Volume 26, Number 2 - December 1979
Microbiology Laboratories Made Easy
by Dr. Rodney Sobieski
ABOUT THIS ISSUE
Published by Emporia State University
Prepared and issued by The Division of Biology
Editor: Robert J. Boles
Editorial Committee: Gilbert A. Leisman, Tom Eddy, Robert F. Clarke, John Ransom
Online format by: Terri Weast
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The Kansas School Naturalist is published in October, December, February, and April of each year by the Kansas State Teachers College, 1200 Commercial Street, Emporia, Kansas, 66801. Second-class postage paid at Emporia, Kansas.
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ABOUT THE AUTHOR
This issue of The Kansas School Naturalist was written by Dr. Rodney Sobieski, Assistant Professor of Biology, Emporia State University. His specialty is in the field of microbiology.
Microbiology Laboratories Made Easy
by Dr. Rodney Sobieski
Classroom exercises dealing with microbiology are probably rarer laboratory undertakings than those in the other major areas of biology, botany and zoology. One can schedule a variety of lab exercises in the macro disciplines which require no major pieces of equipment nor large quantities of materials. For example, living or presevered specimens in those disciplines are of such a size as to be easily handled, manipulated, compared and even dissected. Very little equipment and materials are needed to have students dissect a fetal pig, yet the class may spend multiple periods studying this former living system or that of a starfish, cat, tulip or sunflovver head.
Microbiology, by contrast, requires a variety of preparatory materials (culture media, bacteria or other microbial cultures), equipment (autoclaves, incubators) and instruments such as oil immersion microscopes in order to accomplish almost any teaching objective in the laboratory area. Thus, students in many biological laboratory courses never have the opportunity to work with microbes because of these technical requirements. Thus, they do not gain the hands-on experience leading to the realization that bacteria, fungi, yeast and blue-green algae possess the main characteristics of life; namely, reproduction, irritability and metabolism.
Because the microbial world is unseen to our eyes without magnification it is often neglected as a laboratory topic, yet the numbers of individuals in this invisible ecosystem exceed those in the living world by orders of magnitude at least 1013 or more1. I In spite of this predominance of microbes most introductory labs in microbiology begin by giving students a perception of this world which is dependent on technology enabling students to visualize the organisms occupying it. Thus, even to begin at the traditional starting point requires oil immersion microscopy, thereby allowing a first band comparison of the sizes and shapes of bacterial and other members of the invisible world.
(1) Human GI tract has 1011 bacteria/gram fecal material; 105 - 107/cm2 of skin, up to 109/g fertile soil and 105/ml unpolluted water.
This issue of the Naturalist will present several things that can be done in the area of microbiology that will convey aspects of the biology of bacteria using a minimal amount of equipment, yet using living organisms. In these labs the more involved equipment normally used in microbiology such as large capacity sterilizing autoclaves and incubators are avoided or substituted for, as are the often used and microscopes. Easily substituted alternatives are also presented when certain needs cannot be replaced, for example, several of aluminum foil make convenient test tube caps when are needed.
These exercises have several objectives: (1) to convince students of the ubiquity of microorganisms; (2) to
have them appreciate the inherent reproductive and metabolic capacities of microbes, and (3) to realize how disease resistance occurs without the use of vaccines.
The exercise will be described in terms of its significance to the student's learning experience along with methods of overcoming technical investments in little used, difficult to justify equipment. Finally, sources of supplies and materials will be listed for the essential materials.
One easy manner of spurring student interest and enthusiasm is to incorporate health or health-related aspects into an experiment. This interest is further amplified if the lab requires a sample from the student to be tested against a microbe. The disease-causing capabilities of microbes are well known and most students possess enough common knowledge about them to enable the teacher to easily introduce microbiology laboratories from that point of view. One unusual consideration of microbes resides in our ability to remain free of microbial diseases. Many students and readers at this point will think of receiving vaccinations to diseases like influenza, polio, diphtheria, whooping cough, tetanus to name a few that we routinely acquire in this country. We are discussing a further division of the field of microbiology, that being immunology, which is a discipline in its own right but is considered in most courses in microbiology. Thus, the first exercise deals with immunity, but not the type obtained through vaccinations.
Vaccines generate in the recipients a form of immunity called "acquired immunity." In acquired immunity individuals are first exposed to the killed or nonpathogenic, disease-causing microbe or its altered toxins, protection is then built up or acquired once the immune system in us manufactures antibodies. Antibodies are very specific molecules, which explain the fact, for that tetanus antibodies protect us from that disease
and no other.
The first experiment deals not with acquired immunity, but with an area not often considered in beginning immunology -- "innate immunology." This is the form of immunity an organism possesses because of its inborn genetic make up and not on its prior exposure resulting in antibody protection. All animals express innate immunity to diverse microorganisms due to inherent characteristics of anatomy and/or physiology. For example, humans are inherently resistant to dog distemper, feline pneumonitis and other exclusively animal diseases. By the same token, chickens and frogs are resistant to polio, a virus disease of primates. Likewise, the bacterium causing soft rot of vegetables, Erwinia carotovora, causes people no problems because this bacterium's disease production is due to its elaboration of amounts of the enzyme pectinase. This destroys the firmness of vegetables which is due to the polymer substance pectin. Once pectin is hydrolysed a mushy consistency results in our descriptive term for the spoiled food product.
Lytic Action of Tears
This exercise was first reported in the American Society for Microbiology News by Walter Fluegel. It uses tears obtained from students the use of a freshly cut strong onion, and the tear following experimental evaluation demonstrates one of the innate immunity of our eyes. As we walk around various microbes in the air2 are impinged onto the eye with dust and other debris. Few of us have diseased eyes resulting from this particular source of exposure to the microbal world because the majority of microbes we encounter are not pathogenic for people. However, the eye does have two important innate protective mechanisms used
against all bacteria. Lysozvme, an enzyme in tears which constantly bathes our eyes is one reason. The mechanical flushing of the eye by tears is the other. The constant washing of the eye by tears removes
impinged material from the eye by drainage into the oropharynx through the lacrimal ducts and represents a mechanical dilution or flushing of the potentially harmful microorganisms. The enzyme, lysozyme, on the otherhand, cleaves the chemical bonds which maintains the shape and rigidity of the bacterial cell wall. Once the bonds are broken, the microbe loses the support of its rigid structural girdle and undergoes lysis
since the intracellular pressures cannot be contained solely by the easily deformed plasma membrane. Thus, the two innate machanisms operating in the eye are at the same time mechanical and chemical. This
exercise shows the beginning student that tears are one of our constitutive or innate immune mechanisms.
(2) Actual numbers vary from a few/cu. ft. to thousands depending on the sample, fresh air on outskirts of a town vs. air in a crowded room. Sec Chapter 46, Fundamental of Microbiology, Frobisher, Hinsdill, and Goodheart, 9th ed., W.B. Saunders, or Chapter 19, Introduction to Microbiology, 2nd ed., Anderson and Sobieski, 1980, The C. V. Mosby Co.
The sterile items needed for each pair of students are: one six-well spot plate (or six microscope slides), two medicine droppers, two cotton swabs, any size test 80% filled with water, and a nutrient agar petri plate. Figure 1 shows an unsterile swab, dropper and two 3-well spot plates. A pure broth culture or aqueous suspension of the bacterium Micrococcus lysodeiktieus and a beaker or glassful of disinfectant are also needed. A later section will describe the fairly uncomplicated preparation of these materials.
Fig. 1 An unsterile swab, dropper, and two 3-well spot plates.
In the classroom the students use one of the swabs and a broth culture of the bacteriurn to make a "lawn" inoculation on a nutrient agar plate having no visible surface moisture. Aseptic technique3 must be used to innoculate the plate but Bunsen burners or alcohol lamps are not rigid requirements and can be eliminated if unavailable. The one precaution on the lawn inoculation is to remove excess culture fluid from the swab by gently wringing it out against the inside of the culture tube near the upper end. Inoculation of the plate is accomplished by swabbing the entire surface of the medium first in one direction and then at a 90 degree angle to the first streak. After students make the bacterial lawn they should mark the underside of the dish into six pie-shaped sectors and number them 1 to 6. Later each sector will receive a small amount of diluted tear.
The single onion induced tear drop that is needed from the donor for testing is drawn up into the sterile medicine dropper. The donor facilitates sampling by tilting the head to one side. This drop is then put into the first well of the spot plate or onto the first slide which in either case already has nine drops of sterile water added to it using the second dropper. This first mixing represents a 1:10 dilution of the tear. The second dropper is used to mix the mixture by pumping back and forth a few times and then a single drop of the 1:10 is added to the next slide or well thereby making a 1:100 dilution. Continuation of these 1 drop transfers following mixing into nine drops of diluent to the sixth slide or well results in a 1:1,000,000 dilution of the active principle in tears. The steps are 1:10, 1:100, 1:1000,1:10,000, 1:100,000, and 1:1,000,000. Ideally, a new sterile dropper should be used for each step but results will still be typical if a single one is used throughout.
After dilution of the donor's tear the second swab is used to add each dilution to one of the pie-shaped sectors on the plate. Moisten the swab in the most dilute sample (the 1:106) and then gently touch the surface on the inoculated plate with the end of the swab. Following this the swab is dipped into the 1:105 dilution and it is appled, etc. Again, the swab should not be dripping wet or too much will be applied to the plate and cause running together of results or other anomalies.
The plate is now incubated upside down (lid underneath to prevent any condensate from smearing results on the growing lawn) at room temperature for 1-2 days or at 37C for 14-16 hours. After incubation they
can be stored in a refrigerator for a week or more. Figure 2 shows a typical result where the effect of
lysozyme diminishes with dilution. Here it can be seen that activity was present in the 10-1, 10-2, and 10-3, dilutions as evidenced by the clear zone in these pie-shaped sectors. A flow diagram for this experiment is given in figure 3.
Fleugel mentions that some students' results will have small colonies within the zone of lysis where the diluted tear was placed -- these are probably normal flora bacteria picked up in the process of obtaining the tear from the area near the eye. These microbes are resistant to lysozyme's action.
Fig. 2 The typical result showing how the effect of lysozyme diminishes with dilution.
Results and Extensions
The results nevertheless show that lysozyme in very dilute quantities kills the organism since no growth is evident in the lysed areas of the incubated plate. Eventhough M. lysodeikticus is exquisitely sensitive to
the enzyme, and hence its species name, the exercise shows that the enzyme naturally found in our tears contributes to the innate immunity of the eye. The enzyme is also found in our phagocytic white blood cells where it aids microbial destruction and decomposition, and in egg whites where it helps maintain sterility.
Lysozyme is effective primarily against the gram positive bacteria like Staphylococcus species and Streptococcus species, two major human pathogens, and not toward the gram negative genera like Escherichia and Proteus. A fine discussion of innate immunity can be found in Barrett's Textbook of Immunology.4 Table 1 lists several other elements contributing to innate immunity beside lysozyme.
Fig. 3 Flow diagram of the tear exercise.
This same experiment can be used to introduce characteristics bacteriapossess in common with other living systems. Reproduction is evidenced by the fact that the petri dishes show that much microbial growth occured on the plate following incubation. Their self- possessed metabolism is indirectly demonstrated as a result of the heavy growth also.
Finally, the goal of showing the ubiquity of microbes can be accomplished by having each student use an additional plate or two. They can set them out with lids removed in an area to sample airborne microbes for 5, 10, or 20 minute sampling times. A water moistened swab is an excellent sampler of selected inanimate environmental surfaces. Whether they sample a 3-4 sq. cm. area of a doorknob, floor, leaf or someone's palm they will find microbes present. If swabs are used the plates should be lawn inoculated as performed with M. Iysodeikticus.
(4) 3rd edition, the C. V. Mosby Co.
Foods can also be evaluated by dipping a sterile swab into fresh and 4-5 day or more old aged, but refrigerated, cartons of milk. Plates should be inoculated at room temperature for 3-4 day or longer to allow psychrophiles to grow. Also, a moistened swab sampling of the surface of an uncooked hot dog or ground beef can be another variable to the day's classwork. Many other foods can be tested if a blender or food processor is used to maceratc the sample prior to sampling. Students must be made aware that our world is teeming with the invisible life forms that they are studying and in some samplings large numbers of organisms are expected and these numbers do not reflect any adverse health risk nor does it reflect on the quality of the food. For example, samples of yogurt or acidophilus milk would yield heavy growth if plates were incubated at 55 degrees, yet incubated at room temperature or 37 C may yield few if any colonies. These organisms are thermophiles and grow at elevated incubation temperatures.
Lower Grade Activities
Though most bacteriological exercises are best adapted to be used by a class in bacteriology or chemistry (or that portion of the biology course), there are some interesting activities that may be used by groups as young as the grades. Below you will find some suggestions for your students.
Are girls really "cleaner" than boys? Is that pretty, neat-looking, "kissable" blonde in class also a possible source of bacteria? Do boys have "dirtier" hair than girls? Is there such a thing as " filthy money"? Why is it best to breathe through our noses, so that the filtering apparatus in our nasal passages can do its job? The following are fun, even though they do not actually prove anything.
1. Snip several bits of hair from a number of boys onto a culture plate. Do the same for several girls on another plate. Open the petri dish only long enough to introduce the bits of hair. Label each plate as to what it contains.
2. Snip some bits of finger nail from several boys onto a plate. Do the same for several girls.
3. Have several students slide their finger tips across the surface of the agar plate. Then have these students,
using soap and water, wash their hands thoroughly, and do the sa me to another plate.
4. Place a penny, dime, nickel , and quarter on a plate (or use separate plates for each, if enough are available).
5. Put a well-worn dollar bill (teachers don't usually have any of larger denomination) inside the petri dish, and leave it for a few minutes. Remove the bill, and replace the cover.
6. Have several girls "kiss" (press their lips) to the agar on a plate, and replace the cover. (The girls may
want several boys to do the same on another plate). Label.
7. Keep one of the plates unopened (sterile), to be used as a control.
8. Should you have any more sterile agar plates, have your students suggest where they would like to have them exposed to "pick up" bacteria and fungus spores.
9. Place all the agar plates, including the control, in a warm place. Examine the plates at each of the next two or three class periods.
Observations and questions:
a. How do you account for fewer bacterial and/or fungus colonies growing about the bits of hair from some people than about those from others? Do boys or girls harbor the greatest number of bacteria and/or yeast spores on their hair?
b. Can you see why a scratch is a potential source of infection, especially by a cat or other wild animal, that never "cleans" its nails? Can you see a good reason why you should clean your nails regularly?
c. Does washing your hands carefully reduce the presence of bacteria? Do you now see why doctors even after thoroughly scrubbing their hands before an operation, wear sterile surgical gloves?
d. Remember when your mother told you not to put coins in your mouth? Does what happens on the agar plate tell you why? Do as many bacteria occur on a penny as on a dime, nickel, or quarter? Can you think of a reason?
e. Could "germs" be passed on when kissing a person?
f. Do bacteria and fungi just "start to grow" (abiogenesis), or do they have to come from somewhere (check
your control plate). If bacterial colonies develop on your control, what does that tell you about the sterilization of you agar plates?
A word of caution: Be sure to inform your students that you are not looking for "germs" (disease-causing bacteria!) Rather, you are demonstrating the fact that bacteria and fungus spores are everywhere, even on the "cleanest" of human. Also, be sure to point out that not only are most of these organisms harmless, many are extremely valuable to man.
Preparation of Materials
Aluminum foil is an excellent wrapping for materials requiring and undergoing oven sterilization (spot plates or slides, medicine droppers and swabs). Tubes of water, broth medium and flasks of agar can have foil covers made from 2-3 layers of aluminum bent down over the mouth of the container. All glassware can be easily sterilized in a home oven set at 350 F for 1.5 hours. One should remember not to have the bulbs on the droppers as they will char. Nonsterile bulbs can be placed on sterile droppers without introduction of significant levels of contamination. Alternatively all items requiring sterilization can be processed in a home pressure cooker in only 20 minutes once proper temperature and pressure is reached (here bulbs may be left on the medicine droppers). A small glass serves as a convenient tube holder. A pressure cooker for an identical time will be necessary if you plan to prepare the nutrient agar petri dishes yourself. Another alternative to sterilizing materials is to inquire at your local hospital; most microbiology labs can easily work a load of material into their schedule and are often happy to be able to help out. (In fact, most hospitals are more than willing to give tours of the clinical microlaboratories. Microwave ovens have been shown to be effective in decontaminating materials that are moist; they are not recommended for dry wrapped materials or for preparing new media.
Nutrient agar plates can be purchased already poured from one or more preparation houses listed in the Appendix or one can prepare your own. The former cost between 55-75 cents each, while the latter cost around 15 cents a plate not counting prep time. To prepare your own media the dry medium is weighed out in proper proportions, water added (tap water is fine) then it is sterilized before pouring into sterile disposable petri dishes. Flasks of medium should not be more than 1/2 to 2/3 full to prevent boiling over in the sterilization process.
If a hospital sterilizes your agar medium do not be concerned about pouring the agar while the medium is still molten, it can be redissolved in a boiling water bath after 15 minutes or so. For this, place the flask in water so
that the medium is below the surface but do not have so much water that the flask floats and tips over allowing water to enter the sterile medium and contaminate it. Once the medium completely dissolves it should be tempered to about 42-45 C in a sink of water with occasional swirling. Another altenative is to cool the molten agar under flowing, cool, tap water with swirling until you can touch the flask to your cheek for a second or two without having it burn you. If you swirl too vigorously bubbles will be created in the flask which are difficult to eliminate when plates are poured and give a cratered surface. To overcome this problem, swirl the flask through a wide radius of about two feet or so then reverse the direction. Swirling from the wrist generates many bubbles. The correct volume of agar to put into a petri dish is between 17-20 mls. This can be gauged by noting when the surface of the dish is about 80% covered, then stop pouring and the delay will give the plate sufficient molten agar to fill the dish without overfilling it. It may require a gently side-to-side movement to overcome surface tension to complete the covering. After pouring, the plates should be allowed to sit out (lids closed) on a bench top in order to dry excess moisture for 1-2 or more days depending on local humidity and temperature. Prepared, purchased plates require no drying.
Bacterial cultures and discards
Fig. 4 Tube A shows the desired turbidity:
Micrococcus Iysodeikticus can be purchased from suppliers listed or obtained gratis from the Divison of Biology at ESU if the shipping container is returned. Sterile, wooden sticks can be used to make transfers to an agar plate. Following growth for 1-2 days at room temperature the plate can be sealed with tape and stored in a refrigerator for 3-4 months before a transfer needs to be made. The bacterial culture can be prepared for class use in several ways, but in each case the culture should be turbid but not so heavy that you cannot see your finger when you look at it through the test tube, as in figure 4. The first approach is to inoculate 4-5 mls of nutrient broth with the bacterium and grow the cells for 18-24 hours at room temperature (12-15 hours at 37 C). One can also scrape cells from a heavily inoculated petri dish using a sterile six-inch wooden applicator stick and suspend the cells to evenly disperse them in sterile water to the turbidity depicted in figure 4. Sterile wood sticks are inexpensive transfer devices to make subcultures and scrape growth to tubes. Only 1-2 sticks should be wrapped per package to prevent contamination
once the foil is opened. Discarding of contaminated sticks can be done into a jar or glass with a 10% solution of household liquid chlorine bleach. Contaminated student swabs and slides can also be discarded into diluted chlorine provided a 1-2 hour exposure time is used. This guarantees complete killing. This also makes a very inexpensive desk top disinfecting agent. I find used plastic milk cartons made convenient disinfectant containers. Used petri dishes are best disposed by autoclaving or incerination although the organisms are not pathogenic. One could safely dispose of them by securely sealing the used dishes in a garbage bag and placing them in a trash container. Tubes of contaminated liquids can first have the liquid poured into the chlorine bleach, then pour the material down the drain after at least 60 minutes of contact. Tubes, slides and droppers soaked in diluted bleach for the same time prior to washing would remove the threat or hazards associated with working with any bacterium.
I hope that this issue will provide the reader with sufficient information to carry out at least two or three laboratory sessions in the area of microbiology. Obviously it is impossible to communicate all the nuances of preparing for a laboratory in terms of short cuts and alternatives; however, those presented should make it easier to introduce students to the world of microbiology. If you experience particular problems or have any questions over these exercises, please feel free to write or telephone the author for answers or suggestions.
Table 1. Other elements of innate immunity.
|Element||Mechanisms or Comment|
|1. An intact skin||Prevents microbial entrance into deeper tissues, few bacteria can penetrate the skin, syphilis and tularemia are believed to do so.|
|2. Acidic pH||pH of skin and stomach are destructive to many bacteria; lactic acid is one factor on skin and is lost in diabetics hence their common occurrence of skin infections.|
|3. Fatty acids||Many are bacteriocidal but are destroyed or inactivated by moisture; this is why bandages should remain dry to enhance innate immunity on surface wounds.|
|4. Ciliated cells||The trachea or windpipe is swept clean of debris and microbes by concerted action of ciliated epithelial cells.|
|5. Cough reflex||Helps expulsion of particulate matter from the lungs.|
|6. Hormone balance||Increases in corticorsteroids, decreases resistance to infections by inhibiting phagocytosis.|
|7. Phagocytosis||White blood cells actively devour foreign material in the body where, with the help of lysozyme and other enzymes, they destroy and eliminate the material.|
Appendix of Supplies
The following information was current in 1979. Such supplies may not be available at present.
The cost or approximate estimates given below are prices quoted from catalogs as of June, 1979, and obviously are subject to increase as inflation erodes our budgets.
1. gratis except for postage on return of the shipping container;
Dr. Rodney J. Sobieski
Division of Biological Sciences
Emporia State University
Emporia, KS 66801
316-343-1200, Ext. 312 (In Kansas, 800-362-2578)
Presque Isle Cultures
P.O. Box 8191
Presque Isle. PA 16505
Midwest Culture Service
1924 North Seventh St.
Terra Haute, IN 47804
Indiana Bio Lab
Palmyra, IN 47164
(excellent catalog [$.25J which has many genetics lab packages in Bacteriology, Mycology, Drosophilia, Insects and Bacteriophage.)
medicine droppers 12/$1.05
spot plates $3.00 to $4.00 for a 12-well porcelein plate,
microscope slides 1x3 inch, $5-$6 .00/ gross,
plain 6-inch applicator sticks, $4.85-$5.50/864
cotton tipped applicators,$5.00-$6.00/1000
The above are available from most major science suppliers such as:
Fisher Scientific Co.
711 Forbes Ave.
Pittsburgh, PA 15219
7430 Waukegan Rd.
McCraw Park , IL 60055
Prepared Nutrient Agar Plates
2649 S. Custer
Wichita, KS 67217
$7.08/package of 12
12076 Sante Fe
Lenexa, KS 66215
Midwest Culture Service
Indiana Biolabs $7.20/12
Dry Culture Media and Petri Dishes
Nutrient Agar Unipack, 6x500 ml quantity -$8.70, rehydrating to .500 ml will produce about 22-25 plates
Detroit, MI 48232
Nutrient Agar 1 lb. $26.30 wilJ make about 14 liters of medium, or about 700 and plates and its available either from Difco or from
P.O. Box 243
Cockeysville, MD 21031
Sterile 100mm x 20mm petri dishes usually sold in cases of 500 for $18.20 but several suppliers sell them by the sleeve containing 20 plates each for $1.50-$1.75/sleeve.
1. Carr Microbiologicals
2. Midwest Culture Service
4. Indiana Biolabs
He was as kind-hearted soul -- as rattlesnakes go. In twenty years in the Division of Biology at Emporia State University, he never offered to strike the "hand that fed him", nor did he appear to rescnt the great number of people, especially children, that came to see his white skin and pink eyes. He even learned to associate a crowd in front of his cage with food, and the opening of the lock on his cage with an approaching meal.
Alabaster never killed a mouse or rat except when he was hungry. It was this unwillingness to kill when he didn't want to eat that lead to his untimely demise. A small rat was placed in his cage this summer for food. Unfortunately, it was not removed in time. Instead of being eaten by Alabaster, the rat attacked the albino snake. We found Alabaster dead when we returned to the classroom where he had been kept.
Many people have stopped by Room 50 at Breukelman Science Hall to express their condolences. We thought his many friends among our subscribers would like to know what happened to him.
(See: ALABASTER -- Vol. 17, No.3, February 1971).
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