Week 8 Flashcards
What do you do if you if have Fragmented, crushed and overstained sections from small biopsies and when do these errors occur
-crushing occurs at collection when fiberoptics/forceps are used to grab tissue
-small biopsies are put in fixative at the clinic and processed again at the lab - causing shrinking, dry, brittle tissues
-solution is to process by hand on a short schedule or auto on 2 hour, microwave fix fresh and hand process on 1 hour schedule
how do you know microtomy collected problem is two fold
-if the section is compressed on one edge with extra eosin staining in the compressed edge then you know the problem is twofold and it related to collection and over processing
-FOR HEMORRAHIGC TISSUES LIKE SPLEEN OR THYROID - allow to absorb ice water before sectioning
-for other tissues blocks need to be cold
What do you do when you find a minute biopsy after processing
-small biospies can be hard to see in gauze, filter paper or lens paper
-when you get a small biopsy fix it asap in 50ml of 50% ethyl alcohol added to 5 ml of 0.4% eosin and let fix for 30 mins. So when you embed you see red blob on white background and the eosin will not interfere with the other staining
What can you do if there is Shattering, cracked or folded sections from large blocks processed through paraffin wax.
-occurs because of over dehydration and removal of moisture
-a good section needs a little moisture
-solution -place a cotton gauze over the block face before cutting so the moisture can penetrate 15-20 um so a good section can be cut
how do you Recover tissue that has dried out after a malfunction of a tissue processor. Depending upon where in the cycle the malfunction occurred tissues can become almost mummified, very hard and rubbery
-put dried out tissue in a formal -glyercol solution for 5-10 hours depending on block size. The time isnt critical because the exposure wont hurt the tissues.
-after this treatment tissue can be taken back to the dehydration stage in schedule
-this method softens any hard or dry sample because formal-glycerol is a softening agent
-developed to soften mummified tissue
-enclosed processors are better at keeping tissues moist
What is in a Formol-Glycerol solution.
10 ml formaldehyde, 2 gm sodium acetate and 90 ml tap water.
To produce a working solution from the stock solution 10 ml glycerol is added to 90
ml of the formol-sodium acetate.
how do you obtain a section from tissues which are often brittle after processing
-happens with tissue that has alot of blood or colloid like thyroid or spleen
-clots are hard to cut and end up broken and curled
solution - put cotton wool/gauze soaked in warm water to the surface and then cut
-if that doesnt work use warm soapy water, put on ice and then cut
What causes Crooked or uneven ribbons when sectioning and why does it matter that its straight
-if straight youll get more sections on a slide like when you have small one with several sections on a slide
why is it crooked
-if edges of knife and block are not parallel or if top and bottom are not parallel = ribbon bends
-knife not sharpened properly or is not straight in knife holder
-impurities in wax causing dense areas
-tissue could be varying densities
What causes Wrinkled or compressed sections
-blunt knife: if blunt it can cause different densities in wax
-knife bevel too deep - cutting angle is too shallow which is the same as having a big clearance angle. increased friction as block meets knife edge
-warm block or soft wax
-warm knife - need a hard cold knife - softer warmer more wrinkles
-clearance angle is too great- increasing friction as block meets knife edge = wrinkles
-loose knife - wrinkling, compression or blade can dig into block destroying block face or tissue
-incomplete dehydration - increases friction force in tissue because the tissue is soft and rubbery. Sections are hard to cut
-micrometer too thin for hardness of wax= crumbling
why do Wrinkles appearing in sections floated on a flotation bath. causes?
-floatation bath too cold - if the water is too cold wax around the section will not expand and flatten . The flattening and expanding of the wax flattens the section . Ideal temp 5-10 C below MP of wax
-knife too blunt - stretches tissue as it cut instead of passing through smoothly
why do you cut in thick and thin sections
-loose knife or block
-block to large for microtome - if the clearance between bottom of block and knife top it can be hard to cut causing thick / thin sections . Best to get another microtome with a large clearance or smaller pieces
-small knife clearance angle - acts like blunt knife . The block face is too close to the bevel as as the knife cuts the block face expands causing friction making it hard to cut
-hard wax or tissue cutting with disposable blades because these blades are thinner than regular blades; vibrate more , need to hold firmly even soft tissues will have this problem
-calcification- causes knife to jump or vibrate must decal for 15-30 mins
-bad technique- not sure how to find a steady hand
-microtome needs servicing - feed mechanism
why do Sections adhere to the block on the upstroke of the microtome
-static electricity- when air is dry caused by reverse cycle AC. static can also be caused by friction between block and knife
-knife clearance angle too small - block rubs on knife back causing wax deposits and sections start sticking
-blunt knife - compressed sections tend to stick together and bunch up on the knife
-dirty knife or residual wax on knife
why do Ribbons split vertically or scratch lines appear
-handling of knife badly
-hard material in wax- filter wax before use
-knife clearance too great - causing build up of debris and that buildup causes the lines
-hard material in tissue - calcium (decalcify), crystals ( ID the type and remove ), and sutures
what causes Sections crumble or tear
-dry tissue - due to over processing
-tissue not cleared properly - similar to dehydration
-blunt knife or soft wax
-wrong mp for wax - high MP in cold environment
-wax crystallized from slow cooling - forms large wax crystal, wax no longer homogenous and falls apart. Rapid cool forms small crystal and homo wax that holds up better
-wax contaminated with water or clearing agent - crumbling when floated on warm water. When there is clearing agent in tissue the surface tension difference causes section to crumble. If there is water in the tissue the components will fall apart because there is no wax holding them up .
-denatured paraffin wax- caused by too much heat or reheating. The wax molecule breaks down losing elasticity and ability to adhere to other molecules.
use adhesive on slide to avoid tearing off especially for cellular tissues
What causes chatter - minute high pitch vibration
in a knife edge
-loose knife - keep rigid
- knife clearance too high - should be between 3-5 degrees. for resin section 5-7
-hard wax or tissue - use softening agent, rehydrate with moist cotton , or warm a bit in hands
-calcified section - use decal for 15-30 min but wash under running water before use so the knife does get damaged will cause the knife edge to blunt
