MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #7: MOTILITY, OXYGEN REQUIREMENTS AND CAPSULE STAIN

NAME, ID #:_______________________________________________.

TA Name __________________________

REVISED: 08/04/99

INTRODUCTION

This exercise will introduce you to three more BACTERIAL CHARACTERISTICS that can be used to identify and describe bacteria:

1. The ability to be MOTILE.
2. Their OXYGEN REQUIREMENTS.
3. The ability to produce CAPSULES.

MOTILITY

Many bacteria are able to move actively from one place to another and they are able to "choose" their direction of movement. Basically this means that they can move towards beneficial things like food, oxygen or light or away from deleterious things like toxic chemicals, or oxygen (anaerobes). The ability to move in a direction in response to conditions in the environment is called TAXIS. Consider the IMPLICATIONS of this observation! This means that bacteria can "SEE" or "SENSE" things just like we can. Indeed, some bacteria have primitive "EYES" that see light and primitive "NOSES" that smell chemicals like we smell expelled gut-gasses (which prompts our "taxis" system to move away). Can you think how they might do this? Do bacteria have a tiny brain?

Most bacteria, but not all, that are motile, move by using structures call #FLAGELLA. Flagella are long rigid rod-like structures made OF REPEATING PROTEIN SUBUNITS. They are attached to a MOTOR located in the cell wall that turns them like a propeller. The flagella motor is powered by a flow of PROTONS, sort acting like electricity. As flagella rotate they turn or rotate like a PROPELLER and drive the bacterial cell through liquid. We will use this ability to detect whether the test bacteria are motile or non-motile. You will inoculate tubes of SEMI-SOLID medium with the bacterial samples by a single stab in the center. Only motile bacteria can move away from the inoculation site. After incubation you will examine the tubes to determine which bacteria have spread away from the original stab site.

RESPONSE TO OXYGEN

Bacteria are classified as to their response to GASEOUS OXYGEN. Bacteria that absolutely require oxygen are said to be OBLIGATE AEROBES, whereas those that are unable to grow in the presence of free oxygen because O₂ kills or inhibits them, are called OBLIGATE ANAEROBES. A large group of bacteria have the ability either to use oxygen or to grow in its absence; these bacteria are described as FACULTATIVE AEROBES, because they have the "facility" to grow under either condition. Some bacteria need a SMALL QUANTITY of oxygen, but large quantities inhibit their growth or even kill them. These are called MICROAEROPHILIC bacteria.

How anaerobic jars work:

• The anaerobic jars contain a chemical mixture that produces hydrogen gas after it is wetted.
• The jar is sealed air-tight after the cultures are added and the chemicals are activated.
• The anaerobic jars contains a catalyst.
• The oxygen and hydrogen in the anaerobic jars react on the catalyst to produce water which removes the free oxygen.

CAPSULES

Capsules are structures that lay outside of an organism's cell wall and thus are in direct contact with the environment. Many, perhaps most, bacteria produce capsules under the right conditions. Bacterial capsules are most often composed of long POLYMERS of sugar (or sugar derivatives) known as POLYSACCHARIDES. Some capsules are composed of POLYALCOHOLS or AMINO ACID POLYMERS. Capsules may be absent, thin, thick, well formed or loose. Capsule formation is dependent upon the nutrient conditions. In general, media rich in sugars and low in nitrogen tend to induce capsule formation. Capsules play a number of roles in the life of microbes. Both prokaryotes and eukaryotes form capsules. Capsules can serve the following functions:

1. Protect the cell from desiccation (drying).
2. Protect the cell from phagocytosis (being engulfed by white blood cells).
3. Provide a food reserve when certain organic compounds are in excess.
4. A virulence determinant of pathogenic microbes.
5. They serve as binding or adhesion agents for sticking cells together and/or to a surface such as a rock in flowing stream or a tooth.

We are most interested in the role capsules play in pathogenicity. One of the better understood of these cases is that of the bacterium Streptococcus pneumoniae, the etiological agent (the agent that causes a given disease) of BACTERIAL PNEUMONIA. The virulent form (that form able to cause a disease) of this bacterium always processes a pronounced capsule. Mutants of S. pneumoniae that lack a capsule are avirulent (unable to produce a disease). You will hear later about how a study of this organism and the gene responsible for capsule formation first demonstrated the CHEMICAL NATURE OF GENES and the process of #TRANSFORMATION.

Very few dyes stain capsules, so they are usually viewed using a NEGATIVE stain. However, a slightly different procedure will be used to visualize the cells as well as the capsule. In this procedure Congo Red will be used as the background or negative stain. A basic stain will be used to STAIN THE CELLS so the actual capsule surrounding the cell can be observed.

Figure 3. Polysaccharide chain.   Polysaccharide chains like this make up many, if not most, capsules; the red hexagons represent the sugar molecules and the black lines represent the covalent bonds between the sugar molecules. However, their chemistry is far more complex than shown here between branching and substitutions on the sugar molecules.

PURPOSE OF LABORATORY:

1. To carry out a test for bacterial motility and to learn about various types of flagella arrangements.
2. To demonstrate the effect of oxygen on the growth of various bacteria.
3. To carry out a capsule stain.

RELATIONSHIP TO LECTURE MATERIAL

• NetText101/102: CHAP. III, Bacterial architecture; spores; motility & flagella & taxis; oxygen requirements

MATERIALS NEEDED

1. Cultures:
   • CAPSULE: K. pneumoniae (in milk)
   • OXYGEN REQUIREMENT: Pseudomonas aeruginosa, Clostridium sporogenes, E. coli
     • The Clostridium culture will be at the front desk where you must go to inoculate it into the appropriate tube.
   • MOTILITY: Proteus vulgarus, Micrococcus luteus, B. subtilis, Staphylococcus aureus
2. Tubes of the appropriate media on side bench.
3. Congo red stain for capsule stain on side bench.
4. A-M Capsule Stain Solution on side bench. CAUTION: THIS STAIN CONTAINS PHENOL!
5. Anaerobic jars and N.A. petri dishes.

GENERAL INSTRUCTIONS:

1. Carry out the capsule stain first.
2. While the capsule stain is drying in the air (DO NOT heat fix), inoculate the motility and oxygen-growth tubes with the appropriate bacteria.
3. Place the inoculated tubes in either the 37^oC incubator or in the rack for incubation at room temperature as indicated on the front board.
4. View the capsule stain and draw what you see.

PROCEDURES

CAPSULE STAIN

1. Clean slide with Bom Ami (by sink) and air dry. Do not touch surface of a cleaned slide with your fingers, handle only at edges.
2. Place a drop of Congo red on one end of the slide as in Exercise 4, Fig. 1.
3. Aseptically pick up a sample from a culture of K. pneumoniae with your loop and mix briefly with the Congo red stain.
4. Spread the mixture across the slide as shown in Exercise 4, Fig. 1 so as to produce a thick to thin smear.
5. Air dry ONLY. Proceed to inoculate your tubes while drying.
6. Stain 1-2 minutes with the A-M Capsule Stain Solution.
7. Rinse very gently, air dry, but do not blot.
8. Look for red cells surrounded by a clear, colorless zone on a blue background.
9. Draw the results in the circles below.
10. Compare your capsule stain with that of medical students and on pg. 31 of Atlas. Other examples of capsule stains 1 & 2

Figure 4. Preparation of capsule stain.

MOTILITY

1. Before proceeding read pg. 34 and 64 in A Photographic Atlas for the Microbiology Laboratory. Also look at Fig. 3 in NetText Chap. 3.
2. Obtain an inoculating NEEDLE from one of the cans on the desk.
3. Obtain three tubes of the semi-solid medium and label appropriately with your name, the name of the organism, the section and the date.
4. Using a sterile needle, instead of the loop, aseptically dip it into an appropriate culture. STAB the needle ONLY ONCE into the CENTER of the motility medium all the way to the bottom.
   • Inoculate tubes with S. aureus or M. luteus (each lab partner do just one), with Proteus vulgarus and with E. coli.
5. Incubate the inoculated tubes appropriately (37^oC or room temperature as indicated).
6. At the next lab, examine the growth patterns of your tubes and compare them with the picture in the Atlas. Draw results below. Describe the motility of each of the bacteria tested. Verify your observations with your lab partner.
7. Compare your results with those of a nonmotile and motile organism and with the pictures in your Atlas on pg. 64.

DETERMINATION OF OXYGEN REQUIREMENTS

1. Before proceeding read pg. 7-8 in A Photographic Atlas for the Microbiology Laboratory.
2. Obtain the appropriate number of media plates, label them and inoculate pairs of the plates with the bacteria as indicated by the Instructor.
3. After inoculating the plates give them to the TA.
4. The TA will place one set in an ANAEROBIC JAR. Both sets of plates will be incubated at the same temperature.
5. Draw the growth patterns on the plates and record the data (results) in the table below to determine the oxygen relationship of each of the bacteria tested.
6. Use the data to characterize the oxygen requirements of each of the test bacteria. Verify your conclusion with the Instructor.

CAPSULE STAIN RESULTS

GROWTH UNDER AEROBIC AND ANAEROBIC CONDITIONS

MOTILITY GROWTH PATTERN RESULTS

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

• Draw and name the flagella arrangements discussed in lab.
• Relate the term "repeating protein subunits" to flagella and explain from the lecture material how this relates to ligand/receptor interactions.
• Would you expect to see colonies of obligate anaerobes on your aerobic streak plates? Explain?
• When a yeast cell produces ethanol is oxygen present? Explain.
• Where in/on the human body would you find obligate anaerobes growing? obligate aerobes?
• Why is it not possible to see flagella in the brightfield microscope?
• What is the purpose of the Congo red in the capsule stain?
• What roles do capsules play in the life of bacteria?
• How does the anaerobic jar work to remove the oxygen?
• What is a catalyst and how does it work?
• Why is there the dye methylene blue in the anaerobic jar?
• Draw three tubes 2/3 full with an anaerobic medium and assume that each tube was inoculated (without mixing) with one of the bacterium used in this exercise. Based on the results of this exercise, indicate by shading where the growth of each bacteria would be in each tube after incubation in the air for 24 hours.
• An observant student noted that after the anaerobic jar (see below) was activated and sealed that it warmed up a bit. Explain to this student why this occurred.

Copyright Dr. Ronald E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
E-mail address: hurlbert@wsu.edu
Phone: 509-335-5108
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