Afaf not knowing from where/which source you have that habit of processing...but I - only commenting on this your question very briefly - think the following: it depends perhaps on the volume of your tissue pieces you fix initially ....working attitude to change fixative quite frequently  might help perhaps in the end (regarding good fixation) but: you don't say how long (and at which temperature) your 10%NBF solutions will be applied to what size of specimens until you change/want to change the fixative. IMHO moving   (e.g. by using a rolling or a rotating device= specimen rotator)    your specs in the fixative (sufficient by volume, e.g. 1=spec :20=fix v/v) will reduce change(s) of old fixative to new /fresh solution significantly

(e. g. first solution 1:20, size [cm] 0.5-1 : 1 : 2, using a specimen rotator or at least multiple= 4-6 times gentle shakings / hour):  

Initial fixation step:  at least 3 -6 hrs @RT, then change to fresh fix. solution, additional over night at RT or 4°C....up to 12-18 hours. The regime naturallly also will depend on the task your study must / should fulfill. The same holds for 70% Ethanol-step(s):  usually 1x 20-30 min(or maybe longer if necessary for some known reason), but nothing is lost if you dehydrate 2x 20-30 mins (time also depending on volume/size of specimens to be processed) and then proceed to 96% Ethanol and 100%. 

Hi Afaf, 

You shouldn't need to put your tissues through changes of formalin as long as your original solution isn't too dirty (ie saturated with blood product or diluted out with saline) and you have enough volume (approx 10x volume of formalin to tissue volume). Are you trying to cut down on time or just volume of reagent? 

One container changed on a regular basis depending on how many samples you get through should be adequate. Same for the alcohol though it depends on what you are then doing with the tissue. You may want to then grade into a 90%.  In my experience the alcohol tends to get 'dirtier quicker' so you may need to change that more often.

Hope this is of help,

Sarah

Dear Sarah, you might be right in terms of routine fixation in a Pathology Lab (but I know also the quality of, the problems with, and the consequences for at least human tissue specs - when something went wrong with fixation. So if you have the experience that 1:10 (vol. tissue : vol. fixative) in your institution is enough and sufficient I  will not comment any more on a perhaps "economic factor" in your facility....  but insist that almost every Handbook on histological Techniques will tell you that  1 (tissue) : 20  (fixative) is the rule (and in the Pathology Institute I worked for long I have seen a lot of bulging tissues exhibiting big areas of insufficient initial fixation. When informed by diagnostic report about that fact (e. g. "irrgeular and poor initial fixation might have hampered a correct diagnosis"), the assigning surgeons most often said they had been "sufficiently fixed" the tissue immediately after excision...(but naturally and physically weren't fixed adequately...).   So finally,  for a classical "scientific" application - IMHO-  , it makes no sense to propose or  advise for small volumes of fixative in primary fixation.

thanks a lot for all of you 

Wolfgang H. Muss what i know about how long 10% neutral buffered formalin fixative is applied on specimens is about three changes at every 24 hr at room temperature

concerning the size of my tissues it doesn't exceed 1 gram 

that' s all

thanks in advance 

Dear Afaf Mohammed,

thank you for coming back to tell about the size/ mass volume....if this I understood correctly you said "1 g" (1 gram) which is really few as compared to a whole organ (like heart, brain or lung). I don't think that fish tissue as you informed in your request is heavier or lighter than human tissue. So I guess, only 2x 45' could be sufficient for fixation of your specs. But: Unfortunately you haven't said something about the of your samples to be fixed since the time of fixation depends not only on "g / volume" of specs and temperature of fixative, but also on size at the slightest diameter (=distance which has to be penetrated effectively by the fixative) and density of the tissue. So it makes a big difference whether you want to fix liver (fish or human) or skeleton / teeth (human) or fish bone / teeth. As you know, penetration of  neat buffered formaldehyde based fixative at RT is assumed (and calculated) to be some 3-3.5 mm/h at RT, but the problem is that also the penetration rate is NOT THE ONLY parameter of good fixation.

This is (besides for sure other and more detailed  and /or sophisticated paperwork) stated in the attached short article by Bryan R. Hewlett (2002): "Penetration Rates of Formaldehyde", saying:

So I really do hope that you'll learn a bit more about fixation in Histology  than  

" what i know about how long 10% neutral buffered formalin fixative is applied on specimens is about three changes at every 24 hr at room temperature"

.....Very best wishes and good luck, WM.

Hi Wolfgang,

Thank you for your response. Admittedly there are a number of histopathology handbooks that state the maxim of "20:1 volume to volume ratio', however my statement of using 10:1 is not merely based on my own professional experience but is also in the literature.

If you refer to the highly regarded histological book "Theory and Practice of Histological Techniques" 6th Ed, edited by Bancroft and Gamble, you will find the comment "fixative volume should be at least ten times the  volume of the tissue specimen...".

There are even papers that suggest as little volume as 2:1 is adequate (under controlled temperature and pressure variables). Though this isn't a ratio that I have come across in practice.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Peshkov%20MV%5BAuthor%5D&cauthor=true&cauthor_uid=22483550

Using lower volumes of formalin is more than an 'economic factor' in any laboratory where health and safety is a consideration. 

I would also suggest that some of the tissue specimens you mention that have suffered from inadequate fixation may have suffered in delay from theatre to the lab, or whether the specimen was 'opened up' in a timely manner to allow inner tissue penetration.

I think we can probably agree that understanding fixation and its principles is more useful than sticking to a one rule fits all ratio.

Kind regards,

Sarah

 Dear Sarah, I don't want to go infight...   naturally all is right you were saying...and naturally I know the mentioned Bancroft and Gamble Handbook (and had at least 3 (as last the 8th) edition(s) in my private possession. And the books weren't stored on the shelf...just to look pretty. (I learned about and did all the "specimen preparation and chemical works" in preservation of diagnostic tissue  by myself  in over 35 years of profession). As (during my active work)  a certified safety officer I was and am very familiar with the parameters and priorities for health and safety of employees and colleagues in the Lab, be it "Histology" or a "special lab branch".   Concerning your mentioning: "......may have suffered in delay from theatre to the lab, or whether the specimen was 'opened up' in a timely manner to allow inner tissue penetration."   just as an example I have seen very often (done by the surgical theatre staff...whomever this might concern) e.g. a whole uterus excision specimen tucked into a 200 ml plastic container/vial...no incision....guess how much fixative was in the vial...)

So I agree fully with you that "understanding fixation and its principles is more useful" than...... mybest wishes and regards, WM

thanks a lot for all

Wolfgang H. Muss in fact i didn't put in my mind what is the size or the density of the samples as i thought that my samples will be penetrated by the fixative altogether as a whole and shall be fixed , but really i learnt some new information about fixation and shall seek to learn more evenif  it is n't my specific area of specialization

best regards