Day 1: Syllabus, Safety, and Movie introduction to the microbiological laboratory




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Calculations of Microlengths: 1 um = 10-6 meter (1/1000000 of a meter)




EXERCISE

1) Choose a location in the building and collect a sample with a sterile loop, if you desire to collect from a wet site, or a Q-tip that has been dipped in sterile water to dissolve microbes, if you choose a dry site. Inoculate a nutrient agar plate with the sample.


2) Calculate the number of meters in a micrometer, a nanometer, and a kilometer. Use your text and other resources to solve these problems.


3) View the slides below. For a take-home quiz, search the literature for the organisms below and determine where they live, whether the organism is prokaryotic or eukaryotic, and whether the organism has any medical significance. Turn in your answers during the following lab period.

  1. Bacillus subtilis or Bacillus anthracis

  2. Anabaena

  3. Gloeocapsa

  4. Amoeba proteus

  5. Aspergillus

  6. Treponema pallidum



4)Know the main bacterial shapes - helices, cocci, bacilli.

5) For future reference, look up and know the definitions of the following terms: agar, colony, inoculate, media, pathogen, sterile.


FOR LABORATORY HELPERS

Supplies:

35 nutrient agar plates, microscopes

Sterile Q-tips




REFERENCES


Essential Cell Biology: An Introduction to the Molecular Biology of the Cell, Alberts, Bray, Johnson, Lewis, Raff, Roberts, and Walter, Garland Publishing Inc., New York, © 1998, pages 1-8.


Exercises for the Microbiology Laboratory, Burton E. Pierce & Michael J. Leboffe, Morton Publishing Company, Colorado, © 1999, pages 27-33.


Laboratory Experiments in Microbiology, 6th Edition, Johnson & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 2001, pages 5-17.


Laboratory Manual and Workbook in Microbiology: Applications to Patient Care, 6th Edition, Morello, Mizer, & Wilson, McGraw-Hill Companies, Inc., Boston, © 1998, pages 7-11.


Microbiological Applications, 6th Edition, Harold J. Benson, Wm. C. Brown Publishers, Dubuque, © 1994, pages 3-24.


Microbiology: An Introduction, 6th Edition, Tortora, Funke, & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 1998, pages 3-5.


Day 3: Introduction to Samples, Smears, and Staining

SAMPLES


A sample is all or a portion of an object of interest. For example, when you have a sore throat and go to the clinic, one of the medical staff may need to collect material from your throat (a 'swab') to generate a culture. You may remove, or sample, a portion of your culture to stain. Sources microbial samples include many different bodily fluids, tissues, soil, water, air, outer space?, Mars?, or wherever microbes occur (which is everywhere).


PREPARATION OF SAMPLE FOR VISUALIZATION

HANGING DROP: In this procedure, you will be suspending a drop of water filled with microbes from the bottom of a coverslip and then securing that coverslip to a slide with a well. This procedure will allow you to view microbial motion, which is also termed motility. You may observe another phenomenon called Brownian motion (Einstein won his Nobel prize for accurately describing and interpreting this phenomenon), which presents the appearance of motility when microbes are stuck by water molecules. To view living cells it is best to use a phase contrast microscope. WET MOUNT: In this procedure, a drop of water that has been inoculated with microbes is sandwiched between a coverslip and the slide. This allows you to quickly view microorganisms under the microscope. However, this type of sample dries out quickly, halting microbial movement. SMEARS: Preparing a smear is important if you want to see each individual microbe in detail. This technique allows you to first stain and then view microbes in the absence of microbial motion; you should be able to spend some time with each microbe that you smear and take accurate notes. If your technique is good, you will be able to look at the same slide a week or a year from now and still view the microbes.


Smear preparation:

1) Always sterilize the loop in flame before and after touching the sample. Remove 2-3 loops of broth culture containing the bugs and place on clean microscope slide. If your organism is on an agar plate do the following: place a droplet of water on a microscope slide. Sterilize your loop and touch a colony on the plate, then suspend the material on loop in the water droplet.

2) Allow to air-dry. You can WARM it near the flame, but take care not to cook your bugs! They will lyse and disintegrate if boiled.

3) Heat fix the cells to the glass by slowly passing the sample through the flame 3 times. The slide should be hot to the touch (touch the underside to the back of your hand). Heat fixing both permanently attaches cells to the slide and kills them, which is important if you are working with pathogens.

4) Proceed with staining.


STAINING


Staining is a method of adding light absorbing/reflecting chemicals to a cell to improve the contrast between cell and its surroundings. Basic dyes are positively charged (cationic) and acidic dyes are negatively charged (anionic). Most microbes have net negative charges, so basic dyes are usually employed in staining them.

Simple Stain: A simple stain is the easiest of the stains to use. For instance, methylene blue or crystal violet stain is used to make the microbes on your slide more visible through your microscope. These types of stain can be used to quickly identify the shape of the organism you will be looking at. In general, common shapes are spheres (cocci), rods (baccili), and helices (spiral), but some bacteria can assume star, square, and other odd shapes.

Procedure:

1) Place slide on tray rack and cover smear with methylene blue.

2) Incubate for 1 minute (i.e., allow the stain to soak in).

3) Rinse the stain off of the smear with water.

4) Blot dry with paper towel or bibulous paper and view in scope.


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