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Overview of histological techniques Flashcards

1
Q

What is the first step that enables preservation of tissue structure?

A

Cut up and fixation

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2
Q

Tissue pieces must be cut small enough to allow ____ ____.

A

Chemical fixation

Allows the fixative to penetrate readily

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3
Q

What step occurs after fixation?

A

Tissue processing

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4
Q

What is the purpose of tissue processing?

A

To replace the water in the tissue with paraffin wax

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5
Q

What is the purpose of replacing the water in a tissue with paraffin wax?

A

Paraffin wax is a more rigid structure at room temperature so helps the tissue hold its shape when generating thin sections for staining

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6
Q

What are the properties of xylene relevant to tissue processing?

A

Xylene is a hydrophobic solvent which mixes with paraffin wax and assists in replacing the water in a tissue with paraffin wax

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7
Q

What occurs in a tissue processor?

A

Under pressure a series of increasing concentrations of alcohol and xylene are pumped into the chamber to remove the water from the tissue and replace it with paraffin wax

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8
Q

What is the approximate time frame commonly used for tissue processing?

A

A four to six hour cycle, often set to be performed overnight

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9
Q

What step occurs after tissue processing?

A

Embedding

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10
Q

What temperature are the processed tissue cassettes kept at prior to embedding?

A

63’C

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11
Q

What temperature is the plate that cassettes are placed on following embedding?

A

-4’C

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12
Q

What step occurs after embedding?

A

Microtomy

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13
Q

What is the purpose of microtomy?

A

Generating thin sections of tissue that can be mounted on glass slides for staining and microscopy

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14
Q

What is done with glass slides that have picked up a tissue section from the water bath after microtomy?

A

The slides are baked in an oven to bond the tissue to the surface of the slide

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15
Q

What step occurs after baking the tissue section onto a glass slide?

A

“Taking sections to water”

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16
Q

What is the purpose of taking sections to water?

A

Taking sections to water removes the paraffin wax and rehydrates the tissue

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17
Q

How is the paraffin wax removed from the tissue?

A

The slide is immersed in two changes of xylene then the tissue is rehydrated by passing it through decreasing concentrations of alcohol and finally placing them in the water bath

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18
Q

What step occurs after taking sections to water?

A

Staining

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19
Q

What is the purpose of staining?

A

Staining structures of interest to visualise different tissue components

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20
Q

Most tissue stains are either dissolved in ____ or are combination of ____ and ____.

A

Water; water; alcohol.

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21
Q

What step occurs after staining?

A

Dehydration and clearing

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22
Q

What is the purpose of dehydration and clearing?

A

<p></p>

<p>Removal of water from the tissue in preparation for mounting in plastic</p>

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23
Q

How is the tissue dehydrated following staining?

A

The slide is passed back through the same coupling jars of alcohol and xylene

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24
Q

What step occurs following dehydration and clearing?

A

Mounting in a plastic mounting medium

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25
What is the final step of histology slide preparation?
Mounting in a plastic mounting medium
26
What substance is the plastic mounting medium dissolved in?
Xylene
27
Should a histology slide be allowed to dry out prior to mounting in plastic mounting medium?
No - some xylene must be present on the slide to mix with the mounting medium
28
Name the process by which tissue structure is preserved
Fixation
29
Name the process by which thin sections of tissue are created
Microtomy
30
Name the process by which tissue is oriented in preparation for sectioning
Embedding
31
Name the process by which the water within tissue is replaced with paraffin wax
Processing
32
Name this piece of equipment

 

Tissue processor
33
Name this piece of equipment

 

Embedding station
34
Name this piece of equipment

 

Microtome
35
Name feature A of this piece of equipment
Processing chamber
36
Name feature B of this piece of equipment
Solvent storage area
37
Name feature A of this piece of equipment
Reservoir for molten wax (63'C)
38

Name feature B of this piece of equipment

Cold plate (-4'C)

39

Name feature C of this piece of equipment

Wax dispenser

40

Name feature D of this piece of equipment

Embedding area (63'C)

41

Name feature E of this piece of equipment

Cold plate (-4'C)

42

Name feature F of this piece of equipment

Storage area for tissue moulds

43

Name feature G of this piece of equipment

Storage area for processed tissue (63'C)

44

Name feature A of this piece of equipment

Water bath

45

Name feature B of this piece of equipment

Block holder

46

Name feature C of this piece of equipment

Blade holder

47

Name feature D of this piece of equipment

Hand wheel

48

Name feature E of this piece of equipment

Hand wheel lock

49

Name feature A of this setup

Deionised water

50

Name feature B of this setup

'Wet' microscope

51

Name feature C of this setup

Haematoxylin

52

Name feature D of this setup

Acid alcohol

53

Name feature E of this setup

Forceps

54

Name feature F of this setup

Slide rack

55

Name feature G of this setup

Water bath, used for final rehydration step and for washing steps during staining procedures

56

Name feature H of this setup

Staining rack - slides are often placed here when applying stains

57

Name feature I of this setup

Storage area - stained slides can be stored in these drawers. Once slides are completely dry they can be kept in a slide storage box that can be taken home. 

58

Name feature J of this setup

Xylene

Prior to staining, slides are immersed in this first glass Coplin jar containing xylene for three minutes to dissolve the paraffin wax. 

59

Name feature K of this setup

Xylene

Slides are immersed for a further 3 mins in this second xylene jar to ensure removal of the paraffin wax. 

60

Name feature L of this setup

100% ethanol

AKA 'absolute alcohol'. The first treatment with alcohol for 1 - 2 mins rinses away most of the xylene left on the slide. 

61

Name feature M of this setup

100% ethanol

Slide is immersed for 1 - 2 mins; any residual xylene is removed at this point.  

62

Name feature N of this setup

90% ethanol

This Coplin jar begins the process of rehydrating the slide as it contains 10% water. Slides are typically immersed in this jar for 1 - 2 mins but this time can be reduced by agitating the slide up and down.  

63

Name feature O of this setup

70% ethanol

The slide is further rehydrated in this jar as it contains 30% water. Like the previous jar the slide can be agitated up and down to speed up the process.  

64

Name feature A of this piece of equipment

 

Robotic arm

This moves slides from solution to solution. 

65

Name feature B of this piece of equipment

 

Grip for robotic arm

The end of the arm is designed to grip onto racks of slides. 

66

Name feature C of this piece of equipment

 

Solution container

The required solutions including solvents and stains are contained in these vessels; the first container shown is filled with xylene. The lids covering each container must be manually removed prior to starting a staining run.  

67

How high should Coplin jars be filled?

The standard height for solvents in Coplin jars is the point at which the top bend is reached, with the exception of the 100% ethanol nearest the xylene which should be filled a fraction higher. 

Note: the white arrows indicate the approximate height of solution. 

68
What is the purpose of a 'wet' microscope at a staining station?
A wet microscope is used to view slides at various stages to check the desired level of staining.
69
Why are the forceps in a staining station made of a special plastic?
The special plastic is compatible with xylene
70
What determines the arrangement of Coplin jars when setting up a staining station?
The sink location - the jars are arranged from left to right with the sink on the right side.
71
What is the first step of setting up a staining station?
Ensuring you are wearing the correct PPE: lab coat, safety glasses, enclosed shoes, and nitrile gloves.
72
What is the order of Coplin jars at a staining station?
Jars are arranged in order of increasing water content towards the sink: xylene, xylene, 100% ethanol, 100% ethanol, 90% ethanol, 70% ethanol, then the water bath.
73
After arranging the Coplin jars for a staining station, what should be done next if a bottle of xylene is within reach?
The two xylene Coplin jars should be filled to the required level and covered with their glass lids.
74
After arranging the Coplin jars for a staining station, what should be done next if there is not a bottle of xylene within reach?
Collect the slides you intend to stain during the lab and label them appropriately with a graphite pencil.
75
You are setting up a staining station. You have arranged the Coplin jars and collected and labelled the slides to be stained as there was no xylene within reach. What should you do next?
Collect the bottle of xylene and fill the two xylene Coplin jars to the required level and cover with their glass lids.
76
You are setting up a staining station. You have arranged the Coplin jars and as there was a bottle of xylene within reach you have filled the two xylene jars to the required level and covered them with their glass lids. What should you do next?
Collect the slides you intend to stain during the lab and label them appropriately with a graphite pencil.
77
When setting up a staining station, after filling the two xylene jars and collecting and labelling the slides to be stained, what should be done next?
Immerse the slides in the first xylene Coplin jar to start the process of removing paraffin wax from the slides. This takes three minutes to complete.
78
How long should slides be immersed in the xylene Coplin jars when taking sections to water (removing paraffin wax)?
Three minutes per jar
79
What should be done while slides are immersed in the first xylene Coplin jar?
Commence filling all remaining Coplin jars with the required concentrations of ethanol for the three minutes the slides are in the first xylene Coplin jar.
80
When setting up a staining station, which Coplin jar should be filled slightly higher than the standard height (the top bend)?
The 100% ethanol nearest the xylene
81
What are the four pieces of information that should be included on a slide label?
1. Student name (i.e. patient or subject ID) 2. Date the section was used for staining 3. Name of the tissue (if known) 4. Name of stain applied and any other distinctive staining details
82
How many times should a slide be labelled?
Twice
83
Where and when should the first label be applied to a slide?
The first label should be applied to the frosted/painted end with a graphite pencil, immediately prior to placing the slide in xylene.
84
Where and when should the second label be applied to a slide?
The second label should be applied to an adherant paper label with a ballpoint pen and placed over the original label after the slide has been coverslipped.
85
How long should slides be immersed in the decreasing concentrations of alcohol when taking sections to water?
Approximately one minute in each jar
86
What is the term used to describe the second passing of slides through the solvents in reverse order?
Dehydration, clearing, and mounting (DCM)
87
What does DCM stand for?
Dehydration, clearing, and mounting - the process of passing slides through solvents in reverse order once staining is complete to remove water and and prepare for coverslipping.
88
What is mounting medium?
The clear plastic glue that is used to mount the coverslip
89
Slides should be free of ____ after dehydration and clearing in xylene.
Water

LSB466 (3 decks)

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