University of NizwaCollege of Arts and Sciences

Department of Biological Sciences and Chemistry

CELL BIOLOGY LAB MANUAL

Table of contents

1. Safety in the Lab2. Microscopy3. Electron microscopy 4. Visualizing cell structures 5. Cell division: mitosis and meiosis6. Buffers, dilutions & spectrophotometry (to be done in collaboration with

Chemistry Lab)7. Different types of cells and tissues8. Cell count 9. Cell fractionation10. Cell culture (external Lab visit) 11. Tissue processing for microscopic analysis12. Staining techniques13. Histochemistry (visit to a pathology Lab)14. Examining abnormal cells (genetic diseases, cancer, parasitic infections,

metabolic changes) (there will be a separate handout)

Ahmed Yagi

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Safety in the Lab

1. Do not bring non-essential materials into the lab. The only items you will need in the lab are the lab instructions, pens and pencils, and a notebook for recording your results.

2. No food or drinks are allowed in the lab at any time.

3. Protect your face. The eyes are particularly sensitive to chemicals, and provide a good portal of entry for microorganisms; the best way to avoid contact is to keep your hands away from your face as much as possible.

4. Put on your lab coat once you enter the lab

5. Cover your hair. .

6. Dispose of waste material properly

7. Alert your instructor or TA to any spills or breakage. These accidents need to be handled correctly..

8. Alert your instructor or TA IMMEDIATELY if anyone has a cut that causes bleeding.

9. Know the location of fire extinguishers and the emergency eyewash station.

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MICROSCOPY

Microscopes work by passing light through a sample and then using lenses to focus that light (and the image of the sample that the light forms). In such a way, that the sample seems to be brought nearer our eyes for our careful examination.

With a conventional bright field microscope,

light from an incandescent source is aimed toward a lens beneath the stage called the condenser, The condenser is used to focus light on the specimen through an opening in the stage.

then light passes through the specimen, then through an objective lens, then through a second magnifying lens, the ocular or eyepiece. Then to the eye

We see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are thick enough to absorb a significant amount of light.

A microscope can be a simple microscope, or a compound microscope.

a. a simple microscope uses a single lens to look at the sample.

b. a microscopes uses a series of lenses and mirrors to bring the apparent image much closer than is possible with a single lens.

When using microscopes, we generally think in terms of the magnification it can provide. However, magnification is much less important to microscopy than resolution. Resolution. The resolution of an optical microscope is defined as the shortest distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities

Therefore, the smaller the resolution, the better we see details of the image. More simply, good resolution of two nearby points provides a clear view of two distinct points while poor resolution would result in a single blurry blob. Since magnifying blurry blobs would not serve much purpose, resolution is clearly more important than magnification.

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The resolution of a microscope is dependent on a some factors:

a. most crucially the wavelength of the light being used. The shorter the wavelength, the smaller (and better) the resolution.

b. Another major factor is the speed of light through the various components of the system. It is sufficient to know that these physical constraints limit light microscopy.

- Light microscope resolution is 1000 times better than the naked eye, - therefore the limit of magnification is about 1000-fold. - Electron microscopy has much higher resolution (up to about 1,000,000-fold

magnification) due to the much shorter wavelength of electron beams in comparison to light beams.

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Resolution depends on numerical aperture

In optics, the numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light

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Microscope parts

A compound microscope with mechanical stage

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Eyepiece Lens:  the lens at the top.  They are usually 10X or 15X power. 

Tube:  Connects the eyepiece to the objective lenses

Arm:  Supports the tube and connects it to the base

Base:  The bottom of the microscope, used for support

Illuminator:  A steady light source

Stage:  The flat platform where you place your slides.  It has stage clips which hold the slides in place. With a mechanical stage, you will be able to move the slide around by turning two knobs.  One moves it left and right, the other moves it up and down.

Revolving Nosepiece or Turret:  holds a number of objective lenses and can be rotated to easily change power.

Objective Lenses:  Usually you will find 3 or 4 objective lenses on a microscope.  They usually consist of 4X, 10X, 40X and 100X powers.  When used with a 10X eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. 

Rack Stop:  This is an adjustment that determines how close the objective lens can get to the slide. 

Condenser Lens:  The purpose of the condenser lens is to focus the light onto the specimen.  Condenser lenses are most useful at the highest powers (400X and above).    

Diaphragm or Iris:  This is a rotating disk under the stage.  This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide. 

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Exercise # 1.1:

Parts of the microscope:

1. Examine the microscope and identify all parts2. How many objectives could you find, and how are they labeled?3. What is the magnification of the eyepiece and what does it mean?

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Exercise # 1.2 

The proper way to focus a microscope:

Place a stained slide on the stage

i. start with the lowest power objective lens first ii. turn the lens down as close to the specimen as possible without touching it.  iii. look through the eyepiece lens and focus upward only until the image is

sharp.  If you can't get it in focus, repeat the process again.   iv. Once the image is sharp with the low power lens, you should be able to simply

click in the next power lens and do minor adjustments with the fine focus knob.  

v. Continue with subsequent objective lenses and fine focus each time

- Examine the structures on the slide with different objectives- Record the magnification in each case- What is the difference in the structural details between the lowest and highest

objective?

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Numerical Aperture and Resolution

What has resolution to do with the numerical aperture number of an objective lens (or a condenser lens, for that matter)?

- The numerical aperture number is directly related to the cone of light from the specimen at its vertex which is brought into the lens.

- when light hits an object, it diffracts. A single beam of light will be split into several different diffraction orders bent at increasing angles from the original impinging beam.

- consider what happens when a beam of light is shone through a pinhole onto a dark background. If the image produced on the other side of the pinhole is examined, one finds a light pattern known as an Airy disk. It looks like a negative target with a large central disk of light surrounded by a series of thin concentric circles of light of decreasing brightness the further away from the center they are.

  Airy disk 

Light transmission curve for an

Airy disk

(Redrawn from

Francon)*

- What has happened is that the light coming out of the pinhole has bee, en diffracted into several different orders represented by the concentric circles.

- The same type of thing happens when light hits a microscopic specimen; the diffraction orders spread out.

- The bigger the cone of light brought into the lens , the more of these diffraction orders which can be collected by it, and the more information it forms about the image, and the higher the resolving power of the lens will be .

- The bigger a cone of light that can be brought into the lens, the higher its numerical aperture is.

- Therefore the higher the numerical aperture of a lens, the better the resolution of a specimen will be which can be obtained with that lens. If you are interested in learning how the Airy disk is formed and how the light is diffracted, CLICK HERE.

The advantage of using a higher numerical aperture is that more light is brought into a higher numerical aperture lens producing brighter images.

This becomes a major consideration for darkfield and fluorescence applications where we are imaging a bright object against a dark background. Examine  

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Numerical aperture is defined by the formula     N.A. = i sin q

where I is the index of refraction of the medium in which the lens is working, and q is one half of the angular aperture of the lens.

All high dry lenses work in air which has a refractive index of 1.0.

Immersion oils have a considerably higher refractive index, sometimes even up to 1.56. Using an immersion oil:  i. bends more light into

the lens capturing more orders of diffraction from the object. (Finer details or more closely spaced objects will give much higher angles of diffraction than will larger objects with less fine details).

ii. Will allow a lens to have an N.A. greater than one. It is not possible for a dry lens to have an N.A. greater than one. 

(Redrawn from Gray)*

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Exercise # 1.3

The significance of the numerical aperture (N.A.)

- Examine the objectives of the microscope (unscrew and look at the labels; do not touch the lens surface)

- Record the labels for each objective (the color of the line and NA)- Replace the objectives in the microscope- Draw structures seen on the slide using x 10, x 20 and x 100 (oil immersion)- Write your observations

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Understanding NA:

In the previous Figure, notice the third image in the diagram showing the case of the immersion lens with the N.A. of 1.3.

To understand how the use of immersion oil can allow the lens to gather those outermost diffracted light, remember looking at an aquarium (a glass tank containing fish):

If you look at the corner you can see the same fish from both the end and the side of the tank. How does this happen?

This is because the index of refraction of water is greater than that of air; thus, when the light coming from inside the aquarium hits the air, it is bent at the interface because of the difference in refractive indices allowing you to see the fish from both the end and the side at the same time.

The same thing happens when light in the immersion oil hits the end element of the lens and light is bent inward and the end result is that more diffracted rays are collected by the lens. This provides more image information used to form the resultant image, and the higher the resolution of the lens will be.

The following diagram shows what happens to the Airy disk with increasing numerical aperture. The diffraction maxima are narrowed and more are brought into the lens to contribute to the final image. These curves can be correlated with the previous

diagram. (Redrawn from Francon)*

*Diagrams redrawn from Francon, M. 1961. Progress in Microscopy. Pergamon Press: London  (also Row, Peterson and Co.: Elmsford, NY.) and Gray, P. 1964. Handbook of Basic Microtechnique. McGraw-Hill: New York.

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The Vernier Scale and Vernier Coordinates

- The recording of vernier coordinates is necessary to relocate a particular str This may be necessary to particular structure in a slide.

- It is useful for referral to share your finding with a others. - The coordinates are recorded along an X axis and a Y axis (X,Y). - The X axis running from left to right and the Y axis running from top to bottom

Method:

1. Place the slide in the slide clip.2. Record whether the label is on the left or the right. 3. Record the coordinates of the upper right corner of the cover-slip or the lower left

corner (X,Y). 4. Make a note as to which corner you selected. 5. You may also make a mark on the slide as a reference point and make a note of

it's coordinates6. Record the coordinates of the object (X,Y) centered in the field. 7. Draw a diagram of the field recording any marks that may be useful in relocating

the specimen.

With the above information you can easily calculate relocate the structure on the slide by counting the number of millimeters from the reference.

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Cell Biology LabMICROSCOPY

Dark field microscopy:Dark field optics are a low cost alternative to phase contrast optics.

In dark field microscopy:

an opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye (figure 2).

Instead of coming up through the specimen, the light is reflected by particles on the slide.

Everything is visible regardless of color, usually bright white against a dark background.

Pigmented objects are often seen in "false colors," that is, the reflected light is of a color different than the color of the object.

Better resolution can be obtained using dark field as opposed to bright field viewing.

- A higher intensity light is needed, since we are seeing only reflected light.- At low magnification (up to 100x) any normal optical instrument can be set up so that light

is reflected toward the viewer rather than passing through the object directly toward the viewer

How to set up dark field in a compound microscope- With a compound microscope, dark field is obtained by placing an occulting disk in the

light path between source and condenser.

-

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- Disks can be prepared by cutting circular pieces of black electrical tape ranging up to a diameter that equals the width of the slide, and sticking them to the slide in a row.

- The circles should be spaced well apart. - A specimen is placed on the microscope stage as usual, and the illumination should be made

as uniform as possible. If there is an aperture diaphragm in the condenser (contrast lever), it should be opened up wide.

- After focusing at low power, the slide with occulting disks is placed in the light path between source and condenser, bringing it as close to the bottom of the condenser as it will go

When to use dark field illumination

Dark field illumination is most readily set up at low magnifications (up to 100x), although it can be used with any dry objective lens.

Best for viewing a liquid sample, debris. Even tiny dust particles are obvious. Dark field is especially useful for finding cells in suspension . Dark field makes it easy to obtain the correct focal plane at low magnification for small,

low contrast specimens.

Dark field can be used for:

Initial examination of suspensions of cells such as yeast, bacteria, small protists, or cell and tissue fractions including cheek epithelial cells, chloroplasts, mitochondria, even blood cells .

Examination of lightly stained prepared slides. Initial location of any specimen of very small size for later viewing at higher power. Determination of motility in cultures

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Cell Biology Lab

Phase contrast microscopy: is an optical microscopy illumination technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image

- Most living biological specimens are translucent. - When viewed under transmitted light in a high power compound light microscope, there is

often little or no distinguishable contrast in the image. - The solution came with the advent of the phase contrast microscope. - It is well known that a change in medium will shift a ray of light out of phase, slowing it

down if the medium is denser than what it was travelling in. - It also bends the light, such as a flashlight beam is bent when shining into a body of water at

night. This same principle of light is applied to the discovery of the phase contrast microscope.

When the light travels through a specimen, parts of the specimen are denser than other parts,

so this creates the phase shift in the transmitted light. This phase shift can be detected and transferred to a corresponding change in light

intensity in the phase contrast microscopy system. This gives the ability for a normally translucent specimen to show differing gradients of

light shades, thus resulting in contrast differences.

However the various organelles show wide variation in refractive index (the tendency of the materials to bend light, providing an opportunity to distinguish them)

Principle

- Highly refractive structures bend light to a much greater angle than do structures of low refractive index.

- The same properties that cause the light to bend also delay the passage of light by a quarter of a wavelength or so.

- In a light microscope in bright field mode, light from highly refractive structures bends farther away from the center of the lens than light from less refractive structures and arrives about a quarter of a wavelength out of phase

-- Light from most objects passes through the center of the lens as well as to the periphery.- If the light from an object to the edges of the objective lens is retarded a half wavelength

and the light to the center is not retarded at all, then the light rays are out of phase by a half wavelength. They cancel each other when the objective lens brings the image into focus. A reduction in brightness of the object is observed.

- The degree of reduction in brightness depends on the refractive index of the object

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-

Applications of phase contrast microscopy:

- It is preferable to bright field microscopy when high magnifications (400x, 1000x) are needed- is widely used for examining such specimens as biological tissues. - It is a type of light microscopy that enhances contrasts of transparent and colorless objects by

influencing the optical path of light. - It is able to show components in a cell or bacteria, which would be very difficult to see in an

ordinary light microscope

Figure.

(a) organelles are nearly invisible in bright field although they have different refractive indexes;

(b) light is bent and retarded more by objects with a high refractive index;

(c) in phase contrast a phase plate is placed in the light path. Barely refracted light passes through the center of the plate and is not retarded. Highly refracted light passes through the plate farther from center and is held back another one quarter wavelength.;

(d) The microscope field shows a darker background (in this case the cell cytoplasm has a higher refractive index than the contractile vacuole), with the organelles in sharp contrast

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Using phase contrast:

Phase contrast condensers and objective lenses add considerable cost to a microscope. Images obtainable in phase contrast mode can be very dramatic.

- Altering the Light WavesThe phase contrast microscope uses the fact that the light passing through a transparent part of the specimen travels slower and, therefore is shifted compared to the uninfluenced light. This difference in phase is not visible to the human eye. However, the change in phase can be increased to half a wavelength by a transparent phase-plate in the microscope and thereby causing a difference in brightness. This makes the transparent object shine out in contrast to its surroundings.

- Phase contrast microscopy imparts contrast to unstained biological material by transforming phase differences of light caused by differences in refractive index between cellular components into differences in amplitude of light, i.e., light and dark areas, which can be observed.

-

Figure 1: is a cut-away diagram of a modern upright phase contrast microscope, including a schematic illustration of the phase contrast optical train.

- Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector lens

- and focused on a specialized annulus (labeled condenser annulus) positioned in the substage condenser front focal plane.

- Wave fronts passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures and phase gradients present in the specimen.

- Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by a

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phase plate and focused at the intermediate image plane to form the final phase contrast image observed in the eyepieces.

Figure 2 is a comparison of living cells in culture imaged in both brightfield and phase contrast illumination. The cells are human glial brain tissue

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- The method by which phase differences can be transformed into amplitude differences is known as positive or dark phase contrast.

- An alternate method is known as negative or bright phase contrast- In positive phase contrast the object (e.g., cell component) appears darker than the surrounding

background.- In negative phase contrast the object appears brighter than the background.

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Microscopy

Field of view

- Sometimes it is necessary to determine the size of an object that you are viewing under the microscope.

- There is an easy way to estimate size. If you know the diameter of the field you are seeing in the microscope, you can estimate the size of the object you are viewing. A ruler mounted on a microscope slide is especially designed for accurate measurement.

- You can also do this more simply by placing a piece of graph paper on a microscope slide and viewing it under the microscope.

- Microscopic objects are measured in micrometers. 1 mm = 1000 micrometers

-

Each block on the graph paper is 1 mm wide. It takes about 4.2 blocks to go across the diameter of the field. 4.2 mm = 4200 micrometers

An object that occupies the entire diameter of the field at 40 X total magnification will be 4200 micrometers across

Each time the magnification is increased, the block on the graph paper gets larger. If we try to estimate field size using graph paper with the 40 X objective, the block will be so large that we will not be able see the lines clearly and it will be difficult to estimate the field size.

We know that the higher the magnification is, the smaller the field diameter is. Therefore, the field

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diameter is inversely proportional to the magnification.

We can use a mathematical formula to estimate the field size at 40 X.

The field diameter is 560 micrometers. What is the approximate length of the cell?

………………………………………………………………………………………………………….

Preparing samples to visualize cells and tissues under the microscope:

Dissecting an animal to obtain tissues:

The animal should anaesthetized and laid upon a clean paper towel and have all 4 extremities pinned to thin styrofoam or cork board.

Before dissection, wet the animals' fur with ethanol to minimize contamination with hair. Instruments should be soaked in an ethanol if cultures are to be obtained from tissues.

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Tissue Processing for Light Microscopic examination

Collection of tissues and fixation:

1. Animals should be anesthesized and subjected to cardiac perfusion with saline, followed by a 10% formalin flush. This gives the best morphology.

2. For routine stains tissue is sectioned and drop-fixed in a 10% formalin solution. Fixative volume should be 20 times that of tissue on a weight per volume. Tissues are held in cassettes

3. Due to the slow rate of diffusion of formalin (0.5 mm hr), tissue should be sectioned into 3 mm slices. This will ensure the best possible preservation of tissue and offers rapid uniform penetration and fixation of tissue within 3 hours.

4. Tissue should be fixed for a minimum 48 hours at room temperature.5. After 48 hours of fixation, move tissue into 70% ethanol for long term storage (at 4 C).6. Keep fixation conditions standard for a particular study in order to minimize variability.

The usual fixative for paraffin embedded tissues is neutral buffered formalin (NBF). This is equivalent

to 4% paraformaldehyde in a buffered solution (PBS) plus a preservative (methanol) which prevents the

conversion of formaldehyde to formic acid. Because of the preservative, NBF has a shelf life of months.

Tissues are held in cassettes

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cassettes for tissues

processing

Paraffin embedding:

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Once fixed, tissue is processed for paraffin embedding, using gentle agitation (usually on a tissue processor) as follows:

1. 70% ethanol for 1 hour.2. 95% ethanol (95% ethanol/5% methanol) for 1 hour.3. First absolute ethanol for 1 hour .4. Second absolute ethanol 1½ hours .5. Third absolute ethanol 1½ hours.6. Fourth absolute ethanol 2 hour.7. First clearing agent ( Xylene) 1 hour.8. Second clearing agent (Xylene) 1 hour.9. First wax (Paraplast X-tra) at 58°C for 1 hour.10. Second wax (Paraplast X-tra) at 58°C 1 hour.

Embedding tissues in paraffin blocks:

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Casting the tissue block:

1. Open cassette to view tissue sample and choose a mold that best corresponds to the size of the tissue. Discard cassette lid.

2. Put small amount of molten paraffin in mold, dispensing from paraffin reservoir.3. Using warm forceps, transfer tissue into mold, placing cut side down, as it was placed in the

cassette.4. Transfer mold to cold plate, and gently press tissue flat. Paraffin will solidify in a thin layer which

holds the tissue in position.5. When the tissue is in the desired orientation add the labeled tissue cassette on top of the mold as a

backing. Press firmly.6. Hot paraffin is added to the mold from the paraffin dispenser. Be sure there is enough paraffin to

cover the face of the plastic cassette.7. Paraffin should solidify in 30 minutes. When the wax is completely cooled and hardened (30

minutes) the paraffin block can be easily popped out of the mold; the wax blocks should not stick. If the wax cracks or the tissues are not aligned well, simply melt them again and start over.

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Tissue sectioning:

microtome

microtome

A microtome is used for cutting thin sections. It is just a sharp knife with a mechanism that allow the advancing of a paraffin block standard distances across it.

Cut sections are floated on water in a water bath at 50 C, then picked on glass slides. Sections come as a ribbon

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paraffin sections picked on slide.

The glass slides are then placed in a warm oven for about 15 minutes to help the section adhere to the slide

Staining of sections:

H & E stain is commonly used to stain paraffin sections:

Hematoxylin is a basic stain. It stains basophilic structures such as chromatin and ribosome's a deep purple or blue. The advantage of this stain is that it provides a clear stain of the cell nuclei. Eosin is an acidic stain that stains acidophilic structures red. It provides a good stain for the cell membrane

Reagents :

-Hematoxylin – (Sigma Accustain Harris Hematoxylin)-Acidic Alcohol (396 ml of 95% Ethanol, 4 mls concentrated HCl)-Bluing Agent (1 liter water, 1 gm sodium bicarbonate)-Eosin - Sigma Accustain Eosin Y-75%, 95%, 100% ethanol-Histo-clear (order No. HS 200) -Permount

Staining procedure:

Hematoxylin – 5 minutes

o Rinse in tap water – 30 seconds, to remove excess stain.

o Acidic Alcohol – 5 dips, to destain and differentiate.

o Rinse in tap water – 30 seconds

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o Bluing Agent – 2 minutes. This step is optional. The sodium bicarbonate changes the color of the hematoxylin-stained nuclei from purple to blue.

o Rinse in tap water – 30 seconds

o Eosin – 30 seconds

75% Ethanol – 30 seconds

-Begins the stepwise dehydration.

o 95% Ethanol – 30 seconds

o 100 % Ethanol – 30 seconds

o Histo-clear – 10 seconds (clearing agent makes tissue more transparent)

o Place a few drops of permount ontothe slide before adding the coverslip

………………………………………………………………………………………..

Electron microscopy

The Electron MicroscopeThere are two common types of electron microscopes:

i. scanning (SEM) ii. and transmission (TEM).

For SEM, bulk biological samples are first coated with a metal that readily reflects electrons. This coating also provides a conducting surface for electrons to avoid charging of the sample.In SEM:

a. The incoming electron beam is condensed into a small beam which is scanned over the object. b. An image is formed by the electrons that bounce off the surface of the specimen and are then

collected onto the imaging screen. c. The observer therefore sees a picture of the surface of the sample, but no internal information

In TEM:a. an image is formed that is a projection of the entire object, including the surface and the

internal structures. b. The incoming electron beam interacts with the sample as it passes through the entire thickness

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of the sample. c. Therefore, objects with different internal structures can be differentiated because they give

different projections. d. The projection is two-dimensional against the view screen and relations in the z-axis between

structures are lost. e. It is important that the samples need to be thin, or they will absorb too much of the electron beam

The layout of the electron microscope:

The main parts of the electron microscope:1. The gun (electron source)

a. A wire is superheated by an electric current to emit electrons. The wire is usually tungsten or lanthanum hexaboride (LaB6).

b. Electrons are collated into a tight beam and sent down the microscope column, across the sample, lenses, and apertures

2. Lenses

Electron lenses are magnetic coils that have been designed to focus and direct a passing electron beam. Three primary lenses are used to form and magnify an image:

i. The objective lens is the topmost lens and does the first step of image magnification and

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focusing.

ii. The intermediate lens and its positioning and strength controls the magnification of the

image or the diffraction pattern.

iii. The projector lens used to focus and project the image onto the imaging surface.

3. The aperturesApertures are holes along the microscope column that can limit the size of the electron beam that passes through it.a. The first aperture, the condenser aperture is located near the top of the column. It is used

to condense and maintain the coherence of the electron beam. b. The second aperture, the objective aperture, is located below the sample just after the

objective lens. The objective aperture is used primarily to control contrast in the image4. Fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide

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Tissue Processing for Electron Microscopy: Sections for TEM must be less than 80 nm thick in order to allow at least 50% of the electron

beam to penetrate the sample. These sections are called “ ultrathin sections” TEM sections are cut with knives of glass or diamondThe process of cutting ultrathin section is called Ultramicrotomy

This can only be accomplished by using resins for embedding (epoxy, acrylic or polyester).

Graded alcohol washes are used for the dehydration: typically 20, 40, 70, 90 and 100% in place of the clearing agents used in light microscopy

a "transitional fluid" is used. This fluid is miscible with both ethanol and the embedding resin, and is almost universally 1,2 epoxypropane (propylene oxide). If water soluble embedding resins are used, the sample may be dehydrated in graded baths of the embedding resin instead of alcohol, and a transitional fluid is not needed.

Example of tissue processing procedure:

L.R. White can be used for the LM, EM and the histochemical demonstration of some resistant enzymes as well as for the immunohistochemical demonstration of intracellular immunoglobulins.

Fixation:

1. Fix specimen with a solution of 4% paraformaldehyde – 2 % glutaraldehyde with or without 2.5% sucrose in 0.1M PBS, pH 7.4 for an overnight.

2. Rinse specimen 2x5min with 0.1M PB (or store specimen in 0.2M (8%) sucrose in 0.1M PB for storage and shipping purpose).

Dehydration:

1. Two changes of 70% ethanol for 30 minutes each.2. L.R. White and 70% ethanol (2:1) mixture: slowly add one part of 70% ethanol (drop by drop) to

two parts of L.R. White, and shake gently (otherwise the mixture will become milky). Incubate specimen in the mixture for 1 hour.

3. Incubate specimen in two or three changes of pure L.R. White for 1 hour each, or overnight at room temperature (specimen can be stored in L.R. White at 4 C for weeks if necessary).

Embedding:

1. Embed specimen in Gelatin Capsules. Place specimen in bottom of the capsule and fill up with L.R. White to the brim.

2. Polymerize in 60 C oven for 24-48 hours.

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Sectioning and Staining:

1. Cut ultrathin sections and mount on formvar-carbon coated nickel grids and allow it air-dry overnight.

3. Stain with uranyl acetate for 15 minutes and lead citrate for 1-2 minutes.

Observation:

1. Observe using Transmission Electron Microscopy2. Take pictures

Grids fro mounting ultrathin sections:

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Visualizing cells in different tissues

There are four types of tissue cells in our bodies:

1. Epithelial tissue includes three kinds of cells: squamous , cuboidal, and columnar, Epithelial tissue has an underlying basement membrane. Epithelial tissue can be arranged one of two ways, relative to the position of the

cells on the basement membrane: - simple epithelial tissue , in which all of the epithelial cells touch the basement

membrane,- stratified epithelial tissue, in which there are several layers of cells and not all touch

the basement membrane. Thus, cells may be arranged/classified in the following categories

simple squamous simple cuboidal

simple columnar stratified saquamous

pseudostratifid columnar: The trachea is lined with pseudostratified ciliated columnar epithelium

2. Connective tissue is characterized by a few cells in a non-living extracellular matrix Examples of connective tissue include bone, cartilage, and blood

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3. Muscle tissue: There are three different types of muscle tissue. * skeletal muscles which are under voluntary control and are made of striated muscle tissue, * Smooth muscle tissue (the visceral muscles) which are under involuntary control * Cardiac muscle tissue: which is found only in the heart. Cardiac muscle tissue has properties in common with each of the other two types.

Muscle cells contain filaments of two kinds of proteins, actin and myosin, which slide past each other as the muscle contracts

4. nervous tissue is composed of neurons, which transmit impulses, and the neuroglia cells, which assist propagation of the nerve impulse as well as provide nutrients to the neuron

1. epithelial tissue:- simple squamous epithelium:

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a nucleus of a cell forming the alveolus wall

b nucleus of a cell forming the capillary wall

c red blood cells inside capillary that will carry exchanged gases between the lungs and blood

d cell that forms the large circle of simple squamous forming the alveolus.

line lungs; line blood vessels (endothelium) adapted for osmosis, diffusion and secretion

simple squamous epithelium

- Stratified squamous epithelium: skin-

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- well suited to areas in the body subject to constant abrasion & friction, as it is the thickest and layers can be sequentially sloughed off and replaced before the basement membrane is exposed. It forms the outermost layer of the skin and the inner lining of the mouth, esophagus, and vagina.

- Simple cuboidal epithelium:

The lumen receives the secretion produced by the cuboidal cells

The black circle is superimposed on the basement membrane which holds together a circle of cuboidal cells.

1. line kidney tubules; cover ovaries; glands

2. adapted for secretion of fluid like mucus or enzymes; absorption (microvilli increase surface area)

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- Simple columnar epithelium:

The fuzziness on top of the simple columnar cells is caused by tiny microvilli.

o in GI tract and uterus;

o absorption of foods (microvilli); move sperm (cilia); secretion of mucus (goblet cells).

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- Pseudostratified Columar Epithelial Tissue (ciliated)

a nuclei of pseudostratified columnar cells

1. respiratory tract; paranasal sinuses

2. protection, produce mucus, trap and move dust and other toxins out of the lungs

- Transitional epithelium:

Transitional epithelium is a stratified epithelium. The shape of the surface cells changes (undergoes transitions) depending on the degree of stretch. These cells appear to be cuboidal or round with a domed apex when the organ is not stretched

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2. Connective tissue:

- Areolar CT:

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a dark line structure is an elastin fiber

b broad pink line is a collagen fiber

c fibrobast cell that produces the fibers

d matrix areas that appear empty contain ground substance a gelatinous watery fluid

note: granular mast cells in View 2

found in: subcutaneous layer; mucus membranes; around blood vessels, nerves, organs

function: soppy tissue that lubricates and nourishes epithelial tissue; also provides strength, elasticity, support and immune system protection. Areolar tissue is a common type of connective tissue, also referred to as "loose connective tissue. It is a pliable, mesh-like tissue with a fluid matrix and functions to cushion and protect body organs

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Reticular CT:

a nucleus of one of the many cells found in this tissue b ground substance in the matrix c black reticular fibers which act as strucural support - found: liver, spleen, lymph nodes. - function: structural maze support and slows down blood fluids so cells can perform their metabolic functions

Dense regular CT:

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a long narrow nucleus of a fibroblast (the cell that make the fibers) b many collagen fibers packed together going in the same direction - found: tendons and ligaments - function: provides strength to withstand the pulling of muscles in one direction

Dense irregular CT:

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a collagen fibers are light pink b tiny little dark structures are the nuclei of fibroblasts. c capillary d arteriole

1. found : dense irregular fibers surround the structures found in the dermis

2. function : allows the skin to be resilient and flexible

Adipose CT:

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a swollen cell almost totally full of triglycerides b tiny little dark structure is the nucleus squashed by the cell swelled with fat

found: subcutaneous layer; around organs; in yellow marrow.

function: stores triglycerides; insulates; energy reserve; protects; generates heat in newborns.

Hyaline cartilage:

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a matrix of cartilage is a solid flexible gel; the fibers are invisible at normal magnification b lacuna is a shell like space containing the chondrocyte c cartilage cell called a chondrocyte found: epiphyseal plate; ends of long bones and ribs; rings of trachea; fetal skeleton function: structural and flexible supportElastic cartilage:

a lacuna with chondrocyte inside

b the black material is elastin fibers

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found: framework of outer ear

function: provides elastic shape and support.

Fibrocartilage:

a chondrocyte cell in lacunae b lacunae containing two chondrocytes c fibers in the matrix mainly collagen found: pads between vertebrae; knee cartilage function: withstands tension, pressure and absorbs shock

Compact bone:

a central canal in the center of the osteon contains a blood vessel b concentric lamellae form concentric rings of calcium matrix around the central canal c lacunae with an osteocyte inside d many canaliculi (spidery lines) act as a nutrient conduction system in compact boneBlood:

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a red blood cell (rbc) b white blood cell (lymphocyte) c white blood cell (neutrophil) d white blood cell (eosinophil) e plasma (matrix)

found: in the circulatory system

function: carries oxygen, Carbon dioxide, ions, nutrients and wastes to and from the cells; contains cells for immune response (wbc).

Nervous tissue:

a cell body (soma) b nucleolus in nucleus c axon hillock d axon e dendrite,

Note: Surrounding the neuron many astrocytes with dark nuclei and many tiny light pink cytoplasmic extensions. function: a multipolar neuron is a motor neuron that conducts nerve impluses to muscles

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a Astrocyte nucleus surrounding by many cytoplasmic extensions. b Expanded cytoplasmic extensions of the astrocyte known as end-feet covering the blood vessel membrane helping to form the blood-brain barrier.

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Mitotic division observation: aiming to understand the different stages of the cell cycle [interphase (G1, S, G2)] [M phase (prophase, metaphase, anaphase, telophase)

1. Identifying mitotic stagesMitosis occurs very frequently in cells of the bone marrow, gonads, epidermis, and epithelium of the intestine. However, slides of these structures will often show very few mitotic figures. This could be due to diurnal variation in the rate of mitosis in different tissues; for example, in the skin mitotic figures are seen when biopsies are taken at night when cell division is most frequent. The methods used to obtain and prepare the cells for examination can also influence the number of mitotic figures seen.

1. In prophase, morphologically distinct chromatin threads appear and these shorten and thicken, forming distinct chromosomes. It is the first and last stage in mitosis.

2. At metaphase the chromosomes become arranged at the equatorial plate which is midway between the two pairs of centrioles. .

3. At anaphase, each chromosome pair completes its splitting, and the two daughter chromosomes move toward opposite poles. 

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4. At telophase, the chromosomes have reached the spindle poles and appear as a dense, basophilic mass within which individual chromosomes cannot be defined.

Use root tip of freshly grown onion to observe the stages of cell division:The first step will be to ‘soften’ the roots so that they later can be spread on a microscope slide. 1. Using scissors, cut 2 roots tips about 1 cm long, and transfer them into a plastic micro-tube. 2. Fill the tube about 2/3 full with 1N HCl from a dropper bottle. 3. Place the tube in a 60 ° C water bath , and allow the roots to incubate for 12 minutes. 4. After the 12 minute incubation period, remove the tube from the water bath.5. Rinse the root tips 3 times with water from the dropper bottle Staining the chromosomes.

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1. After removing the water from the third rinse, cover the root with the Feulgen stain. (Feulgen stain does not appear colored but it will strongly stain skin and clothing) 2. Incubate the roots in the stain for 12 minutes. During this time the very tip of the root will begin to turn red as the DNA stains the numerous small actively dividing cells at the tip. 3. Remove the stain and rinse the roots againPreparing the root tip squash. 1. Transfer a root to the center of a clean microscope slide and add a drop of water. 2. Using a razor blade cut off most of the unstained part of the root, and discard it. 3. Cover the root tip with a cover slip, and then carefully push down on the covers lip with the wooden end of a dissecting probe (do not twist or push the cover slide sideways). The root tip should spread out to a diameter about 0.5 – 1 cm. Observations of onion root tip squash.

- Scan the microscope under the 10x objective. - Look for the region that has large nuclei relative to the size of the cell; among

these cells will be found cells displaying stages of mitosis (examples are shown in the figures above).

- Switch to the 40X objective to make closer observations. Since prophase and prometaphase are difficult to distinguish, classify all these cells as prophase

Mitosis and the cell cycle - While making observations, consider the relative number of cells actually

involved in mitosis. - Some of these cells are still involved in the cell cycle, which encompasses all of

the processes involved in cell replication. - Cell that are actively dividing but not yet in mitosis are said to be in interphase,

during which time the DNA is copied and the cell is otherwise preparing for replication.

- Some root cells have ceased dividing and are only increasing in size, whereas others have reached their final, mature size and function, and are said to be in the G0 stage.

Recording data - Identify the stage of mitosis of each cell to be tallied. Since prophase and

prometaphase are difficult to distinguish, classify these cells as prophase. Only count as prophase cells that contain distinctly visible chromosomes.

- Systematically scan the root tip moving upward and downward through a column of cells.

- Tally each cell in a stage of mitosis that you observe, being careful not to record the same cell twice. Tally numbers in the table below. View the cells in columns and simply count the various stages as you move down the rows. Count at least two full fields of view. If you have not counted at least 120 cells, then count a third field of view.

-

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Calculations - 1. Pool your data with that of the class, and then record the class totals in the table

provided below. - 2. Calculate the percentage of cells in each stage. - 3. The relative time span of each stage is equivalent to the percentage of cells

found in that stage.

 Number Of Cells Percent of

Total Cells Counted

Time in Each StageField #1 Field #2 Field #3 Total

Interphase # # # #

Prophase # # # #

Metaphase # # # #

Anaphase # # # #

Telophase # # # #

Total Cells Counted # 1440 min = 1 complete cycle

- 8. Calculate the percentage of cells in each phase. Considering that the average complete cycle in onion root tip cells requires approximately 1440 minutes to complete, calculate the amount of time spent in each phase of the cell cycle from the percent of cells in that stage.

Cell Fractionation

involve the homogenization or destruction of cell boundaries by different mechanical or chemical procedures, followed by the separation of the subcellular fractions according to mass, surface, and specific gravityThe breaking open of cells and separation of the parts into pure fractions requires a large number of cells. The breaking open of cells lysis homogenization

Cell disruption:

Chemicals: alkali, organic solvents, detergents

Enzymatic: lysozyme, chitinase

Physical: osmotic shock, freeze/thaw

Mechanical: sonication, homogenization, French press

CHEMICAL DISRUPTION:

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Detergents such as Trition X-100 or NP40 can permeabilize cells by solubilizing membranes. Detergents can be expensive, denature proteins, and must be removed after disruption

FRENCH PRESS:

Cells are placed in a stainless steel container. A tight fitting piston is inserted and high pressures are applied to force cells through a small hole.

SONICATION:A sonicator can be immersed directly into a cell suspension. The sonicator is vibrated and high frequency sound waves disrupt cells.

HOMOGENIZATION:Cells are placed in a closed vessel (usually glass). A tight fitting plunger is inserted and rotated with a downward force. Cells are disrupted as they pass between the plunger and vessel wall

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HOMOGENIZATION:Mechanical disruption of cell membrane with a homogenizer. Cell membrane is sometimes dissolved with a detergent solution (triton X – 100)

HOMOGENIZER:A tube and a close fitting pestle. The cells are placed in the tube in an appropriate solutions of inorganic ions and low MW organic molecules e.g. sucrose. (disrupt the cells and release the contents without damaging subcellular organelles) in order to maintain the functional and structural properties of the cell parts (once the cells are broken open)The pestle is inserted in to the tube and rotated as it is drawn in and out of the tube. The motion creates a shearing action that breaks open the cell

** for procedure, see note below:

Differential Centrifugation• This is the most common method of fractionating cells• Fractionation is the separation of the different organelles within the cell

• . Cut tissue in an ice-cold isotonic buffer. It is cold to stop enzyme reactions, isotonic to stop osmosis and a buffer to stop pH changes.

• 2. Grind tissue in a blender to break open cells.• Filter to remove insoluble tissue• 4. Centrifuge filtrate at low speeds ( 1000 X g for 10mins )• This pellets the nuclei as this is the densest organelle• 5. Centrifuge at medium speeds ( 10 000 x g for 30 mins )• This pellets mitchondria which are the second densest organelle• 6. Centrifuge at high speeds ( 100 000 x g for 30 mins)• This pellets ER, golgi apparatus and other membrane fragments• 7 Centrifuge at very high speeds ( 300 000 x g for 3hrs)• This pellets ribosomes• Differential Centrifugation allows us to look at each organelle within the cell• We can look at the individual organelles and study them in detail• This helps to determine each organelles function within the cell

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Cell count

Equipment & Reagents

- Haemocytometer plus a supply of cover-slips- 0.4% Trypan Blue stain (fresh & filtered) in phosphate buffered saline- Tally Counter- Cell Suspension- Gilson pipettes or similar- Inverted microscope (preferably phase contrast)

Procedure1. Ensure the cover-slip and haemocytometer are clean and grease-free (use alcohol to clean).

2. Moisten (with water or exhaled breath) and affix cover-slip to the haemocytometer.

3. Look for "Newton&s Rings" which indicate that the cover slip has adhered via suction to the haemocytometer. Newton&s refraction rings are seen as rainbow-like rings under the cover-slip.

4. Mix equal volumes of 0.4% trypan blue stain and a well mixed cell suspension (not too vigorous) e.g. mix 100µl trypan blue stain with 100 µl cell suspension.

5. Pipette trypan blue/cell mix (approximately 10µl) at the edge of the cover-slip and allow to run under the cover slip.

6. Visualise the haemocytometer grid under the microscope, refer to figure 1 for layout of grid. Please note:

i. Trypan Blue is a "vital stain"; it is excluded from live cells.ii. Live cells appear colourless and bright (refractile) under phase contrast.iii. Dead cells stain blue and are non-refractile.iv. To aid accuracy and consistency of cell counts use counting system illustrated in figure 2.

7. Count viable (live) and dead cells in one or more large corner squares and record cell counts.

8. It is advisable to count around 40 to 70 cells to obtain an accurate cell count - therefore it may be necessary to count more than one large corner square.

9. To calculate cell concentration per ml:

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Average number of cells in one large square x dilution factor* x 104

*dilution factor is usually 2 (1:1 dilution with trypan blue), but may need to further dilute (or concentrate) cell suspensions.

104 = conversion factor to convert 10-4ml to 1ml (refer to figure 3 to view a diagram of the arrangement and dimensions)

Calculation of Cell Viability:

No. of Viable Cells Counted x 100 = % viable cellsTotal Cells Counted(viable + dead)

Figure 1. Appearance of the haemocytometer grid visualised under the microscope.

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Figure 2. Counting system to ensure accuracy and consistency. Count the cells within the large square and those crossing the edge on two out of the four sides.

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Basic cell culture procedure

Cells in culture is a useful model for studying the activity of cells in the whole organism in vivo.

Basic Equipment

Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)• Incubator (humid CO2 incubator recommended)• Water bath• Centrifuge• Refrigerator and freezer (–20°C)• Cell counter (e.g., CountessR Automated Cell Counter or hemacytometer)• Inverted microscope

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• Liquid nitrogen (N2) freezer or cryostorage container• Sterilizer (i.e., autoclave)

STERILE TECHNIQUES

Aseptic or sterile technique is the execution of tissue culture procedures without introducing contaminating microorganisms from the environment

Expanded Equipment • Aspiration pump (peristaltic or vacuum)• pH meter• Confocal microscope and inverted microscope• Flow cytometerAdditional Supplies • Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multi-well plates)• Pipettes and pipettors• Syringes and needles• Waste containers• Media, sera, and reagents

• Cells

Nutrient medium cannot be autoclaved. The compounds in nutrient medium are destroyed by the heat of autoclaving. Medium must therefore be sterilized by passing it through a sterile filter small enough in pore size to hold back bacteria and mycoplasmas (Millipore Sterivex - GS 0.22u disposable filter units). Here are some rules of thumb to follow to keep your medium, cultures, and glassware from becoming contaminated:

1. Wipe your work area and hands with 70% ethanol before starting.

2. Never uncover a sterile flask, bottle, petri dish, etc., until the instant you are ready to use it. Return the cover as soon as you are finished. Never leave it open to the environment.

3. Sterile pipettes should never be taken from the wrapper until they are to be used. Keep your pipettes at your work area. Sterile pipettes do not have to be flamed. Pipetting your cells with a hot pipette will kill them.

4. When removing the cap from a bottle, flask, etc., do not place the cap with the open end upright on the lab bench. Do not hold the opening straight up into the air. If possible, tilt the container so that any falling microorganisms fall onto the lip.

5. Be careful not to talk, sing, or whistle when you are per—forming these sterile procedures.

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6. Do not draw from a different bottles with the same pipette. Because such a pipette has been exposed; the chance for contamination is too great; use sterile pipette for each bottle -- especially when pipetting medium.

7. Techniques should be performed as rapidly as possible to minimize contamination.

Assessing the cultures:

Before doing anything with a culture, its general "health" and appearance should be evaluated. This can be done quickly and quantitatively by making the

following observations:

1. Check the pH of the culture medium by looking at the color of the indicator, phenol red. As a culture becomes more acid the indicator shifts from red to yellow-red to yellow. As the culture becomes more alkaline the color shifts from red to fuchsia (red with a purple tinge). As a generalization, cells can tolerate slight acidity better than they can tolerate shifts in pH above pH 7.6.

2. Cell attachment. Are most of the cells well attached and spread out? Are the floating cells dividing cells or dying cells which may have an irregular appearance?

3. Percent confluency. The growth of a culture can be estimat—ed by following it toward the development of a full cell sheet (confluent culture). By comparing the amount of space covered by cells with the unoccupied spaces you can estimate percent confluency.

4. Cell shape is an important guide. Round cells in an un—crowded culture is not a good sign unless these happen to be dividing cells. Look for doublets ordividing cells. Get to know the effect of crowding on cell shape.

5. Look for giant cells. The number of giant cells will in—crease as a culture ages or declines in "well-being." The frequency of giant cells should be relatively low and con—stant under uniform culture conditions.

6. One of the most valuable guides in assessing the success of a "culture split" is the rate at which the cells in the newly established cultures attach and spread out. Attachment within an hour or two suggests that the cells have not been traumatized and that the in vitro environment is not grossly abnormal. Longer attachment times are suggestive of problems. Nevertheless, good cultures may result even if attachment does not occur for four hours.

7. Keep in mind that some cells will show oriented growth patterns under some circumstances while many transformed cells, because of a lack of contact inhibition may

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"pile up" especially when the culture becomes crowded. Get to recognize the range of cells shapes and growth patterns exhibited by each cell line.

Procedure for dissociation of cells for culture (from primary tissue)

1. Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces several times with Hanks' Balanced Salt Solution (HBSS).

2. Add collagenase (50 to 200 U/ml in HBSS).3. Incubate at 37°C for 4 to 18 h. Addition of 3 mM CaCl2 increases the efficiency

of dissociation.4. Filter the cell suspension through a sterile stainless steel or nylon mesh to separate

the dispersed cells and tissue fragments from the larger pieces. Fresh collagenase can be added to the fragments if further disaggregation is required.

5. Wash suspension several times by centrifugation in HBSS.6. Resuspend the pellet in culture medium. Count and seed the cells for culture.

Morphology of cultured cells:

H & E staining procedure

Staining protocol1. Deparaffinize sections in the usual wayand rehydrate

2. Stain in Mayer’s hemalum solution 3minor hematoxylin solution mod. acc. to Gill III 3 min

3. Rinse in HCl solution 0.1% 2 sec

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4. Differentiate in running tap water 3-5min

5. Stain in eosin Y solution 0.5% aqueous, 3mineosin Y solution 0.1% aqueous 3-5minor eosin B solution 0.1% aqueous,erythrosin B solution 0.1% aqueous 3-5min

6. Rinse in tap water 30 sec7. Ascending alcohol series,2 x Neo-Clear® or xylene

8. Mount with Neo-Mount®or Entellan® newTechnical note: Before use, approx. 0.2ml of acetic acid (glacial acetic acid) 100% anhydrous (Merck Cat. No. 1.00063)can be added per 100ml of eosin solution to intensify the red color.ResultsNuclei dark blueeosin Y eosin B erythrosine BCytoplasm red-orange red redCollagen, elastin, erythrocytes yellow-orange red-orange red-orange

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