Handwashing - Conventional Tissue Processing - Autotechnicon

1. Whole mount

2. Sections

3. Smears

Types of histological preparations:

Whole mount

• should be no more than 3-5 mm and cut on microtome

• the entire specimen is mounted on a slide

• whole plant, stem, leaves, flowers, filamentous algae

Smears

• made from blood, bone marrow or any fluid such as pleural or ascitic fluid

• immediately fixed in alcohol to preserve the cellular structures and stained.

• can also be made by crushing soft tissue between two slides in contact with the moist surface of a tissue.

sectioning

• Tissue is dissected in 1.0 cm thick sections

• All hollow structures are opened as part of the initial examination

Fixation

• Preserve the morphologic and chemical integrity of the cell in as life-like manner as possible.

• most widely used

• Harden the tissue to protect it against subsequent processing steps.

10% neutral buffered formalin

most widely used solution for fixation

4%

how many % is formaldehyde in 10% neural buffered formalin

Dehydration

• The process of removing intercellular and extracellular water from the tissue FOLLOWING FIXATION, and PRIOR to WAX IMPREGNATION.

Ethyl alcohol

solution used for dehydration

Acetone/Isopropanol

should be added before the final absolute ethanol with chloroform and trichloroethane as the transition solvent

Clearing

• Process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated.

• the agents used in this must also be miscible with Canada balsam & other resins

Xylene

most commonly used solution in clearing

Infiltration (Impregnation)

• The process of removing the clearing agent from the tissue and replaced by a medium that will completely fill all the tissue cavities, giving the specimen a firm consistency.

Paraffin wax

most commonly used wax in infiltration and is best for routine processing

Orientation

the most important step in embedding

Embedding

the IMPREGNATED tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify.

Double embedding

tissues are first embedded or fully infiltrated with a supporting medium such as AGAR or NITROCELLULOSE, then infiltrated the second time with PARAFFIN WAX in which they are also embedded.

Section-cutting

the process of cutting tissue into thin slices

1. coarse trimming

2. fine trimming

types of trimming

Mounting

is the last step in tissue processing that results in permanent histological preparation suitable for microscopy, after adhesion of the sections on to the slide and appropriate staining of the tissue.

Vacuum

improves processing quality and aid in removal of trapped air from porous tissue

Agitation

Increases the flow of fresh fluids in and around the tissues.

True

True or False: An automatic tissue processing is a machine that fixes, dehydrates, clears, and infiltrates tissues thereby decreasing the time and labor needed during the processing of tissues resulting in a more rapid diagnosis with lesser technicality.

Tissue processor/ Dip and Dunk

• tissue is transferred from one container to another

• the reagents are stationary

Enclosed tissue processor/ Fluid transfer

• specimen is placed on one chamber only

• fluid comes in and out of the chamber

True

True or False: Hand hygiene is one of the most important means of preventing the spread of infection provided that it is achieved properly and when required

CDC/HICPAC

recommend the use of alcohol-based antiseptic hand cleaners (gel, foams, and rinses) in place of handwashing

plain soap and water

A routine hand-washing uses what to mechanically remove soil and transient bacteria?

antimicrobial soap

A 2-minute hand scrub uses what to remove or destroy transient microorganisms and reduce level of normal flora prior to surgical procedures?

downwards

when rinsing your hands, it should be in what motion?

respiratory and diarrheal infections

Washing hands can keep you healthy and prevent the spread of?

40-60 seconds

How many seconds should you do handwashing?

60%

if soap and water are not readily available, you can use an alcohol-based hand sanitizer that contains at least how many % alcohol?

False

True or False: Sanitizers get rid of all types of germs

True

True or False: Hand sanitizers may not be as effective when hands are visibly dirty or greasy

True

True or False: Hand sanitizers might not remove harmful chemicals like pesticides from hands

room temperature

Paraffin wax is solid at what temperature?

65 - 70 degrees Celsius

Paraffin wax melts at about?

56 - 58 degrees Celsius

most common melting point of paraffin wax

1.46

in aqueous mounting media, what is the refractive index of glycerin?

1.47

in aqueous mounting media, what is the refractive index of glycerin jelly?

1.43

in aqueous mounting media, what is the refractive index of Farrant’s medium?

1.52

in aqueous mounting media, what is the refractive index of Apathy’s medium?

1.524

in Resinous mounting media, what is the refractive index of Canada Balsam?

1.532

in Resinous mounting media, what is the refractive index of Dibutyl Phthalate and Xylene?

1.52

in Resinous mounting media, what is the refractive index of Xam?

1.544

in Resinous mounting media, what is the refractive index of Clarite/Clarite X?

1. tissue density and thickness

2. agitation

3. temperature

4. vacuum and pressure

Factors that impact the duration of tissue processing and extent of infiltration

Reduced pressure

increase infiltration rate and decrease the time needed to complete steps in tissue processing protocols

High pressure

facilitates infiltration of dense specimens with more viscous embedding media

False

True or False: in using the Autotechnicon, only 1-2 changes of wax are required to remove clearing agent and properly impregnate the specimen by constant agitation

transfer arm

moves the tissues from one station to another

control panel

sets and monitor processing parameters such as temperature, agitation, and processing time

safety features

alarms, temperature monitoring, emergency shut-off mechanisms

electric clock

controls the time needed for each processing step in Autotechnicon

processing chamber

• where the tissue specimens are placed

• designed to hold cassettes or containers, ensuring proper immersion in the processing reagents

reagent containers and dispensers

equipped with automatic dispensing mechanisms to ensure proper and consistent reagent delivery to the specimens

temperature control system

maintains the desired processing temperature throughout the entire processing cycle

Agitation mechanism

ensures thorough and uniform mixing of the specimens with the processing reagents

Fluid circulation system

facilitates movement and exchange of processing reagents within the processing chamber

Drying system

to aid in the removal of residual fluids from the processed tissues before embedding

1. fixation

2. dehydration

3. clearing

4. infiltration

processes involved with Autotechnicon

True

True or False: Temperatures in the range of 37° to 45°C, for a limited time can speed up fluid penetration and tissue processing protocols.

High temperature

tissues shrink and, become hard and brittle

Low temperature

reduces the rate of diffusion and increasing processing time

False

True or False: Maintain embedding waxes 2C – 4C above their melting points.

vertical or rotary oscillation

mechanism used in agitation factor of the Autotechnicon

True

True or False: Agitation Increases the flow of fresh fluids in and around the tissues. Tissues should be loosely packed in baskets to facilitate exchange of reagents & increase diffusion.

True

True or False: Spongy tissues are usually more rapidly infiltrated than hard and dense tissues

True

True or False: the cassette perforations should be perpendicular to the fluid flow.

4 to 6 um

the cut for routine histologic procedures with fine trimming

1. fixation

2. dehydration

3. clearing

4. infiltration/impregnation

5. embedding

6. trimming

7. section-cutting

8. staining

9. mounting

10. labelling

Steps in tissue processing

3.0 (length) x 2.5 (width) x 0.4 cm (thickness)

Standard tissue cassette dimension

False

True or False: Specimen placed in the cassette should not be more than 0.3 m in thickness