What would not be seen in autolytic tissue?
Nuclear detail lost
Stainability lost
Stain precipitation
Cells coming away from basement membrane
What would be a "normal" specimen received in the grossing room?
Tumor
Placenta
Surgical
Biopsy
Cassettes:
Are 3 cm deep
Must be labeled
Colours indicate day of week the tissue was processed
Has holes to allow the tissue to flow in an out
Fixation:
Functions by creating hydrogen bridges to prevent degredation
Typically shrinks, swells, and distorts tissue
Keeps bacteria and fungi viable
Allows the tissue to be stainable
Which is not a factor affecting fixation?
Time
Density of tissue
Temperature
Type of container
Which is not a coagulant fixative?
Zinc
Picric Acid
Acetic Acid
Alcohol
Which non-coagulant fixative is also non-additive
Osmium Tetroxide
Formalin
Gluteraldehyde
How much concentrated formalin is needed to make 5.5L of 10% formalin?
55 ml
55 L
5500 ml
550 ml
To make 10% formalin, how many ml should be added to 230 ml of 100% formalin?
2300 ml
0.23 L
2070 ml
2600 ml
Which is true about formaldehyde?
It is a coagulant fixative
It is composed of liquid formaldehyde in water
Add methanol to stop the formation of paraformaldehyde
Penetrates tissue slowly but fixes quickly
The best fixative for DNA/RNA is?
Carnoy
Which is false about gluteraldehyde?
Is a non-coagulant, additive fixative
Can be used to disinfect the cryostat
Can cause false positive reactions in period acid schiff
Creates methylene bridges in tissue to preserve it
Mercuric Chloride
Is a component in B5 fixative
Is a common fixative used in the histology lab
Mercuric pigment can be removed using alcoholic picric acid
Is a great fixative when tissue needs to be X-rayed
What is false about osmium tetroxide?
Fixes lipids
Used mostly for electron microscopy
Commonly a secondary fixative
Is a non-additive fixative
Bouin's fixative:
Is composed of picric acid, formaldehyde and mercuric chloride
Is the best preservative for glycogen
Does not stain tissue
Is commonly used as a secondary fixative
Which is known to be a good general fixative?
PAF / Zamboni
Select the correct statement regarding non-aqueous fixatives?
Acetone should not be used for IHC since it destroys enzymes
Acetone is a good fixative since it does not shrink or harden tissue
Ethanol should not be used to fix frozen sections since it inhibits freezing
Ethanol should not be used to preserve glycogen as it is known to dissolve it
Which fixative would be a good choice when RBCs need to be preseved
Carnoy's
Bouin's
Oh no! Your tissue is autolytic! What should be done to prevent this?
Tissue should be placed in fixative 10X greater than the tissue itself
Tissue should have been placed in fixative right after removal from the body
Tissue in fixative should be placed at 4 degrees Celcius
Tissue should have been frozen before fixation
What would be proper fixation procedure for a large tissue?
Large tissue should not be fixed
Large tissue must be cut into smaller pieces
Holes must be poked in the specimen to allow fixative to penetrate
Fixative must be injected into the specimen
Which is true regarding the VIP Tissue Processor?
Uses vacuum technology to infiltrate tissues
Hematoxylin is added to tint the tissue pink for easier embedding orientation
This processor is safer since it does not use heat
This is an open system
Which is TRUE about dehydration?
The dehydration process begins with the highest alcohol concentration then descends
Ethyl alcohol is the best for dehydration
Butanol is a good substitution because it is quick
Alcohol dehydrates tissue by removing xylene
How would you be able to tell if a tissue was improperly dehydrated?
Tissue would smell like xylene
Tissue would be soft
Impossible to tell until staining
Tissue would be dessicated
What is the proper way to fix improper dehydration?
First xylene, then several changes of alcohol, then xylene, then wax
Impossible to fix, request new specimen
First several changes of alcohol, then xylene, then wax
Several changes of wax
After processing; tissue is soft, cloudy, and smells like alcohol. Why?
Improper fixation
Improper dehydration
Improper clearing
Improper infiltration
Which is not a clearing agent?
Xylene
Toluene
Benzene
Chloridine
Which is true considered a universal solvent?
An example is Limonene
A solution that dehydrates and clears
An example is Xylene
A solution that dehydrates and infiltrates
How would you fix improper infiltration?
Increase the temperature of the wax
Remove wax with xylene, dehydrate with alcohol, then clear with xylene, then several changes of wax
Re-fix the tissue
Which statement is TRUE?
Soft wax allows for easy ribboning
Soft wax provides the most support
Hard wax melts at 45 degrees Celsius
Hard wax should not be used if thinner sections are required
What is true about paraffin wax?
Wax should be kept at least 10 degrees over the melting point
Typical melting point is 35-48 degrees
Overexposure to wax will cause tissue hardening and shrinking
Water-bath should be kept 2 degrees above the melting point
What is an advantage to using a microwave for processing?
Clearing step can be skipped
Infiltration step can be skipped
Dehydration step can be skipped
Microwaves would never be used for processing
A technologist is viewing several embedded tissues. Which one would need to be re-embedded?
Several small biopsies are placed together
Red dot is facing towards you when cutting
Two pieces of small bowel are orientated so that the lumens are facing eachother
Transverse cut of tubular tissue
Which of these is proper troubleshooting?
Incorrect orientation of tissue when embedding - discard the tissue and request new specimen
Poor processing of tissues - check solutions and machine
Tissue is over-dehydrated - increase time in alcohol
NBF precipitate is found in the tubing of the processor - perform a picric acid flush
Troubleshoot this cutting situation: what is the most likely cause of crooked ribbons?
Block not chilled enough
Cutting too slowly
Dull blade
Block and knife not parallel to eachother
Select the correct statement about decalcification
Tissue must be > 3 mm in order to be decalcified properly
Calcium is soluble at pH 4.5
Acid methods are usually slow
Decalcification increases stainability of bone tissue
What is the most accurate way to determine the end point of decalcification?
Mechanical methods such as poking
Chemical methods such as ammonium hydroxide and ammonium oxalate which check for the precipitation of calcium oxalate
Cutting the bone tissue
Radiography
When looking at a H&E stained bone under the microscope, the technologist notices that there is many dark blue/purple staining areas. What could this mean?
Bone was stained too long in hematoxylin
Bone was underdecalcified
Bone was improperly fixed
Bone was not stained with the correct stain
What is the incorrect procedure when using the cryostat?
Disinfect daily with sodium hypochlorite
Use cryostat when rapid diagnosis is required or when fat or enzymes need to be demonstrated.
If a tissue with known TB requires cutting, disinfect afterwards with formaldehyde vapor
If tissue is sticking the antiroll plate may be too warm
Which is the correct use for each microscope?
Fluorescent microscope for Acid Fast Bacilli
Polarizing microscope for Oil Red O
Compound light microscope for Acridine Orange
Electron microscope for H&E
Select the correct statement about microtomes:
"Roughing in" is usually done at 20 micrometers
The clearance angle is usually set at 10 degrees
Speed of cutting has no effect on ribbon quality
In a rotary microtome, the knife moves side to side
Select the correct statement about automatic stainers:
With linear stainers, time in solution can be varied by adding more containers
With robotic stainers, the programs cannot be changed
Robotic stainers are good for progressive staining
Linear stainers are good for regressing staining
What is proper water bath procedure?
Water bath should be kept at 5 - 10 degrees above the melting point of wax
Tap water should always be used to fill water baths
Parched earth artifact can result if the water bath is too cold
Water bath should be cleaned after each ribbon
A technologist is cutting many tissues for different areas of the lab. Which of the following is an incorrect procedure?
Slides for IHC were placed on a hotplate for drying.
Slides with biospy were cut using special techniques with 6 tissues per slide
Slides for neurology were placed in a 37 degree oven for drying
Slides for routine H&E were placed in a 60 degree oven for drying
A student is concerned about contracting TB from patient samples. What process would be of the least concern to the student?
Embedding
Grossing
Autopsy
Frozen sectioning
Which is considered to be a correct safe procedure?
Placing used blades in the garbage
Fixing tissue that is known to be TB positive with formalin
Storing glacial acetic acid on the counter top
Fixing CJD positive tissue with formalin
Which is not an element of a dye molecule?
metachrome
benzene ring
auxochrome
chromaphore
What is a correct statement about staining:
A method of nuclear staining is acidic dye
A method of nuclear staining is dye + metal mordant
Cytoplasmic staining occurs from the interaction of dye with neutral molecules
Lipids are stained because the dye has a greater affinity to the solution than the fat
Which is considered a basic auxochrome?
-NH2
-COOH
-OH
-SO4
Which statement is correct?
Basic dyes will have a negative auxochrome
Basic dyes will produce chloride salt
Acid dyes are cationic
Acid dyes produce magnesium salt
Which is a true statement about dyes?
Modifiers will affect staining time
Sulphonics makes acidic dyes basic
Dye binding can be affected by pH
Orthochromic dye will stain a different colour than the dye colour
Which is not a metachromatic component?
AFB
Mucin
Mast Cell
Amyloid
Why are thymol crystals added to dye? ;)
Prevent mold growth
Enhance staining
Makes the dye more acidic
Makes the dye fluorescent
Which is not a natural dye?
Hematoxylin
Orcein
Acridine Orange
Saffron
Which pairing for decolorization is incorrect?
Weak acid differentiating basic dyes ex. PTA/PMA for Biebrich Scarlet
Weak base differentiating acid dyes ex. PTA/PMA for Biebrich Scarlet
Excess mordant differentiating ex. iron alum for Verhoeff
Alcoholic differentiation ex. alcohol for eosin
A technologist needs to prepare a hematoxylin for use in Masson's Trichrome. Which would be the correct procedure?
Oxidize the hematoxylin with sodium iodate
Prepare hematoxylin using aluminum since it can withstand strong acids in the stain
After preparing the hematoxylin, pour directly into the coplin jar for staining
Gill's hematoxylin should be prepared for this stain
A technologist has prepared several different hematoxylins for use. Which one of them is paired correctly for use?
Gill's hematoxylin for use in Alcian Blue
Mayer's hematoxylin for Oil Red O
Harris hematoxylin for Masson's Trichrome
Weigert's hematoxylin for routine H&E
What is true about Hematoxylin and Eosin?
Hematoxylin stains indirectly
Is never a regressive stain
Cannot be used to stain bone tissue
The best substitute for eosin would be phenol red
A technologist is reviewing a manual H&E control slide when they notice that the nuclei have not stained. Which of the following options is likely NOT be a reason for this problem.
Tissue was left in acid alcohol for 20 minutes during coffee break
Aluminum / iron was not present in the hematoxylin
The hematoxylin was not filtered properly
The hematoxylin was stored in a clear glass jar
A technologist reviewing an H&E control has noticed that the cytoplasm is staining very dark. Which is not a likely cause?
Tissue was cut at 3 micrometers
Eosin is too concentrated
Eosin was not left in alcohol long enough
Tissue was left in eosin for too long
A technologist is preparing to perform Masson's trichrome on a kidney slide. Which of the following procedures is INCORRECT?
Places slide in Bouin's for 1 hour at 56 degrees.
Counterstain in Light Green
Places slides in Biebrich Scarlet Acid Fuchsin before PTA/PMA
Uses Mayer's Hematoxylin to stain nuclei
A technologist is reviewing the Masson's Trichrome control. Which of the following stained components is cause for concern?
Collagen has stained blue
Cytoplasm has stained red
RBCs have stained red
Nucleic acids have stained red
A technologist is preparing to perform Gordon and Sweets on a liver tissue. Which of the following is the CORRECT procedure?
Ensure than silver solution is neutralized with HCl before disposal
Place the slides in a thin walled coplin jar to ensure good heating in the microwave
Bleach tissue in Oxalic Acid after Potassium Permanganate
Which staining method is NOT correctly paired with the type of metallic impregnation?
Gomori Burnter - argentaffin
Grocott - argyrophilic
von Kossa - substitution
Gordon and Sweets - argyrophilic
A technologist is reviewing the control for Verhoeff's van Gieson. Which of the following errors is paired with the correct solution?
Elastic fibers cannot easily be seen on the brain control slide so it was placed in Verhoffs stain for a longer period of time
Nuclei are not staining so a new solution of Verhoffs made and used
The stain looks very muddy so it was placed back into gold chloride for a longer time
Elastic fibers are too pale so the slide was placed back into van Gieson stain for longer
Which is the correct statement about elastic stains?
Orcein is specific for elastic fibres
Orcein will stain elastic fibres brown
Aldehyde fuchsin will stain elastic fibres green
Aldehyde fuchsin is a regressive method
A technologist is preparing to perform Toludine Blue. What should the technologist NOT do?
Take the slides down to water before staining
Place the slides in Toludine Blue to stain for mast cells
Place the slides in Light Green as a counterstain
Differentiate with alcohol then clear and mount
A technologist is confused why their Alcian Blue control slide did not demonstrate mucins. Which would NOT be a cause for this to happen?
Stomach tissue was used as the control
The tissue was placed in 3% acetic acid before Alcian Blue dye
The tissue was placed in Acetic Acid following Alcian Blue for 10 minutes
The pH of Alcian Blue was 6.5
Oh no! Our Periodic Acid Schiff's stain didn't work (again).Which of the following would NOT be a possible cause?
Schiff's reagent was tested before use using formalin and the reaction became a deep blue colour
Grocott's method was performed earlier so the periodic acid solution was saved and used agian
Metabisulfite rinse was prepared using 100% HCl straight from the bottle
Tissues were washed for 10 minutes in water after Schiff's
Which statement is true regarding Periodic Acid Schiffs?
Schiff's reagent is composed of Acid Fuchsin and Sulpheric Acid
Periodic acid is the secondary oxidizer
Sodium Metabisulfite rise is what brings the pink colour to the tissue
Fungi will stain pink
Which is FALSE about Mayer's Mucicarmine?
Metanil Yellow is the primary stain
Mucicarmine stains acid mucins
Gill's hematoxylin should not be used as the nuclear stain
Cryptococcus can also be demonstrated with this method
A technologist is preparing to stain for amyloid. Which procedure is correct?
The technologist double checked to ensure sections were cut at 4um
The doctor would like to differentiate between primary and secondary amyloidosis so the slide was pretreated with potassium permangonate
Slides were placed in neutral red to stain for amyloid
The technologist did not know how to use the polarizing microscope so birefringence was not checked on the control
The pathologist has requested an Oil Red O stain for a frozen tissue. Which of the following procedures is INCORRECT?
The tissue was previously fixed in formalin
A frozen section was cut for staining
Tissue was taken down to water before staining
The tissue was placed in hematoxylin after Oil Red O
Which is FALSE about Luxol Fast Blue stain?
Luxol Fast Blue dye is differentiated with an acid
The control tissue is cerebellum
Eosin is the counterstain
Luxol Fast Blue stains myelin
A technologist notices an endogenous pigment on their slides. What is it?
mercury
tattoo
carbon
melanin
A technologist is performing Von Kossa. Which procedure is INCORRECT?
Tissue is placed in silver nitrate and then microwaved for the appropriate time
Placenta is used as the calcium control
The tissues are placed in nuclear fast red or neutral red
The technologist decides the control passed since the calcium stained brown
A technologist is performing Perl's Prussian Blue on a control and a patient tissue. The technologist checks the PATIENT'S slide to find that no iron has been stained! What is the most likely cause for this result?
Slide was placed in eosin
Patient tissue does not contain iron
Slide was taken down to distilled water
Slide was placed in HCl + Potassium Ferrocyanide
Which is a true statement regarding Gomori Burtner stain?
This stain is used to demonstrate argentaffin granules
Gold chloride is a fixing agent
Hematoxylin is the counterstain
Melanin cannot oxidize silver therefore a reducer such as Sodium Thiosulfate is required
A technologist needs to stain for Blastomyces, which stain should they select?
Ziehl Neelson
Gram
Geimsa
Grocott
A sample for AFB is arriving in the lab for staining. What should be done?
After fixation, Gram stain the tissue to detect the bacteria
Wear N-95 for frozen section
Place the specimen in Carnoy's for fixation then stain with Ziehl Neelson
After fixation, take down slides to tap water before staining
Why do AFB stain red with Ziehl Neelson?
Not enough Acid Alcohol was used to decolourize the AFB
Methylene blue stain was missed
Nature of their cell wall
Because they are Gram negative
Which component of Gram stain is correctly matched with its purpose?
Crystal violet - stains Gram negative bacteria
Acetone / Alcohol - decololourizes Gram positive bacteria
Basic Fuchsin - stains acid fast bacteria pink
Picric Acid - stains bacteria yellow
A technologist is having trouble with Grocott stain. Which of the following problems is correctly solved?
Fungi are stained black - the slide is placed back into gold chloride for more toning until fungi are brown
Fungi, nuclei, and other tissues are staining black - the slide is good
No fungi have been stained - the wrong concentration of periodic acid was used
Fungi are brown - the slide is good
Which of these uncommon stains is correctly paired?
MSB stain for bile
Warthin-Starry for H. pylori
Steiner for spirochetes
Hall for fibrin
Which of the following is NOT a correct procedure in IHC?
Deparaffinize and hydrate to water
apply antibody
stain nuclei with hematoxylin
counterstain with hematoxylin
Which is true about antigen sites in tissues for IHC?
Tissue must be washed with water to remove crosslinking from formalin
Proteolytic enzymes will destroy the antigen sites
Heat methods help cleave the bonds
Enzymatic methods will unfold the bonds
Which of these solutions is NOT properly disposed of?
Picric acid in a container
Alcohol in a container
Sodium Thiosulfate down the sink
Silver Nitrate down the sink
Which pairing of control with stain will not work?
Kidney for Masson's Trichrome
Placenta for von Kossa
Cerebrum for Luxol Fast Blue
Stomach for Alcian Blue
Why should gloves be worn when cutting tissues on the microtome?
To prevent the blade from getting scratched
To prevent your hands from getting infected
To prevent skin cells from getting on the microscope slides
To prevent yourself from getting cut
What would not be a likely reason for seeing pale staining elastic fibres for Verhoeff van Gieson?
Left in Sodium Thiosulfate for too long
Left in Ferric Chloride too long
Left in Van Gieson stain too long
Left in Verhoeff stain too long
Which would not be a reason for seeing pale cytoplasmic staining on H&E stained tissue?
pH of eosin is 8.0
Sections were cut at 8 micrometers
Tissue left in alcohol for too long
Tissues were rinsed in water after blueing
