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9. MEDICAL SCIENCES

1

Barbara Conway of Chattanooga, Tennessee, did a project on "Experimental Teratology" which won an American Medical Association award at the Tenth National Science Fair. Then she was invited by Dr. Hans Selye, world au­thority on stress as a basic cause of disease, to work during the following summer in his laboratory at the Institute of Experimental Medicine and Surgery at the University of Montreal. Not only was she the first American to have such an opportunity, but she was permitted to perform surgery herself.

"My next-door neighbor has a malformed little girl five years old who was born blind in one eye as the result of her mother's having a very bad case of flu in the second month of pregnancy. This started me thinking that if I could prevent only one case like this, it would be worth a great deal to mankind.

Early in the same year of 1957, when I was working on a science project entitled "Stress—Basic Origin of Disease," I read in the newspaper two very interesting articles regard­ing stress and cortisone as being causes of cleft palate and harelip in humans and mice. A little later my science club sponsor gave me an article on "Congenital Malformations" in the October, 1957, Scientific American. My exhibit there­fore shows over a year's research on teratology. My objective is to find possible preventions by studying some causes in this new field of preventive medicine.

Teratology is a branch of science dealing with congenital malformations (deformities before birth). Heredity is not the only cause of congenital malformations; environment during pregnancy is also an important factor. This factor has not always been recognized. A few scientists, such as Stockard in 1921, were experimenting with this idea, but it was not generally recognized until after the 1941 epidemic of rubella (German measles). By "environment during preg­nancy" is meant such things as German measles, deep anesthesia, X ray, long airplane flights, certain drugs, diet (vitamin deficiency), accidents and emotional tensions. Any of these environmental factors can affect the fetus.

Here are five possible precautions which can be observed during pregnancy as possible preventions of congenital mal­formations.

1. Some serious eye defects and Mongolism (retarded minds) may be prevented by controlling rubella (German measles).
   
2. Some blindness, Mongolism and cerebral palsy may be prevented by avoiding deep anesthesia (surgery and major dental work).
3. Various deformities may be prevented by avoiding X ray and long airplane flights  (oxygen deficiency).
4. Some cleft palates, skeletal and internal defects may be prevented by paying close  attention to diet   (vitamin  defi­ciency).
5. Some Mongolism may be prevented by close attention to pregnancies of older women.

This is a new field of preventive medicine called "prenatal pediatrics," which concerns unborn children in their mother's wombs.

I then began experimenting with pregnant white rats and mice

Experiment I

One pregnant white rat was given 1 to 3 mg. of hydrocorti-sone for 14 days, from the sixth to the twenty-first day of pregnancy. On the twenty-first day of pregnancy she deliv­ered 4 babies, 3 of which had malformations. There were 3 with undescended testes, and 1 of the babies with the un-descended testes also had a malpositioned heart.

Experiment II

One pregnant white rat was shocked in the refrigerator twice, on the second and tenth days of pregnancy, for 8 hours at 38° F. On the twenty-first day of pregnancy she gave birth to 10 babies. As a result of this environmental factor 8 of the 10 babies were malformed, the rarest of which was a hermaphrodite, an animal which has both the male and female sex organs. This animal had 2 female ovaries and a uterus, and 2 male prostate glands. The proof of this is a pathology report from a well-known pathologist, Dr. Jack Adams, of the Baroness Erlanger Hospital, Chatta­nooga, Tennessee. There were 5 babies with undescended testes and 1 with a malpositioned heart. There was even a liver which was diagnosed as "fatty degeneration of the liver" by Dr. Jack Adams.

Experiment III

One pregnant mouse was shocked in the refrigerator for 8 hours at 38° F. for 2 days, on the sixth and thirteenth days of pregnancy. On the twenty-first day she gave birth to 4 babies. One of these 4 babies had malpositioned testes.

Experiment IV

One pregnant rat was operated on in the ninth day of pregnancy, December 23, 1957. In the operation I removed amniotic fluid from 4 yolk sacs, attempting to cause congenital malformations. During the operation she ceased breathing, but I revived her by inhaling and exhaling through a rubber tube held over her nose. About 13 days later she gave birth to 8 babies. As a result of this operation 3 babies were deformed, 1 with a malpositioned heart and 2 with malpositioned testes.

In 85% of rats the normal descent of testes is 18 to 31 days; the range is 13 to 51 days.

Experiment V

One pregnant mouse was shocked in the refrigerator at 38° F. for 13 days, from the seventh to the nineteenth day, for from 1¼to 4 hours. On the twentieth day, a day early, she gave birth to 9 babies. In this litter, as a result of this environmental factor, 5 babies were born with malformations —2 with cephalhematomas (blood tumors of the brain), and 1 with no skin covering his skull. These babies were stillborn.

Experiment VI

The same mouse in another pregnancy with the same male had no unusual environmental factor. As a result of this all 4 babies had apparently normal heads.

In conclusion I have shown that in four out of four cases, where the pregnant female has been subjected to unusual environmental factors, at least one of her litter has been born malformed, and in one case where the same mother was not subjected to any unusual environmental factors, the litter was apparently normal.

In continuing my work I hope to discover preventions of congenital malformations which may be of great benefit to mankind.

Bibliography

D'Amour, F. E. and Blood, F. R., Manual for Laboratory Work in Mammalian Physiology, University of Chicago Press,  1956.

Griffith, J. Q. Jr. and Farris, E. T., The Rat in Laboratory Investigations, J. B. Lippincott Co., 1942.

Ingalls, T. H., "Congenital Deformities," Scientific American (Oct., 1957), 109-116.

Warkany, J., "Congenital Malformations," Pediatrics, Vol. 19, Part U, No. 4 (April, 1957), 719-792."

2

Diane J. Davis of Whippany, New Jersey, was a finalist in the Ninth National Science Fair. The title of her project was "Cancer Induced by the Rous Sarcoma Virus."

"What is cancer? What causes it? What preventatives and cures are there for this dread disease?

These questions confront many people today, and through my project I set about answering some of these questions for myself. I worked with two main problems. First, what is the latent period for tumor appearance in relation to the various dilutions of the virus which I used? Second, is there any histological difference in the structure of the invaded cells in relation to the virus dilution used?

Dr. Vincent Groupe and Dr. Raushner of the Rutgers Institute of Microbiology instructed me on proper laboratory technique and gave me directions for preparation of the Rous sarcoma virus extracted from tumor tissue by differential centrifugation. I made dilutions up to 10ֿ 5, which are pre­pared in 10 steps by transferring 1.0 ml. of the 10ֿ 1 (4.3 ml. saline, 3 ampoules virus extract) into 9.0 ml. of physiological saline solution. This then is a 10ֿ 2 or 1:100 dilution. To make the 10ֿ 3, 1.0 ml. of the 10ֿ 2 is added to 9.0 ml. of diluent, etc. The placement of saline in tubes and the ex­change of the virus solution from one test tube to another was done with 10 ml. and 5 ml. pipettes, respectively, under extremely sanitary conditions. Flaming of the tubes was im­portant each time the plugs were removed to kill germs around the neck, burn up fragments of cotton from the plug and keep a steady stream of hot air moving outward from the bottles, which prevented germs from entering. Because of the code "if in doubt, throw it out," many pipettes I used while learning the technique of sanitary transfer of solutions were discarded, including the solutions within them. This technique, like others, is mastered through practice.

On completion of the virus dilutions, I was given fifteen white Leghorn chicks and eight white Beltsville turkeys, all one day old, for inoculation with the virus. Five normal chicks and one normal turkey were added to the final group­ing. With the help of Dr. Raushner, I tagged each bird in the right wing web and then injected 0.2 ml. of the virus diluent into the left ventral subcutaneous tissue through the extensor carpi radialis muscle to prevent leakage. I divided the chicks into groups of 5 and inoculated all in each group with either 10ֿ 1, 10ֿ 3 or 10ֿ 5 virus diluent. The turkeys I placed into groups of 4 and used 10 1 and 10ֿ 3 virus diluents.

For about the first month, I raised the chickens and turkeys in my bedroom, because of the need for a constant warm temperature. Each day I checked all the birds for tumor appearance and growth, and kept accurate records of all the data acquired.

The first tumors appeared on January 22, in three chicks and three turkeys. Two thirds of the tumors developed from the 10ֿ 1 and the other third from 10ֿ 3 virus diluents. One chick died from head lacerations caused by the pecking of other chicks. Two turkeys were so badly pecked that I put them in separate brooders until their wounds healed. I made the first sacrifice on January 24 of a bird with a +2 tumor growth (the degrees of classification of tumors by their size ran from +1, which is a group of small growths about the size of a pinhead, to +4, which is usually by then just one huge, hemorrhaging mass of malignant cells) and placed the tissue from the wing web into Zenker's fluid, which I had mixed the previous night. I found no metastases in any of the internal organs. The following day I sacrificed another bird, obtaining the same results.

On the morning of January 25, I visited the Morristown Memorial Hospital to observe a pathologist making slides. I learned proper procedures and a few tricks for quicker and better results in making my own permanent slides.

On February 5, I again visited the Morristown Hospital, this time with my own processed tissue. The stains haema-toxylin and eosin were used in the final steps. Observation under the microscope showed definite invasion of the striated muscle tissue by the cancerous cells.

Upon dissection of a chicken February 8, I found wide­spread metastases in all the organs. Although later I also found metastases in other birds, it was more common for the birds to die before reaching this advanced stage of cancer.

One bird, injected with a -3 diluent, developed what ap­peared to be a huge tumor at the sight of injection. When three weeks later the growth had entirely receded, I wrote Dr. Groupe and received a probable answer to the problem.

.... It is a rather common occurrence that when a a tumor becomes contaminated with certain but not all bacteria, that tumor for an unknown reason may disap­pear to all intents and purposes.

Another bird, although nothing appeared in the wing web, died of cancer in the head, which possibly developed because of cancerous tissue eaten by the chicken. Dissection showed that the tumor had progressed from the left eye to the throat and had closed the gullet, preventing the bird from eating.

Because after a certain age chickens develop a resistance to the Rous sarcoma virus, no birds with the -5 diluent showed signs of tumor growth. It was therefore unnecessary to keep the birds any longer; so on March 15 all the re­maining chickens were killed, leaving only a few turkeys, who were slow in developing their tumors. However, I did section a part of each wing web at the spot of injection and sent the tissue through the regular process for slide making.

To finish the last procedures in making my slides, I went to the National Institutes of Health in Bethesda, Maryland, where I became acquainted with Dr. Albrecht. He advised and helped me in the final analysis of the tissue under study. A center of growth dominated most of the slides, showing signs of deterioration. Both striated and smooth muscle fibers were affected by the invading malignant cells. The 10ֿ 1 growths showed an increase in activity as compared to the 10ֿ 3 tumors. There was, however, no apparent difference as fax as cellular structure was concerned.

Average Latent Period    Virus Dilution

7 days.................................. 10ֿ 1

9 days.................................. 10ֿ 3"

3

"A Study of the Effect of Tranquilizers on the Metabolism of White Mice" was presented at the Ninth National Science Fair by finalist Asa William Bennett of Washington, Georgia.

"I first became interested in experimenting with tranquil­izers in October, 1957. A summer issue of Scientific American discussed methods of assembling apparatus for determining metabolic rate in mice, and about the same time public interest was aroused concerning the effects of new tranquil-izer drugs on the human body. It was revealed that some doctors were using the drugs quite freely and that the total effect of these drugs had not been thoroughly tested experi­mentally. I decided that an experiment to determine the effects of the drugs on metabolism might give valuable in­formation concerning the over-all function of tranquilizers. Many questions began to enter my mind. Do tranquilizers have any effect on metabolism? If they do, is the effect a secondary one resulting from nervous system synaptic inter­ference, or is there cellular level metabolic interference? The last two questions I could not check experimentally, but the first one I could check, and perhaps find information which those who are trained in medical science might use in ex­ploring the other two areas more fully.

My next steps were concerned with collecting more refer­ence material, setting up apparatus for determining meta­bolic rate, obtaining experimental animals and drugs and learning to run accurate tests.

With the exception of a beam balance, all equipment used in my experiment was home-assembled. Cages for white mice were built from orange crates and screen wire. Five flasks, a Mason jar and an aspirator were connected by glass and plastic tubing. Definite amounts of either calcium chlo­ride or soda lime were placed in the respective flasks to serve as absorbers of water vapor and carbon dioxide. A water faucet was fitted with an adapter for the aspirator.

Wyeth Laboratories generously donated a supply of two recently developed tranquilizing drugs. Dr. Duggan, a local physician, gave me a 5-cc. vial of promazine hydrochloride, and several syringes. A medical student, Mickey Standard, gave much assistance, both with information on the drugs and in preparing (with local hospital facilities) a 30-cc. vial of meprobamate.

On. December 4 I made the first trial run. My figures in­dicated that the metabolism of the mouse tested was 1.21, an impossible figure. Normal metabolism in mice ranges from .72 to .97. A total of fourteen runs was made in December. All showed incorrect metabolic rates, some too high, many too low. Finally I located a cause of the inaccuracy. My beam balance had a dull knife edge and was giving erroneous weights, which ranged from one tenth of a gram to as much as one gram. New, highly sensitive triple beam balances were purchased, and tests recommenced January 15, 1958. Still no normal metabolic rate could be obtained. I then observed that much water vapor exhaled by the mouse was condensing on the walls of the animal chamber. The body heat of the animal was causing the temperature inside the chamber to be higher than that on the outside. An electric heating pad (low temperature) thrown loosely over the ani­mal chamber raised the temperature of the outside walls and solved the problem. No more condensed water vapor! Once these difficulties were overcome, I consistently obtained metabolic rates which fell within the normal rate, .72 to .97. I was then ready to inject tranquilizers and make comparisons. Reading had helped me to formulate a hypothesis concerning expected outcomes.

In several scientific publications, I found that both me­probamate (Equanil) and promazine hydrochloride (Sparine) act on the central nervous system, but are classed neither as stimulants nor depressants. Promazine is said to exert "nar-cobiotic" action on subcortical areas of the brain, and to control overt motor and verbal activity. It is also believed to inhibit certain metabolic functions of living cells, particu­larly those of the nervous tissue. Meprobamate seems to act on the interneuronal circuits  as  a blocking  agent and to have a selective effect on the thalamus. High doses have been known to produce subcortical and cortical changes. Me-probamate also relaxes the voluntary skeletal muscles and modifies the number of incoming stimuli. All references show that a relaxed state follows an injection of either me-probamate or promazine hydrochloride. These findings led me to assume that I might expect both drugs to produce an appreciable decrease in the total metabolic rate of my mice.

In testing this hypothesis, I have made 107 metabolic runs since the first test of December 4, 1957, and have found that the tranquilizers, meprobamate and promazine hydrochloride, produce a lowered metabolic rate in white mice. The average decrease following meprobamate injection is 13% in males, 14% in females. In the case of promazine hydrochloride the average decrease is 12% in males and 15%* in females. Ex­citement caused by a placebo injection of physiological salt solution produced a 9% increase in both males and females.

The slowdown in metabolism may be even greater than experimental results indicate, because the injection of physio­logical salt solution produced a metabolic increase. A phys­iological salt solution is not in itself irritating or stimulating to the animal body, so the excitement or nerve tension must have been caused by the needle entering the peritoneal cavity. In tranquilizer injection there was needle involvement and the lowered rate may represent a drop from the needle-ex­cited rate rather than from the normal rate.

Tranquilizers administered to human beings may not have as much effect on metabolism as this experiment indicates for mice. The therapeutic human dose is proportionately smaller than the dose given to experimental mice and the metabolic effect may be proportionately smaller.

Since general metabolism is definitely lowered by me­probamate and promazine hydrochloride, there may be cellu­lar level action of the drugs as well as nerve impulse interference. However, I did not have the equipment necessary to test this theory, so I will leave that for someone else.

With my project I have been extremely fortunate. I re­ceived a trip to the National Science Fair at Flint, Michigan, where I placed fourth in the boys' biological sciences cate­gory. A fourth award carries with it a gift of $25 worth of scientific equipment. This and the tremendously educational experience of the National Fair makes me feel well paid for my 300 hours of work. I can never adequately express my gratitude for the assistance and encouragement I re­ceived. The inspiration of my teacher, Miss Dorothy Wright, has been of inestimable value in helping me carry on this project"

4

Lynda Wallace of Cheyenne, Wyoming, now a student at Creighton University, Omaha, Nebraska, was one of the forty top winners of the 1958 Science Talent Search. As part of her winning entry, she submitted her report on an un­identified bacterium she had discovered. Her tests showed that the enzyme or product given off by this bacterium liquefied carbohydrates  and proteins.

Lynda's science teacher and sponsor, Sister Mary Paulinus, requested and received the first grant ever made by the National Institutes of Health for a research project to be carried out by a high school science club. The high level of their laboratory work and the exciting results of their study of the bacterium, which the club members have christened "Lynda," has drawn national attention.

In September of 1959 the story of this search for a fast-acting anticoagulant which may have important use in treat­ing coronary thrombosis was detailed by James M. Liston, well-known writer and editor, in a major article for the American Medical Association publication,  Today's Health.

A summary of Lynda's paper, "Tentative Identification and Further Studies of an Unknown Bacteria," is presented here.

"In December, 1956, I began work in bacteriology. At first I was interested in it chiefly as a class project. However, a flask of potato agar was left unsterilized in a labora­tory closet for a few days. After this time I found that large gas bubbles had been formed in the agar. To see if any change would occur in it, I decided to leave the agar in the closet and observe it. At the end of a week the agar was completely liquefied. My objective then became the identifica­tion of this bacteria, which differed in its properties from most other types in that it was capable of liquefying agar.

During the following months I ran several tests described in the Manual for the Pure Culture Study of Bacteria concerning identification. My tests and results were based on a period of 24-hour growth at 20° C.

I tentatively arrived at this identification:

Kingdom:	Plant
Phylum:	Thallophyta
Class:	Schizomycetes
Order:	Eubacteriales
Family:	Pseudomonadaceae
Genus:	Mycoplana
Species:	bullata           T >!1"

In the hope that something may eventually be found to keep potatoes from spoiling during storage, tests were also run to determine the effect of antibiotics on this species of bacteria. Those used were Polymyxin B, Chloromycetin, penicillin, Viomycin, Streptomycin and Neomycin. All these antibiotics were contained on a small tab which was placed on a pour plate. It was then noticed after forty-eight hours of growth that Neomycin and Streptomycin had inhibited growth, while Polymyxin B, Chloromycetin, penicillin and Viomycin had not. These last four antibiotics even seemed to encourage growth, but no direct proof was obtained and the study is still to be pursued.

Recently an article in Scientific American described a num­ber of experiments by the California Institute of Technology concerning the effect of coconut milk on the growth of plants. Potatoes were among those being tested and because of the peculiar reaction of this bacteria on potato agar I began to make tests concerning the growth of the bacteria in coconut milk, and on potatoes grown in coconut milk. The coconut milk seemingly contains one of the prime factors for cell growth.

At present I am using dilutions of coconut milk. The dilu­tions are from 1:10 to l:1012. Each solution of coconut milk was inoculated and after growth compared to growth in an equal amount of nutrient broth. This comparison was accomplished by means of plate counts to determine if growth advantages could be secured by the use of coconut milk.

When this investigation was initiated the need for running a combined nutrient-coconut milk broth was not apparent, but this study will have to be made.

An interesting phenomenon occurs in the coconut milk cultures which will be referred to by their key, 107, 108, 109 and 1012. Growth decreased in direct proportion to the dilu­tion in the first six cultures, as is consistent. In the 107 dilution there is practically no growth, if any at all. But the 108 dilution immediately shows an increase almost equal to that of 101 and 102. Growth in 109 again decreases almost to the point of no growth. Gradually small amounts of growth then begin to appear until growth in 1012 is almost equal to that of 108. These results were so startling that several runs were made. The results are recorded in the table below.

With such interesting developments I have also begun to determine the effect of bacterial growth on potatoes in an­other manner, again employing coconut milk. This time, however, I have begun to grow sets of potatoes from seed­lings. One set is grown in coconut milk, the other in water as a control and comparison. From these potatoes I will determine the growth factor of coconut milk on potatoes and later I will take the potatoes grown in this manner, pre­pare them as a medium and inoculate them with the bacteria and compare the growth on this media with that of the regular potato agar and coconut milk solutions.

As the odor produced by this bacteria has proved so chal­lenging and mysterious, I am initiating research on its isola­tion and identification also. Seemingly it is not the bacteria which causes the agar to liquefy but some product which is formed when a particular substance is utilized as food— probably a protein, as is indicated by liquefication of nitrate agar, gelatin, potato agar, bean agar and coconut milk.

GRAPH SHOWING INCONSISTENT GROWTH OF  BACTERIA IN COCONUT MILK

Summary

1. Tests were run on an unknown bacteria, which causes agar to liquefy, according to the methods described in the Manual for the Pure Culture Study of Bacteria and it was tentatively identified as Mycoplana bullata.
2. Preliminary studies of the relationship of this bacteria to antibiotics show it inhibited by Neomycin and Strepto­mycin, but uninhibited or even encouraged by Polymyxin B, Chloromycetin, penicillin and Viomycin.
3. Work  with  growth  of this  bacteria  in  coconut  milk shows an inconsistent rise in a dilution of l:108 and l:1012.
4. Comparison of growth in coconut and nutrient broth shows coconut milk  alone to be  less  good.  The  need  for further  study  on  a   combination  of  coconut  milk-nutrient broth medium presents itself.
5. Seemingly it is a product of the metabolism of the bac­teria  which   causes  the   agar   to  liquefy.   Isolation   of  this product is still in progress.

Bibliography

Bergey, D. H., Breed, R. S., Murray, E.G.D., Hitchens, A. P., Bergey's Manual of Determinative Bacteriology. The Williams and Wilkins Company, Baltimore,  1939.

Difco, Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures. Difco Laboratories, Detroit, Michigan,  1948.

Frobisher, Martin, Fundamentals of Bacteriology. W. B. Saunders and Company, Philadelphia and London,  1952.

Edited by the Committee on Bacteriological Technique of the Society of American Bacteriologists, Manual of Meth­ods for Pure Culture Study of Bacteria. Biotech Publica­tions, Geneva, New York, 1946.

Salisbury, Frank, "Plant Growth Substances," Scientific Ameri­can,  197:   125-134,  April,   1957."

5

David  R.   Brown  of  St.  Louis  Park,  Minnesota,  did   a project on "Humeral Transplants." It won him the top Ameri­can Medical Association Award at the Ninth National Science Fair, as well as several other awards. Three years later David became a top winner in the Nineteenth Science Talent Search.

"Introduction

To the zoologist, anthropologist, anatomist and orthopedic surgeon it is important to know to what degree the shape and size of bone varies due to its own method of growing and the posture action of muscle. Actually a considerable amount is known, by those who have grown early skeletal tissue in glass (tissue culture) and also by those who have transplanted early embryonic bones into a nonmuscular mem­brane in the chick egg. However this tells us only about very early stages and in general has shown us chiefly that bones (before true bony tissue appears) do not need a nor­mal environment to develop their basic shape and size.

It would be important to specialists to know if growth will continue indefinitely in an outside environment. If there are only differences in growth and development, what are the factors responsible? The only way to solve this is to transplant bones of newborn animals to nonfunctional lo­cations. This has been done by several investigators.

To study this matter of growth development and to see how certain tissues survive transplanting, a member of the Department of Anatomy at the University of Minnesota has been working with the mouse, placing small bones under the skin. This technique has answered many questions, but a certain slowing up of the blood supply has been noted. The blood supply of a growing bone, by its rate and volume, determines the rate of growth and makes possible the appear­ance of bony tissue itself.

Whether a rich blood supply is more normal may be tested by the relatively simple process of putting the bones of a newborn mouse into the circulatory system of a chick egg.

The egg is a possible site, because of its rich blood supply and because antibodies are not being produced in the egg. Growth is also limited by the time the blood starts circulating and by the nineteen-day shutoff. The only possible time for transplanting is between nine and nineteen days. Ten-day growth in the egg is being compared with ten-day growth in the mouse, because the age of implant in both cases is fourteen-day embryonic (birth—seven days). This age is chosen because growth is rapid at this stage.

Obtaining the Humeri

a.         The uterus of a fourteen-day pregnant mouse is opened and the embryos removed.

b.         The embryos are then placed under the dissecting micro scope and the humeri removed.

c.         These bones are then placed in saline solution for a short time while the series is being collected.

Placing the Bone in the Mouse

a.         The hair is removed from the side of an adult mouse and an incision about 1 cm. long is made.

b.         A humerus is then placed on the underlying membranes to grow.

c.         A cover glass is then placed in the incision to retract the opening and to make observation possible.

d.         The wound is allowed to heal, causing the window to adhere to the surrounding tissue.

Placing the Bone on the Chorioallantoic Membrane

a.         Exposing  the  chorioallantoic membrane

1. A small square is cut in the shell of a living egg.
   
2. The shell is then lifted and the surrounding tissues are removed.
   
3. The chorioallantoic membrane is now exposed.
   

b.         The humerus is now placed on the membrane.

c.         A cover glass is then placed over the membrane to replace the shell, and sealed with paraffin.

d.         The egg is then placed in the incubator at 103-105° F. and a relative humidity of 60%.

Results

Normal 2-day (control)—4.95 mm. Subcutaneous transplant (mouse)—4.50 mm. Chick egg transplant—5.16 mm.

The above figures are the results obtained from an average of all the grafts. The period of growth in all cases is ten days, during the fourteen-days embryonic to two-days postnatal interval. The average size of the bone at transplantation was 4 mm., ranging from 3.85 mm. to 4.1 mm. in length.

Conclusion

I find that my original hypothesis (as stated in the intro­duction) is conclusive. The possibilities of these techniques in medical research are unlimited. For example, the method of observation employed through the use of a cover glass allows the worker to observe the ossification process, as well as other tissue growth, quite readily. The application of other forms of tissues upon the chorioallantoic membrane is a coming technique. I have thought of using the parathyroid hormone with the subcutaneous transplants in the mouse and seeing the effect of rapid calcium metabolism on the bone. The introduction of acetic acid compounds into the blood stream may produce decalcification of the transplanted bone. The study of dentation through the transplantation of fetal mouse mandibles is now being done at the University of Minnesota."

6

An excellent project that was thoroughly researched won honors for Sheila Marie Most of Gulfport, Florida. Her "Modern Science in Dentistry" was selected by the American Dental Association as one of the two most outstanding ex­hibits in dental science at the Tenth National Science Fair. The purpose of her project was to contrast the condition of the teeth of a prehistoric people with the condition of teeth today, examining the prehistoric and modern diets and taking into consideration the advantages of modern medicine and dentistry. Sheila and Mary Sue Wilson, the other winner, were honored guests at the ADA Centennial Convention in New York in September. Sheila was selected for the second time to represent the Pinellas County Science Fair, St. Petersburg, Florida, as a finalist at the Eleventh National Science Fair-International, where she again won a First Award given by the ADA.

Sheila says, "Now, along with my interest in archaeology is a strong turn toward dentistry, and my future plans are to enter the field of dentistry. . . . Members of the American Dental Association have greatly assisted me with knowledge unattainable through any class or text. ... I know that neither money nor anything else could have bought the edu­cation that the National Science Fair and the Dental Associa­tion meetings have given me."

For her project, Sheila used a great variety of information sources, which she lists in detail at the end of her research paper. The following is an abstract of her project report.

"Findings

While reconstructing aboriginal Timucuan Florida Indian skulls and jaws (1000-1200 A. D.) excavated by the ex­hibitor, a complete lack of dental caries in each and every jaw was noted, although these Indians suffered other dental defects. . . .

These conditions led to research into diet habits of such Indians to determine the reasons therefor and to contrast the effects of their diet on their teeth with the effects of a diet of today on our teeth.

Discussion

Diet habits of the Indians were controlled by what nature provided seasonally. Foods consumed were:

Tobacco was used. (Swanton, 1922, pp. 357-362)

In winter, they subsisted largely on fish and game, which were cooked on wooden racks placed upon  forked  stakes over a fire, then smoked and dried for storage.

Note: The predominant Indian head type has exceptional bony ruggedness of face' and mandible attributed to their marine diet, which was extra rich in phosphates. (Hrdlicka, 1922)

Much grit, remaining in the Indian's staple food—corn— after grinding, had a tremendously abrasive effect on their teeth, causing severe wear. This, in turn, exposed the tooth's nerve, causing it to shrink away. Often the tooth abscessed and, if severe, this sometimes led to death.

Smoking of meats and fish to preserve them tended to toughen the meat. Chewing these foods also caused severe wear. Many female jaws showed the results of chewing leather for softening purposes.

Although it is impossible to determine if gingivitis and Vincent's infection were present, evidence of pyorrhea, ab­scesses, malocclusion, poor alignment, tartar deposits and stains, impaction plus loss of teeth by diseases were much in evidence in almost every jaw.

Present twentieth century diet is usually a matter of per­sonal choice. Most foods are available all year round with the fairly recent development of frozen foods, plus modern transportation. Vitamins, taken on medical advice, help make our proper food and vitamin intake the best in the world. In addition, we have the advantage of many dairy products.

Today, modern parents protect their children's health (before and after birth) and their own with all modern scientific medical and dental aids made available to them. Somewhere along the line, refined sugars (i.e., candy, pastries, candy-coated gum, soft drinks and ice cream) creep into the child's diet—an unnecessary but highly palatable addition. In 1830, the United States per capita consumption of sugar was 12 pounds per year, and we consume 100 pounds per year per capita.

This tremendous increase in our refined sugar consumption leads to the conclusion that refined sugar is largely responsible for our high national rate of tooth decay. The frequency with which sweets are eaten is an even more important factor than the amount consumed. Continued and repeated acid attack is more dangerous to the teeth than occasional attack.

A typical modern family of father, mother and daughter (exhibitor's family), who availed themselves of full dental and medical care, shows the following defects:

Father: missing—3 third molars fillings—9 partial dentures—2 to replace maxillary right and left second bicuspids

Mother: missing—8   (4  third  molars  due  to  impaction; maxillary left second bicuspid, first and second molars, right second molar) fillings—30 partial denture—1 to replace maxillary left second bicuspid, first and second molars crowns—2   mandibular   right   first   and   second molars

Daughter: (deciduous)—missing   1—maxillary left  central incisor nerve killed in blow in accident, subsequent   abscess, extraction   at   age 4½fillings—4—numerous pinpoint caries remaining unfilled at dentist's discretion (permanent)—missing—none fillings—21 orthodontic treatment to correct separation and straighten overlap of maxillary incisors

The hardest part of the human body is the tooth. Teeth remain intact after death long after the bones of the skeleton have turned to dust. Yet, they are among the perishable parts of the body during life because of dental caries. Figures for the United States show:

2-year-olds—50% have 1 or more carious teeth.

6-year-olds—have 3 or more carious deciduous teeth.

16-year-olds—have 7 decayed, missing or filled teeth.  (Less  than 4% of this age group have remained  free from dental caries.)

Summary

It will be noted that in the Indian diet, there were only natural sugars consumed. Therefore, although they had no knowledgeable control over their many dental troubles, they escaped dental caries.

Conclusion

The following care, plus the reduction in the large quantity of refined   sugar-containing   foods,   would   show   a  marked improvement  in America's dental health.

1. Regular visits to the dentist, starting at about age 2.

1. Brushing  after every meal  may help  prevent a certain amount of caries. (Commercial tooth powders and pastes are pleasant to use, but have no special virtue in cleaning. Many  dentists  recommend  finely  precipitated  chalk  or bicarbonate of soda as safe and inexpensive.)
   
2. Removal of tartar and other deposits and stains once or twice a year by dentist.
   
3. Treatment by orthodontist for malocclusion and improper alignment.
   
4. Use  of fluoride  applied directly to teeth by dentist or taken internally through properly treated drinking water (usually 1 part fluoride to 1,000,000 parts water).
   

Experiments at Grand Rapids, Michigan, after 5 years of fluoridation, show caries reduced 66% in permanent teeth of 6-year-old children. Communities with no central water system can have children's teeth treated with applications of fluoride by a dentist on eruption at ages 3, 7, 10 and 13 years."

7

Mary Susan Wilson, Cedar Falls, Iowa, was one of the top American Dental Association winners at the Tenth National Science Fair. Her project, "Bacterial Resistance to Antibiot­ics," is described below.

"Introduction

The purpose of my project was to develop two strains of bacteria: Staphylococcus aureus (pathogen) and Sarcina lutea (nonpathogen) with resistance to penicillin. Having accomplished this, I tested these mutant strains against other antibiotics (Bacitracin, Achromycin, Aureomycin, Terramy-cin, Streptomycin, Dihydrostreptomycin, Triacetyloleandomy-cin and Ilosone) which normally inhibit their growth and observed the effects of these antibiotics on the above or­ganisms. I obtained the pure strains of the two types of bacteria from a bacteriological supply house and transplanted cultures from these two strains. My family physician fur­nished the antibiotics used in this project.

Procedure I used a fairly new method in obtaining the mutant strains of bacteria. Metal slants were made on which to place the petri dishes, as shown in Diagram I.

Nutrient agar was prepared for growing bacteria. I poured one slant consisting of 30 ml. nutrient agar and let it solidify. Then, I placed the petri dish on a level surface and poured a second slant of agar mixed with a standard solution of penicillin and let it diffuse for twenty-four hours. The required gradient of concentration results as the penicillin in the top wedge diffuses into the bottom wedge. The final preparation is pictured in Diagram II.

Originally, I planned to use this procedure throughout the project but as the work progressed, I used other methods.

In working with the nonpathogens, I used two methods. I poured all of those dishes by the wedge method but used different inoculating procedures. The first method was as follows: I sterilized a flask with 150 ml. of sterile distilled water. After cooling the water to body temperature, I in­oculated the water with the organism by using a platinum loop and mixed thoroughly. I poured this solution on the prepared petri dishes and incubated them for forty-eight hours. I noted that the growth of bacteria extended about halfway across the dish from the end of least concentration. As the gradient of concentration of antibiotics became greater, the colony count per square centimeter became less. I as­sumed that those colonies growing in the highest concentra­tion from that test were mutations. I took the colonies that grew in highest concentration and transplanted them, as described previously, to a petri dish of identical preparation. After incubating this for forty-eight hours, I observed that the growth had extended completely across the dish. The results of these tests are summarized at the end of this report.

In the streak method I used the same preparation of agar but a different method of inoculation. With a platinum inoculating needle, I made six to eight streaks from the end of least concentration extending across the petri dish.

I incubated the dish for forty-eight hours, after which I noted that the streaks extended about one fourth of the way across the petri dish. I took the colonies from the outermost ends of these streaks and transplanted them to a dish of identical preparation. After forty-eight hours of incubation, I noted that the streaks had extended about three fourths of the distance out. In each test I used four petri dishes, transplanting each time to four more. The preliminary results  with  this  method  indicate  that  the  organism  was developing resistance to the antibiotic. I did not work ex­tensively with this method; therefore, I am not presenting data at this time.

In investigating Staphylococcus aureus, I used three pro­cedures. I initially tried the water method (as described earlier) with the wedge preparation of agar. I used sterile conditions because Staphylococcus is classified as a path­ogenic bacteria. When grown on nutrient agar, the organism becomes less virulent. However, I used the standard proce­dure for handling pathogens to avoid contamination. This method proved to be inadequate, and I abandoned it.

Then, I used the streak method (described above), but this also proved to be unsuccessful as I was not proficient in the method at that time.

I finally used the filter disk method. I poured 25 ml. of agar into a sterile petri dish, using a platinum loop to inoc­ulate the unsolidified agar with the organism. I allowed this preparation to solidify, placed a prepared filter disk con­taining penicillin in the center of the dish and allowed this preparation to incubate for forty-eight hours. I noted that the organism grew in a circle about 1.5 cm. from the filter disk. Assumption: Bacteria growing nearest the disk are resistent to the antibiotic and therefore mutant. I then transplanted the colonies from the innermost edge of the circle to a dish identically prepared. I incubated this for forty-eight hours and noted that the circle of inhibition had diminished. I took the innermost colonies from this test and transplanted them to a dish of similar preparation. It was noted that in this test, the Staphylococcus grew completely to the disk. This indicated that the organism had developed resistance to penicillin. The results of these tests are sum­marized at the end of this paper.

As my work progressed, I found that I would need a procedure by which I could get an accurate count of the colonies. I designed and built a colony counter as shown in Diagram III. I was able to use this counter with the tests using Sarcina lutea.

The initial upper wedges were prepared, using a standard solution of 200.000 units of penicillin plus 25 ml. sterile distilled water. After testing this solution, I found that the organisms were completely inhibited by this concentration, so I proceeded to dilute the solution by adding 50 ml. water for each test. I finally found that 200,000 units of penicillin in 800 ml. sterile distilled water were adequate for the growth of bacteria.

In testing the mutant strains of Staphylococcus with other antibiotics, I poured 4 petri dishes with 25 ml. of agar. After inoculating the dishes with the mutant strain of bac­teria, I placed a prepared antibiotic (Streptomycin, Ilosone, Bacitracin, Aureomvcin, Terramycin, Triacetvloleandomvcin and Dihvdrostreptomycin) in the center of the petri dishes and incubated them for forty-eight hours. The results taken from the tests with Staphylococcus are given at the end of this report.

I used the wedge method in testing the resistant strains of Sarcina lutea. I used Achromvcin, Aureomycin and Terra­mycin, making a standard solution of each, the same con­centration as the penicillin solution.

The dishes were prepared in the same manner as used in testing the organism with penicillin. I used the water dilution procedure for inoculating.

In order to make a quantitative determination of the mutation rate, it is necessary to determine the number of colonies developing at each level of antibiotic concentration. The information would be more meaningful if the count included the colonies that would develop in a given region but would not develop in a region of greater concentration of the inhibiting agent. The following procedure was devised to obtain this count statistically.

The grid on the colony divides the petri dish into regions, each 1 cm. wide, starting with the least concentration in strip 9. The average number of colonies per square centimeter for each strip is determined by counting the number of colonies in each square centimeter. The strips are divided into square centimeters by the grid. Counting is done at a number of places along the strip to obtain an average number per centimeter for each strip. The colonies counted per square centimeter is the actual count. Let this count in each row be designated by the letter A followed by the number of the row. In Row 1 this would be designated by Al, in Row 2 by A2, etc. To determine the number of colonies that could develop in a given strip but not in a strip of greater concentration, some simple statistical calculations are required. Let B represent the number of colonies that could grow in a given strip, but not in one of greater con­centration. Al — A2 = B1. A2 — A3 = B2, etc. These results have been graphed by assigning B an index number.  This was done in the following manner. Bl was assigned the number 100. Therefore, Bl/100 = B2/X. By finding X in each case, the given B is assigned a number. The strip numbers 1 through 9 constitute the other axis of the graph.

While working on my project, I came upon some results that I felt were interesting, but because of lack of time I was unable to pursue them. My work was dealing primarily with organisms resistant to antibiotics, therefore I was sur­prised to observe that one of the antibiotics (Achromycin) inhibited the mutant strains of Sarcina lutea to a greater extent than the nonmutant strains. This would be an in­teresting aspect of the subject for further study.

Results.

1. A mutant strain of Staphylococcus aureus was developed and resistance to penicillin was evident.
   
2. The mutant strains of Staphylococcus aureus were tested against  other  antibiotics   (Aureomycin,  Terramycin,  Dihydrostreptomycin, Bacitracin, Streptomycin, Ilosone and Triacetyloleandomycin).
   

a.         The mutant organisms showed resistance to Bacitracin, Terramycin and Triacetyloleandomycin.

b.         The mutant organisms were not resistant to Ilosone, Dihydrostreptomycin,   Aureomycin   and  Streptomycin.

1. Mutant strains of Sarcina lutea were developed. Resistance to penicillin was evident.
   
2. These   mutant   strains  were  tested  against  Aureomycin, Terramycin and Achromycin.
   

a. The  mutant strains were not resistant to  any of the above antibiotics.

Summary

This work is an illustration of the evolutionary process that is continually going on in all forms of life. As unfavor­able conditions arise, these organisms must change in order to survive. Thus, the development of effective antibiotics is a never-ending task. Antibiotics today may be ineffective tomorrow, because of changes in the organisms.

This study has been and is of great importance from the medical and clinical standpoint. The effective use of anti­biotics to combat infection depends upon their ability to inhibit all strains of a given species of bacteria.

The ultimate antibiotic would be one which would completely inhibit the growth of a given disease-producing or­ganism, and against which the organism could not successfully mutate.

Bibliography

Epstein, Miracles from Microbes, Rutgers University Press.

Greaves and Greaves, Elementary Bacteriology, W. B. Saun-ders Company.

Irving and Herrick, Antibiotics, Chemical Publishing Com­pany, Inc.

Pratt and Dufrenoy, Antibiotics, Lippincott.

Scientific American, October, 1954, "The Structure of the Hereditary Material," by F. H. C. Crick.

Scientific American, November, 1956, "Transformed Bac­teria," by Rollin D. Hotchkiss and Esther Weiss."

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