Accidental Bacteria Flagellum Stain?

[desertrat]

A few years ago I grew a small hay infusion culture from dry grass collected from our field. After a few days, a bacterial film appeared on the surface. Small pieces of the film were lifted mostly intact with a wide mouth pipette and dropped on a cover slip. Unfortunately I didn't take notes and my memory isn't as good as I thought it was then. After the films dried, some were treated by dropping a little 98% alchohol on them and waiting for that to dry. Some of the others were not treated with alcohol before staining. None of the preparations were passed through a flame. I understand flaming is standard procedure in many labs because it's quick, but some of the old manuals I found online stated that fixing the film with some fluid at room temperature caused less distortion of the specimens.

After drying, the films were stained with Carbol Fuchsin or Methyl Violet for several minutes. The stain was pipetted off with distilled water and the cover slips were left to dry again. When dry, the cover slips were flipped over and mounted on slides with Canada balsam. The preparations were made on cover slips to ensure the films could be reached by an oil immersion objective.

One of the finished slides, stained with Methyl Violet, didn't stain as dark I would like, but I spent some time looking at the various types of bacteria with the microscope. During the staining process, when the drop of stain was sitting on the cover slip for several minutes, some of the drop evaporated and left a circle of concentrated stain on the cover slip. A small piece of bacterial film near the circle of concentrated dried stain was stained a little more darkly than the rest of the preparation.

Looking at that, I noticed some small bacilli had long, slender "tails" or flagella, about 5 times as long as the bacilli. Some checking on the internet revealed many bacteria have flagella, but the flagella are too slender to be resolved by visible light. Special mordanting staining methods are used to build up the thickness of the flagella until they can be seen under the microscope. These methods were developed in the late 19th century, possibly as result of an accidental discovery.

I'm wondering if the flagella I was looking at somehow got built up in thickness by being near where the stain dried, and became more concentrated. It occurred to me these "flagella" might only be artifacts caused by the concentrated stain on the matrix that some of the bacteria seem to have surrounded themselves with. I did find a few isolated artifacts that looked kind of like the flagella, not connected to bacteria. But the majority of these were connected to the ends of small bacilli, not out of the sides or associated with any of the larger bacteria in the field. Also, the larger specimens of the these small bacilli were similar in appearance, darker at each end and paler in the middle. I think there is a good chance these might actually be bacterial flagella.

Below is the field of view with the contrast enhanced slightly in GIMP to look more like what I saw through the eyepiece. Taken with an A/O Spencer 97x oil immersion achromat N.A. 1.25. A 15X A/O Spencer compensating eyepiece was used as a relay lens for the digicam. I have been unable to get images that look quite as sharp as what I can see through the eyepieces, but was able to image the flagella, just barely.

Bacilli_flag1.JPG (246.07 KiB) Viewed 2701 times

Below is the same image with contrast greatly enhanced to make the flagella more visible. Look near the center of the image, and also near the lower right hand corner.

Bacilli_flag2.JPG (326.53 KiB) Viewed 2701 times

Below is a lower magnification image of the mount, taken with an A/O Spencer 5X objective and 10X widefield eyepiece as relay lens. The arrow points to the area where the high magnification images were made. The curved horizontal line at the bottom of the image is where the puddle of stain dried at the edges. This dried line makes a circle all the way around the stained area.

Bact_field.jpg (90.3 KiB) Viewed 2701 times

Comments? Do these look like real bacterial flagella, or do they look like staining artifacts? Can any member with a good background in bacteriology identify the small bacilli?

[Hobbyst46]

An interseting question. I have no experience with bacteria whatsoever. But I can say something about the stain. Stains are large organic molecules that tend to self-aggregate in solution. Especially in the presence of salts. On the border line around the cover slip, water evaporates faster than near the center. So the concentration of the stain increases and it crystallizes out. This effect is enhanced since the salt concentration also increases where water evaporates. These effects explain the dense precipitate of stain around the coverslip, irrespective of organisms under the slip.

The thin twisted stain traces in other regions are, in my inexperienced opinion, flagella indeed, as you think.

[MicroBob]

Hi Rick,
I think that you interpret the image right.
Have a look at this google image search - there are images much like yours:
https://www.google.com/search?client=fi ... VEq4pahtU0

Bob