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Electron Microscope + Hemocytometer

Microbiology cell culture biotechnology microscope hemocytometer

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13 replies to this topic

#1 Celz

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Posted 05 December 2013 - 11:19 PM

Dear all, 

 

I would like to seek for your advice. I noticed that the chamber lines of hemocytometer are not showed clearly under electron microscope. May I know is it only can be view under inverted microscope? 

 

Thank you. 

 

 



#2 bob1

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Posted 06 December 2013 - 01:19 AM

Electron microscope?  Why would you use that on a haemocytometer?  Most people would use them with a conventional upright or an inverted microscope - both light microscopes.

 

The grids are just etched into the glass, so they are just grooves in the glass surface.  You should be able to see this by SEM I would have thought.



#3 El Crazy Xabi

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Posted 09 December 2013 - 04:48 PM

Why do you want to use the haemocytometer in the EM? huh.png  just wondering...

 

as bob1 said, if you use SEM the "lines" on the glass should be really easy to see



#4 Celz

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Posted 09 December 2013 - 04:53 PM

Hi, 

 

There are 2 reasons that I am using EM,

 

first, is because currently only EM is available in my lab,

 

secondly, is because of the protocol that showed in this link http://www.youtube.c...h?v=pP0xERLUhyc

 

Thank you. 



#5 El Crazy Xabi

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Posted 09 December 2013 - 05:48 PM

Using EM to count cells!? Wow wink.png

By the way, SEM? You only don't see the haemocytometer lines or you don't see the cells either?

 

Leaving aside the suitability of using the EM for counting cells... of it is SEM, do you sputter-coat your sample/haemocytometer? A lack of a conductive layer/compounds makes the imaging of any EM rather difficult. It also depends a lot on the type of EM you have



#6 Celz

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Posted 09 December 2013 - 05:53 PM

I can see the haemocytometer line clearly without loading any sample in. However, after I load the sample (even just pure distiller water) it also will make the line become invisible. 



#7 El Crazy Xabi

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Posted 09 December 2013 - 07:44 PM

That's normal due the light refraction..

 

Wait, you don't place water in the EM, do you!?!?



#8 Celz

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Posted 09 December 2013 - 08:06 PM

Place water in the EM? Nope. I didn't do it. 



#9 Phil Geis

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Posted 10 December 2013 - 05:36 AM

So what vehicle will you use to install cells?

 

Also, please recall hemocytometers are validated with water as carrier and examination via light microscopy.  You can't assume that a major change in protocol will maintain that validity so you'll need to validate your protocol.



#10 El Crazy Xabi

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Posted 10 December 2013 - 07:54 PM

Could you post what do you exactly do since you load the sample onto the haemocytometer until you place it in the EM?

Didn't mention before but haemocytometer counting is only valid when you use the coverslip provided, as it creates a layer of liquid of known depth that plus the known area of the squares on the glass you can calculate the concentration of cells. So, it can only be effectively used with liquids... and you cannot place them in the EM chamber unless it is a special EM such environmental EM or low vacuum models. In any case, cells are not electron -dense material nor conductive so you will have problems to look at them... and you won't be able to sputter-coating as they will be inside the haemocytometer



#11 Celz

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Posted 12 December 2013 - 12:46 AM

First of all, you are right, I am using the cover slip which came with haemocytometer. 

 

My case is to count for gram +ve bacteria, which means cannot use the trypan blue as staining dye since it is not human cells. Therefore, I have create a protocol by using crystal violet, iodine and safranin to stain out the cells first before I load to the haemocytometer. 

 

First of all, I was dilute the sample into 10^-4 dilution factor, 1mL from the tube was transferred to 10^-5 tube (contains 8mL of water and 3 drops of crystal violet), incubate for 1 min, 3 drops of Iodine was added into the same tube and incubate for another 1 min. 1mL of sample from 10^-5 tube was transferred to 10^-6 tube (contains 7mL of water and 2mL of ethanol), incubate for 30 sec; transfer 1 mL of sample from tube 10^-6 to 10-7 (contains 8.5mL of water and 3 drops of safranin), incubate for 1 min. Small amount of sample was loaded into the haemocytometer and view under EM. 

 

Thank you. 



#12 Phil Geis

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Posted 12 December 2013 - 05:19 AM

This is so far from the methodologies modified that you can't assume validity.    It's not obvious that is can be done with any accuracy but in any case you need to validate.



#13 bob1

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Posted 12 December 2013 - 11:47 AM

I also don't see how you can viability from that stain (that's what trypan blue is for), and it won't show up under the EM.  The stains you have indicated are all for light microscopy.  I really really don't see how you can be in a micro lab and not even have a single light microscope.



#14 El Crazy Xabi

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Posted 12 December 2013 - 05:37 PM

No wonder why you didn't see the lines... Do you know how does the EM works?

In any case, if you place the haemocytometer with the liquid sample in the EM you are going to break the EM sooner or later...

Why do you stain the cells? huh.png If you stained them with some metallic salt I would understand but safranin? crystal violet? You don't see colors on the EM and unless those compounds create precipitates (unlikely given the protocol) you won't see anything... and so many dilutions

 

If you want to count teh cells with the EM anyway, use Isopore filters, vacuum filter a know amount of culture suspended in a total of 20 mL PBS, saline or similar. Carefully remove filter. Freeze dry or just dry it in the oven about 60-70° C (unless you want to keep structural info of the cells). once dry, Sputter coating the filter. If you can see an even distribution of cells on the filter then is ok. You should be able to measure some areas with the EM software and counting the cells. You can backcalculate the real concentration after that...

 

In any case, are you working with pure cultures? Why don't you just make a growth curve base on absorbance/CFU? If you have an spectrophotometer, though. That would be way easier...







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