Fixative volumes RE: [Histonet] tissue processing

Gayle Callis gcallis <@t>
Wed Apr 25 13:04:52 CDT 2007

We have always opt for the 20:1 volume of fixative for research 
purposes.  Fixative is cheap so we like the "rule" that more is 
better.   In the past, the "plunk and dunk fixation syndrome" was alive and 
well here.  The worst example was a whole (unsliced) femoral head sitting 
in 100 mls for over a month (not in this lab), sent to us, and when 
bisected, it was still bloody.  Needless to say, the fixation was horrible.

We encourage submission to this core histology lab as soon as possible, 
prefering the next day, and IF the tissue is put into less than a 10:1 
ratio, it is changed to fresh fixative to replenish the active fixing 
ingredient.  Too little fixative happens way too often especially when the 
people who initiate the tissue fixation do NOT and sometimes never seem to 
understand the concept of good fixation, or don't want to bother.

  If immunostaining is required, time constraints for optimal fixation are 
explained but that doesn't always work either. Fortunately, most 
researchers here do their own immunostaining, so they have to deal with 
their fixation problems.

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

At 10:26 AM 4/25/2007, you wrote:
>Sheehan states "at least 15 to 20 volumes of fixative should be used for 
>every volume of tissue"
>pg. 49---in the  "Tissue-Handling fixation chart".
>At 07:53 AM 4/24/2007, Rittman, Barry R wrote:
>>René raises an interesting point.
>>While we all accept the 20:1 ratio for fixation as our standard to aim 
>>for, I do not believe that this has actually been studied or published as 
>>original paper. Please let me know if I am incorrect in this statement.
>>The 20:1 ratio is one of those items that are always passed along.
>>In fact it has been suggested that the concentration of the main fixing 
>>agent (within certain limits) and the time of fixation are more important 
>>considerations. This principal is used in tanning leather when the amount 
>>of glutaraldehyde is carefully matched to the quantity of the proteins in 
>>the leather. The aim is to have just the right amount of glutaraldehyde. 
>>Excess glutaraldehyde would end up permeating various parts of our 
>>anatomy close to the billfold for example.
>>In most labs we only carry out a partial fixation. This is necessitated 
>>by time constraints, immunohistochemistry etc.
>>An alternative to immersion fixation for small or thin sample sis vapor 
>>fixation. This has the added advantages that no carrier vehicle is 
>>necessary, small amount of sometimes expensive fixing agents can be used.
>>-----Original Message-----
>>From: histonet-bounces <@t> 
>>[mailto:histonet-bounces <@t>] On Behalf Of Rene J Buesa
>>Sent: Tuesday, April 24, 2007 7:18 AM
>>To: histonet <@t>
>>Subject: Re: [Histonet] tissue processing
>>If you are using a tissue processor (TP) you do not have to worry about 
>>this problem but since you asked it seems that you are going to process 
>>   If that is the case you should maintain the same volume ratio of 20:1 
>> as when fixing.
>>   The other thing (if processing tissue manually) is that you will not 
>> have agitation, pressure, vacuum or controlled temperature, so time has 
>> to be increased (as compared with an automatic TP) for each step.
>>   Volume alone will not determine proper processing without the other 
>> advantages of a TP and these disadvantages have to be compensated with 
>> longer steps.
>>   René J.
>>mwstarbu <@t> wrote:
>>   Hello,
>>Is there a standard tissue volume to liquid volume ratio that you use when
>>dehydrating samples with ethanol?
>>Thank you in advance.
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