Has anyone seen anything like this growth? It was streak inoculation, The colony surface is dry, but inside is wet, the odor is putrefactive.
Proteus is well known to not producing colonies and overgrowing all the others on the plate, like in your case. However, typically, the bacterial growth is producing "waving" - the surface looks like a troubled see surface.
Did you try Gram staining? But if it is not G+ you have not won, yet, Bacillus in exponential phase of growth is Gram-labile, showing false Gram negative staing (KOH test would be a traditional solution). You must find cyst-forming cells.
Cheers
Tatyana Tarasova, The growth and colony could very well be a species of Bacillus. If so do a endospore stain it would be revealing along with a gram stain and put the bacteria in a API20E battery of physiological tests this should give a good ID. Also a genetic blast analysis would give a very good assessment of the species identification. Many Bacillus have a distinctly ammonia odor which coupled putrfactive and are dry outside and juicy inside. Let me know your results best wishes. Paul Reed Hepperly
Tatyana Tarasova,
Find here more precise information on the bacterial identification system I recommended for your information and potential use.
An Introduction to
Bacterial Identification
Page 2: The API-20E® Enteric
Identification System
JOHN L's BACTERIOLOGY PAGES >
SELECTED GENERAL TOPICS >
BACTERIAL IDENTIFICATION:
• Page 1 – General Principles
• Page 2 – API-20E Enteric ID System
• Page 3 – Genotypic Identification
The API-20E test kit for the identification of enteric bacteria (bioMerieux, Inc., Hazelwood, MO) provides an easy way to inoculate and read tests relevant to members of the Family Enterobacteriaceae and associated organisms. A plastic strip holding twenty mini-test tubes is inoculated with a saline suspension of a pure culture (as per manufacturer's directions). This process also rehydrates the dessicated medium in each tube. A few tubes are completely filled (CIT, VP and GEL as seen in the photos below), and some tubes are overlaid with mineral oil such that anaerobic reactions can be carried out (ADH, LDC, ODC, H2S, URE).
After incubation in a humidity chamber for 18-24 hours at 37°C, the color reactions are read (some with the aid of added reagents), and the reactions (plus the oxidase reaction done separately) are converted to a seven-digit code which is called the Analytical Profile Index, from which name the initials "API" are derived. The code can be fed into the manufacturer's database via touch-tone telephone, and the computerized voice gives back the identification, usually as genus and species. An on-line database can also be accessed for the identification. The reliability of this system is very high, and one finds systems like these in heavy use in many food and clinical labs.
Note: Discussion and illustration of the API-20E system here does not necessarily constitute any commercial endorsement of this product. It is shown in our laboratory courses as a prime example of a convenient multi-purpose testing method one may encounter out there in the "real world."
In the following photos:
•Note especially the color reactions for amino acid decarboxylations (ADH through ODC) and carbohydrate fermentations (GLU through ARA).
◦The amino acids tested are (in order) arginine, lysine and ornithine. Decarboxylation is shown by an alkaline reaction (red color of the particular pH indicator used).
◦The carbohydrates tested are glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose. Fermentation is shown by an acid reaction (yellow color of indicator).
•Hydrogen sulfide production (H2S) and gelatin hydrolysis (GEL) result in a black color throughout the tube.
•A positive reaction for tryptophan deaminase (TDA) gives a deep brown color with the addition of ferric chloride; positive results for this test correlate with positive phenylalanine and lysine deaminase reactions which are characteristic of Proteus, Morganella and Providencia.
In the first set of reactions:
•Culture "5B" (isolated from an early stage of sauerkraut fermentation) is identified as Enterobacter agglomerans which has been a convenient dumping ground for organisms now being reassigned to better-defined genera and species including the new genus Pantoea. This particular isolate produces reddish (lactose +), "pimply" colonies on MacConkey Agar which exude an extremely viscous slime as may be seen here; this appearance is certainly atypical of organisms identified as E. agglomerans or Pantoea in general.
•Culture "8P44" is identified as Edwardsiella hoshinae. The CDC had identified this culture (in 1988) as the ultra-rare Biogroup 1 of Edwardsiella tarda which may not be in the API-20E database. This system probably would not be able to differentiate between these two organisms. Note that 8P44 shows H2S production which is probably typical of Edwardsiella tarda Biogroup 1. Clinical laboratories usually run this test in Triple Sugar Iron Agar in which the organism's fermentation of sucrose (with consequent high acid production) tends to negate the H2S reaction, and – as a result – the organism is mis-characterized throughout the literature as H2S negative even though it shows a positive reaction in KIA and other H2S-detecting media.
culture
no. O
N
P
G A
D
H L
D
C O
D
C C
I
T H
2
S U
R
E T
D
A I
N
D V
P G
E
L G
L
U M
A
N I
N
O S
O
R R
H
A S
A
C M
E
L A
M
Y A
R
A identification
8101 + – + + – – – – + – – + + – + + + + – + Escherichia
coli
5B + – – – + – – – – + – + + + + + – – + + Enterobacter
agglomerans
8P44 – – + + – + – – + – – + + – – – + – – + Edwardsiella
hoshinae
culture
no. O
N
P
G A
D
H L
D
C O
D
C C
I
T H
2
S U
R
E T
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A I
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L
U M
A
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O
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H
A S
A
C M
E
L A
M
Y A
R
A identification
8030 + – + – + – + – – + – + + + + + + + + + Klebsiella
pneumoniae
8068 – – – – – + + + + – + + – – – – + – + – Proteus
vulgaris
8P14 – – + + – + – – – – – + + – + + – + – + Salmonella
sp.
These general microbiology pages have copyright by John Lindquist
and found their permanent sanctuary on www.jlindquist.net circa 2001.
Copies found elsewhere are neither authorized nor up to date.
Page content was last modified on 6/1/10 at 9:00 PM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison
The characteristic of the colony suggests a bacterium of the genus Bacillus. So we suggest you to perform two tests that are essential to initiate a characterization of bacteria process: Gram (taking the opportunity to register the cell type) and the endospore test. All featuring the colony should be on nutrient agar.
Thank you all for very useful information
It is Gram-labile culture and I don't find cyst-forming cells.
I'll do some additional tests. However has anyone advise how to induce sporulation? Cooling to 8°C did not help
Hi Tatyana, You can proceed directly with genome DNA, as suggested above. Further, please have a look of your culture under a microscope, if it is a pure culture or a mixed one. If it a mixed one, then try to get single colony by continuous stricking of the cells on an agar plate. To induce sporulation, you better use a poor or low enegy medium, by reducing either carbon source or nitrogen source. With a carbon source (for example, glucose of 10 g/l or so, you may expect sporulation within 24 hours. Thank you and good luck.
Dear Tatyana,
I am 100% sure it is Bacillus. It might be belonging to B. cereus cluster, if this is on Tryptic soy agar plate. The culture is very fast growing and secrets lots of mucilage.
But please confirm this with 16S rRNA gene sequence analysis and phenotypic tests. You will definitely find endospores.
As I have seen, It may be Bacillus sp. You can check by simple doing endospore staining before proceed further.
This growth is characteristics of several Bacillus species. You can check it by 16S RNA sequencing or by MALDI-TOF MS.
Yeah May be Bacillus enumeration ll also help you identify the species.
You can not indentify ever bacteria by visual criteria, do a molecular analysis of it genome to confirm especie/s.
I confirm that it is the typical growth of Bacillus sp. on agar substrate. The Bacillus Group comprises organisms that are widely distributed in the environment and are of health and economic interest. We find these colonies when environmental samples, especially soil, are analyzed.
As everybody else this is typical of Bacillus sp. but if you think it could be Proteus which I don´'t think you can do an urease test which definitively be positive por Proteus sp. and have you made Gram stain? Bacillus should be a positive rod one and Proteus a negative one. Besides spore forming is typical of Bacillus sp. not Proteus. Then you can proceed with molecular tests if the confusion persists which I really don't think. Good luck !!!
1- To induce sporulation add MnSO4 (5mg/L) to culture medium such as TSA (Trypticase Soya Agar), we add it to enhance sporulation. incubate at 30 C 24-48h, prepare hanging drop to check if there is spore(s) (use phase contrast microscope). the following link is useful.
http://dzumenvis.nic.in/Taxonomy/pdf/Aerobic%20endospore.pdf
or you could do a simple experiment :
2- Suspend a colony in 1 mL saline (sterile) or Dis H2O (sterile), incubate at 80 C for 10 min then streak on TSA incubate at 30 C, check the results after 24-48h, if it grows then its sporeformer that could be a Bacillus. Please remember that you cannot tell by naked eye that its Bacillus or not. if you have access to phase contrast microscope then do a hanging drop test for to see spores (position & shape) and even take images of them.
Hope this helps.
Dear Sir, what is this Hanging drop method? How does it show spore staining? Please send me the protocol or share the PDF
Dear Anjali
You need a microscope slide and cover slip, take a drop of dis H2O by loop and put it in the centre of the slide (don't spread it), then suspend (gently) a colony in it, cover it with cover slip and check it under the microscope, this will allow you to see spores if any, it also show the motion of bacteria with out the need of USING ANY SPORE STAIN.
Good luck
Thank you all!
It is obviously Gram-positive rod shaped mobile big rods.
Biochemical tests in the attachment. According ABIS-online service in can be Bacillus cereus. But I am still working on spores detection and some biochemical reactions
This answer is meant for Anjali Nair, who wanted to know about hanging drop experiment. As far as i know, this is carried out to determine motility of a bacterium. for this a cavity slide and a cover slip are required. a freshly grown cell suspension of the bacterium in the question is placed at the centre of a cover slip whose corners are deposited with vaseline (the drop must face you) place the cavity slide in such a way that the drop is placed exactly within the cavity slot. the vaseline will fix it. Observe the slide under light microscope at 40X you would be able to monitor motility. observation must be made targeting the edge of the water drop. If you detect any one cell to be motile, the culture mus be concluded as motile.
Certainly does not resemble B. anthracis. I'm not so sure Its B. cereus either.
Agar was probably wet...or humid. It explains the dispersion. Might want to just re-streak it and see if you can get some colonies.
Much have been discussed on the topic without an answer on spore formation by this spp. so far. So please confirm sporulation, its shape & spore position central or polar. Then try a penicilin susceptibility test or lecithinase test for B. anthracis or cereus. .For further differntiation among bacillus spp. there are some tests like crystal formation etc but 16S rDNA sequencing seems to be the gold standard. If suspected to be an anthracis sp. better talk to an reference lab. rather than further handling. Good luck. Please refer to the link below: http://www.google.co.jp/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0ahUKEwi9qvzKxeXPAhVJopQKHfB9AL4QFggoMAE&url=http%3A%2F%2Fwww.sfam.org.uk%2Fdownload.cfm%3Fdocid%3DA8649AF0-52D3-4098-A5FF68A2D1A0978B&usg=AFQjCNFWtuTvEuffzeIsIj6llGIx6adqCA
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