Connective tissue Prelim Exam Requirement:

INDIVIDUALLY Submit a transcription of this video on connective tissue: on December 16, 2016

Connective tissue

1. Remember that all connective tissue contains

• -cells 
• -fibers 
• -collagen 
• -elastic 
• -reticular 
• -ground substance (extracellular matrix) 

2. In photomicrographs, identify and give the location of all the types of connective tissue:

• -fetal mesenchyme 
• -mucoid connective tissue 
• -loose, areolar fibrous CT (e.g., mesentery) 
• -dense, irregular fibrous CT 
• -dense, regular fibrous CT 
• -elastic CT 
• -reticular CT 
• -adipose CT 
• -white (unilocular) fat 
• -brown (multilocular) fat 

3. Give the basic functions of the two major resident cells of connective tissue:

• -fibroblasts 
• -adipocytes 

4. Explain the role of the transient cells of connective tissue:

• -macrophage 
• -mast cell 
• -plasma cell 
• -leukocytes 

5. Define the terms “agyrophilia and metachromasia.”

6. List the general functions of connective tissues 

7. Name the germ layer(s) from which connective tissue cells derive and the embryonic tissues containing undifferentiated connective tissue cells  

8. List the two major classes of macromolecules that constitute ground substance

9. Describe adipose tissue as a connective tissue in terms of its cells, fibers, and ground substance.

10. Compare the three types of cartilage in terms of:

1. Type, amount, and arrangement of cells, fibers, and ground substance
2. Location in the body 
3. Histogenesis 
4. Function

11. Compare osteocytes with osteoblasts in terms of their shape, filopodia, amount of RER, location, and rate of matrix synthesis.

SUBMIT ANSWERS PER GROUPS OF 8 MEMBERS EACH TO apbautista@ceu.edu.ph before 2 pm today (12-7-16)

Additional points will be given to:

1st - 20 pts to CP and 5 pts to the quiz on Friday

2nd - 15 pts

3rd - 10 points

Post lab discussion will be on Friday (12-9-16) and a short quiz will be given on connective tissue and epithelial tissue (30 pts)

PRELIM EXAM WILL BE ON FRIDAY NEXT WEEK (12-16-16) 

Lab Activity Sheet: Epithelium, Skin and Glands

Illustrate/sketch/printout of photomicrographs (b/w) of all the tissue sections listed below. Label all photomicrographs with the given description for each tissue section.

Objective: Differentiate between the different types of epithelium; whether they are keratinized, stratified, and whether they are ciliated or contain microvilli.

 

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1.  Simple squamous epithelium (isolated) – Label Cell Membrane;ICS;Nucleus

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2. Stratified squamous epithelium non-keratinized from the vagina.  The portion lining the lumen contains the epithelium.  Identify the types of cells that make up this lining.  The innermost layers of cells are cuboidal, but towards the surface they become squamous.  Remember that in stratified epithelia, the outer layer determines the classification of the epithelium.  Below the lining of the epithelial cells lies the region of connective tissue.

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3.  Stratified squamous from the esophagus  

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4.  Simple cuboidal epithelium surrounding thyroid follicles  

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5.  Simple cuboidal to simple columnar in kidney.  Look for a large Y-shaped region.  This is the urinary space of the minor calyx.  Thin tubules enter/drain into this space, having radiated through the light pink medulla region from the darker red, cortex region.  Microscopically, the medulla contains tubules with cuboidal (cube-shaped, with a squarish profile) or short columnar (taller than it is wide) epithelium.  You will also find some very narrow tubules with squamous (flattened cells) epithelial walls.  The cortex has glomeruli with squamous epithelium, and tubules with cuboidal epithelium.

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6.  Simple columnar epithelium of the pyloric stomach.  Label the unique cell types that are present in the tissue section.

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7.  Pseudostratified columnar ciliated epithelium in the respiratory passages (trachea).  Note that this is a slice of a tube.  Look at the inner lining to find the epithelial cells.  These are pseudo-stratified (falsely layered) because the nuclei are at different levels and the cells are of different heights, but all cells sit on the basement membrane (which is not obvious in this slide).  Thus, there is only a single layer of different-sized cells.  These epithelial cells are ciliated, and this differs from the brush border of the gut epithelial cells.  Make sure you can tell the difference between cilia and microvilli of the brush border.  You will also see goblet cells here.  These are the flask-shaped cells sitting on the outer 2/3 of the epithelium.  The base of the goblet cell also sits on the basement membrane.  What is the purpose of the cilia?

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8. Transitional epithelium of the urinary bladder. The bladder has a folded lumen (central space).  This lumen is lined by transitional epithelium.  Note the layers of epithelial cells. The outer layer of transitional epithelial cells (sometimes called umbrella cells) has an outer lining of dark red material.  This dark lining is formed by the invagination of the membranes to accommodate stretching of the cell when the lumen is expanded (when the bladder fills).

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9. Stratified squamous epithelium keratinized palm) .   Compare this with the stratified squamous epithelium of the vagina.

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10.   Tongue. The upper surface has papillae, which are projections of the stratified squamous epithelium which create friction.  The papillae are keratinized.  This means that the epithelial cells have died and produced a non-cellular layer of keratin on the surface.  Keratinized epithelium lines the body surface for protection.  For now note the intact cells under the keratinized layer. 

Spikes are false intercellular bridges.  These do not connect the cytoplasm of adjacent cells, but are really sites of cell membrane (junctions, desmosomes) that connect the adjacent epithelial cells.  When the material was prepared, there was shrinkage, and the cytoplasm pulled away from the next cell, leaving only the strong desmosomal attachment, creating this appearance.  “Stratified squamous” means that the uppermost cells are squamous in appearance, and stratified means there is more than one layer of cells.

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11.  Mitochondria from liver.   Look for the little dark dots which are the mitochondria.  Identify the nuclei and nucleoli.

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12.  Golgi Apparatus. In the electron microscope, this is seen as a system of parallel membranes arranged around the nucleus.  It is seen as black splotches in the cytoplasm of large round nerve cells.  These nerve cells gave a large, light nucleus with a dark dot (nucleolus).

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13.  Scalp.  The skin consists of stratified squamous epithelium, keratinized.  The basal layer is cuboidal, but becomes squamous in the upper layers.  The cells die and form keratin, which are layers of protein that slough off regularly.  Identify the hair follicles, sebaceous (oil) glands, sweat glands and smooth muscles making up the arrector pili muscle.

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14. Gut epithelium / Small intestine (duodenum) simple columnar cells. Identify finger-like processes (villi). The lining of these villi contains a simple columnar epithelium with a striated or brush border (the electron micrograph).  The brush border can be seen as a thin amorphous pink layer at the top of the epithelial cells.  In most preparations some cells are different from the simple columnar epithelium.  These appear as dark shadows or clear spots on the luminal surface of the epithelium as you focus up and down.  These are the goblet cells, which are unicellular mucous secreting cells.  .

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15.  Parotid gland. Identify the secreting portion and the ducts.  Most of the gland is make up of serous secreting alveoli (sacs of cells), which have characteristic round, rather light, central nuclei and pink cytoplasm.  The ducts look like necklaces, with the cells being cuboidal, stratified cuboidal, to columnar.  Identify the large ducts in the spaces between lobules – some of these are stratified cuboidal.

Pre-lab Activity on Microscope, Cell, Mitosis and Stains

INTRODUCTION 

TO GENERAL MICROANATOMY AND EMBRYOLOGY

The body contains at least 200 distinct cell types. These cells contain essentially the same internal structures yet they vary enormously in shape and function. The different types of cells are not randomly distributed throughout the body; rather they occur in organized layers, a level of organization referred to as tissue.

The variety in shape reflects the many different roles that cells fulfill in your body. The human body starts as a single cell at fertilization. As this fertilized egg divides, it gives rise to trillions of cells, each built from the same blueprint, but organizing into tissues and becoming irreversibly committed to a developmental pathway.

HISTOLOGIC METHODS.

LIGHT MICROSCOPY method - Allows to see only cell NUCLEUS and only CYTOPLASM, because magnification of light microscope is not sufficient for revealing of ORGANELLES and

CHROMOSOMES.          
 

 If objective lenses have magnification x40 and ocular lenses - x7,

TOTAL magnification of LIGHT microscope is: 40 x 7= 280 times.

Steps for slide manufacturing in method of light microscopy:

a)  Fixation of specimen in FORMALIN solution.

b)  Dehydration – is removal of water from specimen. Put it in ETHYL ALCOHOL.

c)  Embedding – is infiltration of specimen by melted. PARAFFIN.

d)  Sectioning – is cutting of specimen by histologic knife with aim to get SECTIONs with thickness 7 micrometers. Device for this – is MICROTOME.

e) Staining of section by histologic Dyes.

Dyes are classified on 2 types: 

1) COMMON dyes

2) SPECIAL dies

Common dyes
reveal only pink cytoplasm and blue nucleus, without chemical identification of cellular content. Common dyes are classified on:

a) BASIC dyes.  It is HEMATOXYLIN. It stains NUCLEUS in BLUE colour.

b) ACIDIC dyes. It is EOSIN. It stains CYTOPLASM in PINK colour.

Special dyes reveal only ONE CHEMICAL of cellular content.

It may be: ORSEIN for revealing of ELASTIC fibres;
SUDAN BLACK for staining of LIPID droplets into BLACK colour.

ELECTRON MICROSCOPY method (=EM). Allows to see: ORGANELLES, CHROMOSOMES, CHROMATIN, because electron microscope gives magnification x5.000 times up to x30.000 times.

There are 2 types of EM method:

A) TRANSMISSION EM (TEM method). It gives PLANE picture of organelles and chromatin;

B) SCANNING EM (SEM method). It gives 3-D image of cell.

HISTORADIOAUTOGRAPHY method. It allows to evaluate RATE of DNA duplication. If in microslide number of SILVER GRAINS on phone of nucleus is big, it signifies about AST rate of DNA duplication.

IMMUNOCYTOCHEMISTRY method. It allows to see proteins-RECEPTORS on the surface of PLASMA MEMBRANE. If after injection of LABELED MONOCLONAL ANTIBODIES into rat, plasma membrane is stained into pink colour (into colour of LABEL), it means :proteins-RECEPTORS present.

PHASE CONTRAST MICROSCOPY method. It allows to see

ALIVE and NON-stained cells, to evaluate RATE of SPERMATOZOA movement.

THIS IS TO BE DONE INDIVIDUALLY.

NAME: ___________________________     

· Always begin examining microscope slides with which power objective? ____________

· The lens that is within the eyepiece of the light microscope is called the: ____________

·What must be done to a specimen to increase the contrast of the structures viewed? ____________

· The wheel under the stage that adjusts the amount of light is called the: ____________

· At high power, always use which adjustment knob to focus the image? ____________

· To focus a specimen, it is best to start with which objective: ____________

· Which objective provides the greatest depth of field? ____________

·The scanning, low, and high power objectives are mounted on the: ____________
If the ocular of a microscope is 10X and the objective is set at 100X, then what is the total magnification of the microscope? ____________

Fill in the blanks

1. This ____  (organelle) functions in cellular respiration.

2. The ____  (organelle) functions to package and deliver proteins.

3. Cell organelles are located within the ____ of the cell.

4. The endoplasmic reticulum functions to____________________    

5. Genetic material is contained within the ___ of the cell.

6. This organelle is responsible for destroying worn-out cell parts___

7. The _____ controls what enters and leaves the cell.

8. The rough endoplasmic reticulum has ____ located on it.

9. Located within the nucleus, it is responsible for producing ribosomes:

10. Which structure is directly responsible for the formation of proteins within the cell.

Pre-test on Mitosis

·         What is mitosis?

·       Beginning with mitosis, what are the other parts of the cell cycle in order in which they occur?

·         What occurs in the cell during the G2 phase?

·         Beginning with interphase, what are the phases of mitosis in order in which they occur?

·         In which phase do the chromosomes align on a plane in the center of the cell?

·         What occurs during anaphase?

·         What is the part of the chromosome that joins the two chromatids?

·         What is the structure in cells that divides the cytoplasm into two cells.

·         What is a blastula?

·         What is the term that describes a nucleus with two of each type of chromosome?

A quiz on this assignment will be given on Friday. DO YOUR HONEST BEST!

STAINS (Study in advance)

Aldehyde Fuchsin

• 
• Used to stain pancreatic islet beta cell granules. 
• This stains elastic fibers purple/black.

Alician Blue

• 
• Stain mucins and mucosubstances blue. 
• Copper in the histology stain is what ultimately is responsible for the blue color. 
• Nuclei will stain pink/red. 
• Cytoplasm stains a lighter pink. 
• Mucins stain blue.

Alkaline Phosphatase

• Stain endothelial cells. 
• Sites of alkaline phophatase activity will appear red. 
• Nuclei will stain blue.

Bielschowsky Stain

• Silver is used in this histology staining process. 
• This histology stain shows reticular fibers. 
• This histology stain is also used for showing neurofibrillary tangles and senile plaques. 
• Neurofibrillary tangles and senile plaques will stain black.

Neurofibrillary Tangles associated with Alzheimer's

Cajal Stain

• 
• This histology stain is used on nervous tissue

Congo Red

• Congo red histology stain is used to stain amyloid. 
• Amyloid will stain orange/red.

Giemsa Stain

• This is a histology stain for peripheral blood smears and bone marrow. 
• It is also used to visualize parasites and malaria. 
• This is a Romanowski type stain. 
• Methylene blue and eosin are used. 
• Erythrocytes stain pink/red. 
• Platelets and leukocytes stain blue.

This micrograph depicts the appearance of a well-stained slide using the Giemsa staining technique. 
Note that the acidic components of the cellular constituents such as the cytoplasm and chromatin, pick up the basic methylene blue azure compliments of the Giemsa stain, which reveals the characteristic blue 
coloration of this stain.

Golgi Stain

• 
• This histology stain will stain neurons.

H&E

• 
• This is a standard histology stain. 
• "H&E" stand for hematoxylin and eosin. 
• Hematoxylin and eosin stain is used for routine tissue preparation frequently. 
• This is the most often used combination in the histology lab for general purpose staining.
• Hematoxylin can be thought of as a basic dye. 
• It binds to acidic structures, staining them blue to purple. 
• It will bind and stain nucleic acids. 
• Therefore, the nucleus stains blue.
• Eosin is an acid aniline dye. 
• It will bind to and stain basic structures (or negatively charged structures), such as cationic amino groups on proteins. 
• It stains them pink. 
• Cytoplasm, muscle, connective tissue, colloid, red blood cells and decalcified bone matrix all stain pink to pink/orange/red with eosin.
• With an H&E stain, mucus and cartilage will stain a light blue color.

Iron Hematoxylin 

- 

• stain nuclei bluish/black
• one of a number of stains that allow one to make a permanent stained slide for detecting and quantitating parasitic organisms

Oil Red O

• This is a histology stain used for lipids. 
• Lipids will stain red. 
• Nuclei will stain blue/black

Papanicolaou Stain

• 
• This histology stain is used mainly on exfoliated cytological specimens. 
• Cells in smear preparations can be stained with Pap staining. 
• Gynecological smears (Pap smears), sputum, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, semminal fluid and fine needle aspiration samples can all be stained with a Pap stain. 
• This staining technique involving five dyes in three solutions.

Periodic Acid-Schiff (PAS) 

• This histology stain is particularly useful for staining glycogen and other carbohydrates, but is useful for many things. 
• It is often used to show glomeruli, basement membranes, and glycogen in the liver. 
• PAS stains glycogen, mucin, mucoprotein, and glycoproteins magenta. 
• The nuclei will stain blue. 
• Collagen will stain pink.

Prussian Blue

• 
•  used to stain iron (ferric iron and ferritin).
• demonstrates the blue granules of hemosiderin in hepatocytes and Kupffer cells.-  Hemochromatosis 

Safranin O

• 
• This histology stain will stain mucin, cartilage and mast cells. 
• It stains them orange/red. 
• Safranin O is sometimes used as a counterstain.

Sudan Stains

• 
  
• Sudan histology stains are used for staining of lipids and phospholipids. 
• Examples of such histology stains are sudan black, sudan IV, and oil red O.