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EFFECT OF CECECTOMY ON THE.
FECAL FLORA. OF WHITE RATS

Lmesh: {or ths Degree of M. S.
MICHIGAN STATE COLLEGE

Mulkh Rai Dhanda
1947

This is to certify that the

thesis entitled

"Effect of Cecectomy on the Fecal Flora
of White Rats."

presented by

unlkh Raj Dhanda

has been accepted towards fulfillment
of the requirements for

_£:_Ss__degree Wow

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EFFECT OF CECECTOMY ON THE FfiCAL FLORA OF WHITE RATS

By
W RAJ 9mm

A {HES IS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Bacteriology and Public Health

19h?

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17

18

INTRODUCTION

The influence of diet on the bacterial flora of the intestinal
tract has been the subject of intensive research from the time Herter
and Kendall (1909) first showed that a change in the diet from one domin-
ating in proteins to that of milk and sugar is followed by a gradual but
rapid substitution of an acidOphilic non-proteolytic type of flora for
a strongly proteolytic type. Later researches (Rettger and Horton, l9lh;
Hull and Rettger, 1917; Torrey, 1919; Porter and Rettger, l9h03‘Winblad,
l9hl; iitchell and Isbell, l9h2) have all confirmed this and established,
beyond doubt, a relationship between the type of flora predominating in
the intestinal tract and the type of food ingested.

Of late years, intestinal flora have come to be regarded as useful
to the well being of the individual harboring them and one of the factors
under consideration is their ability to synthesize accessory food sub-
stances. This view has changed the trend of research from the original
line of search for harmful metabolic products, and normal flora of the
intestinal tract have now come to be regarded as important symbionts
rather than merely incidental, much less harmful.

Guerrant, Dutcher and Tamey (1935) observed cecal enlargements
in white rats fed on diets rich in dextrinized corn-starch and indicated
that the cecum of the rat is a seat of synthesis of B vitamins. These
workers also discovered the presence, in the cecum of these rats, of large
numbers of yeast cells and believed them to be the specific agents of elabo-

ration of these vitamins. Abdel-Sqlam and Leong (1938) demonstrated the

2.

synthesis of thiamin by a mixed flora taken from the rat cecum and grown
‘in 33232 although they failed to detect any diffusion of the synthesized
vitamin B1 from the bacterial cells into the broth medium in which the
organisms were grown. Similar evidence of the synthesis of B vitamins by
the intestinal flora in the rat cecum has been put forward by Mitchell and
Isbell (19h2), who think that the amount synthesized is markedly influenced
by the type of flora and their numbers. Their investigation has also
emphasized the marked effect that diet has on both the quality and quantity
of the intestinal flora. Dam and associates (l9hl) considered E, 22;;

as a probable organism responsible for the synthesis of vitamin K. The
work of Taylor, Pennington and Thacker (l9h2) indicates, further, that the
cecum contributes to the rats' supply of some of the B vitamins depending
on whether the diet is of the type that will support a cecal flora of the
right quantity and, or, quality. Day and associates (l9b3) have also shown
this by experimenting on rats deprived of their ceca.

In their work on the study of the influence of diet on the distri-
bution of bacteria in the various parts of the gastro-intestinal canal of
rats fed on stock diet, Porter and Rettger (19h0) have shown that whereas
the normal bacterial flora of the young white rat is comparatively simple
in the upper segments of the alimentary tract, it becomes increasingly more
abundant in the lower sections of the small intestine. These workers found
the cecum to harbor the highest number of bacteria.and, with the exception
of the animals fed on a high protein diet, all rats were shown to harbor
large numbers of lactobacilli in their ceca. Coliform organisms were always
feund to be present in the cecum of these rats, regardless of the diet, as
also other forms such as streptococci and anaerobes.

From the works cited above, it would naturally follow that the

cecum in rats forms an important part of the alimentary tract and with

3.

its large bacterial populations, it is not surprising that it has been
shown to be an active portion of the intestinal canal where synthesis
of B vitamins has been noticed to take place.

Rats fed on purified diets to which sulfonamides were added were,
discovered by Gant‘gt'gl (l9h3) to bring about a marked diminution in their
intestinal flora. These workers discovered that this reduction in the
numbers of microflora had a definite effect on their rate of growth and
resulted in the development of symptoms of vitamin deficiency.

During her study of the bacterial flora of the feces of rats,
Miller (l9h5) observed that the number of organisms present varied from
rat to rat and in the same animal from day to day. She did not find any
significant change in the fecal flora following change of diet from Purina
chow to a highly purified diet containing adequate amounts of all the
growth factors. A drop in the number of coliform organisms as a result
of addition of succinylsulfathiazole and phthalylsulfathiazole to the
purified diet was, however, noted although this did not affect the number
of aerobes present and it was presumed that the decrease in colifbrm
bacteria resulted in the corresponding increase of other organisms. Rats
fed on these diets containing succinylsulfathiazole and phthalylsulfa-
thiazole were noted to exhibit symptoms usually associated with dietary
deficiency and are a further indication of the role of these bacteria in
the synthesis of some of the accessory growth factors.

The work of Taylor and associates (l9h2) on the vitamin require-
ments of cecectomized rats already referred to above has indicated that
a diet deficient in several of the B vitamins when fed to cecectomized
rats produces a severe effect on their health, whereas rats subjected to

no cecectomy kept on the same diet thrive well. They have further concluded

h.

from their experiments that where the diet supports the right intestinal
flora, the cecum contributes to the rat's supply of several of the B
vitamins. thich type or types of bacteria inhabiting the large intestines
would be the most active in this reSpect has yet to be ascertained and is
a matter for further research. Further work is also necessary to deter-
mine whether, in the event of cecectomy, any of these bacteria would
establish themselves in other parts of the large intestine in sufficiently
large numbers to be able to carry out the function of vitamin synthesis

as indicated by these workers. That there is such an attempt on the part
of the neighboring portions of the colon and the junction of the colon
‘with the ileum in the cecectomized rats is evidenced by the changes of

a compensatory nature observed by these workers in their rats.

Nutritional studies in pnogress at the Michigan State College under
the auspicies of the Department of Home Economics have recently revealed
the develOpment of enlargements of the ceca (probably similar to those
observed by Taylor gt al, 19h2, in their experimental animals) in rats
fed on a diet consisting of Carnation Milk supplemented by a mixture of
minerals containing iron phosphate, magnesium sulfate and c0pper sulfate,
while no such abnormalities were observed in the control group of rats

fed on a stock diet having the following composition:

Yellow corn meal 5,000 parts
Linseed oil meal 1,600 parts
Alfalfa meal 200 parts
Salt 50 parts
Calcium carbonate 50 parts
Casein 500 parts
Wheat germ 1,000 parts
Yeast 500 parts
Powdered milk 500 parts
PURPOSE

The work reported in this paper was undertaken to study the

relative numbers of the various groups of microbes in the feces of

5.

cecectomized and uncecectomized rats with a view to get an idea of the
effect of cecectomy on bacterial population. Since the counts in these
determinations varied considerably from rat to rat, the work was further
extended to study their numbers in the feces of young rats before and
after cecectomy to obtain a more comprehensive picture of the changes in
the intestinal flora that might result from the removal of their ceca.

A comparative idea of the relative numbers of different important species
of intestinal microbes in the cecal contents and feces of normal rats was
obtained by making tests on samples of relevant material from two of the

rats collected at the time of cecectomy.

MATERIALS AND METHODS

A total of 32 weanling male albino rats was used in the feeding
experiments referred to above. Of these, a group of 16 rats was fed on
Carnation Milk supplemented by the minerals and the second group, com-
prising the remaining 16 animals, was fed on the stock diet as per for-
mula given above. Half the number of animals in each group were cecec-
tomized and the remainder of the animals in the group served as normal
controls which were not cecectomized.

Two rats from each of these four groups were selected at random
and counts of the principal groups of bacteria in the feces of the
cecectomized and control rats fed on the two diets were determined at
varying intervals after cecectomy.

'gggia:-The choice of media for this investigation was made
comparatively simple as a result of the recent work of Evenson and
others (l9h6) who have reported a very useful set of media for use in
this kind of work. They have developed a medium for each type of
important microbes that commonly occurs in the intestinal tract, particu-

larly of white rats. Liver infusion broth made from bactotryptone,

6.

glucose and liver extract, as used by these authors, has proven excellent
for getting a "total count" of viable bacteria and is very simple to make.
The only modification made in this medium has been the use of dehydrated
bacto-liver powder in place of fresh liver, which is often difficult to
obtain. Six grams of the powder in 100 ml of water steamed for 30 minutes
in the Arnold Steamer gave sufficient liver extract for a litre of the
medium. This liver powder also replaced the dried liver chunks intro—
duced to provide anaerobic conditions in the medium and a quantity of the
powder sufficient to cover the flat end of a small sized spatula was all
that was necessary to be added to each tube before dispensing the infusion
broth. The medium was finally sterilized by -auteclaving at 15 pounds for
15 minutes.

E. C. Medium for growing the coliform organisms was at first made
after the formula of Hajna and Perry (l9h3). .This medium was subsequently
discovered to be available in a dehydrated form.

The S. F. Medium for growing enterococci and Vinblad's acetic acid
medium were adopted for use without any modifications. There being no
facilities available for incubation of the culture at hS degrees C, the
S. F. cultures were incubated at 33 degrees C. This allowed the growth

of some other organisms besides the Streptococcus fecalis. Examination

 

of smears from all the tubes showing a change in the color of the medium
and a few following dilutions showing no color change, however, was
enough to decide the presence of streptococci. It was noticed from the
results of smear examination that all tubes showing a change in color
revealed the presence of streptococcal chains and all the others were
fbund to be negative. As a result of this conformity between the color
change and smear examination, the practice of examining smears was later

abandoned to save time.

7.

The Winblad's acetic acid medium used for growing lactobacilli
and yeast-like organisms proved equally efficient as claimed by Evenson
33 El (l9h6). It was noted, on microscopic examination, that all tubes
showing gas in the inserts revealed the presence of yeast-like cells in
the smears. This was not true of the tubes in which gas was not trapped
in spite of shaking the culture tubes several times during incubation as
recommended by the original authors, and smears from such tubes always
proved negative for these micoorganisms.

The formulas of the media used in this work are listed in Appendix I.

Animals:-—As mentioned previously, young albino rats, about 3 weeks
of age, were fed on the two diets. Half the number of animals in each
feed group were cecectomized and the other half left as normal controls.
Two rats from each of the four groups (each group consisting of eight
animals) were used for bacterial examination of their feces. For making
a comparative count of the various groups of flora in the cecum and feces,
cecal contents were collected from two rats at the time of cecectomy by
squeezing into sterile petridishes, under aseptic conditions, the contents
of the ceca immediately after their removal from the body. All fecal
samples were collected in sterile petridishes by gently squeezing, with
all possible precautions of asepsis, a little of the fecal material from
the rectum of the rats held from the tail.

For the determination of counts, a small amount of the material
(about 0.1 gram) was transferred to previously weighed sterile test tubes
containing glass beads and the actual amount of material used determined
by weighing the tubes with the samples in them. Sufficient sterile dis-
tilled water was added to give a known dilution (usually 1:100) and a
homogeneous suspension was obtained by a thorough and vigorous shaking.

Further decimal serial dilutions of the material to 10-11 were obtained

8.

by transferring 10 ml of the suspension into stOppered bottles containing
90 cc sterile distilled water. By inoculation of 1 ml quantities of each
dilution (10'3 to‘lo-ll) into sets of three tubes of each kind of medium
and incubation at 37 degrees C for 3 days, the number of tubes showing
growth in each dilution was determined. Counts of various types of
organisms per gram weight of the material examined were obtained by the
dilution count method. Growth was judged by the presence of turbidity,
acid production and gas production. Examination of smears after staining
by Gram'a method from each type of medium after growth had been obtained
showed the predominance of the particular type of organisms for which
the medium was intended, thereby proving the suitability of each medium
used. The actual counts were obtained by a reference to the Dilution

Count Tables (Buchanan and Fulmer, 1928).

RESULTS

Table I shows the counts of the various types of bacteria in the
cecal contents and feces collected from two rats at the time of cecectomy.

The average counts of the fecal flora obtained in cecectomized and
uncecectomized rats belonging to the two groups fed on the Carnation Milk
diet and stock diet are recorded in Tables II and III reapectively. The
graph (Fig. 1) shows the composite growth curves of the two groups of
rats covering the entire period of observation of the feeding experiment.
Table IV is included to show the counts as determined on a group of four
white rats 10, 5 and 0 days before cecectomy and 6, 11 and 17 to 18 days
after the operation. This table also shows the average counts of the

whole group of four rats.

DISCUSSION

It will be observed from Table I that bacterial counts of most

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1’.

of the Species in the feces are comparatively higher than the correspond-
ing counts in the cecal contents. it is difficult to draw any conclu-
sions from data obtained from only two rats and attribute any significance
to these higher fecal counts. These findings are, however, contrary to
those of Porter and Rettger (l9hO), who found cecum to harbor the highest
number of bacteria and indicate that bacterial proliferation does continue
in the intestinal tract beyond the cecum.

The higher incidence of total numbers of bacteria, coliform
organisms and enterococci in cecectomized rats fed on the Carnation Milk
diet as compared with the corresponding counts in the control animals in
the same feed group (see Table II) is a striking feature of this study.
The number of lactobacilli which was lower in the cecectomized rats in the
first two determinations also rose about 100 to 200 times in the last
two determinations. It is surprising to note that in spite of being
devoid of their ceca these rats were observed to maintain the same rate
of growth as those with their ceca intact. In the case of the group of
rats fed on stock diet, the "total counts" were not markedly different
in the cecectomized rats as compared with the uncecectomized, and the
enterococci counts were variable, but the coliform counts were through!
out higher and so were the lactobacilli except in the first sampling.

Even in this group the growth curves of the cecectomized and uncecec-
tomized rats are exactly identical. hhether higher bacterial counts
would have any'bearing on the rate of growth of these animals is purely
a matter of conjecture and hard to conceive. But since these two
groups of rats were kept under absolutely identical conditions with the
absence of cecum as the only variable factor besides the variation in
bacterial population, the higher numbers of bacteria might influence

the growth through their ability to synthesize some of the accessory

15.

food factors of which they would otherwise be deprived by the removal of
the cecum which has been shown to be a seat of synthesis of these factors
‘ by some of the microbes in the intestinal tract.

The effect of cecectomy on the fecal flora is further illustrated
by the results of determinations carried out on a group of four rats at
varying intervals before and after cecectomy (Table IV). It will be
observed from this table that the total counts in these rats were fairly
constant throughout the period of observation and were in close proximity
‘with those obtained by some recent workers (Everson 23 al, l9h6) in
their control group of rats. The counts of lactobacilli, enterococci
and the yeastnlike organisms have been quite variable both before and
after cecectomy and give no indication of the effect of the removal of
ceca. The coliform organisms, however, have been found to show a definite
increase in numbers in all the rats in tests made subsequent to the
Operation as compared with the counts obtained in the same rats before
cecectomy. These higher counts appear to have been maintained in all
the tests made during a period of 15 days after the removal of the cecum.
The number of animals used in the experiment, however, is rather small
and the results cannot be accepted as generally true unless the experi-
ment is repeated on a larger number of rats fed on different kinds of
diets to make the data statistically sound. The fact that these rats
continued to maintain a good general level of health fellowing cecec-
tomy as indicated by their general appearance and their composite growth
curve (Fig. 2) leads one to presume that this increase in the coliform
counts might have a favorable influence on their health. This presump-
tion would be supported by the observations of Miller (19h5) on her rats
which exhibited symptoms of dietary deficiency when a drop in the number

of coliform organisms was brought about by the addition to their purified

16.

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l7.

diet of succinylsulfathiazole and phthalylsulfathiazole.

SUKIARY

A study of the bacterial populations of the cecal contents and
feces of two young albino rats revealed the numbers of various groups of
intestinal bacteria to be larger in the feces than in the cecal contents.

Ybung cecectomized rats fed on Carnation Milk showed higher
"total bacterial counts" as well as coliform and enterococci counts in
their feces as compared with the corresponding counts in uncecectomized
rats. The lactobacilli counts were lower in cecectomized rats in second
and fourth week samplings but increased considerably in the subsequent
determinations. In the group of rats fed on stock diet, the "total
counts" were comparable in the cecectomized and uncecectomized rats,
the coliform and lactobacilli counts were definitely higher in the
cecectomized, but the numbers of enterococci and yeast cells were very
inconsistent. The composite growth curves of these animals for a period
of 12 to 13 weeks were much better in rats fed on stock diet than those
fed on Carnation Milk but the cecectomized rats showed growth curves
identical with those with their ceca intact, thereby showing that cecec-
tomy has no untoward effect on the rate of growth.

Examination of feces of a group of four young rats for different
groups of microbes before and after cecectomy has shown the "total counts"
to be fairly consistent and other groups to be quite variable. The animals
showed a definite increase in their coliform counts in determinations made
subsequent to cecectomy.

It is suggested that the increase in the number of bacteria in
the intestinal canal of rats following cecectomy might have a favorable
influence on their health and might possibly be a compensatory mechanism

which provides a substitute for lost physiological function.

18.

BIBLICGFAEHY

Abdel-Salam, A., and Leong, P. C. 1933. Synthesis of vitamin 81 by
intestinal bacteria of the rat. Biochemical Journal, 22, 956-963.

Buchanan, R. E. and Fulmer, h. I. 192d. Physiology and Biochemistry
of Bacteria. vol I, p. 10. william and Lilkins Company,
Baltimore.

Dam, B., Glaving, J., Orla—Jensen, 8., and Orla-Jonsen, Ama L. l9hl.
Bildung von vitamin K in celibacterien auf synthetis-chem
substrat. haturwiss., 29, 267-260. Cited by Avenson and
associates in Journal of Bacteriology, l9ho. 21, 513-521.

Day, H. G., hakim, K. G., Krider, M. M., and O'Banion, E. E. l9h3.
Effects of cecectomy, succinylsulfathiazole and p-aminobenzoic
acid on vitamin K synthesis in the intestinal tract of rats.
Journal of Nutrition, 29, 585-600.

Evenson, A., thoy, Elizabeth, Geyer, B. B., and leehjem, C. A. l9h6.
The cecal flora of white rats on a purified diet and its modifi-
cation by succinylsulfathiazole. Journal of Bacteriology, E1,

513-521.

Gant, Ola K., Ransome, Beverly, HcCoy, Elizabeth, and Elvehjem, C. A.
19b3. Intestinal flora of rats on purified diets containing
sulfonamides. Proceedings of the Society for Experimental
Biology and hedicine, £2, 276-279.

Guerrant, N. B., Dutcher, R. A., and Tomey, L. F. 1935. The effect of
the type of carbohydrate on the synthesis of B vitamins in the
digestive tract of the rat. Journal of Biological Chemistry,
112, 233-210.

Hajna, A. A., and Perry, C. A. 19b3. Comparative study of presumptive
and confirmative media for bacteria of the coliform group and
for fecal streptococci. American Journal of Public Health,

.3_3_’ 550-5560

Herter, C. A., and Kendall, A. I. 1909. The influence of dietary and
carbohydrate feeding on the characters of the intestinal flora.
Journal of Biological Chemistry, 1, 203-236.

Hull, T. J., and Rettger, L. F. 1917. The influence of milk and
carbohydrate feeding on the character of the intestinal flora.
Journal of Bacteriology, g, h7-71.

19.

killer A. Kathrine. 19b5. The effect of succinylsulfathiazole and
phthalylsulfathiazole on the bacterial flora of rat feces.
Journal of Nutrition, 22, 1h3-15h.

hitchell, H. K., and Isbell, E. R. 19h2. Intestinal bacterial synthesis
as a source of B vitamins for the rat. University of Texas Publi-
cation NO. 14237, 123-1311.

Porter, J. R. and Rettger, L. F. 19hU. Influence of diet on the distri-
bution of bacteria in the stomach, small intestine and cecum of
the white rat. Journal of Infectious Diseases, éé, 10h-110.

Rettger, L. F., and Berton, G. D. 191h. A comparative study of the
intestinal flora of white rats kept on experimental and ordinary
mixed diets. 'Tentr. Bakt. Parasitenk, I, 0rig., 73, 362-372.
Cited by Evenson 33 a1 19h6 Journal of Bacteriology, 51, 513-521.

Taylor, A., Pennington, D., and Thacker, J. 19h2. The vitamin require-
ments of cecectomized rats. University of Texas Publication

No. b237,.135-1uh.

Torrey, J. C. 1919. The regulation of the intestinal flora of dogs
through diet. Journal of Kodical Research, 22, h15-hh7.

Winblad, Sten. 19bl. variations in the bacterial flora of the intentine
of white rats in different carbohydrate diets. Actas Pathologica
Microbiologica Scandinavica, lg, 20h-22h.

APPENDIX

FORHULAS OF ELDIA USED IN THIS WORK

Liver Infusion Broth

bacto-tryptone 5 gm
Glucose 5 figm
Liver extract 100 m1
Tap water to make 1,000 m1

Tubed with added liver powder (Bacto)
Autoclave at 15 pounds steam pressure for 15 minutes

Liver Extract

Bacto-liver powder 60 g
Tap water 1,000 ml

Steam in Arnold Steamer for 30 minutes; filter through
guaze

E. C. Medium (Bajna and Perry, l9h3)

Distilled water 1,000
Bacto tryptose 20
Sodium chloride 5
Lactose 5
Dipotassium hydrogen phOSphate (K2HPOu) h
Monopotassium hydrogen phosphate (KHzPOh) l
Bacto bile salts No. 3 l

0‘
o
\O

Titration of pH and filtration not necessary; final pH
Autoclave at 15 pounds steam pressure for 15 minutes

S. F. Medium (Hajna and Perry, 19h3)

Distilled water 1,000
Bacto tryptone

Sodium chloride

Glucose

KzHPOh

KHzPOh

Sodium azide

Brom cresol purple, 1.6% alc. sol.

NOHC’U‘IU‘IB
EEEBEQEE

Autoclave at 15 pounds steam pressure for 15 minutes

QEEEBEE

21.

 

 

ninblad's acetic Acid Medium
for Lactobacilli and least Cells

nth-3114141412 {00014
Beef extract 3 gm
Bacto tryptose 10 gm
Bact dextrose 5 gm
Sodium chloride 5 gm

Acetic acid sufficient to bring the concentration to 0.12%

Dispense with enclosed fermentation tubes up to a level of the
inserts
Autoclave at 15 pounds steam pressure for 15 minutes

 

A C KNO? :1 h”). C} 3} EN

The author wishes to record his
indebtedness to Dr. E. J. Stafseth for
his valuable advice and guidance in the
prosecution of this work. Thanks are
due to the Department of Home Economics
for the facilities provided in connec-

tion with this work.

mm 49 .
. _ mg 3 I49 '
. ROOM USE ONLY.
m2?) ‘3‘}- - .

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