Lab 1

Objectives:	Reading:
1. Check in	1. Tortora, Chapter 3, pages 56-75
2. Lab safety	2. Pierce, pages 2, 5; 28-33
3. Sampling the environment
4. Use of the microscope

Elephants never forget...humans, however, often benefit from short reviews!

A Review of the Metric System

Meter:

Standard unit of length; its units are related to each other by factors of ten.
Latin prefixes (deci, centi, milli) generally refer to measurements less than one.
Greek prefixes (deca, hecta, kilo) generally refer to measurements greater than one.

Micrometer:

Also known as a micron.
Equal to .000001 of a meter or 10^-6 meter, which is also written as 1 x 10^-6 meter.
It is abbreviated as mm, often written as um, because of the difficulty in typing the Greek letter m (mu).
Most bacteria are between one and ten mm in length. White blood cells are between ten and twenty mm in diameter.
Using the oil immersion lens, a light microscope can easily magnify objects of this size with sufficient detail to allow for general observations to be made.

Nanometer:

Also known as a millimicron.
Equal to .000000001 of a meter or 10^-9 meter, which is also written as 1 x 10^-9 meter.
It is abbreviated as nm.
Viruses are less than 100 nm in length (less than 0.1 mm).
Proteins are less than 10 nm in length.
Atoms, the building blocks of all matter, are just over 0.1 of a nm.
The electron microscope is necessary to view objects within these size ranges.

A lesson in metric perspectives:

The white bar in the black box above represents 10 microns (0.01mm).

The green dots in the black box above the white bar are viruses (0.05 to 0.1 microns).

The purple oval bodies in the black box below the white bar are bacteria (0.5 to 1.5 microns).

Relative sizes of virus (on left) and bacterium (on right).

This is a scanning electron micrograph of a human macrophage (gray) approaching a chain of Streptococcus pyogenes (yellow). The red sphere on top of the macrophage is a lymphocyte, which interacts with the macrophage to help eliminate infection.

Thanks to http://www.cellsalive.com/frameset.htm!

A Review of Scientific Notation

Precision in Communication

Within the laboratory, it is customary to write numbers using scientific notation, also known as exponential notation. There are many advantages to using this system, especially as the field of microbiology deals with very small numbers.

Numbers written in scientific notation take the form of M x 10ⁿ.

M represents a number between 1 and 10 and includes all the significant figures that are known about the number.
The exponent, n, locates the decimal point and is obtained by counting the number of places between the original decimal point of the number and the new decimal point that is present in the scientific notation format.
When writing the value for n, the location of the decimal point is represented by a positive sign if the number is greater than 1, and a negative sign if the number is less than one. When the number is equal to one, it is written as 1 x 10⁰.
For example, 1000 is written as 1 x 10³; .001 is written as 1 x 10^-3.

Summary

To express numbers in scientific notation, remember that every number consists of the product of:

a number between 1 and 10 containing the proper number of significant figures, and
a power of 10, indicating the location and number of decimal places.

One of the advantages to using scientific notation is that addition, subtraction, multiplication, and division of very cumbersome numbers become comparatively simple.

Addition and Subtraction

All exponents must be the same before the values of M can be summed or subtracted.
Example: 6.5 x 10² + 2.0 x 10⁰- 3.5 x 10^-1 =
Change all numbers such that the exponents are the same.
One way to do this is 6.5 x 10² + .02 x 10² - .0035 x 10² = 6.5165 x 10²
Another way to do this is 650 x 10⁰ + 2.0 x 10⁰ - .35 x 10⁰ = 651.65 x 10⁰
To put this number back into standard scientific notation, simply re-write it as 6.5165 x 10².

Multiplication and Division

In multiplication, exponents of like bases are added (we use base 10 for M); in division, exponents of like bases are subtracted. The values for M are multiplied or divided, as indicated.
Multiplication example: (3 x 10⁵) x (2 x 10⁴) = 6 x 10⁹
To square, square the number M and multiply the exponent by 2; then continue:
(3 x 10⁵) x (3 x 10⁴)²= (3 x 10⁵) x (9 x 10⁸) = 27 x 10¹³
Division example: (6 x 10⁴)/(3 x 10²) = 2 x 10²

Compound Light Microscope

Illuminator: Visible light rays are used as light source (usually a light bulb)

Lens System: Composed of condenser, objective, and ocular lenses

Condenser: Directs the light rays onto and through the specimen

Objective lenses: Lens found immediately above the specimen; light rays continue to move from specimen into lenses, where magnification of object occurs

Ocular lenses: Light continues to move from objective lens into ocular lens (eyepiece), where a final magnification occurs before light hits the microscopist's eyes

Total magnification: To determine just how much your specimen is magnified, multiply the magnification of the objective lens by the magnification of the ocular lens

Resolution: This is the ability of the lenses to distinguish fine detail and structure and is defined by the distance between two points that can be distinguished as separate objects; maximum resolution using oil immersion lenses is around 0.2 mm, meaning that two small objects that are 0.2 mm apart are seen as separate entities. To get maximum resolution:

Use a blue filter over light source (blue light's short wavelength maximizes resolution)
Keep condenser at highest position to allow maximum amount of light into objective
Adjust diaphragm so that contrast is best (less light) without hurting resolution (more light)
Use immersion oil between the slide and the 100X oil immersion objective lens

Refractive index: Different media refract (or slow down and bend light) at different angles. The refractive index is the ratio of the speed of light in a vacuum to the speed of light in a particular medium.

Because no medium allows light to move faster than when it moves through a vacuum, refractive indices of all media will be greater than one.
Staining of the specimen causes a change in the velocity of the light due to its bending (refracting) as it crosses the boundary of the different medium.
Once light moves through different media, its rays begin to scatter, rather than continue in the straight line they started out with.
Consequently, in order to preserve as many light rays as possible (the more that are lost, the less we can see), immersion oil is used to completely seal the rays.
Immersion oil has the same refractive index as glass, so the oil essentially becomes part of the optics. This prevents a final refraction of light as it moves from the slide, through the specimen, and into the air before hitting the objective. The "into the air" step is eliminated by the oil.

Refraction of light as it moves from the illuminator (incident ray) through the stained material on the slide (refracted ray)

Brightfield:

Light rays pass directly through microscope to eyes without being deflected by an intervening opaque plate in the condenser.

Darkfield:

Darkfield condenser is used that contains an opaque disc that blocks light from directly entering the objective.
Light reaches the specimen at an angle, and when this light reflects off the specimen, it enters the objective.
Organism appears light against a black background.
Used to examine live microorganisms.

Phase contrast:

Special condenser is used that allows a ring of direct light to pass through the condenser, focusing light on the specimen and a ring-shaped diffraction plate in the objective lens.
One set of light rays is directly from the light source; the other is from reflected light from a particular structure in the specimen.
Phase contrast greatly increases the apparent contrast between cell organelles.
Used for detailed examination of internal structures in living microorganisms.

As seen in the prostate cancer cell (right) the phase contrast background is a mid-line gray, phase-dense organelles appear dark, and there is a bright halo around the cell.

Take Out Food for the Brain:

Bacteria, unicellular organisms that they are, reproduce with abandon. For example, the common E. coli bacterium, through cell division, becomes two bacteria in a matter of 20 minutes. Twenty minutes after that, the two are four, and so on. At this rate, within 43 hours that one bacterium would have produced enough progeny to fill the entire volume of our planet (1,090,000,000,000,000,000,000 cubic meters-can you express that in scientific notation?). After two more hours, these bacteria and their offspring would weigh as much as our earth (6,600,000,000,000,000,000,000 tons).

So, why aren't we wallowing in prokaryotic putrescence? You tell me!

Take Home Thought

The consequence of underestimating the prokaryote's powerful proclivity for propagation is potentially pejorative to our patients.

Lab 1

A Review of the Metric System

A lesson in metric perspectives:

A Review of Scientific Notation

Compound Light Microscope

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