1.3: Procedure

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SPECIMENS

Prepared slides of the following bacteria:

Staphylococcus aureus
Escherichia coli
Treponema pallidum
Spirillum species

On-line demonstration slides of the following bacteria:

Micrococcus luteus
Neisseria gonorrhoeae
Streptococcus pyogenes
Bacillus megaterium

Broth culture of Saccharomyces cerevisiae

Human hair

PROCEDURE

TIPS FOR MICROSCOPIC OBSERVATIONS

Move the slide around until you see an area representing the true arrangement of each organism. Also remember:

In the process of making the slide, some of the arrangements of cocci will clump together and others will break apart. Look carefully to determine the true arrangement.
Small bacilli (such as Escherichia coli) that are dividing or have just divided by binary fission will look similar to cocci. Look carefully for bacilli that are not dividing and are definitely bacillus-shaped as well as bacilli in the process of dividing to confirm the true shape.
Bacilli do not divide so as to form clusters. Any such clusters you see are artifacts from preparing the slide.
Look carefully to see the spirochetes as they are the thinnest of the bacteria. When seen microscopically, spirochetes resemble extremely thin, wavy pencil lines.

1. Using oil immersion microscopy (1000X), observe and measure the bacteria listed below.

a. Spirillum: Spirillum species appear as thick, rigid spirals. Measure the length and width of a typical spirillum.

When finished, remove the oil from the prepared slides using either a paper towel or a Kim wipe and return them to their proper tray.

2. Observe the on-line demonstration slides of the following bacteria:

a. Micrococcus luteus: Micrococcus luteus can appear as tetrads or cubes of 8. This strain is a coccus usually exhibiting a tetrad or a sarcina arrangement. Note the shape and arrangement.

b. Neisseria gonorrhoeae: Neisseria species are cocci usually having a diplococcus arrangement. Note the shape and arrangement.

c. Streptococcus pyogenes: Streptococcus species, as the genus name implies, are cocci that usually possess a streptococcus arrangement (cocci in chains). Note the shape and arrangement.

d. Bacillus megaterium: Bacillus megaterium appears as large bacilli in chains (a streptobacillus). Note the shape and arrangement.

3. Prepare a wet mount of baker's yeast (Saccharomyces cerevisiae).

a. Using a pipette, put a small drop of the yeast culture on a microscope slide and place a cover slip over the drop.

b. Using your iris diaphragm lever, reduce the light for improved contrast by moving the lever almost all the way to the right.

c. Observe using oil immersion microscopy. Measure the diameter of a typical yeast cell.

d. When finished, discard the coverslip in the broken glass disposal container, wash the slide with water and dry with a paper towel, and use the same slide again for step 4.

4. Prepare a wet mount of your hair.

a. Remove a small piece of a hair from your head and place it in a small drop of water on a slide.

b. Place a cover slip over the drop and observe using oil immersion microscopy.

c. Measure the diameter of your hair and compare this with the size of each of the bacteria and the yeast observed in steps 1-3.

d. Discard the slide and coverslip in the broken glass disposal container.

5. At the completion of the lab, remove the oil from the oil immersion objective using lens paper and put your microscope away.

Contributors and Attributions

Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)