1014MSC LAB Reviewer PDF

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LAB NOTES LIGHT MICROSCOPY PARTS OF THE MICROSCOPE

Total Magnification = power of ocular lens (10x) x power of objective lens used (4x,10x,40x,100x)!

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4x -> 20x! 10x -> 100x!

40x -> 400x! 100x -> 1000x! Focusing Technique • Turn on the microscope, check that the light is on! • Have the OBJECTIVE LENS at 4x • Begin with the STAGE as far down as possible! • Slowly adjust the COARSE FOCUS KNOB, until the slide is visible, and adjust the FINE FOCUS KNOB until the slide is completely in focus! • Switch to 10x OBJECTIVE LENS, use the FINE FOCUS KNOB to completely focus the lens! • Repeat for 40x OBJECTIVE LENS • See video for 100x (Immersion oil needed)!

Microscope Part

Description and function

Base

The bottom of the microscope. Provides a sturdy flat surface to support and steady the microscope

Substage light

Located in the base. The light from the lamp passes directly upwards through the microscope.

Light control

Located on the base or arm. This dial allows you to adjust the intensity of the light passing through the specimen.

Stage

The platform that the slide rests on while being viewed. The stage has a hole in it to allow light to pass through the stage through the specimen.

Mechanical Stage

Holds the slide in position for viewing and has two adjustable knobs that control the precise movement of the slide.

Condenser

Small non-magnifying lens located beneath the stage that concentrates the light on the specimen. The condenser may have a knob that raises and lowers the condenser to vary the light delivery. Generally, the best position is to close to the inferior surface of the stage.

Iris diaphragm lever

The iris diaphragm is a shutter within the condenser that can be controlled by a lever to adjust the amount of light passing through the condenser delivery can be moved to close the diaphragm and improve contrast. If your field of view is too dark, you can open the diaphragm to let in more light.

Coarse adjustment knob

This knob allows you to make large adjustments to the height of the stage to initially focus your specimen.

Fine adjustment knob

This knob is used for precise focusing once the initial course focusing has been completed.

Head

Attaches to the nosepiece to support the objective lens system. It also provides for attachment of the eyepieces which house the ocular lenses.

Arm

Vertical portion of the microscope that connects the bass in the head.

Nose Piece

Rotating mechanism connected to the head. Generally, it carries three or four objective lenses and permits positioning of these lenses over the whole in the stage.

Objective lenses

These lenses are attached to the nose peas usually a compound microscope has four objective lenses scanning (4X), low-power (10X), high power (40X), and oil immersion (100X) lenses.

Ocular Lenses

Binocular microscopes will have two lenses located in the eyepieces at the superior end of the head. Most accurate lenses have a magnification power of 10X. Some microscopes will have a pointer and or reticle (micrometer) which can be positioned by rotating the ocular lens.

MAGNIFICATION: image is magnified and inverted twice

TISSUES EPITHELIAL TISSUE • STRUCTURE!

- Simple (one layer) or Stratified (two or more layers)! - Squamous (scalelike), Cuboidal (cube-like), or Columnar (column-shaped)! • FUNCTION: Protection, absorption, filtration, excretion, secretion! • LOCATION: The lining between the external and internal environment and cavities throughout the body! SIMPLE SQUAMOUS

- Key characteristics: single layer of flattened cells!

- Location: alveolar (air sac) walls of lungs! - Function: allows material to pass by diffusion and filtration! STRATIFIED SQUAMOUS!

- Key characteristics: thick membrane composed of several cell layers; surface cells are squamous!

- Location: lining of oesophagus! - Function: protects underlying tissues in areas subjected to abrasion! SIMPLE CUBOIDAL

- Key characteristics: single layer of cube-like cells!

- Location: kidney tubules! - Function: secretion and absorption! STRATIFIED CUBOIDAL! - Key characteristics: two layers of cube-like cells! - Location: salivary gland duct!

- Function: protection! SIMPLE COLUMNAR!

- Key characteristics: single layer of tall cells! - Location: digestive tract — small intestine! - Function: absorption and secretion of mucus, enzymes; ciliated type propels mucus by colliery action! STRATIFIED COLUMNAR!

- Key characteristics: several cell layers; superficial cells are elongated and columnar! - Location: male urethra

- Function: protection and secretion! PSEUDOSTRATIFIED COLUMNAR!

- Key characteristics: single layer of cells of differing heights!

- Location: trachea! - Function: secreted substances, particularly mucus; propulsion of mucus by ciliary action!

MUSCLES SKELETAL MUSCLE!

- Key characteristics: long, cylindrical, multinucleate cells, obvious striations!

- Location: skeletal muscles attached to bones! - Function: voluntary movement; locomotion; manipulation of environment; facial expression; voluntary control! SMOOTH MUSCLE!

- Key characteristics: spindle shaped cells with central nuclei; no striations; cells arranged closely to form sheets; uninucleate!

- Location: walls of hollow organs! - Function: propels substances (food/urine) or a baby along internal passage ways; involuntary control! CARDIAC MUSCLE! - Key characteristics: branching, striated, generally uninucleate cells that interdigitate at specialised junctions called intercalated discs

- Location: walls of the heart! - Function: as it contracts, cardiac muscle propels blood into circulation; involuntary control!

CONNECTIVE TISSUE — living cells in a nonliving matrix! FUNCTION: protection, support, binding of tissues! LOCATION: everywhere! Cartilage

Bone

Blood

Cells

Chondroblasts and Chondrocytes

Osteoblast (immature) and Osteocyte (mature)

Red blood cells, White blood cells and Platelets

Matrix

Collagen fibres and elastin fibres

Bone = collagen and bone minerals

Plasma

Function

Support and protection

Support and protection

Transportation of nutrients, gasses, waste, cells, and signalling molecules

BLOOD

BONE

- Key characteristics: red and white blood cells

- Key characteristics: hard calcified matrix

in a fluid matrix (plasma)!

- Location: contained within blood vessels! - Function: transport of respiratory gases, nutrients, waste, and other substances!

- RBC: carry oxygen and carbon dioxide! - Platelets: clotting and wound repair! - WBC: fight infection; many subtypes for different roles (PURPLE NUCLEI)!

- Plasma: non-living matrix; medium that transports living and nonliving throughout the body! CARTILAGE - ELASTIC CARTILAGE

containing many collagen fibres; osteocytes lie in lacunae; very well vascularised!

- Location: bones! - Function: support and protects; provides leavers for the muscles to act on; stores calcium and other minerals and fat; marrow inside bones is the site for blood cell formation (hematopoiesis)!

- Compact bone: osseous tissue! - Spongey bone: cancellous bone$

- Key characteristics: high density of chondrocytes and more elastic fibres! - Location: external ear (auricle)! - Function: maintains shape of a structure while allowing great flexibility! - HYALINE CARTILAGE - Key characteristics: less chondrocytes than elastic; smooth matrix! - Location: costal cartilage of a rib! - Function: supports and reinforces; serves as resilient cushion; resists compressive stress! - FIBROCARTILAGE - Key characteristics: least amount of chondrocytes; fibrous matrix; banding effect! - Location: intervetebral disc! - Function: tensile strength with the ability to absorb compressive shock!

CONNECTIVE TISSUE PROPER • LOOSE CONNECTIVE TISSUE • AREOLAR: binds to skin and muscles!

- Key characteristics: fibres of different sizes; gel like a matrix with all three fibre types; cells: fibroblasts, macrophages, mast cells, and some white blood cells!

- Location: widely distributed under epithelia of body; e.g. forms lamina propria of mucous membranes; packages organs; surrounds capillaries!

- Function: wraps and cushions organs; its macrophages phagocytise bacteria plays important role in inflammation holds and conveys tissue fluid! • RETICULAR: soft supportive structure for cells! - Key characteristics: many cells; reticular fibres are curvy and branching like roots and blood vessels!

- Location: spleen, lymph nodes, bone marrow! - Function: fibres form a soft internal skeleton that supports other cell types including white blood cells mast cells and macrophages.! • ADIPOSE: stores lipids and regulates metabolism! - Key characteristics: look like droplets!

- Location: under skin; around kidneys and eyeballs; within abdomen; in breasts! - Function: provides reserve fuel; insulates against heat loss; supports and protects organs! • DENSE CONNECTIVE TISSUE! • REGULAR: tendons and ligaments — parallel collagen fibres! • IRREGULAR: covering of muscle and bones! • ELASTIC: elasticity and recoil in lungs and vessels!

NERVOUS TISSUE • Collection of specialised cells that make up the nerves and the nervous system! • Central nervous system — brain and spinal cord! • Peripheral nervous system — nerves leaving the spinal-cord! • The two main cell types of the nervous system! • Neurons: electrical signals! • Neuroglial —support nourish protect!

OSMOSIS AND DIFFUSION" OSMOSIS AND TONICITY: Movement of water through a semipermeable membrane. This movement of water is driven by the difference in tonicity between solutions.! DIFFUSION: The movement of molecules from a region of higher concentration to a region of lower concentration moves down its concentration gradient ! Osmosis and diffusion are types of passive transport (do not require energy)! OSMOSIS

• Process! 1. Weigh the eggs! 2. Place one egg in each beaker and leave for an hour! 3. Weigh the eggs! • Deshelled egg in pure water!

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Solution 1: water (hypotonic)! Solution 2: egg (hypertonic) 10% solutes! Water will flow from solution 1 to solution 2!

Egg will gain weight! • Deshelled egg in 30% sucrose (hypertonic)!

- Solution 1: sucrose water (hypertonic) 30% solutes!

- Solution 2: egg (hypotonic) 10% solutes! - Water will flow from the egg into solution 2! - Egg will lose weight! • movement of pure water from a diluted solution through a semipermeable membrane to a concentrated solution!

• How do we know osmosis occurs? — change in water! • 1mL water = 1g! • weight after - weight before = water weight!

• water will move from a high water potential (more water per solute) to a low water potential

EXPERIMENT 2:

• Goal of osmosis is to have equilibrium !

Osmosis in osmometer

• Direction of water movement is dependent on OSMOTIC PRESSURE!

• Solution 1: Molasses (80% solutes) (hypertonic) — inside osmometer!

• Osmotic pressure = [solutes]/[water]!

• Solution 2: water (0% solutes) (hypotonic) — beaker!

• Osmolarity (all solutes) = [solutes]/[water]! • Tonicity (non-penetrating only — cannot pass membrane) = [solutes]/[water]! TONICITY • “Hypo” — less than (diluted) • “Iso” — equal • “Hyper” — more tha (concentrated) • Water will flow from hypo to hyper Example 1: same amount of water! Solution 1 — Hypertonic! Solution 2 — Hypotonic!

• Water will flow from the beaker into the osmometer (gain height because water is entering! Osmosis in red blood cells • Solution 1: Red blood cells ! • Solution 2: Plasma (0.9% salt)! • Process! 1. Mix blood with solutions of different tonicity (9%, 0.9%, and 0% NaCl)! 2. Observe under a microscope !

Example 2: same number of particles!

• 0.09% NaCl solution — normal RBC morphology! • 9% NaCl solution (hypertonic) RBC: 0.09% (hypotonic)!

Solution 1 — Hypotonic (more water)! Solution 2 — Hypertonic (less water)!

- Water will flow from of RBC to salt solution! - When RBCs are in a hypertonic solution it

- Water from solution 2 will move to solution 1!

- Water from solution 1 will move to solution 2! Example 3: Isotonic! Solution 1 — 5 particles and 100mL water! (5/100 = 1/20)! Solution 2 — 10 particles 200mL water! (10/200 = 1/20)! EXPERIMENT 1: Osmosis in a non-living membrane!

will CRENATE (shrink! • 0% NaCl solution (hypotonic) RBC: 0.09% (hypertonic)!

- Water will flow from solution into RBC! - When RBCs are in a hypotonic solution it will swell and explode (haemolysis) — RBC ghosts

• Beaker 3! HYPOTONIC

DIFFUSION • Net movement of anything from a region of higher concentration to a region of lower concentration until equilibrium is met! • Factors that affect the rate of diffusion!

- Concentration gradient! - Mass of the molecules (smaller molecules diffuse quicker!

- Temperature (Higher temperature; particles are moving quicker — diffuse quicker!

- Permeability of membrane! EXPERIMENT 3: diffusion and mass (size of molecule) Potassium permanganate (molar mass = 158g/mol) ! - diffuse more!

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Methylene blue (molar mass = 319.85g/mol)! % - diffuse less! EXPERIMENT 4: Osmosis and Diffusion in Dialysis! • Process! 1. Fill the beakers with solutions! 2. Fill capillary sacs with solutions! 3. Weigh the sacs (before)! 4. Place sacs in beakers for one hour! 5. Weigh sacs (after)! • Beaker 1!

- Dialysis sac: 40% glucose! ✓ Low water potential! ✓ High solute concentration! ✓ Hypertonic ! - Beaker: water! ✓ High water potential! ✓ Hypo-osmotic! - Osmosis: water will move from the beaker into the sac

- Diffusion: glucose moved from the sac into the beaker • Beaker 2!

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Dialysis sac: 40% glucose! Beaker: 40% glucose! Iso-osmotic and Isotonic! There is no net change but molecules still move between the sac and the beaker!

- Dynamic equilibrium!

- Dialysis sac: 10% NaCl! ✓ Low water potential! ✓ High solute concentration! ✓ Hypertonic! - Beaker: water! ✓ High water potential! ✓ Hypo-osmotic! - Osmosis: water will move from the beaker into the sac

- Diffusion: NaCl moved from the sac into the beaker! • Beaker 4!

- Dialysis sac: 40% sucrose with red dye! ✓ Low water potential! ✓ High solute concentration! ✓ High dye concentration! ✓ Hypertonic! - Beaker: water! ✓ High water potential! ✓ Hypotonic! ✓ Hypo-osmotic! ✓ Low dye concentration! - Osmosis: water will move from the beaker into the sac

- Diffusion: sucrose moved from the sac into the beaker

- Dye molecule is too large = no movement of dye • How do you know osmosis occurred? — water weight! • How do you know diffusion occurred? — reactions with other solutes! • Benedict’s test: test for sugars! • mix benedicts with sample fluid then apply heat — turn red (colour change)! • Beaker 1: both dialysis sac (red) and beaker (orange) tested positive but not the same colour = not the same concentration of glucose = sac still has slightly more sugar = not left in long enough to reach equilibrium! • Beaker 4: beaker fluid tested negative and dialysis sac is negative — benedicts does not detect sucrose because of the oxygen that reacts with benedicts is being used to connect glucose and fructose! • Benedicts tests for monosaccharides ! • Silver nitrate’s test: test for chlorides! • Put a drop of silver nitrate into a solution that has chloride — it will turn white (chemical reaction between silver and chlorine)!

• Beaker 3: when sodium enters water, Na and Cl are pulled apart — when silver nitrate enters water, Ag and NO3 are pulled apart — when these are mixed Ag and Cl attach = AgCl forms a solid in liquid (white); Na and NO3 attach! OVERVIEW! OSMOSIS: net movement of water from a diluted solution to a concentrated solution! — dependent on osmotic gradient (solutes concentration)! — hypotonic>hypertonic! TONICITY: how much solutes are in the solution that give its concentration! WATER ALWAYS FLOWS FROM HYPO TO HYPER EGG (non-living)

- Egg in a hypotonic solution gains weight! - Egg in a hypertonic solution loses weight! RBC (living) (has a semipermeable membrane and a cytoskeleton that forms its shape

- RBC in a hypotonic solution swells and explodes (haemolysis)!

- RBC in the hypertonic solution shrinks and crenates! DIFFUSION: net movement of anything from a region of concentration to a region of lower concentration !

- solutes it's gonna be detected by testing the chemical reactions !

- Benedicts solution will react with nonreducing sugars!

- Silver nitrate will react with chlorine ! REVIEW QUESTIONS! 1. What is the relationship between molecular weight and the rate of molecular and movement (diffusion)?! 2. Draw diagrams that represent three microscopic fields containing RBCs that are isotonic, hypertonic, and hypotonic. Explain or draw the direction of osmosis.! 3. Think about your results from activity three. What conclusions can you make about the relative size of glucose, sucrose, congo red die, NaCl and water molecules?! 4. Summarise the results from activity five. Diffusion or osmosis through living membranes? !

GRAM STAINING MICROORGANISMS! • Food source/digestion! • Nitrogen cycle! • Oxygen Cycle! • Decomposition! • Maintaining human health!

CELLULAR MORPHOLOGY! Characterised under a microscope!

- Arrangement: how do individual cells arrange with each other!

- Shape: shape of individual cells! - Cell wall: what type of cell wall does it have! Arrangement + shape = give morphological name!

TYPES OF MICROORGANISMS! • Bacteria! • Archaea! • Viruses! • Protozoa! • Fungi! • Algae! • Multicellular Animalia! BACTERIA! • Unicellular organisms that exist in the form of colonies ! • prokaryotes — lack membrane bound organelles! • Ways to identify and characterise bacteria! • Colony morphology ! • Cellular morphology ! • Metabolism! COLONIES Culturing bacteria • Allows you to grow bacteria for further testing! • Viewing under microscope! • Gene analysis! • Antibiotic testing! • Allows for observation on colony morphology! • Colour! • Shape/form! • Edges! • Elevation! • Allows for observation of growth characteristics! • Colony forming units: how quick and how many! • Blood agar: to see if bacteria has the capacity to haemolyse red blood cells! • Normal agar: growth medium! • Allows for observation on metabolism! • Can’t grow in the presence of oxygen or sugar!

CELL WALL! • Thick peptidoglycan layer = GRAM POSITIVE — stains purple • Thin peptidoglycan layer = GRAM NEGATIVE — stains pink

GRAM STAIN EXPERIMENT !

Common Errors

1. Fixation!

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- Safety flame: yellow; used to create an aseptic environment!

- Working flame: blue; used to fix bacteria to the slide!

- Create an aseptic area using the working flame!

- Add a drop of sterile water to a microscope slide!

- Sterilise the inoculation loop (working flame)! - Pick up a small amount of bacterial colony an place in sterile water!

- Dry the slide! - Fix (melt) ...