Hi. I have only worked with Vibrio species so far in the case of spirochetes, but from the picture, the rods do not seem to be all very spiral in shape. Is this a mixed culture?

Regarding the endospore-look-alike, why don't you try to go ahead with endospore staining? It'll only take about 5-6 minutes max. Maybe it will work, maybe not. But in few cases, if you are working with an old stain (to make the stain as you attached) or if your culture is more than 48 hours, the staining may have limitation, that it gave you an idea that the culture is spore-forming when it is actually not.

Dear Heba,

What is the spiral bacteria you are working with? 

Sometimes bacillary contaminations (aerobic laboratory contamination) look like quite long. Those are gram positive and they may have spores too, but what I see in your picture is quite clear in spiral shape and surely not Bacillus sp. So, I suggest you to do a broth culture followed by a good Gram's staining and of course an endospore staining. I suggest you more that while preparing the smear or film on glass slide please just put a drop of that broth on glass slide (or along with a drop of normal saline) and don't tease the drop to make the smear. Rather, you just gently spread the drop on glass slide and do both the staining.

Teasing may distort the morphology of spirochetes.

Please do the Endospore staining before saying it a spore.

consider spirochetal L-form and cyst forms if your used media can contribute their formation.

Good luck

This bacteria is environmental,  I isolate if from salt marshes , thanks for replying

surajit and Nuria? 

These bacteria are gram negative, I will do spore stain.... but do bacteria that live in salt environment may be curved? Dr surajit

Ok generally, does spiral bacteria  produce spore or no ???

I think you may be dealing with  than one bacterial species. and spiral bacteria dont produce spores at all

No. Atleast no reports there....

I don't think these are spiral bacteria. Since you isolated from a salt marsh, this could easily be a Vibrio sp. Or another common marine bacterium. The reason why you see some spirals is because when bacteria are starved or stressed, they tend to grow (elongate) but don't divide. So those may simply be elongated cells.

Most of the spiral bacteria  don't produce endospore, 

I test to grow it on tcbs medium but there is no growth so it isnot vibrio, so may be spiral , how can I deferentiate if it is spiral or another stressed bacteria elongated , Dr mesrop

The second photo seems to have true spiraled bacteria.  They do not look pleomorphic from stress to me.  I assume this is from a broth culture by its looks.  I believe it is likely a Spirillum.  Much could be deduced from what medium supports its growth.    What are you calling spores or endospores?  If there are clear spots inside the bacteria, they might be gas vacuoles.  If you are seeing refractive objects outside of the cells, they might be deposits of end products.  Aerobic and anaerobic endospores are resistant to shock heating at 80 degrees Celsius for 15 minutes.   If you heat a small sample for this time and temperature, and then recover growth, it would confirm the presence of endospores.  If you cannot grow the bacterium from a heated sample, the particles you are seeing are not endospores.  Judging by your photos, I do not believe you have endospores. 

Hola, te comento que las bacterias espirilares no producen esporas, esta es una estructura de resistencia de algunos bacilos gram positivos. Además la mayoría de las células de bacterias espirilares no se tiñen con métodos tradicionales y por su morfología es dificil observarlos en microscopios de campo claro (brillante) y se prefiere ne microscopios de campo oscuro donde incluso observas su motilidad, la forma de sus extremos. además son bacterias que no crecen en medios convencionales. Pudes además de estresar termicamente una porción del cultivo y hacerlos crecer en otro medio para determinar estructuras de resistencia. Pudes incluso solo sembrar en medios sólidos como triptona soya ya que las espiroquetas no desarrollan crecimiento en medios sólidos simples.

From your images it doesn't appear to be endospores. The objects are too large to have emerged from the nearby mother cells. Heating and re-sampling as suggested would be a good route, and you could also do a side by side analysis of nutrient rich growth cells, and nutrient deprived cells (best way to induce endospores). Also I'm not sure your species are vibrio or perhaps your culture is mixed, but it's hard to tell from the BF images. If you have the ability a dark-field view should provide better cell wall definition and confirm.

Sorry, but from your images, I cannot see any of the endospores you're talking about. To me they seems normal Spirillum... Maybe some deposits or dirt?

Regardless of your images, there only two genera that able to form endospores; Bacillus  and Clostridium. There are no vibrio or spiral bacteria have this character. To be sure that your bacteria  are able to form or not endospores, stain the bacterial film with malachite green stain (the culture bacteria from which you will prepare the  bacterial film  should be of 2-3 days old to be able to distinguish between the spores and bacterial cells. Please see the attached malachite green stained Bacillus  image (from our special work).

No known vibrio or spiral bacteria have demonstrated this ability, but there are other genera capable such as Paenibacillus, which is a Firmicute like Bacillus and Clostridium (safer to say this Phylum has shown this ability). We know about these two genera because one causes horrible diseases and the other is ubiquitous. I think it's unlikely that we will find any non-rod species that do form endospores, but considering that we have only identified less than 1% of the estimated bacterial diversity on Earth it is still possible. In this case the species remains unidentified as it's an environmental sample without genomic data to date.

It may be polyphosphate granules, which are quite common for Spirillum species. You can check it by using Loeffler staining. For this stain, you should use saturated methylene blue alcoholic solution mixed with 1% KOH solution in water (1:3) - it is called Loeffler's methylene blue solution. The procedure is as follows:

Cover a fixed smear with the stain. Wait for 10 minutes. Then wash the smear with water, then differentiate with 1% H2SO4 solution for 1 minute. Finally wash the acid with water and leave to dry. Use oil immersion. The cytoplasm appears light blue, the polyphospate granules look violet or even reddish-violet (metachromic effect).

It is a spirillum and they do not form spores, your images appear to be stained is this the case? If so try DIC it would show any refractile granules.

Can't visualize any spores. Could be stain deposits or granules.

It seems the picture was taken at 40X magnification. Moreover, this does not look like Malachite-safranin stained (what we normally do for endospore observation & detection)

Please do the same under oil immersion lens (at 100X), i am sure this will definitely look like Bacillus.

You may also do a TEM study to detail investigation about the structure.

To confirm carry out malachite staining in water bath for 20-30min followed by counter staining in safranin (follow any microbiology practical text for this). Please take a positive control (3 to 4 days old culture Bacillus subtilis) and a negative control (3 to 4 days old culture of E. coli) to ensure that your protocol and reagents are correct. Endospores will appear green within red outline.

There are large number of exceptions among bacteria. There are Gram negative bacteria producing endospore (Serratia marcescens subsp. sakuensis), You must keep yourself open to discover new phenotypes and new things. Never underestimate the diversity of prokaryotes.

Hope it helps