BNRICHMENT MEDM EN THE
ISOLATEON OF PATHC‘GENIC

ORGANXSMS BELONGING TO THE

COL.ON"TY FHOiD GROU P

T‘fizsais for {he Dagraa of M. 3:
MlCHiGAN S'E'ATE COLLEGE
("x " ‘ '

mace Annabelie Tenmga

2947

THESIS

 

4‘ ._VA._r-v

This is to certifg that the

thesis entitled

Enrichment Media in the Isolation of Pathogenic
Organisms Belonging to the Colon-Typhoid Group

presented b1]

Grace Teninga

has been accepted towards fulfillment

of the requirements for

__ 3129 So degree in .___BE_1_C_ £9111 0 1053/

 

Date _ WEE, _131£Z-- .._

| M—796

 

EJRICHJJL” MEDIA IE TEE ISOLaJ oH CF PJJIDJELIC DEGAMISJS
' EJLULdIhj TC ”K3 CLLOh-TYPHUIU ;RQUP

Grace Annabelle leninga

A THESIS
Submitted to the School of uraduate Studies of Uichigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIElCE
Department of Bacteriology and Public Health

1947

1H E815

AC KlJU . .‘LED Cit-1.41. ‘f S

I an very grateful to Dr. H. :. Cepe,
Pathologist and fiery Bronson, b. 8., Technician,
at Michigan Department of Health for their ad-
vice and assistance during the planning and
completion of my experimental worn-z.

I am grateful to the Lichi an Department
of Health for the equipnent and media used While
0 mpletin; this work.

I also wish to thank Dr. H. J. Stafseth

for his kindly advice during ny course of study
and the writing of this thesis.

193304

m.1r;1 _" f‘lr‘ 2'."\1 =1."
IAULJIIJ (JI‘ \JUIeL4Jl.&D

I. Introduction . . . . . . . . . . . . . . . . . . . . .

II. Lx

perilnenta.l ".0141; o o o o o o o o o I o o o o o o 0 0

III. Experiments and Results . . . . . . . . . . . . . . .

.12 .

b.

Use of beces Showing no Growth . . . . . . . .
l. Tetrethionate Broths . . . . . . . . . . . .
2. Selenite F and wandelic Acid Broths . . . .
3. The Zone of Inhibition . . . . . . . . . . .

use of liegvtive il‘eCeSo o o o o o o o o o o o o 0

TJ. UiSCUSSion O 0 a I O O O O O O O I O O O O O O O l O

V. Results of Specimens from Patients . . . . . . . . . .

Carriers and Carrier Suspects . . . . . . . . .
Shigella parcdjsenteriae carriers . . . . . . .
Knox Tetrathionate and Selenite.3‘used for
Sixteen Speciuens . . . . . . . . . . . . . .
Routine Specimens Tested with Knox fetrathionate
and Handelic Acid . . . . .'. . . . . . . . . .
haller's Tetrathionete, Knox Ibtrathionate,

and Selenite F used for Routine becal Specimens.

VI. Appearance on 85 Plates . . . . . . . . . . . . . . .

VII . Still-IL L‘LLlI‘y O O O 0 O O I O O O O O O O I O O O O O 0

VIII. Formula . . . . . . . . . . . . . . . . . . . . .

IL; . like fCl‘el’ice S o o o o I o o o o o o o o o o o o o o o o

Pnéu

; ‘3‘1

0)

O3

10

12

13

l3

l4

17

18

19

20

IXTHODUCTILH

Often negative results have been obtained in bacteriological
examination of fecal Specimens containing pathogenic bacteria, because
the size of the inoculum was too small. by increasing the size of the
inoculum, positive results were increased, as was evidenced by the use
of Jilson and Blair medium where growth was found on the plate contain-
ing 5 ml. of a saline suspension of l g. of feces and not on the plate
containing 1 drop. This method, however, could not be applied to a
streak plate.

Attempts were made to obtain a liquid enrichient medium in
which a large inoculum could be placed. ihis medium should support
the growth of the few pathogens present while inhibiting or delaying
the growth of the non-pathogens during an incubation period of 12 to
2% hours. Then, fron this culture, streaked inoculation could be
made on solid agar for colonial isolation.

hdller in 1923, used a broth medium containing sodimn thic-
sulfate to which was added an iodine solution. This resulting
tetrathionate medium was kept basic with chalk, and increased the
positive results when used with routine media.

Schaeffer employed the above medium with less chalk. He
was able to isolate four times as many fecal pathogens of the
paratyphoid and thyphoid group as were found by ordinary methods.

Kauffnan found that hdllcr's and Schaeffer's modification

ii
of Muller's tetrathionate medium allowed many lactose fermenters

and Proteus to grow. In an a tempt to limit this he added ox bile
and brilliant gr an to huller's broth. Later workers, Knox, dell
and Pollack, found that this medium of Kauffnan's also inhibited
Salmonella typhi.

Jones also added dyes to the tetrathionate broth in an
attempt to control the lactose fennenters. he added a conbination
of brilliant green and eosin which he titrated to a point where it
would grow fi, ting; but not Escherichia coli. This he added to a
tetrathionate broth titrated in the same way. He claimed very good
results with this mediun.

Knox, Gell, and Pollack published many articles on their
worx with tetrathionate broth when used for growing Salmonella
organisms. They found that the Saluonellae reduced tetrathionate
rapidly to thiosulfate. They advocated the use of a balanced aediun.

'jsvn m. 09 .. ‘1‘ '1 5'” ' ' 1’ - ' ‘

H

an excess of iodine kills all bacteria and an excess of thiosulfate

inhibits g, tvuhi. huller, in his original research work, used a

balanced medium but recomnended an increase of thiosulfate five times
greater than iodine for routine work.

In a comparative study by Knox, Sell and Pollack, a conbination
of tetrathionate broth and brilliant green, methylene blue agar gave I
better results than Uilson and blair's medium or bosin methylene blue
agar alone.

Tetrathionate broth was used successfully in several epidenics

by Jones, Sell and Knox and by Holt, Vaughan and Jri ht.

 

 

 

 

There were workers, however, who were not 8 tisiied with

4’.

tetrethion te broth. Jollov, Jinter end Steincerg found Uflfit al-
though tetrithion te broth increfised the total nuicer of positives,
more were picked up by SS alone than by tetrathionete and Mchonkey's.
Invonics found that six strains of Aero eigs inhibited the grouti of
.i- tvohi in tetrathion te broth more t-un in plain broth. jut synn
and ”illians found the Opgosite to be true.

H

In Holland, Pot (19i3) used guller's tetrethionete.success-
fully,r but had to SDI-11151011 it in Cuiracozi, .etmilinc’ ..est Indies
because of the Spoilage by Ploteis.

Anotherl _iquid medium which h1s been used extensively in this
country. and which h.s been recox ended 03 hdr3lo nd State Health
Department, is a Selenite broth as suggested by Sinar Leifson (1936).
In his research work, he found that typhoid becilli,cholera organises,

Proteus, and most parath3rihoid organisms grew in a solution of l»

sodiun selenite. Enterococci, Aeroogngg, Shigello sonnei and

 

SsL1onell- gellin rum grew in a O. 5 but not a 13 sodium selenite

solution. ‘5. coli grew in a .2% but not a .5; sodiwo selenite solution

 

while -rucoll: grew only in a 1; solution.

In 1343, Gohur also worked with a delenite broth mediui. he

 

:88 not aware of Leifson's work. He found that Salmonella U‘rhthvohi
”*- fi.‘h
.é and g, S:lnonell.s. ont itiii and Strggtococcu fe colic were oore

 

 

 

resistant to selenite than 3. coli. Saloonollt res sli latl more so.

He recommended a 0.2% Sodium Selenite broth or peptone water. Ihis

a.

solution was weaker than the medium recomhenoed by Leifson.

' I I'V)‘

EKPJHIJJLEAL .cnn
In order to find mhich was the best enrichment medium for
the isolation of the pathogens found in fecal Specimens, a comparative
otudy of the efficiency of the following four media has been made:

Selenite - a dehydrated product produced by the
Digestive lensents Co;peny

mendelic Acid - Also a dehydrated product from the
Digestive Ierments Conpsny

H
Tetreuhionzte broth - nuller's fornulee
Tetrathionete broth - suggested by Knox, cell and Pollack
This research was divided into two p rts:

l. A comparative study of human feces inoculated ertifically
\fi 1011:

lnonella tyohi
b. Sfilmonelle enteritidis
c. gglnonelle typhiniriui
d. Salnonella orcnienoer;
Si‘zigelle p; radysentcriae ieflgg

 

 

 

 

 

2. A comparative study of fecal specimens from carriers,
Carrier suspects, feces from patients suffering from
Salmonella (food poisoning and typhoid fever.

5

human ICCBS ertificelly inoculated with pathogens.

hethod: l. A suSpension of the pathogen We made from an agar slant
by rubbing the surface gently with a sterile cotton swe‘
and immersing it in sterile saline solution. The result-
ing saline suspension was diluted to match the Kaolin
standard of 250 o.c.u. or 1000 million organism per ml.

a. L

Serial dilutions were made from this suspension: 1-50,
1-100, 1—200, 1-400, 1-300 and 1600.

N
o

3. One tenth ml. quantities of the above dilutions weze
inoculated into 103. of pooled hunch feces found
negative on routine examinat'on for enteric organisms
in the michigan Department of health Laboratory.

4. Apgr x‘mately 0.1 gram of the seeded feces was in-
oculeted into 10 ml. enrichment medium and into 10
31. saline.

5. One tenth ml. of the saline was streaked onto an

SS plate. The next day colonial counts were made.
This gave an approximation of the number of organises
present per 0.1 ml. of enrichment mediwn.

6. The enrichment medium were incubated for 12-24 hours.
Then 0.1 m1. of the enrichment broth was streaked onto
33 and MacConkey's agar. yilson and Elli? poured
plates were made free 0.1 m1. and 5 m1. of the broth.

7. The SS and mecConhey plates were incubated 24 hours.
Then colonial counts were made and compared with the

original counts.

8. Wilson and Blair poured plates were incubated for 43
hours and counted.

9. The organisn was identified by biochenicel and rapid
slide agglutination tests.

Method of Making Serial Dilutions

 

 

 

Am't. of Dilution 0.1 ml. in 0.1 41. in theoretical
Saline 10 g. feces 10 ml. broth organisms
in 0.1 ml.broth
l'l of s 0 ml 1-50 1-5000 1-500,000 2F0
2=5 of l 5 ml 1-100 1-10,000 1-100,000 100
3'5 of 2 V m 1-200 l-20,000 l-200,000 50
é=5 of 5 5 ml 1-400 1-40,000 1-4,000,000 to
5'5 ; of 4 b m1 1-8 0 l-o0,000 l-u,ooo,ooo 12
6 5 of 5 0 ml 1-1600 1-150,000 l-ld,000,000 6

 

 

A=organisns diluted to standard 250 ppn or 1000 million
organisms per milliliter
B31 n1 of A and 4 31 saline

figperiments and Results. At first attempts were made to use pooled
feces, negative to pathogens, but shoeing bacterial growth on anConke5's

or 83 plrtes. These feces were seeded with g. tvohi and put into the

 

" \
tetrethioneto broth ’nuller'3° and inox Pollack and sell) incuodted
K . s
and plrted on hacConkey's, 33 and bilson and blgir media. 83 and
neoconhey plwtes were all overgrown with lactose fermenters or

Pseudonones. (ilson and Blair medium gave negative results.

 

Jse f Leces showing pg grontg. Because of this overgrowth, feces

.-

fir

showing no growth on routine exsiinetion_Ly the hi nigen Lepsrtnent of
Health were used. These were pooled and mixed. xeny of the specimens

were several days to severe weeks old and meiy contained bile.

\

Tetreohionete Broths. Again serial dilutions of,§ tvohi were made and

 

0.1 m1. quantities were seeded into 10 g. feces. One tenth gran

H
seaples of these seeded feces were inoculated into huller's and Knox'
tetrethionete broth. These broths were then incubated for twenty hours.
Then 0.1 ml. w;s plated onto 83 and MncConkey's solid media. Poured
plates of ”ilson and Blair :edium were made with 0.1 and 5 m1. unounts
of the broths. 511 SS and Mchonhey plates showed a marked increase in
the number of organisms, the colonies being too numerous to count.
Uilson and Blair plates had counts of 100 to too many to count on the
plates containing 0.1 ml. of broth and one to ten colonies on plates
having 5 ml. tetrnthionnte broth. These plates were discolored, there-

fore, it is probable that there were too many organisns ior the nedium

to supgort a good growth.

y-‘f -~. ‘ 7'1"“ 1 ‘- l'". “ "- f \‘L‘fl‘ n n 1 V} HUITTT‘
J‘J‘JoLJhI ahA—lJ E . “Mk-d .CJMIDH—JIU “V E.) fichU--&~J:

The above experiment was reoeuted using Selenite b‘(uifco)

and Mendelic acid broth (Difco) as the enrichment media. The control

['7‘

counts showed that the suspension contained clumps. lne ounts ranged

C)

from 200 to no colonies. Plated Selenite E‘nediun showed a marked in-
crease in organisms, the colonies being too numerous to count. This
was not the case with Handelic Acid broth; the increase vns great but
countable as seen by the Table. There was no growth on qilson and
Blair medium at any dilution.

Maidelic Acid 3 Selenite F inoculated with feces seeded with S.Typhose.
December 6, 1946

 

Plate Counts. 43 200 120 105 - -
Orgenism/O.l ml broth
0.1 ml d ndelic Acid plated

 

 

 

on 33 a ter 20 hrs.incubntion Many Many 64 13 200/ 55
0.1 ml dandelic acid

glated on Jilson and blair - - - - - -
0.1 ml Selenite broth on

SS after 20 hrs.incubrtion Many Many Lsny any many many
0.1 ml Selenite F broth on

Jilson and Blair - - - - - -

 

It Was noted that there mas a zone of inhibition on the 33 plate
where Selenite F and Mnndelic acid broths diffused into the 33 medium.
There was a slight change in color at this point from red to orange. It

is ossible that there was a reaction between tne 33 medium and the

’d

Selenite and unndelic acid broths nnking it more inibitive to the
organisms, or that the double action of the Selenite-~35 and dandelic

acid--SS heat the organism from growing.

5

A second test was run using the SeLenite F and dandelic acid
enrichment media. The inoculum vas smaller than that used in the
first experiment on Selenite F and Mandelic acid broths. The broths
were incubated for six, twenty and forty-four hours to find if the
period of incubation he d anys Mic -nce on the arenth of the
organism. The inoculwn seemed to be too small for the handelic scid
broth. he organisms COlld oe isole ted on 33 plates in 20 nours.
Selenite plates on 33 showed a decrease in six hours but an increase
in twenty and forty-four hours.

Feces sh Wing no growth with routine examination seeded with §. tyohi

and inoculated into Selenite E and Landelic nCid enrichment broth.
January 7, 1947

 

 

 

 

Plate counts 41 13 6 2 2 2
colonies in one tenth ml broth

Mandelic acid 01/31 on SS - - - - - -
after 6 hrs. incubation - 4 - l - -
after 20 hrs. incubation - - - - - -
after 44 hrs. incubation - - - - - -
Selenite B‘O.l ml on 53

after 6 hrs. incubation - d - 2 - -
after 20 hrs. incubation - many 1 300% 37 19
after 44 hrs. incubation 77 anv 12‘ many hany many
rm- *1 '7.‘\ H CT‘“ “7.1121"

4.4-.4 UV- -J
_ n

L .‘A‘J-~-‘I: V‘I
-m

Because Mandelic acid broth and Selenite F broth showed this
zone of inhibition of all organisms where the broth media diffused into
the 83 plate, a spec'al test was made. bne tenth ml of sterile Selenite
medium was pipetted onto one— —half pl te of tvo lots of SS a3? and two

lots of macConl eyr's agar. “he plates were allowed to stand until the

broth had thoroughly diffused into the solid media. Then two strains of
,§.tynhi, one of an unidentified Salmonella and one of Shi.elle, Blexner
strain, were streaked across the plate. on the 83 medium there was
complete inhibition of all organisms on the experimental side but good
growth on the control side of the plate. mchonley's medium showed a
slight inhibition of the organisms where the Selenite diffused 'nto the
solid medium and good growth on the rest of the plate. Those colonies
present were smaller and there was a reddening of the center of th
coloni. A nrw lot of Selenite F median showed the same results.

The Landelic acid nedium was tested as was Selenite F and the
results were the sane.

Since it was possible tint the organisms were-present in the
Selenite B‘and Mandelic acid broths but were inhibited by the double
action of the SS -- Selenite or 33 --'handelic acid media, attemgts
to dilute out the enrichnent media were made. Three organisns were
used with this experiment: a Salmonella, an i. tynhi, and a Elexner
organism.

Serial dilutions of these were seeded into pooled humrn feces
showing no growth on routine examination, and these feces vere in-
oculated into Selenite r and handelic acid broths. after twenty hours,
one tenth milliliter of the broth wa transferred into 10 ml. sterile
saline. one tenth ml of the saline was transferred onto 35 and

MacConKey plates and streaked. Colonial counts were made the next day.

The Flexner and tynhoid bacilli showed no ;rowtn. The original counts

.

10

showed that the inoculum was very small. Itenty-three orgénisa were
0.1 ml of broth was the heaviest inoculum of Elexner organism, and
seventy-two was the heaviest of the thyphoid bscelli. It is possible
that growth occurred but Was snail and was diluted out.

In the case of the Salmonella the inoculwn was heavier through-
out. The resrlts show hat the Selenite r broth supported the .rotth
of the pathoges to s greater extent than the nondelic acid broth. no
zone of inhibition was noted.

Salmonella seeded into feces showing no gFQWDH with routine eth nation

"\

and inoculated into handelic “Cid end Selenite 2 broth

 

Originul Colonial counts 125 4a 20 16 8 2
organisms /O.l ml broth

Counts after 20 hrs.ineub1tion

dandelic acid ureth -

0.1 ml in 10 ml saline 120 - - - - -
0.1 saline on 65

Minoelic RCid

0.1 ml in 10 ml saline 40 78 - - - -
0.1 saline on MacConkeys

Selenite E

0.1 in 10 ml Saline

O.lr:1l Seligehonwééu 92 26:9 to} - - -

UV
-mo‘ 0 -0“..- ‘-‘--.‘C. -. -.r-‘»-u - m D .. - --... -‘

 

”I

Selenite :
0.1 nl in 10 ml Saline 134 3 3 66 - - -
0.1 ml saline on JacConkeys

m m -.-.—.--o “‘90—.- .- -4..-

 

Use Legetive geces. beces negative ior potho;ens but showing growth

I...

 

on macConkey's or 33 pistes with routine examinations were then used.
These were pooled :nd ten gran saigles were seeded with the test organism.
Three attempts to isolate g. tvohi from seeded negative feces inoc~l ted
into the tetrethionrte broths resulted in overgrowth on the 63 plates

1

and Mchonkey plates by Pseudononus or lactose fermenters. allson and

11

Blair poured plates were negative.
Selenite F, and dendelic acid broths were inocul:ted with

negative feces seeded with g. tvohi, 2 Saloonell: and o Shi'ella

 

Flexner strain. ho pathogens were isolated frog vny plates in-
oculated with 0.1 ml of the broths. All non-lactose fer enters
proved to Le spore formers. ’l‘l’iere were not many lactose fernenters

present. The Salnonellee; ejnritidig, tvohiuuriu: and cronienberg

‘ -—.— #4

 

were then seeded into feces negative to pathogens. These feces
were inoculated into all four broth media; Knox tetrethionate, huller's
tetrathionnte, Selenite F and dandelic acid. Only §. gaggggggggg
could be isolated from the tetrnthionnte broths due to the extrege over-
growth of lactose fennenters. Lne colony of g. QELEESQEQES was isolated
from hendelic acid broth plated on uscConkey's nediun. All other plates
were negative. all Selenite plates vere negotive. There were very few
lactose fernenters on plates inoculated with dandelic acid and Selenite
F nedie.

Beef extract broth was inoculated at the sane time from the
some seeded feces, to see if any liquid media would increase positive
results. As a whole, the broth inoculated plates were grossly over-
grown with lactose fermenters. Swerdin; was much more noticeable than
was noticeable with tetrnthionste broths. An sessionel orznienberg
could be isolated.

é. enteritidis was again seeded into negative feces and these

 

.0

in turn were inoculated into all iour enrichhent media and into

tetrathionate broth prepared by the michiyan departient of Health.
All plates inoculated with the tetrathion te broths were overgrown
with l ctose fermenters. The Jilson and Blair medium was negative
vith all enrichment media. There were very few lactose fenaenters

on the 33 plates inoculated with Selenite B‘and Janeelic acid broth.
Eight to ten uncolored translucent colonies apoe red on each 35 plete
inoculated with Selenite and handelic acid broths. These proved to
be very slow lactose fermenters ;nd not §. egtgg;tidis, seeded into

the feces.

DlSCUSSIcJ

From the evidence gathered thus far it appears that nornal
feces seeded with pathogenic organiSns was not a good test for the
comparison of the efficiency of enrichnent media. heces shoving no
growth during routine examination often contained antibiotics such
as bile and possibly penicillin which further inhibited or counter-
acted the inhibitory substances already present in the enrichient
media. beces negative for psthogens but showing gronth on routine
media, often contained rapidly groting non-pathogens, such as lactose
fenienters, spore for ers, end coliform org: isms, in proportions
much greater then found in the freshly passed specimen. Ihe consis-
tency Ind possibly the chemical composition were not the same as the
pathogenic stool. The isolsted or; nisms inoculuted into tLese

specimens vere not as protected by >rotein and f:ts and by other

13

organisms, as were the non-pathogens present in the stool.

The second part of this research nroblem was therefore in order.

"I LI| I \f“

REDULIS LE SPECIndha :nUJ PnILLLlD
Carriers 3nd Carrier Susnects. Beeal specimens from carriers of
Salmonella and thyphoid bacilli were then tested. about one gran of
feces was inoculated into each broth. ”hese were incubated for la to
24 hours. One drop was placed on an 5d plate and spread with a glass
spreader. This Spreader was then rubeed over the surface of a plate
of Mchonkey's agar. One dro; was placed in a Petri dish and a poured
Jilson and Blair bismuth sulfite plate was made.

The results of sixteen specinens were tabulated. Selenite and
tetrethiondte broths proved to be superior to Mdrfielic acid broth for
these sixteen Specimens. Selenite showed 33;, Mandelic acid lG.£, Knox

H

tetrathionate 28.4% and Muller's tetrsthioncte 3d.o; more positive

than routine media.

I

I" 9‘11!" !--v~> _q 7 r41. {7"} . 1 w‘ a .“F - v -' a ,v 1-
Dd -JU «0.4-0 PUUIuLDvo—U 1U 361-L~AV$..J'_JLJ41

 

~- ‘--—9—

 

 

Enrichment Jedis Positive Positive Total 3 increase
on on on on any on routine positive over routine
33 macConkeys bi.3. of the analysis feces

nedifi

_.. .‘_.—~_—.—--‘-—_ .—._-—._—

 

0

Selenite 16 15 4 l6 lO l6 33s
Linndelic acid 12 9 l 13 lO 16 3.6 .541,
Knox tetra-

hioncte 12 13 9 14 lo 14* 24.5;
afiller's tetra-
__t_hiontzte 12 12 4 14 10 14

to
C;
o

(:1
‘0 ".

l4

1

Snigella para dvsenteri e c: rriers Fecal Specimens from carriers of
Shigella paradisenteriae, Fl xner mere tested by these four enrichnent
media. The res lts for ell the uledia were poorer thgn the results found
with routine media. This is uncerstanc able \.hen it is realized that

.0 J1.

thee media were made for the isolation of the Salmonella group.

 

Shigella P radysenteri e Flexner Carriers

 

 

Pos it ves found on Percent Tot l

33 hVCSOnkegs either Secreose Speci1ens
Selenite l O 1 53.3 lo
a: ndelic acid 3 l 3 40 15
Knox tetrathionate 2 O ” 45 13
mullcr's tet'athionate 2 l “ 46 15

u.) l‘
H
(LI

Routine media

01

Y”GX T23‘1Thlonsr3 Ann SLth :3 XELIA U33D .en SIKJJI“ 33g31h3;
Approximately a gran of feces fron sixteen selected routin
specimens were inoculated into Knox tetrathionnte and Selenite media.

These media were incubated for 20 hours a1d then one drOp was placed

'1‘

on 33 medi‘n and spread with a g ass spreader. inis soreader was then

.0

rubbed over the surface of the agvr 01 a second 33 plate. A poured

q o

Eilson and Blair plate was made with one drop of the enrichnent media.

L

Beth enrichment media shoved xiore positives for Sphnonella

 

than were found on routine media. Three nore positives were found
with K1103. ' tetrathionate broth plz‘ted on 33 and Bisz.1uth sulfite 1:11". two
more were found with Selenite. One Speci en has found positive with
routine media but not with Selenite nedia. A Boyd 103 strain of

C

dysentery b cilli was found on routine media but not with the enrich-

15

ment media. r"here were many more lactose fermenters on plates in-
oculated with the tetrathionnte broth than those inoculated with
Selenite broth. Selenite was found to be inhibitory enough to find

isolcted colonies on the first 63 plate making ne second one un-

necessa“y.

 

 

 

Positives 6n Total Percent
33 nilson & hither Speci- Increase
1.3.1.111" 1161.118.-- _
Selenite 3 2 3 l6 6
Knox tetrathionate 5 4 5 l6 9
Routine 2 16

 

houtine specimens tested with Knox tetrathionate and dandelic acid

 

enrichment media. A comparative study of Handelic acid and Knox

 

tetrathionate was made on routine specimens. There was no increase
in positive results for either of the media but all Specimens found

positive for Sahnqnella with routine culture were positive with Knox

 

tetrathionate plated with JilSOM and nlnir medium. hone of these were

H

found positive with dandelic acid 0‘ with Knox tetrathion te plated
on 83 mediun. Two paradysenteriae Boyd 103 and one paradysenteriae
Flexner were found in Specimens on routine examination but not with
the enrichment media. Twenty-two Specimens (five of wiieh mere patho-
genic) showed no growth on 6” plates inoculated from the Lnndelic acid

broths. Only six of these gave no growth on 66 plates inocul‘ted with

tetrathionate.

Routine Speciaens Tested for Salmonella

 

 

 

Positive for Salmonella on Total Relcent

SS Bi 8 either §pgc§igns Deereeee
Knox tetrathionate l 6 6 43
Mandelic acid 0 O O 43 14
Routine examinatign 6 43 _ ___

 

ll .-
Juller's tetrrthion'te, Knox tetrathionate, and Selenite i used or

 

Routine Becal Specimens. Eighty-three routine specinens sent into the
Michigan Lepartnent of He lth were inoculated into Knox tetrathionate
broth, muller's tetrathionate broth and Selenite F.media. Salmonella
Was found in four and S. tyohi in six of these eighty-three Specinens
when cultured with routine media. ‘ho more Salmonellae were found on
iilson and Blair media plated with ooth tetrathionate broths. ‘here
was no incr see in the number of positives with Selenite broth culture.
ive specimens were positive with routine and not with Selenite E;
two were positive with routine and not with Knox tetrathionate. One

II
was positive with routine and not with Muller's tetrathionate. All
specimens were one day to a peek older than when exanined with routine
culture. One Specinen positive in routine examination was overgrown

with Pseudonones on the 35 plates of the tetrathionate hroths but was

 

H
positive on Jilson aid Blair median plated from duller's tetrathionate
broth and on 33 mediwn streaked with Selenite broth. A Flexner strain
of dysentery bacilli which Wes isolated with routine media was not

found with any of the enrichment media.

17

Eighty-three Specimens Tested with Enrichment hedia

 

 

 

Positives Un Relative 3 Total 5
SS Bi 3 Either Increase Increase
Knox tetrathionate 5 10 10 O 2.4
Muller‘s tetrathionate 8 ll 11 1.2 2.4
Selenite F (oifco) 5 5 - .6 - .6
Routine 10
Total Positives 12

 

DISCUSSION OF‘PART II

appearance f 33 plates. mandolic acid is very inhibitory; only a few

 

 

colonies appear on the plate. There were very few lactose fernenters.
Many plates showed no growth. There is a zone of inhibition where the
broth medium diffused into the SS plate if there was drowth on the plate.

Selenite F was less inhibitive than handelic acid but much more
inhibitive than was the tetrathionute broths. Most of the plates con-
tained isolated colonies. Except in rare instances there were very few
lactose fermenters present. Sometimes there was a pure culture of the
pathogens. There was a zone of inhibition there the Selenite diffused
into the SS plate. Many plates contained only lactose fermenters with
tetrathionate broth but plates from the same Specimen had no growth
with Selenite broth.

Huller's tetrathionate supports the growth of the pathogens
very well but it allowed the non-pathogenic lactose fermenters to grow.
Often these overgrow the pathogens. Sometimes it is hard to find an
isolated colony on the second plate containing a very small inoculum.

Knox tetrathionate supports the growth of the pathogens equally

18

as well as ufiller's but there is not quite as many lactose fermenters.
Some plates show no growth with Knox tetrathionate wnen there are

. ..u , . . L,
lactose fennenters on plates inoculated with nuller 3 containing the

same specimen. The colonies do not swarn quite as much.

S‘;J.u1;£{

l. feces showing no growth on routine examination, seeded
with a pathogen and inoculated into four enrichment media proved that
Mandelic acid medium was more inhibitive to the pathogen than were Knox,
Gell and Pollack's tetrathionate, huller's tetrathionate and Sélenite
F broths.

2. Most pathogens were overgrown by the non-pathogens when
artifically seeded n-gative feces were used.

3. Selenite F medium was more inhibitive to the non-pathogens,
especially lactose fermenters, than were the tetrathionate broths.

4. A zone of inhibition to all organisms Was noted where
Mandelic Reid and Selenite F media diffused into the 83 solid medium.

5. Mandelic acid broth inhibited many pathogens as well as
the non-pathogens from routine stool specimens.

6. There was little difference in the results in Knox, dell
and Pollack tetrathionate broth and nfiller's tetrathionate broth. both
produced more positive results than the other two media.

7. Selenite F medium incr ased the total number of positive
Specimens. However, it failed to give positive results with several
specimens that were positive with routine media'and the tetrathionate
broths. It has an advantage of giving more isolated colonies on 83

medium than are found with tetrathionate broths on 33 medium.

1.331.311! S

19

FLEJULaB

I“!

.L. .TRnTEiI‘ul .5323

To 180 ml sterile nutrient broth with 9 grans chalk was
added 20 ml sodium thiosulphate solution. (50 g sodium
thiosulphate in 100 ml water, sterilized by steaning)

4 ml Lugols solution (25 g. iodine, 20 g. potassium Iodide

in 100 ml water

KNOX, CELL AND POLLuuK_§ALALCSD TLTRATEIVLATE

To 80 ml broth containing 4% g. chalk was added 5.5 ml
thiosulphate (24.8g. Sodium thiosulfdte to 100 ml water)
and 5.5 ml Iodine solution (205. Potessiun Iodide and

12.7 g. Iodine to 100 ml water)

A ““r‘t '5‘ "'f“' Fr“! 1
Ulzou 0.04.4.1“; _

DI

w-vwfl

7

2.3 g. dehydrated product made to 100 ml was not sterilized

This media was very unstable even in the dehydrated state

Ingredients per liter: Sodium and Selenite 5 g.
Bacto-Tryptone 4 g.
Bacto-Lactose 4 g.
Lisodium Phosphate lO 3.

n w ;.._._.-.;;-V2-::.Ic ACID

 

3.3 g. dehydrated product made to 100 ml. This was not

sterilized. (Fornulde not known)

‘1“

IQI‘CELLLCno

COOper, K. E., hood, h., C swell, 3., Eliot, and Small: The
1942 Bacterium ParatyphOSLn b fron Faeces. Jour. of Path. and
Bact. Qfiz345-35

Gohdr, H. A.: Sodium Selenite as a bacteriostatic Substance and
1943 its use in the Isolation of Psratyphoid bacilli. Jour.
Trop. fled. and Hyg. 46. Lo. 3 29-32

Hohn, J. and Hermann 4.: Die jrreger der Typhus - Paratyphus

1940 Enteris Uruppe in Untersuchungs material in Lssen.
Anahrend der thr 1933-1939. Zentrbl. bkt. I abst.
Orig. 1453 4,5 2Q9-22o, Biol. antP. 11736, 1943

Holt, H. L., Vaughn, 4. C. T., :right, h. D.: epidemic of

1942 Paratyphoid fever in Liverpool. Luncet 133
Jones, D. J., Knox R., dell, P. G. H. Experiments with
1936 Brilliant Green - Losin Agur. Jour. of Path. and

Bact. 42: 45
Jones, D. J., L811, P. e. 5., Knox R. A water borne outbreak
1942 of Paratyphoid lever, ancet 1942, 362

Knox, R. T. The ejfect of Setrnthiondte on Bacteriul wreath,
1945 Brit. Jr. of 233213913 Perth. gt“; (3):l46-50

Knox, h., dell, a. e. H., and P011303, 3. h., Isolation of
1942 Intestine Peth05ens Conpwretive Study of Media.
Jour. of Path. and bact. 54, 463-433

Knox, h., Gell, P. d. H., and Pollack, n. h. Selective action of
1943 Tetrathionnte in bacteriological Media J. Hygiene 4§,(3):
147-158

Leifson, Einnr. Lew Selenite Enrichment Media for the Isolation of
1936 Paratyphoid (Salmonella) Bacilli am. Jour. of Hygiene 24, 423

Levine, Victor C. Enducing PrOperties and nicro-orgdnisns with
1925 special reference to Selinum compounds. Jr. tact. 19, 217

Massey, Kathleen n. Lotes on Tetrathiondte broth and mncConkey's
1943 agar media in the isolation of Salmonella Hnteritides
var duhlin from Bovine bdeces and nilk. Jr. Conp. Path.
and Therap.,2§ Lo. 2, 151. Jan. abstr. Bul. Hygiene 1g, 867

Hedical Research Council; A Brilliant drecn acid Buchsin median

1942 for isoletion of Salnonella. (Lonthly Bull. Energency
Pub. Health Lab. Service. March Kg, 26-5) Bull. Hygiene
‘13, 505

Medical Research Council; Growth of Typhoid Bacilli on Different

1942 Selective Media (Monthly bull. Emergency Puo. Health Lab.

Service. Sept. 7-d) Bull. Hygiene 13, 74

Hollov, Hollie; Uinter, Jeanette; Steinberg, Phillip: 33 agar

 

 

1942 for the isoL tion of inert ell~,3a1nonella and :31 ella
groups fron reces. Jour. Lab.nnd Clin. Med. 2:, 1021-1027

Pollock, l. B., linox, h., 0e11, P. 3. h. Bacterial reduction of
1942 tetrathionite. Hature 150 (3794): 94

Pollack H. b. The influence of temperature on a.a)tion of
1945 Tetro.t1:ionrse in ashed Suspensions of Parntyghosun B
Bri. Jour. of ixaer Path. 2‘ _(o ): 410-

 

g.
15

Pot, n. n. Difco SS A;r.r in D1 :nosis of Bacillcry DyS‘ntery,
1941 Bul. Hygiene 17:721-

Pot, A. , The isolation of Bacterium typhosun. Jour. Path. and
1913 dot. pg (1): 100-103

Buys, 2., Charlotte, H. D. The 13019 Ition ofI 'Irp1oid Paratyphoid and

1940 Dysentery Bacteria from Faeces andb I.e. Brit. med. Jour.
1940. Vol. 1, 606

Schafer, .. Bin enfaches V rfahrnen Zur Hexiuung, der Proteusuberwuck-
1944 erung in Stuhlkulteren. (Ztschr. f 323;. in Infektionskr.
125 L0. 6: 55 1-3) Public Health 21 (7):459

Schrieh, Jalter. Die Vernendun; von Saccharosesplatten Zur Isolierung
1940 pathogener Brreger ous den Stuhl. (Zentra ol Ba.:t. I not. orig.
143 (3/4) 213-219) 1:37 Biol. Abst.
ll
Steur J. and Schindler, E. Uber die Leistun sle r;s:eit Des Brilliant
1940 green Pr nolrot a;ar zur Typhus and Piratyphu us Diagnose.
Zentralbl. Baht. J. Abt. Orig. 146 (2):4J-53, Biol. abstr. 1942

Jynn, B. S. and Jillian, 0. B. Growth of ;bertnella tyuhosa and
1945 Aerobactcr aero;cnes in association in Tetrathionate Broth.
Jour. of Bact. 49:629-632

 

 

.rm

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