Benson: Microbiological Applications Lab Manual, Eighth Edition
III. Microscope Slide Techniques
19. Motility Determination © The McGraw−Hill
Companies, 2001
• Label each slide with the name of the organism.
• By manipulating the diaphragm and voltage con-trol, reduce the lighting sufficiently to make the organisms visible. Unstained bacteria are very transparent and difficult to see.
• For proof of true motility, look for directional movement that is several times the long dimen-sion of the bacterium. The movement will also oc-cur in different directions in the same field.
• Ignore Brownian movement. Brownian move-ment is vibrational movemove-ment caused by invisible molecules bombarding bacterial cells. If the only movement you see is vibrational and not direc-tional, the organism is nonmotile.
• If you see only a few cells exhibiting motility, con-sider the organism to be motile. Characteristically, only a few of the cells will be motile at a given moment.
• Don’t confuse water current movements with true motility. Water currents are due to capillary action caused by temperature changes and dry-ing out. All objects move in a straight line in one direction.
• And, finally, always examine a wet mount imme-diately, once it has been prepared, because motil-ity decreases with time after preparation.
Hanging Drop Slides By referring to figure 19.1 prepare hanging drop slides of each organism. Be sure to use clean cover glasses and label each slide with a china marking pencil. When placing loopfuls of or-ganisms on the cover glass, be sure to flame the loop between applications. Once the slide is placed on the microscope stage, do as follows:
1. Examine the slide first with the low-power objec-tive. If your microscope is equipped with an auto-matic stop, avoid using the stop; instead, use the coarse adjustment knob for bringing the image into focus. The greater thickness of the depression slide prevents one from being able to focus at the stop point.
2. Once the image is visible under low power, swing the high-dry objective into position and readjust the lighting. Since most bacteria are drawn to the edge of the drop by surface tension, focus near the edge of the drop.
3. If your microscope has phase-contrast optics, switch to high-dry phase. Although a hanging drop does not provide the shallow field desired for phase-contrast, you may find that it works fairly well.
4. If you wish to use oil immersion, simply rotate the high-dry objective out of position, add immersion oil to the cover glass, and swing the oil immersion lens into position.
Motility Determination • Exercise 19
Benson: Microbiological Applications Lab Manual, Eighth Edition
III. Microscope Slide Techniques
19. Motility Determination © The McGraw−Hill
Companies, 2001
5. Avoid delay in using this setup. Water of conden-sation may develop to decrease clarity and the or-ganisms become less motile with time.
6. Review all the characteristics of bacterial motility that are stated on pages 72 and 73 under wet mounts.
Tube Method Inoculate tubes of semisolid or SIM media with each organism according to the following instructions:
1. Label the tubes of semisolid (or SIM) media with the names of the organisms. Place your initials on the tubes, also.
2. Flame and cool the inoculating needle, and insert it into the culture after flaming the neck of the tube.
3. Remove the cap from the tube of medium, flame the neck, and stab it 2⁄3of the way down to the bot-tom, as shown in figure 19.2. Flame the neck of the tube again before returning the cap to the tube.
4. Repeat steps 2 and 3 for the other culture.
5. Incubate the tubes at room temperature for 24 to 48 hours.
Plate Method Mark the bottom of a plate of soft agar with two one-half inch circles about one inch apart. Label one circle ML and the other PV. These circles will be targets for your culture stabs. Put your initials on the plate also.
Using proper aseptic techniques, stab the medium in the center of the ML circle with M. luteus and the cen-ter of the other circle with P. vulgaris. Incubate the plate for 24 to 48 hours at room temperature.
S
ECONDP
ERIODAssemble the following materials that were inocu-lated during the last period and incubated.
Materials:
culture tubes of motility medium that have been incubated
inoculated Petri plate that has been incubated Compare the two tubes that were inoculated with M. luteus and P. vulgaris. Look for cloudiness as evi-dence of motility. Proteus should exhibit motility.
Does it? Record your results on the Laboratory Report.
Compare the appearance of the two stabs in the soft agar. Describe the differences that exist in the two stabs.
Does the plate method provide any better differ-entiation of results than the tube method?
L
ABORATORYR
EPORT Complete the Laboratory Report for this exercise.Exercise 19 • Motility Determination
Wire with organisms is brought into tube without touching walls of tube.
Wire penetrates medium to two-thirds of its depth.
Wire is withdrawn from medium and tube. Neck of tube is flamed and plugged.
Figure 19.2 Stab technique for motility test
Culture Methods
All nutritional types are represented among the protists. This diver-sity requires a multiplicity of culture methods. An attempt has been made in this unit to present those techniques that have proven most successful for the culture of autotrophic and heterotrophic bacteria, molds, and slime molds.
The first four exercises (20, 21, 22, and 23) pertain to basic techniques applicable to both autotrophs and heterotrophs. The other four exercises are concerned with the culture of specific types. In performing the last four experiments, you should be just as concerned with understanding the growth conditions as with the successful growth of a particular isolate. For example, the use of an enrichment medium, such as in Exercise 27 (Anaerobic Phototrophic Bacteria), has direct application in the culture of many other autotrophic bacteria as well.
This unit culminates the basic techniques phase of this course.
A thorough understanding of microscopy, slide techniques, and culture methods provides a substantial foundation for the remain-der of the exercises in this manual. If independent study projects are to be pursued as a part of this course, the completion of this unit will round out the background knowledge and skills for such work.
P ART
4
Benson: Microbiological Applications Lab Manual, Eighth Edition
IV. Culture Methods 20. Culture Media Preparation
© The McGraw−Hill Companies, 2001