HISTOLOGY
Practical Manual
BACHELOR OF SCIENCE IN RADIOGRAPHY AND IMAGING TECHNOLOGY (BS-RIT)
BY DR. AAMIR SHAHEEN
Certificate of Completion
This is to certify that _________________________________________ Reg No. ______________ of
BS-RIT/BS-MUT First Semester (Fall 2024) has carried out all the necessary practical work for the
subject of Basic Anatomy for the session 2024-2028.
Remarks
Dr. Aamir Shaheen
Senior Lecturer
Ibadat Internation University, Islamabad
Pakpattan campus, Pakistan
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Index
Sr. No. Practical Date Teacher Signature
1. Basic Principles of Histology- Tissue Processing and Staining
2. Introduction and using Microscope to view Histological Slides
3. To draw and view Histological slides of Epithelial tissue
4. To draw and view Histological slides of Connective tissue
5. To draw and view Histological slides of Muscle tissue
6. To draw and view Histology Features of Cartilage
7. Studying Histology Features of Stomach
8. Studying Histology Features of Small Intestine
9. Studying Histology Features of Tongue
10. Studying Histological Features of Spinal cord
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BASIC PRINCIPLES OF HISTOLOGY
SYSTEMIC ANATOMY
COURSE CODE: RIT-421, MUT-2393
CREDIT HOURS: 01
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Practical No. 1
Basic Principles of Histology - Tissue Processing
and Staining
Histology (microanatomy) is the study of the human body at a tissue or sometimes at a cellular
level. As disease processes occur at the molecular/cellular levels, manifestations of the disease
processes are readily and economically observed at the tissue level using a microscope.
To examine tissues under a microscope, several steps to acquire, fix, and stain the samples are
necessary in each of the preparatory steps, a variety of artifacts may be introduced to the tissue
samples.
Aim of Tissue Processing: The aim of tissue processing is to embed the tissue in a solid medium
firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut,
and yet soft enough not to damage the knife or tissue
Major steps in Tissue Processing
1. Fixation - preservation of tissues in its original condition.
2. Dehydration - removal of water from tissues.
3. Clearing -infiltration of paraffin solvent.
4. Embedding -infiltration of paraffin wax
5. Microtomy - preparing thin slices of tissues.
6. Mounting & Staining - arranging tissues on slides and colouring of tissues.
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Hematoxylin & Eosin Staining:
Aim: To perform Hematoxylin & Eosin stain for observation histological studies
Principle: The acidic component of the cells has the affinity to basic dye and basic component of
the cells has the affinity to acidic dye. In hematoxylin and eosin stain, Hematoxylin stains the
acidic part of the cell, i.e. Nucleus. So, hematoxylin is called as nuclear stain. While eosin acts as
acidic stain and bind with the basic part of the cell, i.e. cytoplasm of the cells staining pink.
Materials required: H&E Reagents, Staining Racks, Sodium Bicarbonate, HCI, 1% Acid
Alcohol, DPX, Xylene, Glass Slides, Cover Slips, PPE
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Procedure:
1. Sections are first deparaffinised (removal of wax) by placing the slide in a jar of xylene for 10-
15 minutes.
2. As haematoxylin is a water-based dye, the sections before staining are rehydrated which is done
by passing the sections in a series of descending grades of alcohol and finally bringing the
section to water.
3. Place the slide in haematoxylin stain for 8-10 minutes and then rinse in water.
4. Differentiation (i.e. Selective removal of excess dye from the section) is done by putting the
slide in a solution of 1% acid alcohol for 10 seconds and then rinse in water.
5. Counterstain with 1% aqueous solution of eosin for 30 seconds to 1 minute and then dip in tap
water.
6. Before mounting, the sections have to be dehydrated which is done by passing the sections in
a series of ascending grades of alcohol and finally cleared in xylene, 2-3 dips in each solution.
7. Mount and coverslip.
8. Currently, modern laboratories employ automated programmable auto-stainers.
Results
• Nuclei appears Blue
• Cytoplasm appears Pink
• Muscle, collagen, RBCs, keratin appears Pink
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Practical No. 2
Studying and Using Microscope
Aim: To study parts of a compound microscope.
Principle: Magnified and real images of minute objects are obtained using combination of lenses.
A compound microscope is a complex assemblage of such lenses enabling highly magnified images
of the microscopic living organisms and intricate details of cells and tissues. A Binocular compound
microscope is normally used in laboratory.
Requirement: A compound microscope, glass slides, cover slip, prepared histological slides of
human specimen.
Components of the Compound Light Microscope
(A) Ocular Lens (also known as an eyepiece): located at the top of microscope. It is 10X
magnifying lens. A monocular microscope has one whereas a binocular microscope has two ocular
lens or eye-piece.
(B) Revolving Nose-piece: located above the stage. It holds the objective lens together.
(C) Objective lenses: Held in place above the Used stage by the revolving Nose-piece, used to
magnify the specimen place on stage. Objective lens is of 4 types in a binocular compound
microscope having magnification of 4X, 10X, 40X and 100X.
(D) Stage: Flat surface on which specimen is place is named as stage. It is located directly beneath
the Nose-piece and objective lenses. Stage adjustment knobs are used to move the microscopic
slide on the stage.
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(E) Condenser: Present beneath the stage. Contains a lens system that is used to focus the light
from light source onto specimen.
(F) Iris diaphragm control arm: It is present on the condenser. It is used to adjust the amount of
light passing through the condenser.
(G) Base: the lower most part of the microscope which carry light source is known as Base of
microscope.
(H) On/off switch: Present on the side of the base. Turns the light source on and off.
(I & J) Fine and coarse adjustments: on the arm of the microscope near the base which are used
to focus the objective lenses
(K) Arm: Supports the binocular body and the revolving nosepiece; held with one hand when
carrying the microscope, with the other hand beneath the base to support the weight of the
microscope.
Procedure:
1. Place the microscope on the working table and remove dust by wiping the body with a silk
cloth.
2. Clean the lenses with lens cleaning fluid and lens cleaning paper.
3. Identify the various parts of the microscope.
4. Take a prepared histological slide of human specimen or a temporary preparation made by you.
5. Keep it on the stage, fix the slide with stage clips.
6. Turn on the light source by on/off switch, adjust the amount of light through condenser and
light adjustment knobs.
7. Select the objective lens through nose-piece of a particular magnification.
8. Focussing is done by moving the coarse adjustment and fine adjustment knobs.
9. Observe the specimen through eye-piece.
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10. Gradually increase the magnification by changing objective lens through nose-piece.
Magnification:
Magnification by a microscope is a multiple of the X value of the lenses of the eye-piece and
objective. For example, a 5X eye piece and a 40X objective will magnify the image 5 x 40 = 200
times the size of the object. Similarly, when a 10X eye piece and a 40X objective are used,
magnifying power would be 10 x 40 = 400X. Generally, 1n a compound microscope, the eye piece
lenses are 10X or 15X and the objective lenses are 4X, 10X, 40X and 100X.
Precautions:
1. Always clean the lenses before and after using the microscope
2. For cleaning lenses, always use lens cleaning fluid and lens cleaning paper.
3. While observing, the objective lens should be carefully adjusted so as to avoid touching the
slide lest it breaks the slide.
4. Always put back the microscope in its case after use.
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Practical No. 3
To draw and view Histological slides of
Epithelial tissue
Aim: To observe different types of Simple epitheliums
Epithelium: A special type of tissue in which cells are in so close apposition that, a very small or
no extracellular matrix or space is present.
Simple Epithelium: It is a type of covering Epithelium in which there is only single layer of cells
are present. An inert membrane on which epithelial cells rest is named as Basement membrane.
Types of Simple Epithelium – On the basis of shapes of cells resting on basement membrane
Simple Squamous Epithelium – A single layer of thin flat cells with Nucleus is flat and central
Examples: Endothelium of blood vessels, Lymph vessels and Alveoli of Lungs.
Simple Cuboidal Epithelium – A single layer of cube or square shaped cells with Height and width of cells are
equal and Nuclei of cells are spherical and present in the center.
Examples: Small ducts of exocrine glands, Distal Convoluted tubules of Nephrons in kidney etc.
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Simple Columnar Epithelium – A single layer of pillar or column shaped cells with the height of cells is greater
than the width. Nucleus is oval and located towards base Sometimes this type of Epithelium has cilia.
Examples: Epithelial lining of Gastrointestinal Tract, In Gall bladder, bile duct etc.
Pseudostratified Epithelium – A Modified form of Simple Columnar Epithelium, in which nuclei of cells lie at
different levels giving false impression of having layers of cells.
Examples: Conducting part of respiratory system, such as Nasal Cavity, nasopharynx, trachea and bronchi.
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Aim: To observe Stained Smear of different types of Stratified Epithelium.
Stratified Epithelium: Stratified epithelium also known as multilaminar epithelium. A type of
covering epithelium which consists of 2 or more layers of cells is named to be stratified
epithelium.
Stratified Squamous Non-Keratinized Epithelium
Contains many layers of cells with basal layer resting on a basement membrane.
Has Basal layer of columnar cells, middle layer of polygonal cells & superficial layer of flattened cells.
Examples: This type of epithelium is present in Oral cavity, esophagus etc.
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Stratified Squamous Keratinized Epithelium
Has Basal layer of columnar cells, middle layer of polygonal cells & superficial layer of flattened cells. flattened
cells which are present in the superficial layer have keratin protein accumulated in them, and nucleus and other
organelles are lost due to this accumulation.
Examples. This type of epithelium is present in Epidermis (outmost layer of skin), Hair and nails etc.
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Transitional Epithelium
Microscopic structure of this type of epithelium varies greatly depending on the states of bladder. As Number of
layers of cells in this epithelium vary.
3 to 6 layers of cells are present.
Basal layer of cuboidal cells, middle layer of polygonal cells and superficial layers of dome shaped cells with
single nucleus.
Examples. This type of epithelium is present in Urinary tract i.e. Ureter, Urinary bladder and urethra.
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Practical No. 4
To Draw & View Histological Slides of
Loose and Dense Connective Tissue
Aim: To observe Stained smear of different types of Connective tissue.
Loose Connective Tissue: For loose connective tissue, another name “areolar” connective tissue
is also used. The word "areolar” means a small open area. Fibers and cells are far apart in larger
amount of ground substance, giving the impression that there is a lot of space present fibers and
cells. Such type of connective tissue can be named as loose connective tissue.
Dense Connective Tissue: This is category of connective tissue proper, having a close packing
of fibers, with fewer cells as compared to areolar connective tissue and lesser amount of
amorphous ground substance.
Loose Connective Tissue
Larger amount of ground substance, giving the impression that there is a lot of space present fibers
and cells.
Common cells types include, Fibroblasts, Macrophages, Mast Cells and Adipocytes.
Fibers include, Collagen Fibers are most prominent, Elastic Fibers are also present and Reticular Fibers are
scarce.
Examples: Superficial fascia and Deep Fascia, Mesentery and Omentum.
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Dense Connective Tissue
Have less amount of ground substance having a close packing of fibers, with fewer cells as compared to areolar
connective tissue.
Cells are found in rows between bundles of Collagen fibers.
Collagen fibers are densely packed either exhibit a random or regular orientation.
Fibroblasts are the most prominent cells.
Examples: This type of connective tissue is present in Tendons, ligaments, vocal cords and Perichondrium.
Loose Connective Tissue Dense Connective Tissue
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Practical No. 5
To Draw & View Histological Slides of
Skeletal, Cardiac & Smooth Muscles
Aim: To observe Stained smear of Skeletal Muscles, Cardiac Muscles and Smooth Muscles.
Skeletal Muscles: The skeletal muscles are named as such because of the fact that they are
attached to skeleton of the body (which are bones and cartilages). These muscles show rapid and
unsustained contraction. These muscles are controlled by Somatic Nervous System.
Cardiac Muscles: Those muscles which are present in the heart myocardium and in some larger
blood vessels are called as cardiac muscles. These muscles show automaticity.
Smooth Muscles: Those muscle which appear smooth mostly associated with viscera are smooth
muscles. These cells show slow but sustained contraction. They are under control of Autonomic
Nervous System.
Skeletal Muscles
Identification Points
1. Unbranched long cylindrical fibers.
2. Multiple peripherally placed flattened nuclei.
3. Dark and light cross striations seen.
• Each cell is long cylindrical without branching.
• Fibers run parallel to each other.
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• Skeletal muscles have transverse striations which are seen as light (1) and dark (A) bands. Hence, they are
called striated muscles. Striations are not easily seen under low magnification.
• Dark and light bands are due to regular arrangement of actin and myosin filaments.
• Center of I – band is called Z line. Center of A – band is light H zone. Dark M line is seen in the center of
H zone.
• The part of myofibril between two adjacent Z lines is called sarcomere which is the functional unit of the
muscle.
• Skeletal muscle fiber or cell is covered by sarcolemma (cell membrane) and is multinucleated with
flattened peripherally positioned nuclei.
• Connective tissue covering of each muscle fiber is endomysium. Bundles of such fibers (fascicles) are
covered by perimysium. Entire muscle covered by epimysium.
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Cardiac Muscles
Identification Points
1. Short cylindrical fibers with central oval nuclei.
2. Branching of fibers seen.
3. Intercalated discs seen.
• Cardiac muscle fibers are small and cylindrical compared to skeletal muscle fibers
• Nuclei are central, single and oval.
• Muscle fibers are branched.
• Intercalated disc one of the unique features of cardiac muscle is the junction between adjacent cardiac
muscle cells. It is zig zag in appearance and made up of three types of cell junctions- desmosomes, gap
junctions and tight junctions. Allows electrical impulse to pass from one cell to another and makes the
cardiac muscle fiber a functional syncytium.
• Cardiac muscle is also striated but striations are no prominent under lower magnification.
• Cardiac muscle is involuntary, seen only in heart.
• It is highly vascular and innervated by autonomic nervous system.
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Smooth Muscles
Identification Points:
1. Smooth muscles cells are long, spindle shaped contractile cells.
2. When seen microscopically you will not find any striations.
3. Each smooth muscle fiber or cell contains single, rod shaped, central nucleus.
• The cytoplasm of smooth muscle cells or fibers contains, Contractile apparatus of thick and thin filaments,
Cytoskeleton of Intermediate Filaments. In addition, Few Mitochondria, endoplasmic reticulum, free
ribosomes, a small Golgi apparatus and some glycogen granules are present.
• On the surface of smooth muscles cells dense bodies are present.
• 2 types of smooth muscles are there on the basis of nerve supply multiunit and unitary smooth muscles.
• The arrangement of smooth muscles is like that, end of one muscle lie over the central component of lower
muscle cell.
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Practical No. 6
To Draw & View Histological Slides of Cartilage
Aim: To observe the stained smear of different types of cartilages
Hyaline cartilage
Identification Points
1. Typical chondrocytes in nests of 2-4 cells.
2. Homogenous basophilic matrix
3. Perichondrium present.
• Hyaline cartilage has typical ground glass appearance with homogenous bluish matrix.
• Chondrocytes lie within the lacunae. They are arranged in groups of 2-6 cells called cell nest. Apposing
of chondrocytes are flattened surfaces
• Matrix is basophilic due to presence of large number of proteoglycans and homogenous because the
refractive index of collagen fibres and ground substance is same.
• Matrix around the cell nest looks denser and dark staining and is called territorial matrix. It is the newly
formed matrix secreted by the chondrocytes.
• In between the nests is the inter-territorial matrix. which is pale staining.
• Hyaline cartilage is surrounded by perichondrium which is a two layered structure - Inner cellular and
outer fibrous layer.
o Fibrous layer made of collagen fibres.
o Cellular layer is made of chondrogenic cells responsible for appositional growth of cartilage.
Example: Articular cartilage, fetal skeleton, costal cartilage, epiphyseal plate.
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Aim: To observe the stained smear of Fibrous cartilage
Fibrous Cartilage
Identification Points
1. Predominantly made of bundles of collagen fibers.
2. Scanty chondrocytes arranged in rows.
3. No perichondrium.
• Chondrocytes are very few and small in size
• Cells are arranged in rows located inside lacunae in between bundles of coarse collagen fibers
• Perichondrium is absent.
Example: Intervertebral disc, cartilage of pubic symphysis, menisci of knee joint, articular disc of
temporomandibular and sternoclavicular joints
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Aim: To observe the stained smear of Elastic cartilage
Elastic Cartilage
Identification Points
1. Numerous chondrocytes but fewer cell nests.
2. Matrix with elastic fibers.
3. Perichondrium present
• Chondrocytes are large arranged singly or in groups of two within the lacunae.
• Number of chondrocytes is more in elastic cartilage.
• Matrix in elastic cartilage is eosinophilic with lot of branching elastic fibers.
• Elastic cartilage is surrounded by perichondrium which is a two layered structure-Inner cellular and
outer fibrous layer.
o Fibrous layer made of collagen fibers.
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o Cellular layer s made of chondrogenic cells
• Responsible for appositional growth of cartilage
Examples: epiglottis, Eustachian tube, external acoustic meatus, external ear, corniculate and cuneiform
cartilages of larynx etc.
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Practical No. 7
To Draw & View Histological Slides of Stomach
Aim: To observe the stained smear of Fundus of Stomach
Histological view of Stomach
Identification Points
1. Mucosa lined by Simple columnar epithelium.
2. Mucus secreting oesophageal glands in the submucosal layer.
• Normally oesophagus lumen is collapsed with longitudinal mucosal folds. Functionally it
is a passage for bolus of food.
• Four layers of Gastrointestinal tract-
o Mucosa,
o Submucosa,
o Muscularis externa.
o Adventitia /serosa
• Mucosa
o Epithelium made of Simple columnar epithelium.
o Lamina Propria- Loose connective tissue with blood vessels
o Muscularis mucosa is two layered—inner longitudinal, outer circular (smooth
muscle)
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• Submucosa =-loose connective tissue with blood vessels and nerves. Also contains mucus
secreting oesophageal glands The secretions from the glands help in lubrication of lumen
and protect the mucosa
• Muscularis externa - inner oblique layer, middle circular layer, and outer longitudinal layer.
• Adventitia/Serosa - loose connective tissue called adventitia with blood vessels and nerves.
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Practical No. 8
Studying Histological Features of Small Intestine
Aim: To observe the stained smear of Fundus of Small Intestine
Histological view of Small Intestine
Identification Points
1. Mucosal villi lined by columnar epithelium with microvilli (brush border) and lots of goblet cells,
2. Peyer’s patch in mucosal layer extending to submucosa
• Four layers mucosa, submucosa, muscularis externa, serosa.
• Mucosa-lined by simple columnar cells with microvilli and goblet cells. Mucosa thrown into folds
called villi for increasing the surface area--small finger like.
o Each villus has a core of lamina propria with a central lacteal and blood vessels.
o Lamina propria filled with intestinal glands- crypts of Lieberkühn
o Cells present are columnar cells for absorption -- enterocytes.
o Mucus secreting goblet cells, Paneth cells which secrete lysozymes, undifferentiated cells and
neuroendocrine cells.
o Lamina propria shows aggregations of lymphoid follicles called Peyer’s patches each
containing 10-200 follicles
o Muscularis mucosae made of inner circular and outer longitudinal layers of smooth muscles
• Submucosa - loose connective tissue with numerous blood vessels, lymphatics and nerve fibers.
• Muscularis externa - inner circular, outer longitudinal smooth muscle
• Serosa -- peritoneal covering made of flat squamous cells.
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Practical No. 9
Studying Histological Features of Tongue
Aim: To observe the stained smear of Human Tongue
Identification Points:
1. Th epithelium on the dorsal surface of the tongue is irregular or rough owing to numerous elevations or
projections called papillae.
2. All papillae on the tongue are covered by stratified squamous epithelium that shows partial or incomplete
keratinization
• The tongue is a muscular organ located in the oral cavity.
• The core of the tongue consists of connective tissue and interlacing bundles of skeletal muscle fibers.
o Three types of intrinsic muscles exist i.e. Longitudinal, Vertical and Transvers
o The dorsal surface of the tongue is divided into an anterior two thirds and a posterior one third
section by a V-shaped depression called the sulcus terminalis.
• Papillae
o These are indented by the underlying connective tissue called lamina propria.
o All papillae on the tongue are covered by stratified squamous epithelium that shows partial or
incomplete keratinization. In contrast, the epithelium on the ventral surface of the tongue is
smooth and nonkeratinized.
o There are four types of projections or papillae on the dorsal surface of the tongue:
▪ Filiform,
▪ Fungiform,
▪ Circumvallate,
▪ Foliate
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o Filiform Papillae - The most numerous, narrow, conical, or pointed. They cover the entire
anterior dorsal surface of the tongue and are. They do not contain taste buds.
o Fungiform Papillae - The less numerous but larger, these papillae exhibit a mushroom-like
shape, more prevalent in the anterior region and tip of the tongue.
o Circumvallate Papillae - Circumvallate papillae are much larger than the fungiform or filiform
papillae. About 8 to 12 circumvallate papillae are located in the posterior region of the tongue in
humans. Numerous taste buds are located in the stratified epithelium on the lateral sides of each
papilla.
o Foliate Papillae - Foliate papillae are well developed in some animals but are rudimentary or
poorly developed in humans.
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Practical No. 10
Studying Histological Features of Spinal Cord
Aim: To observe the stained smear of Spinal Cord
Identification Points
1. Central H shaped gray matter with large anterior horns, narrow posterior horns.
2. Surrounding white matter with anterior deep median fissure
• Spinal cord extends from outer margin of foramen magnum to lower border of first lumbar vertebrae.
• It is part of CNS.
• Has three layers of meningeal coverings, dura mater, arachnoid, pia mater
• Median anterior fissure is deep
• Made up of central gray matter and outer white matter.
• Central H shaped gray matter divided into anterior and posterior horns.
• Lateral horns are seen in thoracic segments.
• Anterior horns are larger than posterior.
• Contains multipolar motor neurons and interneurons,
• The gray matter of two sides is connected by gray commissure containing the central canal.
• White matter contains ascending and descending tracts. Are grouped as anterior, lateral and posterior
columns.
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