Bio206 Microbiology

Lab 1: Laboratory Orientation

Updated 16 December '04

I. Safety:

The microbiology lab is a place for handling and examining cultures of microorganisms. Any culture of any organism should be handled with respect for its potential pathogenicity. It is important to avoid any risk of contaminating oneself or one's neighbors. It is also necessary to avoid the contamination or cross contamination of the cultures studied. Asepsis and proper disinfection must be a part of all activities.

The guidelines for the microbiology laboratory are designed to:

1. Restrict microorganisms present in specimens or cultures to the vessels in which they are contained or studied.
2. Prevent environmental microorganisms from entering specimens or cultures and interfering with results of studies.
3. Facilitate the successful completion of assigned laboratory activities.

Guidelines and precautions:

1. Any handouts or web pages should be reviewed before the laboratory period.
2. Hands and bench tops are cleaned before and after laboratory activities. Take special precautions with any open wounds on the hands. Culture spills or other accidents should be given the proper attention immediately. Wipe up culture spills with paper towels and disinfect spill area. Place contaminated towels in a "kill" container.
3. Protective clothing (lab coat) is recommended.
4. Long hair is controlled and no jewelry is worn during laboratory activities.
5. Nothing is placed in the mouth! (No food, drink, pencils, gummed labels, pipettes, fingers, etc.)
6. Coats, excess books and other personal items are left outside the lab.
7. Keep a careful record of all your laboratory activities, and answer any questions in a lab notebook. For each lab activity note date, provide a description of starting materials, give expected results, record observations, make explanations, and give your conclusions. If you use information from a source other than your own work, make an appropriate citation.
8. Properly dispose of all cultures and glassware. Do not remove any contaminated materials from the lab.
9. Immediately report any accident or malfunction of equipment to the instructor.

II. Use of microscopes (see chapters 1 & 3 in the text)

A. Learn to identify the important parts and their functions. The functions will be demonstrated by the lab instructor.

B. Microscopic examination of a slide preparation.

1. Place a slide preparation of a microorganism in position on the stage. Position it so the light from the condenser passes through a specimen area. The smaller the light beam the better the view.
2. Bring the low-power objective into position and lower it to the slide with the coarse adjustment, observing from the side.
3. Look through the ocular and raise the objective slowly upward with the coarse adjustment until you see cells in the field of view.
4. Use the fine adjustment knob to get the image in sharp focus. This should give you an overview of the preparation and enable you to select an interesting area for closer observation. Draw a representative area of what you observe. Make sure an interesting area is near the center of the field.
5. Position the high-power objective without adjusting the focus and examine the image. Adjust the fine focus to get a sharp image. Note the difference in magnification with the different objectives. Draw a representative view of what you observe. Position an interesting area near the center of the field.
6. Move the high-power objective a little to one side and place a drop of oil on the slide, directly over the stage opening. While watching the oil-immersion objective, bring it carefully into position without adjusting the focus making certain it does not touch the slide. Carefully lower the objective into the oil. Look through the ocular and carefully adjust upwards with the fine focus until the image is sharpest. If you have trouble ask the instructor for help. Draw a representative view of what you observe. ***Do not pass the 40X objective back through the oil. It would be damaged.***
7. When you have finished your observation, raise the objective out of the oil and gently clean the lens with a piece of lens paper.
8. Place all slides of bacteria in a designated disinfectant sloution.

C. Care and handling of the microscope

1. Always use both hands to carry the microscope.
2. Before each use, examine the microscope carefully and report any unusual condition or damage.
3. Keep the ocular(s), objectives, and condenser lenses clean with lens paper.
4. After each use, remove the slide from the stage, wipe away the oil, and place the low-power objective in vertical position.
5. Return the microscope to its storage area.

D. Questions

1. Which objective focuses closest to the slide?
2. What does it mean when a microscope is parfocal?
3. What controls the amount of light reaching the ocular lens?
4. What is the recommended method of carrying a microscope?
5. Name three ways to enhance resolving power.
6. What advantage does immersion oil provide? Why?
7. What bacterial shapes did you observe? Sketch several representative of each (see p 13 in your text as an example).

Handling and observing cultures

I. Procedures

A. Transfer of a culture

1. Pick up the inoculating loop as you would a pencil, loop down. Hold the wire in the flame of the Bunsen burner until it glows red. Remove the loop from the flame and hold it quietly for a few moments until cool. Do not lay it down or touch it to anything.
2. Pick up a slant culture with your left hand. Still holding the loop like a pencil in your right hand, use the little finger of the loop hand to remove and hold the closure of the culture tube. Pass the neck of the open tube rapidly through the flame two or three times to sterilize the mouth of the tube.
3. Insert the loop into the open tube. Touch the loop to the cultured material and remove a loopful by scraping gently.
4. Withdraw the loop slowly and steadily without touching the sides of the tube. Hold it steady and do not touch it to anything while you replace the tube closure.
5. Still holding the loop in one hand. use the other hand to pick up a tube of sterile medium. Remove the tube closure, flame the neck of the tube, insert the loop into the tube and gently rub the loop on the surface of the medium to remove the culture material. Carefully remove the loop from the tube.
6. Flame, close and replace the tube in the rack; then sterilize the loop as before.
7. Label the freshly inoculated tube with your name, the name of the organism and the date.
8. Describe and sketch the bacterial growth on the original culture in your lab notebook.

B. Incubation

1. Check that all new tubes are properly and fully labeled with the organism, the date, and your name.
2. Place the transferred cultures in an assigned area of the proper incubator. Record the the reading of the incubator thermometer at the start and at the end of your culture period. Let the instructor know immediately if it is not at the expected temperature.

II. Results

A. Examination

1. Carefully note the characteristics of the cultures provided. Record information on the appearance, size, color, density, consistency, surface texture, and shape of colony.
2. Sketch in your lab book representative bacterial colonies from each culture.
3. After 24 and 48 hr of incubation observe the cultures you inoculated and record their appearance. If you have made successful transfers and achieved pure cultures, the color and appearance of your cultures should match that of the ones from which you transferred. See your notes and sketches from the original cultures. Note: Unless otherwise indicated, after 48 hr cultures should be removed from the incubator and stored at room temperature for future use.
4. Sketch in your lab book representatives of the bacterial colonies you observe.

B. Questions

1. What constitutes a proper and full label of a culture for you?
2. What is the purpose of flaming the loop before use? After use?
3. How can one tell if the media available for use is sterile?
4. Can you determine whether a culture is pure (all one species) by visual inspection without a microscope? Explain.
5. Why should long hair be controlled in the laboratory?
6. How did the colonies you observed from your 24 hr cultures compare with those from the original cultures provided?
7. What differences do you note between the 24 and 48 hr observations?