HISTOLOGY AND ITS METHODS Flashcards by Mark James Mateo | Brainscape

Q

is an instrument that magnifies an image and allows visualization of greater detail than is possible with the unaided eye

A

Microscope

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Q

 Single lens
 Magnifying Glass

A

Simple Microscope

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Q

 Systems (multiple) of lens
 Used in Laboratories

A

Compound Microscope

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Q

is the ability of a microscope lens or optical system to produce separate images of closely positioned objects

smallest detail a microscope can resolve

A

Resolving Power

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Q

ability of the microscope to distinguish details. To
detect 2 objects as different objects.

A

Resolution

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Q

ability of microscope to see small objects seem larger

A

Magnification

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Q

(EYE VS INSTRUMENT RESOLUTION)

Human eye

A

0.2 mm

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Q

(EYE VS INSTRUMENT RESOLUTION)

Bright-field microscope

A

0.2 μm

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Q

(EYE VS INSTRUMENT RESOLUTION)

Scanning Electron Microscope (SEM)

A

2.5 nm

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Q

(EYE VS INSTRUMENT RESOLUTION)

Transmission Electron Microscope (TEM) - Theoretical

A

0.05 nm

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Q

(EYE VS INSTRUMENT RESOLUTION)

Transmission Electron Microscope (TEM) - Tissue Section

A

1.0 nm

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Q

(EYE VS INSTRUMENT RESOLUTION)

Atomic Force Microscopy

A

50.0 pm

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Q

Distance Between Resolvable Points

A

Human Eye > Bright-field microscope> SEM> TEM (Theoretical) > TEM (Tissue Section) > Atomic force Microscopy

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Q

: 1 x 10 ^-1000 in M

A

Micrometer

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Q

1 x 10 – 9000 in M

A

Nanometer

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Q

The most used microscope of students and researchers

A

Bright-field Microscope (LIGHT)

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Q

the one providing source of light

A

light source

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Q

allows you to adjust the intensity of the light coming into the object of interest

A

condenser lens

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Q

holds the microscope slide that you are viewing

A

stage

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Q

further magnify the image that you
see in the ocular lens

A

objective lens

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Q

The eyepiece, where we observe our specimen. Has a magnification of 10x.

A

Ocular lens

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Q

Scanning Total Magnification

A

4x * 10x = 40x

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Q

Low Objective Lens Total Magnification

A

10x * 10x = 100x

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Q

High Objective Lens Total Magnification

A

40x * 10x = 400x

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Q

Oil Immersion (OIO) Total Magnification

A

100x * 10x = 1000x

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Q

enables examination of unstained cells and tissues and is especially useful for living cells

A

Phase Contrast Microscope (light)

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Q

Allows quantification of tissue mass

A

Interference microscope

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Q

Useful for assessing surface properties of cells and other biologic objects

3D Image

A

Differential Interference Microscope

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Q

Object will appear bright in a dark background

Is useful in examining autoradiographs, in which the developed silver grains appear white in a dark background.

A

Dark-Field Microscope

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Q

Clinically it is useful in examining urine for crystals and in demonstrating specific bacteria particularly Treponema pallidum.

Beneficial in Microbiology: allow you to appreciate the shape of the bacteria (rods or curve rods shape) and their movements.

A

Dark-field Microscope

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Q

Indicator of syphilis

A

Treponema pallidum

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Q

is a sexually transmitted infection (STI) that can cause serious health problems without treatment.

A

Syphilis

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Q

Indication of cystinuria

Piatos like appearance of crystals

A

Cysteine Crystals

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Q

is an inherited disease that causes stones made of the amino acid cystine to form in the kidneys, bladder, and ureters.

A

Cystinuria

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Q

Used to display autofluorescent molecules (Fluorochromes) such as vitamin A and some neurotransmitters

A

Fluorescence Microscope

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Q

act as stains to view antibody and antigen complex

A

Fluorochromes

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Q

Widespread application is in the detection of antigens or antibodies in immunocytochemical staining procedures

Antibodies can either be polyclonal or monoclonal to detect antigens in the tissue.

A

Fluorescence Microscope

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Q

-Tolerant to antigen epitope change since these heterogenous antibodies are directed against different types of epitopes in an antigen.

-prone to cross-reactivity

A

Polyclonal

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Q

More specific than polyclonal antibodies. Monoclonal antibodies are binding specific or single epitope type in an antigen unlike with polyclonal antibodies.

-with minimal or less chance of cross-reactivity.

A

Monoclonal

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Q

The direct fluorescence antibody test of brain tissues from animals suspected to be infected with rabies.

A

Positive dFA: Apple green fluorescence
Negative dFA: No bright objects

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Q

the one utilized to detect the
presence of rabies antigen in the tissues. This is particularly called Anti-rabies Antibodies

A

Reagent antibodies

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Q

against the rabies antigen

A

Anti-rabies antibodies

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Q

utilized to label the antibodies to visualize if there is really a binding that happened.

A

Fluorochromes

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Q

if the antigen is present in the tissue. Indicating infection of rabies.

A

There is a binding

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Q

if there is no antigen present in the tissue.

A

There will be no binding

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Q

Orange fluorescence

A

Acridine Orange

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Q

Yellow fluorescence

A

Auramine O

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Q

Bright red fluorescence

A

Rhodamine B

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Q

Apple green fluorescence

A

Fluorescein Isothiocyanate (FITC)

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Q

Using one antibody to detect antigen.

A

Direct Fluorescence Assay

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Q

Using two antibodies to detect antigen

A

Indirect Fluorescence Assay

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Q

Allows visualization of a biologic specimen in three dimensions (3D configuration)

It combines components of a light optical microscope with a scanning system to dissect a specimen optically allowing you to visualize the minute organism

A

Confocal Scanning Microscope

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Q

uses quartz lenses with an ultraviolet light source.

The image depends on the absorption of UV light by molecules in the specimen

A

Ultraviolet Microscope

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Q

may achieve a resolution of 0.1 m and resembles the workings of a spectrophotometer;

results are usually recorded photographically.

is useful in detecting nucleic acids, specifically the purine and pyrimidine bases of the nucleotides

A

Ultraviolet Microscope

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Q

It is also useful for detecting proteins that contain certain amino acids

Also use to determine quantitatively the amount of DNA and RNA in individual cells.

A

Ultraviolet Microscope

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Q

Identify crystals to identify the exact dxs

It uses the fact that highly ordered molecules or arrays of molecules can rotate the angle of the plane of polarized light

A

Polarizing Microscope

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Q

(POLARIZING MICROSCOPE)
located Between the light source and the specimen.

A

Polarizer

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Q

(POLARIZING MICROSCOPE)
located between the Ocular and the
Objective lens.

A

Analyzer

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Q

has an additional feature as compared to the Light microscope. Which is the addition of 11 filters

A

Polarizing Microscope

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Q

can provide morphologic and analytic data on cells and tissues:

A

Electron Microscope

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Q

Two kinds of EMs

A

Transmission electron microscope (TEM)
Scanning electron microscope (SEM)

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Q

uses the interaction of a beam of electrons with a specimen to produce an image. Enables you observe structures found in the inner portion of the organism, it’s organelles.

A

Transmission Electron Microscope

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Q

[T for through (nakikita through the specimen)

A

Transmission Electron Microscope

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Q

the electron beam does not pass through the specimen but is scanned across its surface. Allows you to appreciate its appendages.

A

Scanning Electron Microscope

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Q

[S is for surface (nakikita lang surface ng specimen)

A

Scanning Electron Microscope

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Q

PREPARATION OF TISSUES FOR STUDY

A

Fixation
Dehydration
Clearing
Infiltration
Embedding
Trimming
Section-Cutting
Staining
Mounting
Labeling

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Q

To preserve cell and tissue structure in a life like manner.

Small pieces of tissues are immersed in a solution called fixative after removal from the body

A

Fixation

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Q

best general fixative

A

10%neutral buffered formalin

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Q

Self Digestion

A

Autolysis

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Q

Preserve tissue in a life-like manner

To avoid tissue digestion by enzymes present within the cells or bacteria

A

Fixation

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Q

Fixative ratio

A

20:1

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Q

Remove intracellular and extracellular water. To prepare it in absorbing solutions used in subsequent steps

Tissue is transferred through a series of increasingly concentrated alcohol solutions called as dehydrating agents , ending in 100%, which removes all water

A

Dehydration

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Q

Most common series (DEHYDRATION)

A

70%, 95%, 100% (absolute alcohol)

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Q

Small specimen (DEHYDRATION)

A

30% - pataas

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Q

Why is it in an increasing concentration? (DEHYDRATION)

A

to avoid tissue distortion

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Q

most commonly used alcohol in dehydration

A

Ethanol (Ethyl Alcohol)

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Q

Also known as Dealcoholization

removal of alcohol from the tissue

A

Clearing

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Q

Alcohol is removed in the tissue by immersing in a clearing agent.

Solutions makes tissues clear

A

Clearing or Dealcholoization

Q

also known as Impregnation

filling the spaces between tissue to make the tissue firm to facilitate cutting

A

Infiltration

Q

most commonly used for impregnation

A

Paraffin wax

Q

Tissue is then placed in melted paraffin until it becomes completely infiltrated with the substance.

To facilitate cutting, make tissue stable, and fill spaces

A

Infiltration or Impregnation

Q

tissues that are fresh and stained. Used
siya for living cells

A

Vital stain

Q

ang purpose is to remove the unbound
antibodies. Diba, we have a single epitope of rabies Ag, so yung mga other Ab don’t have any Ag to bind to kasi iisa lang naman yung rabies Ag. Dahil naka bind yung anti-rabies Ab sa Ag even after washing, andun pa rin. So ang matitira na lang would be the Ab na nagbind sa rabies Ag. So ang result would be positive.

A

Washing

Q

medium used for large hollow organs

A

Celloidin

Q

medium used for brain tissue

A

Wet Celloidin

Q

medium used for eye organ

A

Dry Celloidin

Q

used for delicate specimens such as prostrate tissue

A

Gelatin

Q

Also known as casting

The paraffin- infiltrated tissue is placed in a mold with melted paraffin and allowed to harden

The medium used in infiltration of tissue is the same medium used for this.

A

Embedding

Q

contains the paraffin infiltrated tissue and
the paraffin mold

A

Tissue block

Q

Trim the edges to create a perfect block to fit in the microtome

The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome

A

Trimming

Q

cuts the tissue block into thinner section

A

Microtome

Q

Most common, invented by Minot

A

Rotary Microtome

Q

– more dangerous because the knife is the one that is moving across the tissue block

A

Sliding microtome (Adams)

Q

Rocking microtome

A

Trefall

Q

obsolete and replaced by cryostat

A

Freezing microtome (Queckett)

Q

Cut the tissue block into thin films

Tissue block is sliced into thin films using a microtome which are placed on glass slides and allowed to adhere, deparaffinized, and stained.

A

Section-Cutting

Q

thin films that has impression of the tissues

A

Tissue ribbon

Q

straighten the tissue ribbon

A

Water Bath

Q

remove excess paraffin and prepare for staining

A

Oven

Q

Why is dewaxing important?

A

Because paraffin wax is not miscible to the stain. If paraffin wax is is not removed, the stain will have difficulty to penetrate the tissue.

Q

The concentration of alcohol in this step is in decreasing concentration. From absolute alcohol going down to up to 70% alcohol

A

Rehydration

Q

Methods of staining have been devised that not only make the various components conspicuous but also permit distinctions to be made between them.

A

Staining

Q

cell components with a net negative charge (anionic), they have affinity and stain more readily with basic dyes. (ex: nucleic acid, nucleus)

A

Basophilic

Q

cationic components, they stain more readily with acidic dyes (ex: proteins)

A

Acidophilic

Q

Commonly used staining technique

A

Hematoxylin and Eosin (H&E)

Q

stains DNA, RNA-rich portions of
cytoplasm, and matrix of cartilage. It produces a dark blue or purple color. Color of the nucleus would be blue-black

A

hematoxylin

Q

acts as a counterstain, meaning it’s a single dye applied separately to distinguish additional features more. It stains the cytoplasm pale pink

A

eosin

Q

special stains used for lipids

A

Sudan dyes

Q

used for carbohydrates in the demonstration of glycogen

A

Periodic acid-Schiff (PAS)

Q

used for nucleic acids

A

Feulgen technique

Q

DNA and RNA

A

Methyl Green Pyronin (MGP)

Q

Stained tissue slides are mounted with a cover slip using a mounting media

A

Mounting

Q

Purpose of mounting

A

to cover and protect the tissue, to avoid fading of the stain

Q

can be natural or synthetic

A

Mounting Medium

Q

(MOUNTING MEDIA)
is used in the lab

A

Synthethic Medium

Q

(MOUNTING MEDIA)
– include Canada balsam from Abies balsamea

A

Natural Medium

Q

higher refractive index (1.518); avoid fading and protection

A

Cover slip

Q

resinous mounting media and the most commonly used mounting medium

A

Canada balsam

Q

Canada balsam refractive index

A

Refractive index: 1.54-1.55

Q

slides are labelled on the frosted areas with assigned tissue numbers or codes called as “accession number”

A

Labelling

Q

(FLUID TO TISSUE RATIO)
Dehydration

A

10:1

Q

(FLUID TO TISSUE RATIO)
Clearing

A

10:1

Q

(FLUID TO TISSUE RATIO)
Infiltration

A

25:1

Q

Is a method for localizing newly synthesized
macromolecules in cells or tissue sections

It makes use of a photographic emulsion placed over a tissue section to localize radioactive material within tissues

A

Autoradiography

Q

With either______________________, autoradiography permits unique studies of processes such as tissue growth or cellular pathways of macromolecular synthesis

A

TEM or light microscopy

Q

indicate the cells or regions of cells in which specific macromolecules were synthesized prior

A

Silver grains

Q

Cells can be grown from newly explanted tissues or as long-established lines and can be examined in the living state by __________

A

Phase-contrast microscopy

Q

It allows the direct observation of the behavior of living cells

A

Cell and Tissue Culture

Q

refers to a preparation of cells and tissues that are obtained directly from a tissue or organ and placed in a clear dish under sterile conditions

A

Primary cell cultures

Q

refers to a process by which certain changes, often related to oncogenes, can promote the cell immortality

A

Transformation

Q

has been widely used for the study of the metabolism of normal and cancerous cells and for the development of new drugs

A

Cell culture

Q

is also useful in the study of microorganisms that grow only within cells such as viruses, mycoplasma, and some protozoa

A

Cell culture

Q

Cervical cancer cells from a px later identified as__________, who died from a dsx in 1951, were used to establish one of the first cell lines, _______, which are still used in research or cellular structure and function throughout the world.

A

Henrietta Lacks; “HeLa cells”

Q

can be used to confirm the identity of the stained material such as glycogen, DNA, or RNA

A

Enzyme Digestion

Q

removing of something to confirm the presence of something particularly RNA or DNA.

A

Enzyme Extraction

Q

Enzyme extraction of dna

potassium dichromate and will inhibit digestion, and should be avoided

A

Fixation

Q

enzyme extraction of dna

deoxyribonuclease, 0.2 M Tris buffer, pH 7.6, distilled water

A

Reagents

Q

Enzyme extraction of dna
Results after staining with Fuelgen method:
Test Section

A

DNA - negative

Q

Enzyme extraction of dna
Results after staining with Fuelgen method:
Control Section

A

DNA -red

Q

Methyl green will stain your DNA _____

A

green

Q

Pyronin will stain your RNA ___

A

red

Q

a tissue section known to have RNA and DNA.

A

Control Section

Q

If there is NO green and red staining the control section, your control section and stain may be ________

A

deteriorated

Q

Enzyme extraction of rna

fixation

A

: potassium dichromate and mercuric chloride will inhibit digestion and should be avoided

Q

Enzyme extraction of rna

reagents

A

RNAse, distilled water

Q

Enzyme extraction of rna

Results after staining with methyl green-pyronin method:

Test section:

A

RNA-negative ; DNA-green

Q

Enzyme extraction of rna

Results after staining with methyl green-pyronin method:

Control section:

A

RNA-red ; DNA-green

Q

Stain the glycogen with Periodic acid-Schiff (PAS)
Color:

A

Magenta

Q

enzyme that removes RNA in the cell leaving the DNA, when stained by the Methyl Green-pyronin, the result would be RNA negative and DNA Green.

A

RNAse

Q

digests glycogen

A

Amylase

Q

high in homogentisic acid

absence of homogentisic acid oxidase causes

A

Alkaptonuria

Q

Used to look into the activity of enzymes, where exactly in the cell or tissues does this enzyme do its activity or its function (localization of the enzyme)

Used to detect enzymatic products

Uses frozen tissue sectioned in a specialized microtome called cryostat also known as freezing microtome (the conventional counterpart of cryostat, it is like a rotary microtome inside a refrigerator)

A

Histochemical Techniques

Q

Enzyme classes

A

phosphatases,
dehydrogenases,
peroxidases

Q

remove phosphate groups from macromolecules

A

phosphatase

Q

transfer hydrogen ions from one substrate to another

A

Dehydrogenases

Q

promotes the oxidation of substrates with the transfer of hydrogen ions to hydrogen peroxide

A

Peroxidases

Q

Detects iron storage diseases (hemochromatosis/ hemosiderosis)

Detects which type enzyme has missing/decreased activity

A

Perl’s Prussian blue reaction

Q

Used to detect glycogenosis and mucopolysaccharidosis

A

PAS-amylase and alcian blue reactions

Q

Used to detect sphingolipidosis

Lacking enzyme activities results to the accumulation of a certain substance that is needed to be converted by an enzyme. That is why precursor substances are stored and fail to undergo metabolism, hence, accumulation.

A

Reactions for lipids and sphingolipids

Q

Using primary antibody to detect presence of an antigen

A

Direct Immunohistochemistry

Q

Using secondary antibody to detect the binding of the primary antibody with the antigen

A

Indirect Immunohistochemistry

Q

Detecting a mic and an infecting microorganism using complementary nucleotidases

A

Hybridization Techniques

Q

is based on specific reactions between an antigen or antibodies labeled with visible markers, often fluorescent compounds for light microscopy and gold articles for TEM

A

Immunohistochemistry

Q

Primary Antibody

we can detect the presence of an antigen through the labelled primary antibody

A

Direct IHC

Q

primary (not labelled) and secondary Antibody (labelled)

It is the secondary antibody that is labelled that denotes the presence of the antigen through detecting the antibody-antigen complex.

A

Indirect IHC

Q

Highly sensitive compared to direct technique

Gives more intense result than direct technique

A

Indirect IHC

Q

2 types of antibodies used in immunocytochemistry

A

Polyclonal Ab
Monoclonal Ab

Q

Mixture of heterogenous which are usually produced by different B cell clones in the body of an immunized animal

Two of the different B cells are against the antigen but they produce antibodies that attack different epitope

A

Polyclonal Antibodies

Q

Are generated by identical B cells which are clones from a single parent cell (immortalized Ab-producing cell lines)

A

Monoclonal Antibodies

Q

an immortalized cell line capable of producing monoclonal antibodies.

A

hybridoma

Q

APPLICATIONS OFIMMUNOCYTOCHEMISTRY

A

Prognostic markers in cancer
Tumors of uncertain histogenesis
Metastasis
Response to treatments
Infections
Neurodegenerative diseases
Muscle diseases
Brain trauma

Q

allows the specific identification of sequences of DNA or mRNA by hybridizing the sequence of interest to a complementary strand of a nucleotide probe

Commonly performed with nucleic acids in solution

A

Hybridization

Q

solution of nucleic acid are applied directly to cells and tissue sections

A

In situ hybridization (ISH)

Q

the label that is attached to identify the nucleic acid sequence

A

Reporter Molecule

Q

Methods that will be used to view the result depends on the type of reporter molecule:

Fluorochromes
Radioisotopes
Chemiluminescent

A

Fluorochromes – fluorescent microscope
Radioisotopes – autoradiography
Chemiluminescent – emission of light or Chemiluminescence assay

Q

Several nucleotide probes used in ISH:

A

Oligonucleotide probes
Single- or double-stranded DNA probes
RNA probes

Q

Labels used for complementary probes:

A

radioactive isotopes (e.g., 32P, 35S, 3H)
digoxigenin
biotin

Q

a specifically modified nucleotide

A

digoxigenin

Q

(a commonly used covalent multipurpose
label)

A

biotin

Q

fluorescent dyes that are combined with nucleotide, making it possible to visualize multiple probes at the same time

A

Fluorescence in situ hybridization (FISH) procedure

Q

is used to simultaneously examine
chromosomes, gene expression, and distribution of gene products such as pathologic or abnormal proteins

A

Fluorescence in situ hybridization (FISH) procedure

Q

Many steps in tissue processing, slide preparation, and staining can introduce minor artifacts such as spaces and precipitates that are not normally present in the living tissue and must be recognized

A

Interpretation of structures in tissue sections

Q

read using Fluorescence Microscope, the result will be in fluorescence.

A

Fluorescent labels

Q

can be read using Colorimetric Assay- result is indicated by a colored change.

A

Enzyme label

Q

read using Radioimmuno Assay technique

A

Rage reactive particles label (Iodine 125)

Q

active sites of antigen. Site where antibodies are binding the antigen

A

Epitope

Q

is the one that is being detected, the antigen in which the primary antibody is binded

A

Desmin

Q

: probe nucleotide sequence should be complementary to the sequence of the unknown.

A

Hybridization Positive

Q

probe is not complementary to the sequence

A

Hybridization Negative

Q

read the result using Autoradiography
32p – labelled probe
Detected by black areas on film

A

Radioactive Reporter

Q

: read the result using Colorimetric Detection
Biotin– labelled probe
Detected by changes in color

A

Biotin-Avidin Reporter

Q

read the result using Chemiluminescent Assay
Acridinium – labelled probe
Detected by capturing emission of light

A

Chemiluminescent Reporter

Q

orange probe (LSI 21) is locus specific for
chromosome ___

A

21

Q

____ probe (LSI 13) is locus specific for
chromosome 13

A

green