratia is a common cause of opportunistic infections in hospitals, especially intensive care units. In a study I was involved it, it was the leading cause of intravenous catheter associated infections in dogs kept in intensive care.
I agree that referral laboratories are geared to identify unusual micro-organisms and should be used. Stating that I am one of those people that love to find things out, ask advice, and use this advice to solve my own problems. A scientific forum such as this one is ideal for one to share one's ideas and experiences.
Your welcome Jackie,
Yes and its motile but i didnt do the KOH gel test.
Thank you and i will consider your suggestion.
Many thanks Khalid
All that needs to be done is to make up 3% potassium hydroxide in water. Mix a colony or two in a drop (50-100ul). If it forms a gel within 1 min. it is Gram negative if not it is Gram positive. You can take a known gram-negative and Positive as controls.
dear, contact BIOLOG and BIOMIC, they have solution of your problem, If there is not in their database u can patent it. INSHALLAH
Yeah it may be serratia,but you can do biolog it is not so expensive and once you able to get data of carbon utilization patter it will be easier..........................
Thank you guys for your replays and its not serratia for sure because it is gram positive bacilli
i agree with Khalid Abdalla it may not serratia by the gram stain serratia is GNB but here Khalid Abdalla strongly saying that is gram positive bacilli.
Hi Khalid
The reason why I believe that it is a possible Gram-negative are the other test results as well as colony appearance. I have learnt that one must not hang everything on one test as no test is 100% specific. Did you every try the KOH test?
What I can get from your findings is that the bacteria is rod shaped and is giving non lactose fermenting colonies on Mac Conkey agar.
We know that only Gram negative bacteria can grow on this medium besides Enterococcus and some species of Staphylococcus i.e. Staphylococcus aureus(We don't have cocci here,it is rods as mentioned by you).
Secondly the colonies are apparently visibly as Serratia.
You can do the biochemical test for confirmation of Serratia.This organism is also a temperature sensitive mutant so grow the organism on two Nutrient agar plates/Slants and incubate at 25 degree and 37 degree temperature,if you get the pigment only at 25 degree, then it would again confirm Serratia.
There can be some problem with the Gram staining giving variability with the results.
Yes, It is different,showing mucoid appearance.
Perform capsule staining & get the sequencing done for confirmation.
If the microbe is a Gram negative rod shaped (bacillus) bacterium, one of the diagnostic tests most frequently employed is the API 20E test strip manufactured by bioMerieux, Inc., a biotech company that produces a variety of different test strips for identification of yeast, Staph, and anaerobic bacteria, to name just a few.
The API 20E system consists of a plastic strip of 20 individual, miniaturized tests tubes (cupules) each containing a different reagent used to determine the metabolic capabilities, and, ultimately, the genus and species of enteric bacteria in the family Enterobacteraceae.
API-20E Bacterial Identification Test Strip: Identifying Gram-negative Enteric Bacteria Using API 20E System
I think pathogenic bacteria differential in species Iam sure pigment producted different from serratia marscens and ser liquifaciase whiche is diagnosis in API E
If you have money, sequence its DNA and that´s all.
In the other hand, I think that if you can´t made a basic search information on pubmed or in a bacteriology books (as many persons that had the time and patience to respond your eternal question), only means that you don´t have intention to respond that. In my opinion, we have many work to do and not time to colaborate to your idleness
I thank information from khalid not correct about baclli and positive gram stain probably colonie old taken to smear fixation decrease fixation stain in cell appear incorrect result
This days the organism start to produce pigments(red pigments) also on Nutrient agar and MacConkey agar medium.
Cause produce pigment because bacteria suitable grow on macconkey agar and possible content medium to bacteria have resistance to environment
The Best way to identify this organism is to use API 20 strips by biomerieux Inc. Technology uses conventional Biochemical test miniaturized and standardized in strip form. they are very easy to use and testing can be performed by yourself in your lab.
different of the sources from clinical specimens or from vectors example cockroaches Iam isolated from insect and diagnosis by API 20 E but isolate khalid probably mixed with staph or strepto bacteria required pure reisolation
I have to concur with the fact that If it grew on McConkey agar, its a Gram negative. McConkey agar has crystal violet in it, no Gram positives should grow
It with red dye and CATALAS test positive and the growth of the MEDIA MACCONKEY intestinal bacteria and bacteria rarely if intended to be re-isolated and diagnosed tape API E
Make sure your oxidaise is tested from colonies growing on blood agar not Macconkey. A gram negative rod, non-fermenter, oxidaise positive is probably a pseudomons species. Hope this helps!
entero bacteria growth on macc and blood so pseudo cultured on TSI agar if change colour not pseudo because bacteria not fermentation of glycose
dear colleague.I think you had better have a standard chart for identification so again test oxidase with a plastic or glassy material because oxidase is a important test.these standard chart for a correct diagnostic found in Baily and scott and mahon.again you need check your pigment production in nutrient agar or...
It looks like Cellulomonas; do a 16S rRNA gene sequence analysis
This intestinal bacteria. Because of the growth center of macconkey agar. And negative for dye gram. The best diagnosed by Bar API 20 E. Special for the diagnosis of intestinal bacteria. I believe that this bacteria may. Serratia marscens or ser liqufaceus
Specifications of the red pigment produced by the bacteria. Serratia. And specifications of this bacteria is similar to the unknown bacteria serratia. But there are some mistakes that this positive bacteria to dye Cram. This does not. Possible error in stain . Bacteria and dab a specific time. Possible colonies of bacteria are old and lose their ability to keep the dye, then the result is wrong. Coli bacteria and positive. So is the bacteria's bacillus, but that does not produce a red dye. As a result, there is the possibility of two types of bacteria. Must work to isolate on a platter by the planning of transplantation purification.
Rule out the bacteria positive for the dye. This sample of bacteria is the negative of the dye. If diagnosed in the bar API20 E. Does not appear as a result. Means can not be cleansed of bacterial type. Must be the work of purification and reservation. We must work and knowledge of genes. Any work PCR. Possible isolation is rare. Or unknown
PCR the 16S and sequence it will give you at least a rough idea...
Go for Biotyper ... and if you can manage do 16s rRNA sequencing (I guess it;s not a big deal nowadays) , as anyway you need to do it for publishing your research ..
From which location / sample was isolated this bacterium?
It IS serratia marcescens !!!!!!!!!!!!!!!!!!!... SIMPLE..........U can try sequencing of 16SrRNA... but simple is try out these biochemical test.... Gram neg, Motile, Catalase pos, oxidase neg, nitrate & citrate pos, indole neg, pigment production, Glucose fermented with gas, sucrose & maltose fermented, lactose not ferment.... Check these reaction and then tel......
Go for routine identification by doing a Gram stain of this growth and using the appropriate API strip 20E probably in this case
Check these reaction :gram stain (branched or filamentous),Complete Acid Fast Staining,Partial Acid Fast Staining,Urease and Motility
First we have purified bacterial isolation. Be any one type of bacteria and not mixed. Second diagnosis tape API 20 E.
Bergey's Manual of Determinative Bacteriology is primary basic method for classification, proceed accordingly, it will help u alot
Khalid Abdalla, from which location/sample was isolated this bacterium?
What's news about diagnosis of that isolate?do u check your oxidase and gram stainning?
For the Past seven months this guy want to know stuff from others, not checking at all , single suggestion..many answered most correct
Since intestinal bacteria produces a red dye. And grow on macconkey agar. So is the serratia bacteria, but to determine the type. Must be diagnosed by tapes api 20 e intestinal bacteria.
Dear all thank you for your kind replays and i would like to i did all you mention above API and also i process the sample in Vietck machine and both give me nothing!!! Unknown result !!
Try to isolate genomic DNA and amplify the 16S rRNA. The sequencing will give you conclusive evidence about the identification of this organism.
Khalid
Unusual bacteria don't identify well in both the API and Vitek systems. However, I do find that I can use the biochemical test results. So possibly you can provide them.
A Gram-positive bacilli/rod growing on MacConkey agar? Are you completely sure about the Gram properties of your microorganism?
You should go for the molecular level like 16S rRNA sequencing for completely identification of this organism. Biochemical methods are not sufficient to identify.
Maldi tof, is the last one and modern way to identify, bacteria.
Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined via a time measurement. Ions are accelerated by an electric field of known strength.[1] This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will depend on the mass-to-charge ratio of the particle (heavier particles reach lower speeds). From this time and the known experimental parameters one can find the mass-to-charge ratio of the ion.
It is a bit tough to identify a bacteria without doing 16s rRNA sequencing. But u may also use specific commercially available test kits (eg, HiMedia KB001...13) for basic identification purpose if ur budget is low (cost of a set of 10 kits ranges from rupees 2000-5000 only). Or else u may send amplified genomic DNA or simply the pure culture to some of the labs such as Xcelris Labs Ltd., Genei Bangalore etc. for identification purpose. They will charge rupees 1500-2000 for amplified samples and 8000-9000 rupees if u send the pure culture. But u will get the results within 2 weeks after receiving the samples. Till now I have sent so many samples to the above mentioned labs and got satisfactory results.
diagnosis with API strip specially to bacteria gram negitive and bacteria produce red pigement its from group of serratia
Hi,
I agrree, the easiest solution of the problem would be the anlaysis of at least a fragment of the 16S rRNA. Of course you need to extract a small amount of DNA (easily done by 3 cycles of freeze thawing a loopful of biomass in 100 µl H2O, spin it and the supernatnat is ready for PCR amplification. Appropriate primers are 27f and 1492r (Lane, 1991). The product often is pure enough to send it to a commercial sequencing service. One sequence run here in Austria costs not more than 5 Euro which is approximately 250 Rupies. You get the sequence, do a sequence comparison in the internet applying FASTA, BLAST and/or http://eztaxon-e.ezbiocloud.net/. From the result you will know which the next related species is.
How was Gram-behaviour carried out, only by standard staining or was also the KOH test applied. Some bacteria belonging to the phylogentic lineage of Gram-negatives do stain Gram-positively and vice versa. Did you stain young and old cells. The result may vary. The same is true for the KOH test. The third option to determine the Gram-behaviour is the commercially available aminopeptidase test.
If you used API, ok you got no result. But often it helps which bacteria show a similar code. Your bug may be related to them.
Much bette than API is the Biolog system that is using 96 substrates.
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