1. OVERVIEW OF METHODS USED IN HISTOLOGY

microscope

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

simple and compound

Two types of microscope

Resolving power

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

bright-field microscope

The microscope used by most students and researchers is the ______________________

– light source

– condenser lens

– stage

– objective lens

– ocular lens

The bright-field microscope has the ff parts:

bright-field microscope

in this type of microscope the object/specimen appear dark in bright background

-scanning - 40x

-LPO - 100x

-HPO - 400x

-OIO - 1000x

types of objective lens and their total magnification

phase contrast microscope

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

1. interference microscope

2. differential interference microscope

Two modifications of the phase contrast microscope are:

interference microscope

(modifications of the phase contrast microscope)

allows quantification of tissue mass

differential interference microscope

(modifications of the phase contrast microscope)

(using Nomarski optics), which is especially useful for assessing surface properties of cells and other biologic objects.

there is a buldging on the specimen and it it embosed.

dark-field microscope

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

dark-field microscope

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

Treponema pallidum

the bacterium the causes syphilis

Fluorescence microscope

is used to display autofluorescent molecules such as vitamin A and some neurotransmitters

Fluorescence microscope

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

antibody

if we’re looking for antigen; we use ____________ to detect

antigen

if we’re looking for antibody; we use ____________ to detect

Fluorochrome Fluorescein Isothiocyanate (FITC)

most common stain used in fluorescence microscopy

green

Fluorochrome Fluorescein Isothiocyanate (FITC) emits the color ________

confocal scanning microscope

allows visualization of a biologic specimen in three dimensions. It combines components of a light optical microscope with a scanning system to dissect a specimen optically

ultraviolet microscop

e uses quartz lenses with an ultraviolet light source. The image depends on the absorption of UV light by molecules in the specimen. results are usually recorded photographically.

ultraviolet microscope

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

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

• Also use to determine quantitatively the amount of DNAand RNAinindividual cells.

polarizing microscope

is a simple modification of the light microscope in which a polarizing filter (the polarizer) is located between the light source and the specimen, and a second polarizer (the analyzer) is located between the objective lens and the viewer

polarizing microscope

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

it is important in disease to differentiate crystal arthritis

1. transmission electron microscope

2. scanning electron microscope

two kinds of EMs

TEM

light passes through the specimen. uses the interaction of a beam of electrons with a specimen to produce an image

scans the surface, if there are appendages. the electron beam does not pass through the specimen but is scanned across its surface

viro: nano : electron microscopy

viro: ______: ________

bact : _______ : _________

bact : micro : bright field

1. Fixation

2.Dehydration

3. Clearing

4. Infiltration

5.Embedding

6. Trimming

7. Section-Cutting

8. Staining

9. Mounting

10. Labeling

enumerate histopathologic techniques:

Fixation

(histopath techniques)

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

• To preserve cell and tissue structure

Fixation

(histopath techniques)

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

10% neutral buffered formalin

(histopath techniques)

best general fixative that is used in fixation

20:1

(histopath techniques)

ideal fluid to tissue ration in fixation

Dehydration

(histopath techniques)

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

alcohol-ethyl alcohol (ethanol)

(histopath techniques)

common agent in dehydration

10:1

(histopath techniques)

ideal ratio in dehydration

Clearing/Dealcoholization

(histopath techniques)

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

Clearing/Dealcoholization

(histopath techniques)

it nakes he specimen clear and higher refractive index

xylene

(histopath techniques)

common agent in clearing

10:1

(histopath techniques)

ideal ratio in clearing

Infiltration / Impregnation

(histopath techniques)

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

-paraffin / melted paraffin

-celloidin

-gelatin

-ester wax

(histopath techniques)

common agent used in infiltration

25:1

(histopath techniques)

ideal ratio in infiltration

Embedding/ Casting

(histopath techniques)

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

false

true or false

the same medium is used in infiltration of tissue is the same medium used for embedding.

Trimming

(histopath techniques)

The resulting paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome. Furthermore, it is used to fit to the tissue block holder of microtome.

tissue block

(histopath techniques)

the product in embedding / casting is called

Section-Cutting

(histopath techniques)

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

tissue ribbon

(histopath techniques)

product in section-cutting

-rotary

-base-sledge

-rocking

-sliding

-freezing (cryostat) = used in enzyme; doesn’t need preserving: frozen section

(histopath techniques)

types of microtome

Staining

(histopath techniques)

to make various components conspicuous and permit distinctions to be made between them

basophilic

(histopath techniques)

Cell components with a net negative charge (anionic) stain more readily with basic dyes and are termed

acidophilic

(histopath techniques)

Cationic components have affinity for acidic dyes and are termed as

hematoxylin and eosin

(histopath techniques)

common agent in staining

hematoxylin

(histopath techniques)

is a stain used for negatively charges or basic specimens. it dyes the DNA or nucleus blue to black.

eosin

(histopath techniques)

is a stain used for acidic or positively charged specimens. it dyes the cytoplasm pale pink

Mounting

(histopath techniques)

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

canada balsam = derived from Abies balsamea

(histopath techniques)

commonly used mounting media

Labeling

(histopath techniques)

Tissue slides are labeled on the frosted areas with assigned tissue numbers or codes

pencil

(histopath techniques)

in labeling use

a. marker

b. pencil

Autoradiography

•is a method of 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

Cell and Tissue Culture

allows the direct observation of the behavior of living cells under a phase contrast microscope

Cell culture

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

false

true or false

all microorganism can grow in petra dish

Enzyme Digestion

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

periodic-acid-schiff

diastase or amylase

give the stain and enzyme to identify glycogen

fuelgen stain (red)

DNAse

give the stain and enzyme to identify DNA

basic dyes (methyl green-pyronin)

RNAse

give the stain and enzyme to identify RNA

DNAse, 0.2 M Tris Buffer, pH 7.6, distilled water

reagents needed in enzyme extraction of DNA

potassium dichromate

in enzyme extraction of DNA avoid the fixative ___________

RNAse, distilled water

reagents used in enzyme extraction of RNA

potassium dichromate and mercuric chloride

in enzyme extraction of RNA avoid the fixative ___________

Enzyme Histochemistry

used to diagnose storage diseases and answers the question “why is there accumulation?”, thus it uses cryostat

phosphatases, dehydrogenases, and peroxidases

enumerate enzyme classes:

Perls' Prussian blue reaction

for iron (used to detect the iron storage diseases hemochromatosis and, hemosiderosis)

PAS-amylase and alcian blue reactions

for glycogen and glycosaminoglycans (to detect glycogenosis and mucopolysaccharidosis

Immunohistochemistry

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

1. direct immunohistochemistry

2. indirect immunohistochemistry

two types of immunohistochemistry

Direct immunohistochemistry

uses primary antibody

Indirect immunohistochemistry-

uses secondary antibody. the secondary antibody is labeled

1. polyclonal antibodies

2. monoclonal antibodies

Two types of antibodies used in immunocytochemistry:

polyclonal antibodies

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

monoclonal antibodies

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

monoclonal

polyclonal

advantages polyclonal antibody

• Short production time and low cost.

• Highly stable

• High affinity Tolerant of minor changes of antigen.

• are less sensitive to antigen changes

disadvantages of polyclonal antibodies

• Prone to batch variability.

• Multiple epitopes make it important to check immunogen sequence for any cross-reactivity.

advantages of monoclonal antibodies

• Highly specific recognition of only one epitope of an antigen

• Immortal hybridoma cell lines have the ability to produce unlimited quantities of antibodies

• Minimal cross-reactivity

disadvantages of monoclonal antibodies

• it takes time and requires high technical skills.

• They can produce large amounts of specific antibodies but may be too specific to detect in across a range of species.

• Vulnerable to the change of epitope

Hybridization

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

In situ hybridization (ISH)

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

– Oligonucleotide probes

– Single- or double-stranded DNA probes

– RNA probes

Several nucleotide probes used in in situ hybridization

– radioactive isotopes

– digoxigenin

– biotin

Labels used for complementary probes: