The principle of embedding is where tissues are embedded in paraffin wax so that they are hard enough to withstand cutting into thin sections. On the other hand, it also makes them soft enough to allow the blade to cut through, with little or no damage to both the blade and tissues. The equipment used is an embedding centre, which is shown in the picture below.
Picture of a tissue embedding centre
There are three main compartments, which consists of the wax dispenser, cold plate and the heated storage area for metal moulds. Wax dispenser as the name implies, dispense appropriate amount of melted wax into the metal mould used. Cold plate allows the wax to solidify, the heated storage area stores 4 different sizes of metal moulds, depending on the size of tissues to be embedded. Also, in the picture above, there is a black ‘knob’ on top of the metal cover. Lift it up and there is a space to contain metal racks which is used to put cassettes. (The cassettes contain tissues inside).
The procedure for embedding is very straight forward.
1) One cassette is taken out from the racks and opened
2) The lid is detach and thrown away into a biohazard bag
3) A metal mould of the appropriate size is chosen and filled with melted wax
4) Tissue is transferred into the mould using a heated forcep
5) It is held down by the forceps to prevent it from floating around
6) A metal press is used to press down the tissue so that the surface is on even
plane.
7) The mould is covered with the other part of the cassette and press down to
secure it
8) The top is partially filled with melted wax again
9) It is then left on the cold plate to allow the wax to solidify
Picture of some cassettes
Picture of metal moulds
‘The procedure looks pretty easy, so what is so hard about embedding’, is my first thought when I observed the procedure. Only when I attempt it did I realize that it is not as easy as it seems. The trick to embedding is that every little thing that you do during the process has a tiny principle behind it, which will eventually reveal whether the tissue has been embedded properly. For example, the orientation of the tissue is especially important. This is because it will determine if the relevant areas of the tissue that needs to be studied has been exposed fully. Different types of tissues need to be orientated in a different way.
If there is more than one tissue, it has to be arranged in the same way/pattern for easier studying under microscope. Another thing to take note when there is more than one tissue is that they must be placed closed together. The reason being that wax cannot expand but tissues can. Thus, when this happens, the tissue is constraint to a limited space because of the wax and it will be ‘squeezed’ together, forming a lot of folds.
Another tricky part is where to place the mould during orientation of the tissues. Can it be placed on the cold plate? The answer is no. This is because the wax will solidify very fast on it. If there is only one big tissue, it will not really pose as a problem. However, if there are around three or more tissues, by the time the third or fourth needs to be put in, the wax is already partially solidifying. Thus, when you attempt to press down the tissues, the whole thing will be something like a ‘mash potato’ (it really looks like that).
Although it is not preferred on the cold plate, it can still be carried out nicely, provided that the person has lightning speed (no joking really). In conclusion, speed plays a major factor during embedding and practice will make perfect. There are still other precautions, but I will stop here for now.
Any questions pertaining to embedding will be gladly welcome.
Cheers,
Zi Shuang (0703383J)
TG01
References:
Picture of tissue embedding centre, Retrieved on 18th July 2009 from
http://images.google.com.sg/imgres?imgurl=http://www.gmi-inc.com/images/EG1160_duv_pro_b_sh%255B1%255D.jpg&imgrefurl=http://www.gmi-inc.com/Categories/histology.html&usg=__UeZDfSiguhmad3l39c-ohrfU3O0=&h=350&w=500&sz=20&hl=en&start=8&um=1&tbnid=cHWzwlCJJKy2kM:&tbnh=91&tbnw=130&prev=/images%3Fq%3Dshandon%2Bembedding%2Bcenter%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26um%3D1
Picture of cassettes, Retrieved on 18th July 2009 from
http://images.google.com.sg/imgres?imgurl=http://www.proscitech.com.au/cataloguex/img/R/rch42.jpg&imgrefurl=http://www.proscitech.com.au/cataloguex/online.asp%3Fpage%3DR4&usg=__hd28LbblhsHqxdMYYtEjE8qNDaQ=&h=219&w=320&sz=36&hl=en&start=35&tbnid=g1i_6q4zg1QQ1M:&tbnh=81&tbnw=118&prev=/images%3Fq%3Dcasettes%2Bhistology%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26start%3D18
Picture of metal moulds, Retrieved on 18th July 2009 from
http://images.google.com.sg/imgres?imgurl=http://www.largro.com/MBMPic1.jpg&imgrefurl=http://www.largro.com/mbm.html&usg=__xqeT05lsc160vtvJrEhkCumuia8=&h=141&w=200&sz=6&hl=en&start=25&tbnid=MS50N43X1Tkc9M:&tbnh=73&tbnw=104&prev=/images%3Fq%3Dmetal%2Bmoulds%2Bhistology%26gbv%3D2%26ndsp%3D18%26hl%3Den%26sa%3DN%26start%3D18
Hi Zi Shuang,
ReplyDeleteIt seems fun doing embedding just like how we did during HTECH lab (:
Anyway care to name a few precautions to note while embedding?
QINGLING
To Qingling:
ReplyDeleteHihi, thanks for commenting on my post :)
Yup, embedding is fun as you get to see different types of tissues everyday, but most of the time it is quite hard to identify them :S
Some of the other precautions are avoiding over exertion of force during pressing, using a heated forceps instead of the usual ones, and also the pressing of tissues must be done on the cold plate.
Over exertion of force during pressing will distort the tissue structure where the whole thing will look flat under the microscope. This will lead to inaccurate results.
Using a heated forcep is normally preferred and also easier to use. This is because it is heated at around 65 degree celsius which helps to melt the wax, thus, preventing it from sticking to the forceps, which in turn prevent tissues from getting stuck to it. The usual forceps are also heated, but at a lower temperature.
Pressing down of tissues must be done on the cold plate as wax will tend to solidify and prevent the tissues from moving around during that process.
zi shuang :)
hi zi shuang,
ReplyDeleteif the mould could not be placed in the cold plate when orienting tissues. then where should it be placed?
-nyzah
Hi Zi Shuang,
ReplyDeletehow do you determine the size of the metal mold to be used? Is there a certain ratio of specimen to wax to follow or it is just according to sizes of the specimen? Thanks.
Hui Juan
0702012F
To Nyzah: The metal mould can either be placed on the edge of the embedding centre, where it is neither too hot or cold, or it can be held in hand. However for me, i would prefer the former as the metal mould is quite hot. Holding it in hand for too long would scald the finger
ReplyDeleteTo Hui Juan: There is no specific ratio to follow. It is kind of a challenge to determine which size of mould to be used. For larger specimen, a bigger and wider metal mould have to be used. For small ones like pigments, the mould with the smallest square in the middle is choosen.
helo0o0o0o0o zi shuang..
ReplyDeleteerrmmm.. we were taught to place the tissues close together, because wax would not expand but tissues will..
err...im sorry, i dun quite understand the reason behind this..
isnt it better to place it far from each other, to allow them to expand. if there is no space, folds will appear. And isnt it going to affect the results on the slides later on..?
heeheheee
see u at 11am!!
Nadiah
0705365E
hihi nadiah! haha next week only left the 2 of us doing routine :)
ReplyDeleteBasically when you placed the tissues far from each other, there would be a lot of wax in between them right? So when the tissues expand, these wax cannot expand. Since wax cannot expand, the tissues have no space to expand also, and they would be 'squeezed'. However, they should not be placed too close to each other as they may overlap. i suggest around 2 mm apart will do. :)
zi shuang
HUllo zi zhuang,
ReplyDeleteregarding your procedure point6:"A metal press is used to press down the tissue so that the surface is on even
plane."
when i was attached to histology, after the med tech uses a forcep to place the tissue into the metal mould,she will then used the forcep to press around the 4 sides of the tissues to ensure the tissues are fully embed and will be close to the surface of the wax so that during trimming, you wouldnt have to trim so much to expose the tissue. however,i didnt remember my colleague uses a metal press..What is a metal press? Can elaborate more about it? THK you.
happy histo-ing!
Yong Herng
0702243G
hihi yong herng (sesame),
ReplyDeleteusing the metal press is also another option besides using forceps. however, from my observation, the people in my lab prefers to use metal press because it has a flat surface, thus, it can also ensure that the tissue is also on even plane. besides that, the metal press is also warmer, preventing the wax from solidifying on it.
basically it looks something like a screw :D
picture explains a thousand words (you can refer to the pic below)
http://physicsgeek.mu.nu/archives/screw-thread.gif
HELLOS
ReplyDeleteyou mentioned different types of tissue need to be orientated in a different way, can give a few examples to illustrate the difference?
also, why are folds formed when the tissue is contraint by the wax? are the folds caused by the wax or by the tissues? how would these folds affect the presentation of the tissue later on when fished onto the slides?
thanks~ =)
Ang Yu Hui Jacelyn
0702632A
Hihi Jacelyn,
ReplyDeleteFor example, if tissue with epithelial surfaces such as skin, gall bladder, urinary bladder, intestine, they should be embedded on edge. Which means the tissue should be 'standing', to expose the wall.
For tubular structures such as vas deferens, veins, arteries, they must be embedded in a way that the blade is able to cut across the lumen(hole).
The folds are caused when tissues expand and there is a lot of wax around them. However, if you placed the tissues close together, the very little amount of wax in between them would not really affect the results.
regarding the cause of the folds, it is quite hard to arrive at a conclusion between the two because both affects each other, where the main reason is more on how you orientate the tissues.
regarding the presentation of the tissues, we are more concerned about their appearance under the microscope. if the tissues have a lot of folds, there will be a lot of overlapping of cells under the microscope. This will in turn hinders the diagnosis and leads to inaccurate results.
zi shuang :)