Critical Thingking 1. Why are microorganisms hard to see in wet preparations?

In a wet mount the microorganisms are hard to see because they blend in with their surroundings or the liquid they are grown in. An example of this would be in a hay broth where the water is cloudy and the microorganisms are transparent. Plus, they move quickly and is hard to bring into focus. 2. Can you distinguish the prokaryotic organisms from the eukaryotic organisms (see figure 8)?

3. Why isnt the oil immersion lens used in the hanging drop procedure? Oil immersion would make things worse because it would refract the light even more considering that the refraction index of the oil immersion and the slide [depression slide+drop] are different. 4. From where did the organisms in the infusions come? Protozoans (e.g. Paramecium sp) come from rehydrated cysts. Bacteria can come from bacterial cysts or endospores. Fungi and water molds come from spores. Animals (e.g. rotifers, worms, daphnia) come from eggs or cysts. Function of petroleum jelly : So that the cover slip adheres to the slide and to keep the water in the depression Question 4: The human eye is only sensitive to amplitude (intensity) and wavelength (color), which are observed in a normal bright field microscope. Small, transparent objects like a cell do not change these parameters much, but due to their different refractive index from the surrounding medium, they slow down the light that passes through them. The light gets diffracted and has a phase change of approx. 1/4th of the wavelength (depends on the object thickness). Phase contrast microscopes have two rings, one that provides a hollow cone of light that illuminates the specimen and a second (so called phase plate) which lets the unaltered light pass through a thinner part and the bent light through a thicker part .This introduces another relative phase shift of 1/4, causing a net phase shift of 1/2 of the wavelength. Now this results in destructive interference, resulting in a dark object on a bright background. Question 6:

The hay infusion is a time-honored method for securing bacteria for study. Pour hot water on a handful of hay, and filter the fluid through

blotting paper. Place the fluid in a glass dish, and cover with a piece of glass to keep out the dust. When the fluid begins to appear turbid, bacteria will be abundant. The active movements are easily observed in a mount from the turbid water. As the bacteria pass into the resting condition, they form a scum on the surface of the water. Usually, the first to appear is a somewhat rod-shaped form, the Bacterium termo of the older texts. Spirillum and Coccus forms often appear later. Fine preparations may be obtained by inoculating a mouse with Anthrax, and then cutting paraffin sections of favorable organs. For making mounts of a dangerous form like Anthrax, secure properly fixed material from a bacteriologist. Stain in gentian-violet or crystalviolet. The following schedule gives good results with Anthrax and many other bacteria: 1. Gentian-violet, 5 minutes. 2. Rinse in water a few seconds. 3. Gram's solution (iodine 1 g., potassium iodide 2 g., water 300 c.c.) until the color is almost or quite black; this will generally require 1 or 2 minutes. 4. 95 per cent alcohol until the color has nearly disappeared. 5. Rinse in water and examine. If the bacteria are well stained, a counter-stain may be added. 6. Light green or erythrosin, 5 seconds; or Bismarck brown, 5 or 10 seconds. 7. 95 and 100 per cent alcohol, dehydrating as rapidly as possible. Not more than 5 or 10 seconds can usually be allowed. 8. Xylol, 1 to 5 minutes. 9. Balsam. After the rinsing in water of stage 5, the preparation may be dehydrated rapidly in 95 per cent and 100 per cent alcohol, and then stained for 5 or 10 seconds in orange dissolved in clove oil. From the clove oil, transfer to xylol and mount in balsam. With sections not more than 5( thick, excellent results can be obtained by staining in iron-alum

haematoxylin. The following rapid method gives fairly good results: 1. Place on a clean cover a drop of water containing the bacteria and dry completely in a flame or on a hot plate. 2. Stain 2 to 5 minutes in gentianviolet or methyl violet. 3. Rinse quickly in water. 4. Dip into 95 per cent alcohol to reduce the stain. 5. Remove most of the alcohol by touching a corner of the cover with filter paper and then dry completely by passing through a flame. 6. Mount in balsam.

Question 1: The hanging drop and wet mount techniques allow for observation of living organisms. The wet mount tend to dry out quickly under the heat of the microscope light; it is simpler to perform than the wet mount, but it is useful for short-term observation only. The hanging drop is a more complex technique, but it allows for longer-term obervation and more reliable observation of motility. These techniques are usually performed without the addition of any stains; therefore, the organisms can be difficult to see. Reduce the illumination on your microscope as much as you can while still allowing yourself enough light to observe the organism. If you use these techniques to observe motility, be sure you can tell the difference between motility and brownian motion. Vibration of the cell is caused by the cell colliding with water molecules. True motility allows the cell to move in different directions and across larger areas. Question 2; Brownian motion is the bombardment of cells by water molecules. The cells appear to be
vibrating. True motility is movement of the cell by appendages. The cells move to and fro and the movement is more obvious. True motility is movement in a specific direction. Brownian movement is just random motion in any direction. c.t :

Eukaryotic Cell
Nucleus: Number of Present More than one

Prokaryotic Cell
Absent One--but not true chromosome:

Eukaryotic Cell
chromosomes: Cell Type: True Membrane bound Nucleus: Example: Telomeres: Genetic Recombination: Lysosomes and peroxisomes: Microtubules: Endoplasmic reticulum: Mitochondria: Cytoskeleton: DNA wrapping on proteins.: Ribosomes: Golgi apparatus: Mitosis: Chloroplasts: Multicellular Present

Prokaryotic Cell
Plasmids Unicellular Absent

Animals and Plants Present (Linear DNA) Mitosis and fusion of gametes

Bacteria and Archaea Circular DNA doesn't need telemeres Partial, undirectional transfers DNA

Present

Absent

Present Present

Absent or rare Absent

Present Present Yes

Absent May be absent No

larger Present Yes Present (in plants)

smaller Absent No---but has binary fission Absent; chlorophyll scattered in the cytoplasm Submicroscopic in size, composed of only one fiber

Flagella:

Microscopic in size; membrane bound; usually arranged as nine doublets surrounding two singlets

Eukaryotic Cell
Permeability of Nuclear Membrane: Plasma membrane with steriod: Cell wall: Selective

Prokaryotic Cell
not present

Yes

Usually no

Only in plant cells (chemically simpler) 10-100um

Usually chemically complexed

Cell size:

1-10um

the difference is Brownian movement is that first of all it is not a true form of motility and is simply the result of vibrating particles that make the bacteria seem like they are moving on their own. This usually occurs in dead bacteria. True motility requires a flagella and is a true form of motility it is also seen through rotations in spiral movements by the bacteria. True motility is only seen in live bacteria.

Brownian motion is a phenomena whereby small particles suspended in a liquid tend to move in psuedo-random or stochastic paths through the liquid, even if the liquid in question is calm. It is the result of asymmetry in the kinetic impacts of molecules that make up the liquid. The liquid phase, by definition, must have some temperature, meaning its molecules or atoms must be thermally excited, bumping into each other and objects suspended within them. To picture this phenomena, a person can imagine the motion of golf balls on a table filled with thousands of ball bearings moving in quick trajectories. The phrase Brownian motion can also refer to mathematical models used to describe the phenomenon, which have considerable detail and are used as approximations of other stochastic motion patterns. The mathematical motion is related to, but more structured than, the random walk, in which the displacement of a particle is entire randomized. The phenomena has the Markov property, a term from probability theory which means that the future state of the particle is determined entirely by its current state, not by any past state. Used in this sense, the mathematical concept is slightly different, but very similar to, physical Brownian motio