Microscopic View of Sperm: Examining Technique, Heat & Motility

[red01]

I have been using a relatively cheap microscope (My first Lab Microscope) to look at a sample of my semen. Under 40x magnification I see a lot of motionless sperm and generally not enough sperm floating about compared to what I see from other microscope videos of sperm floating (no pun) on the internet. It got me a little worried although I might be doing something wrong. I am wondering if the heat from the lamp is not killing the little guys before I get a chance to peek at them. I use slides with the cover glass, but sometimes I just place a sample on the slide without the cover glass because I want to see if I am doing something wrong.

I would be interested to know from anybody more knowledgeable if there is something that can be improved in my steps. It is hard to give advice this way but I will answer any questions.

 

[Evo]

I have been using a relatively cheap microscope (My first Lab Microscope) to look at a sample of my semen. Under 40x magnification I see a lot of motionless sperm and generally not enough sperm floating about compared to what I see from other microscope videos of sperm floating (no pun) on the internet. It got me a little worried although I might be doing something wrong. I am wondering if the heat from the lamp is not killing the little guys before I get a chance to peek at them. I use slides with the cover glass, but sometimes I just place a sample on the slide without the cover glass because I want to see if I am doing something wrong.

I would be interested to know from anybody more knowledgeable if there is something that can be improved in my steps. It is hard to give advice this way but I will answer any questions.

And there is a meaningful purpose for this?

 

[Fra]

Hmm I've never tried to look at sperm, just yeast cells so I don't know but my hunch is that x40 sounds like on the low end! Can you see also the tails? I wonder if your magnification is too low and fail to resolve tail movement. Wether they actually propagate perhaps has to do with the medium? Just an idea.

I've got a "hobby microscope" at home I used for yeast counting and it's reasonably focues up to x600 (although the spec is up to x1000, but the quality of the hobby scope optics makes the higest mag useless).

You can also try to locate some stainings. There are a range to stains out there. Some chemical suppliers also sell to schools and private persons. I used metheylene blue for viability counts on yeasts, I think that with and addition of something else is used of semen, to colour the head read and tail blue. You hould be able to locate it if you google.

Apart from that I have no experience with looking at semen. Good luck.

/Fredrik

 

[Ken Natton]

Okay red01, I am not any kind of expert, but I can offer you these comments that might be helpful for you. Firstly on the specific matter of sperm, it is true that they have limited time at higher temperatures but that does not necessarily mean that they die immediately. If your microscope is a very cheap one, I suppose it might be possible that it is so poorly designed that the light generates a lot of heat on the slide – I suppose the clue would be that the slide itself quickly becomes hot to the touch. But if the slide remains at a reasonable temperature for long enough, I would think that ought not to present too many problems. However, I think you’ll find that, even for young, virile, healthy men, it is quite normal that a surprisingly high proportion of sperm produced are mal formed, unable to swim or swim uselessly in circles. You shouldn’t expect to find veritable armies of purposeful, directive sperm. I really wouldn’t read too much into it. Obviously, it would require someone of much greater expertise and experience to make any kind of assessment of the relative health of your sperm.

But as Fredrik has suggested, the obvious question is going to be on the matter of the optics in your microscope. Serious optics, as used in serious scientific laboratories, cost the kind of money that would make your eyes boggle. However, in response to your post I just did a quick search and was quite surprised by the results. I found one National Geographic microscope with magnifications up to x1200 that cost £15. Another one, with an LCD screen so that multiple people can view the slide at the same time, and with an in-built digital camera cost less than £150. I don’t know if anyone with greater experience of microscopes would care to comment on how good we should expect these microscopes to be, at those kind of prices I might even consider investing in one myself.

 

[Fra]

I've used my microscope mainly for looking at yeast cells. A yeast cell is 8-10 um in diameter. However, I did mainly cell counting, viability counting and assessing cellular glycogen levels using staining methods.

To actually look INTO the cells, study cell membraners you really need an electron microscope. And I'm not aware of anyone having a electronmicroscope at home :) I payed only 120$ for my microscope, but it was one of those "noname" brands.

The main issues which cheaper microscopes are
- limiting optic quality. This you typically notice at the highest magnification. Mine goes up to x1000, but really only decent up to x600, which happens to be just enough.
- the other factor is poor mechanics, meaning it gets kind of shaky or drifty at high magnification. So stay away from plastic. A heavy solid model is always better.

As for digitalization I mounted a regular high end dig cam on the eye piece of hte microscope. This gave me much better pictures that the low res cam that came with the microscope. This is also how I tried to assess glycogen levels. I tried to quantify the colour intensity after staining with iodine with the RGB codes. Worked well enough for my purposes. This also works with video cameras. It's a bit of tweaking to get it aligne and focus, but for mos eye peices it actually works and the result is decent. Even for cell counting, I never counted live in the microscope, I used a cell chamber - took a digital shot and counted in photoshop. Then you also have documentation.

Toystore microscopes are typically plastic crap (I know I tried them to. But some lab companies focus on hobby levels work. They often have decent hobby level microscipes that are enough for counting.

I just googled, and it seems a sperm is quite small. In particular to see clearly the tail movement, I am quite sure that x40 sounds insufficient. My guess is that you only see only dots. To count and stain cells you can do with less microscope quality than if you want to assess shape and looks, then you need detail.

I know I t ried to look for bacteria in the yeast, but with my microscipe that was really hard. The blurring resolution of my scope was a bout the size of the bacteria.

/Fredrik

 

[Fra]

This is an attached of a picture I took long time ago. I found sperm like presumed bacteria in spent grains I left to rot for 3 months (just to see what happened). It was taken with my cheap microscope.

It gives you an idea of what to expect from a "100$ microscope".

/Fredrik

 

[Ken Natton]

Actually Fredrik, that is quite interesting. It is clear that you were doing something altogether more serious than anything I had in mind. It can be fascinating to look at things under a microscope – I have a couple of youngsters at the start of their secondary school (high school) years and it could be good to try to stimulate their interest. It might well be that I will succeed for a couple of hours after which they’ll go back to the X Box, but who knows? But if it is possible with a reasonably economic microscope to achieve the levels required to see bacteria, for example, or perhaps to see red and white blood cells, that sort of thing, then that does sound very interesting to me.

 

[Fra]

Yes you can certainly do that. The cell count chamber I have used is a hemacytometer bought from a medical shop, it's designed to give a well defined volume in the view so you can determine also cell counts in blood. It's a grid where you place your blood drops and you can view the grid in the microscope, and you know exactly the volume in each grid.

Human blood cells are the same order of size as yeast cells so it's fine.

You can see cells and some internal organells at limited resolution though.

to make it more fun, various staining can provide more information. Some stains are used to determine which cells are dead, since the metabolism of the dye and thus colour shifting depend on the cell beeing alive.

Iodine are for glycogen testing (glycogen in cell forms a starch-iodine complex), the colour reveals how much glycogen deposits it has. In yeast management this is the first indication of poor health as glycogen are the energy reserve for yeast during storage and dormancy. Depleted glycogen pools means the cell has slim chance of surviving longer times.

/Fredrik

 

[Andy Resnick]

I have been using a relatively cheap microscope (My first Lab Microscope) to look at a sample of my semen. Under 40x magnification I see a lot of motionless sperm and generally not enough sperm floating about compared to what I see from other microscope videos of sperm floating (no pun) on the internet. It got me a little worried although I might be doing something wrong. I am wondering if the heat from the lamp is not killing the little guys before I get a chance to peek at them. I use slides with the cover glass, but sometimes I just place a sample on the slide without the cover glass because I want to see if I am doing something wrong.

I would be interested to know from anybody more knowledgeable if there is something that can be improved in my steps. It is hard to give advice this way but I will answer any questions.

Can you provide some extra details- what is the numerical aperture of your 40x objective? Is this your scope?

http://www.xump.com/science/MyFirstLabMicroscope.cfm

Heat could definitely cause problems- when I look at bacteria, they swim out of the field of view so fast it's hard to get a clear picture. 40X is sufficient to see E. Coli (1 x 3 microns) swimming around, although I use phase contrast to help the visibility. One way to block the infrared (if your scope is upright) is to place a shallow dish of water over that window where the light comes out.

You are also going to have problem due to the lack of a condenser lens. That will limit your ability to resolve fine detail.

This may help as well:

http://www.uhmc.sunysb.edu/urology/male_infertility/SEMEN_ANALYSIS.html

 

[Fra]

Can you provide some extra details- what is the numerical aperture of your 40x objective? Is this your scope?

http://www.xump.com/science/MyFirstLabMicroscope.cfm

Heat could definitely cause problems- when I look at bacteria, they swim out of the field of view so fast it's hard to get a clear picture. 40X is sufficient to see E. Coli (1 x 3 microns)

I agree that microscope probably works fine, it looks similar to mine in construction.

But note that the scope is not only x40 - it's x400 total as you have to multiply it with the x10 eyepiece.

/Fredrik

 

[red01]

Thank you to all who responded. A few have commented about the microscope lacking detail. At x400 magnification (x40 x 10) I can see the little guys clearly, tail movement, length and all. X400 is not small enough to see only dots. The sperm can be clearly seen at this magnification. My issue is not lack of detail – I am trying to understand why so many sperm are motionless. As I said, I am trying to figure out if I am doing something wrong.

Fredrik has suggested staining the sample. I have not been using any staining – I wonder if that would help to keep the sperm alive? As for the heat of the lamp, I placed my hand on the illuminator and it was quite warm. However, I don’t think that it warms the slide considerably. The microscope uses a 15W bulb.

Dr. Resnick, to answer your question, the link that you provided seems to show the microscope that I have. I got mine from Amazon: https://www.amazon.com/gp/product/B00005J377/?tag=pfamazon01-20. It is not of professional quality, but it seems to be better than a toy. And thank you for the link.

Fredrik, yes, the total magnification is x400. I was only writing about the objective lens magnification.

 

[rizwan_89]

Hi, you need to magnify your sample in electron microscope with high magnification, that is over x400. So in this way you can clearly seen the organs moving around. The basic magnification is not enough. The micro objects in the semen need to be covered with slides.

 

[red01]

Hi, you need to magnify your sample in electron microscope with high magnification, that is over x400. So in this way you can clearly seen the organs moving around. The basic magnification is not enough. The micro objects in the semen need to be covered with slides.

As I wrote in my original post and my reply, my issue is not magnification. I can see the guys, I can see them swim, etc. My issue is with the lack of movement in some of them. And I used slides (cover glass over slides) as mentioned above.

 

[Fra]

Ok, that seems to leave out the technical details.

Like mentioned, I have not experience of knowledge of sperm cells but staining should hardly help mobility of sperms. Staining is either for simple visibility, or to make tests; ie. to see how certain cells metabolize or complex with dye.

My issue is not lack of detail – I am trying to understand why so many sperm are motionless. As I said, I am trying to figure out if I am doing something wrong.

All I know from yeast cells is that cells are very responsive to ambient conditions. Temperature, temperature shocks, pH and of course medium composition matters alot. Somehow who is expert on sperm cell physiology could probably tell you what factors that impact movability. I'm not sure if sperms move all the time, or just as a response to certain ambient conditions. I figure that from st a flat reproduction evolutionary point if view, it may seem rational that the sperm could actually sense that it's in the female, and then change behaviour. Before ejection I'm not sure if it's in some kind of dormancy? I have no clue.

Did the link Andy posted on methodology not contain any hints?

/Fredrik

 

[Andy Resnick]

<snip>
Fredrik has suggested staining the sample. I have not been using any staining – I wonder if that would help to keep the sperm alive? As for the heat of the lamp, I placed my hand on the illuminator and it was quite warm. However, I don’t think that it warms the slide considerably. The microscope uses a 15W bulb.
<snip>

Like mentioned, I have not experience of knowledge of sperm cells but staining should hardly help mobility of sperms. Staining is either for simple visibility, or to make tests; ie. to see how certain cells metabolize or complex with dye.

All I know from yeast cells is that cells are very responsive to ambient conditions. Temperature, temperature shocks, pH and of course medium composition matters alot. <snip>

I would not try staining the cells if you want them kept alive. While there are several fluorescent dyes made for live-cell imaging (FM1-43, Fura-2, Fluo-4 and calcein come to mind), AFAIK all non-fluorescent stains (for examlpe H&E staining) will either kill the cells or their use requires the cells be killed and permeabilized.

It's entirely possible that the ambient temperature is too low- or that the pH changed. The link I posted indicated that initially the seminal fluid will become thickened due to a protein kinase present (I think it's a pH defense mechanism). After 20-30 minutes, the sample should liquify, presumably permitting motility analysis.

 

[Mk]

And there is a meaningful purpose for this?

Uh! Human investigation!
Don't criticize curiosity!

 

[Moonbear]

I'll put your fears to rest. Sperm do not become fully motile until diluted. Some dilution occurs in seminal fluids, but mostly those are providing the energy for the sperm and pH adjustments to keep them alive. Full dilution and motility normally happens in the female reproductive tract. In a fertility clinic, other fluids are used to mimic the conditions of the female reproductive tract before testing sperm motility. (Though, yes, you could also be cooking the little guys with the lamp if it's getting too hot, but more than likely, that's not the main issue. And, your "collection method" may not be ideal for sperm survivability either.)

 

[red01]

Thank you Moonbear. I had a hunch that my methods were a bit crude, and they are indeed. I use a cheap plastic pipette to suck the little guys and place them on the slide. Is this 'collection method' not good? Any tips for improving my chances?