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TESTS FOR INCIPIENT PUTREFACTION
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THESIS
:1:

Submitted to the faculty of the Michigan State College
of Agriculture and Applied Sciences partial
fulfillment of’the requirements for the

degree of Doctor of Philosophy.

By

Ralph E; weaver
*

June 1926

THESlS

I34
24-5"

”"115

 

Contente
Introinction
Review of Literature
Part I. Study'of Organiene
Experimental Work
Iecletion
Study of Physiological Characterietiee
Bannery
Bert II. Ieete for Incipient Putrefectien
‘Experinentel work
Innei Teeth
Hydrogen Bulfid Teet
Hydrogen Baltic feet Compared with.lethy1en
Blue Reduction Tent
Sumnery
Acknowledgment

Bibliography

INTRODUCTION

In V10I of the fact that neat ie one o: the moat
important articlel in the diet of man it in evident that the
aarketing ehould be under etriot eupervieion in order that it
nay be in the beat poeaible aanitary condition when it reachee
the coneuner. Thie is particularly true when it ie ooneidered
that neat ie an ideal mediu- tor the Growth of nioroorgenieae.
lI'oxine and deoonpoaition produote formed by theee
nioroorganiane are undoubtedly uore or leee harmful to the
human being. ror theee reaeone it ie inportant that lone rapid
teet be available, which can be applied to determine whether or
not neat hae been handled under eanitary oonditione and to
deternine whether the meat ie etrictly freeh or whether it ia in
the beginning etagee of putrefaction. It ie the purpoee of thie
work to etudy eone or the teete which can and have been applied
to neat and to determine, if poeeible, one to neet the above

requiremente. ~
Review of Literature

In thie diecueeion it will be inpoeeible to review
all or the papere which have been publiehed pertaining to the
eubieot because the entire tielde of protein deco-poaition and
nitrogen netabolien or uicroorganiene are concerned. The nore
important papere dealing directly with neat and a few or thoee
concerned with the other two i'ielde will be dieoueeed under the

following heade;

 

-2-

a. Bacterial count. A linited number of workere have
atteapted to apply the plate count to the inveetigation of the
eanitary condition of neat. Harxer (I), 1903, euggeeted a
etandard of 1,000,000 bacteria per gran for the purpoee of
condo-nation. Hie etudiee were all nade on hanburger eteak.

leineirl and llewtcn (2-3), 1914, publiehed two
papere giving reporte of the bacteriological etudy of hanburger
eteak. They found that the Itandard of 1,000,000 per gra-
eetabliehed by lerxer wee toe low, and advanced 10,000,000 ea
being a fairer liait. i'hey ocnpared the bacterial count with
Iber'e (a) a-cnia teat and with the organoleptic teat. In
their eonclueion appeare the following etatenent, "i‘here ie no
cloee egreeaent between the nunber of bacteria preeent and the
f degree of ”011m ae Judged by thie teat.” The reference 1'. to
the organeleptie teet.

Garey (5), 1916, etudied 34 eenplee of eaueage. 0f
theee 31 cane within the lilit of 10,000,000 bacteria per gran.
. Hie counte varied widely and did not oorreepond cloeely to the
eanitary conditicne of the ehope. He concluded that the bacterial
count wee very inadequate and wee of little eignifioence.

In 1917 Le l‘evre (6) purchaeed 22 eanplee of
hamburger eteak fro: the earkete at Waehington mo. and etudied
then bectericlcgicelly. Hie reeulte checked cloeely with thoee
of .Ieineirl and newton, except for the fact that he found the
etandard eet by Ierxer to be preferable.

In the etudy of "fecdy eel-on" Hunter (7), 1920-22,
eaid, "in attempt to correlate the total count with the phyeioel

condition of the eel-en feile." He thought that a count of

-3-

16,000,000 per gran wee eufficient to explain deocmpceition,
bat placee the greeteet eephaeie on the etudy of the typee of
niorcergenieae preeent.

Hoffetadt (8-9), in 1924, ueing ground beef
purchaeed fro- publio eerkete, attempted te eeeooiate the
bacterial count with the eanitary ccnditicne of the ehepe and
with the crganoleptic teat. In her ocnolueicne ehe etated, "i'he
total aerobic bacterial count or count of dextreee fernentere
ie unreliable ae a etandard for neat analyeie', and "fhere wee
no relation between initial counte and counte in eubeequent
epcilege of eemplee."

Brewer (10), 1925, nude counte on a variety of
different neate. He found that the counte from different
eemplee of the cane piece of neat varied acre than the counte
from different phone of neat.

In unpubliehed work by the writer (111.1983,
bacterial counte were none on eanplee of huburger eteak ueing

the plate, the direct eioroeocpic, end the haeeceytcmeter
eethode. 0f theee the plate aethod wee found to be the beet,
ae an indicator of the condition of the neat. None of then
wee found to be of euffioient value to warrant extended uee.

b. chemical Teete. lumercue workere have atteapted to
apply oheaical teete for cone of the intermediate or end
producte of the deccnpoeition of proteine, to the detection of

the firet etagee of decoapeeiticn or putrefaotion.
Ottolegnhi ( 13),1913, found that the determination

.4-

of aaino acide wee valuable ae an index of the beginning of
active deoompoeition. the method ueed wee the Soreneen (13)
formol titretionlmethcd. Itanc (it), 1916, in a etudy of the
proteolytic activity or My; eubfllgg, need the eene
method with good reeulte. Bokman (16),' in 1918, aade uee of
the method in the examination of fieh, with equally good
reeulte. He rethdcd that the name teat be need for meate.
Van brie (16) applied it to eaueagee and cured meate with fair
reeulte. Riffort (17). in 1932, need the "ninhydrin" color
reaction and advocated the eetimeticn of aninc acide to be of
value. he Bord (18) concluded that, ”The amino nitrogen found
in bacterial culturee may be taken ae an approximate index of
proteolyeie under certain conditione." Hoagland and hie
aeecoiatee (19), in work publiehed in 1917, etated that the
inoreaee in nine nitrogen afforded the beat meeeure of the
extent of autclyeie having taken place in cold etored meate.
0n the other hand Scare (80), in 1916, inveetigated
the amino acid and ammonia producing abilitiee of a number of
organieme ueing peptone and meat peptone eclutione ae media.
He found that neither alone wee a good index of deoompoeition
becauee it wee poeeible to get an accumulation of amino acide
with a very alight production of ammonia, or on the other hand,
a deoompoeition of the amino acide ae faet ae formed with a
eubeequent high concentration of ammonia. atrohecker (81), in
1980, etated that the determination of aaino acide by the
"ninhydrin' reaction wee very uneatiefaotory. He found that
oxygen-coneumed teete, nitrate reduction teete and methylen

blue reduction teete were much more applicable. Wakemen and

Lcmanite (22),l925, made an extended etudy of the chemietry

-5-

of the deoompoeition of amino acide and of proteine. With
regard to amino ocmponnde they eaid, "the determination of
amino nitrogen an an index of deoompoeition of proteine ehould
be need, however, only‘ when the particular proceeeee carried
on by the different organiene are properly underetccd....fhe
amine compounde are therefore formed and deco-pond; they will
accumulate in the medium only if the particular crgeniem ie
unable to nee theee compounde or the larger part of then,
formed from the protein; they will dieappear only when the
organiem attacko then an readily an it dcee the protein or
when another organiem ie preeent that attacko theee ccnpcunde
ae econ no they are formed by the other crganieme from the
protein molecule.”

mania teete, generally epeaking, have met with
more approval than the nine acid teete becauee ammonia in an
end product while amino acide are intermediate prodnote. Eber
(d) propoeed a qualitative teet for ammonia in 1893. He claimed
thie teet to be .a good criterion of the point of beginning
, pntrefection. The teat proved worthlece becauee it wee not
delicate enough. lent dechOped eufficient odor at about the
came time or before the ammonia teet wee poeitive, to condom
it without the nee of any artificial teat. It proved to be an
indicator of advanced, rather than one of beginning or incipient
putrefacticn. *

Biohardeon (23), in the chapter on "Meet and neat
producte" in Allen'e commercial Organic Analyeie eaid, "Nitrogen
in ammonium ealte, together with other eubetencee eaeily

deconpoeed by neane of weak alkalie probably afforde the boot
available chemical nethode at the preeent time for the detection

-6-

of deoompoeition in fleeh foode."

Palk, Baumann and lloGnire (84), 1919, made
determinatione of total nitrogen, non-protein nitrogen, ammonia
nitrogen, total creatinine nitrogen and purine nitrogen, to
ace (if any of then would be indicative of meat epcilage. i'heir
reeulte ehowed ammonia nitrogen to be the only one which
coneietently increaeed with epoilage. In a continuation of thie
work Bulk and lchuire (85) found that in order to interprete
the reeulte it wee neoeeeery to know the hietory of the eanple
examined. i'hey diecovered that the amount of anemia preeent
in a given eanple of neat wee much greater at the time of
epoilage if the meat had been kept at a low temperature than if
it had been kept at a higher temperature. Ernie difference they
attributed to autclyeie.

Among othere who have found the a-onia teet
valuable are Van Brie (16) and Kendall and walker (26).

Scare (80), an etated above, diecovered that both
the amino acid and the ammonia teete not be run on the cane
eanple to be of any value. DeBord (18) concluded that, "The
ammonia found in bacterial oulturee ie not a reliable index of
bacterial proteclyeie." wakeman and Lcmanite (82) found that
the ammonia teet wae the moat valuable of the chemical methode;
however, they aleo etated that, "Annie accumulation can serve
an a good index of proteclyeie only when no available carbohyd-
ratee are preeent. In the preeenoe of the latter thie method
ehould be eupplenented by the etudy of another proceee, either
the dieappearance of the original protein, or the formation of
amino nitrogen.... rho term ie only relative and ehould not be

made tonean any more than it doee.” fhie eparing action of

-7-

oarbohydratee hae been demonetrated by numerone other
inveetigatore. Since oarbohydratee are preeent in the ordinary
eanple of meet it can only be concluded that the ammonia tent
in comparatively unreliable.

lber (27), in addition to hie ammonia teat, worked
on a hydrogen eulfid teat intended to give einilar reeulte.
In thie teat be partially decompoeed the meat with dilute
eulfnric acid and meeeured the amount of hydrogen eulfid given
off by neane of a etrip of filter paper ecaked in lead nitrate
eeluticn. Eber died before the completion of hie work and the
teat wae never applied enough to determine ite value. a method
wee elaborated by renere, Bhoetrom, and clerk (as), in 1934,
for the determination of hydrogen eulfid in bacterial oultnree
and in certain canned foode. they found that in general the
non-proteclytic typee of bacteria including the cocci, aerobic
eporing rode, yeaete and inactive water and coil bacteria fail
to liberate hydrogen eulfid from proteine. Under the conditione

of the experiment , no hydrogen eulfid wee liberated from

decompoeed ealnon er ehrimp. . I

eleugh m), 1922, made a etudy of the formation
of 1...: and ekatel during the deoompoeition of ealnon. He
concluded that, "Although the determination of indel cannot
eupplant odor and phyeioel appearance in the examination of
canned ealnon, it ie nevertheleee of value and afforde coneid-
arable information ac to the previoue hietory of the eanple."

do a generalisation, it may be etated that attempte
to apply chemical methede for the determination of incipient

‘putrefeoticn have met with moderate succeed. dome of’theee

b '.-“$~2'(:w P.»

 

-8-

methode are of value but good.reeulte are limited by ac many
factore that their nee ae einple, practical teete ie queetionable.
.lany of them indicate advanced rather than incipient putrefaction.
Home of then indicate greee contamination of the meet by

careleee handling, unleee enfficient tine hae elapeed after
contamination to allow active patrefaction to eat to.

c. Biological feete. Certain German inreetigatore have
attempted to apply biological teete baeed on the number and
kinda of microorganieme preeent, me well an on the producte of
their activity. The firet report wee publiehed by fillmane and
Hildner (50) in 1916, the eeoond by Strohecker (81) in 1980,
and the third by fillmane, atrohecker and Bohmtee (31) in 1981.
fhey found three teete which were of value in chewing the firet
etagee of deoompoeition. fheee were the oxygen-ccneumed teet,
the nitrate reduction tent, and the methylen blue reduction
teat. from data publiehed it would appear that theee teete are
worthy of further inveetigation. They are baeed however, on the
number and activity of the common aerobic organieme preeent,
rather than on the number and activity of the epecifio organiene
reeponeible for the greater part of putrefnotion. rhne it would

appear that they muet be enbiect to many of the name criticieme
which have been applied to the bacterial count.

Brauer (32) found that incipient deccnpoeition in
eaueagee, preserved foode, etc. could be detected by the
inoculation of glnocee fermentation tubee with a emall portion
of the eanple and noteing whether gee wae formed or not. fhie

ie in oppoeition to the etatement of’Hoffetadt (8) that the

count of dextrcee fennentere ie unreliable an a etandard for

-9 ..
meet analyeie.

d. Orgauieme Concerned in Pntrefaotion. Humeroua
inveetigatore have aecribed the phenomenon of putrefacticn to
aerobic organieme. Chief among theee aerobic organieme ie
Bacillue (Proton!) vulggie. Hoot later inveetigatore have been
unable to find any aerobic crganiem capable of canning
putrefacticn. Bienetock (as) attributed it to Bacillue pgtrificue.
He defined true putrefacticn co the bacterial deoompoeition of
albuminoue matter accompanied by the formation and elimination
of foul emelling eubetancee.

' fieeier (34) in a recent review of the eanect,
baeed on twenty yeare work by ficeier and Hartelly, found that
the main role in putrefacticn wae played by anaerobee but that
certain aerobee prepared the way for them and deetroyed the
intermediate producte of deoompoeition remaining after their
action.

Rettger (38) hae ehown repeatedly that only
anaerobee can initiate putrefacticn. He dietinguiehee between
digeetion or deoompoeition ae oaueod by the aerobee, like
gagillng,(zzntnnn) rule-hie and ngillne enbtilie, and true
putrefacticn. The former ie very much elower and in not
accompanied by foul odore. He haa found that hydrogen eulfid in
formed early in putrefacticn ae canned by the anaerobee. 0n the
other hand he hae found little or no indel, ekatcl or phenol
formed. fghe rate of putrefacticn, according to hie work, depende
eonewhat upon the aerobee aeecoiated with the proteclytic
anaerobee. -

Hunter (7, 35-36) found that the deoompoeition of

-10-

ealnon wee due chiefly to the organieme deeoribed in the
literature ae water, eewage and coil organiema. He added however
that, l".l'he bacteria reeponeible for the deoompoeition of the

fich are not, in the etrict eenee, putrefactive and there in no
rapid digeetion of the fleeh of the fieh in the culturee.....
fhie makee it apparent that the fleeh of fich ie more eneceptiblc
to deoompoeition by ordinary water bacteria than the fleeh of
higher animale."le found that no contamination with.anaerobee
took place during the handling of fieh.

Hoffetadt (8-9) concluded that epoilage of ground
beef wee due to the anaerobee preeent. She icclated three typee
of anaerobee; proteclytic, medially proteclytic and eaccherolytio
and found that the type of epoilage wee due to the type of an-
aerobe preeent. She etated that, "The preeenoe of proteclytic
anaerobee indicatee a definite way by which the keeping qualities
of meat can be predicted."

weineirl (39), 1924, confirmed the work of
Bienetook, Hettger and othere and ehowed that anaerobee do
canoe putrefacticn. He proved, by the nee of a modified
Weineirl Anaerobic Spore teat (40), that theee anaerobee ere
preeent. In hie oonclneione he euggeeted the deeireability of
a teat beecd on the anaerobee preeent; "Aerobic and facultative
bacteria eeeiet in the epoilage of meat, but the putrefactive
odor ie mainly due to anaerobee. The aerobic bacterial count
in, therefore, only indirectly an indicator of pntrefaction....
a teet for anaerobee in meat, comparable to the aggillgg‘ggli
teat in water enalyeie, appeare highly deeireable."

Ob.

 

Part I.

A Study of Organieme Ieclated from Preeh Hamburger
Steak and from Hamburger Steak in the firet Stagee
of Putrefact ion.

Experimental Data

In thie inveetigation it eeened expedient to attempt
to baee a teat for incipient putrefacticn of meat upon the
reeulte of the phyeiological activity of the organieme preeent.
In order to do thie it wee neceeeary to know eomething of the
type of flora in the meat at thie etage. d eeriee of organieme
ieoleted from comparatively freeh meat and another ieoleted
from meat in the early etagee of putrefacticn were etudied in
order to note what typee developed during the eo-called etage
of incipient putrefact ion. Only aerobee were included in thie
etudy.

Hamburger eteak wee choeen for theee inveetigatione
becauee it wee eaeily obtained and worked with and becauee it ie
one of the neat producte which in moat frequently groeely
contaminated and one which neede clcee enpervieion. for the firet
eeriee of organieme a eanple wee collected from a local market.
fhie eanple wee ground in the preeenoe of the inveetigator and
wae made of meat which appeared comparatively good and freeh.
The eanple wee plated immediately on etandard meat infueion
agar. The organieme were freed from the meet by grinding in
eterile eand, according to the method of weineirl and Newton (3).

-12-

4 count of 3,000,000 bacteria per gram wee obtained. Two platen
of the 1 to 100,000 dilution were eelected which contained 87
and 44 coloniee reepectively. Traneplante were made from all of

the coloniee on theee pletee. Due to the fact that many of the
coloniee were very email and aloe, undoubtedly, to the fact that
many of them would not grow readily on ordinary media, only 61
culturee were obtained. lo attempte were made to eliminate
duplicatee becauee the main reenlt to be obtained wae the
proportion of the different 'phyeiolcgical typee preeent.

The eeoond eeriee of organieme wee ieoleted from a
eanple of hamburger eteak purchaeed from the ammo market, under
me nearly identical conditione ae poeeible. The eanple wee etored
at 80' for a period of two daye and wee then plated according
to the eame method. a moderately putrid odor wee given off by the
meat at the time of examination. a count of 178,000,000 wae
obtained. dttente were made to traneplant 77 coloniee, which
repreeented one half of theme preeent on a 1 to 1,000,000
dilution. Tron theme on culturee were obtained.

' 4 microecopic examination wae made of each culture
in order to claeeify them according to morphelogic groupe. The
reaulte, together with their phyeiological characterietioe,

are recorded in Table I and Table II. Only theee characterietioe
were included in the etudy which have been or which might eaeily
be applied ae an indication of the preeenoe and activity of the
groupe concerned.

Hydrogen eulfid determinatione were made by
euepending etripe of filter paper ecaked in lead acetate eolution
along wide the cotton plnge. Standard infueion broth wee need

becauee it wae noted that it wee more efficient than ordinary

-13-

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rooordod so portiol roootions. fho nuobor o1 doys nooossory for
tho production or hydrogon.sultid, sud of soid and goo tron
gluooso woo rooordod. This givos sn.indiostion of tho
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protons-liko rods, 1 produood distinot inrolution for-s cod 1
produood o fluorosoont oolonyt at tho 68 cultures fro-usost in
tho bogining stsgos or putroiootion only 13 woro ooooi. This is
o poroontsgo or 81 so oosporod with 88 frou.tho frosh.noot.
13 woro dosoribod so short rods, 1 so s.sporo tor-or, 8 ss lsrgo

rods without sporos Ind 32 so protons-liho rods, @515 would "

indiosto‘thot thoro is on inorssso in tho proportion of tho

(.3

F‘,

.7".

"-22-

nu-bor of rods to tho nunbor of ooooi during tho poriod of tho
boginning or putrofootion..i group of organisms dosoribod os
protons-liko rods, including all ohort rods with oooosionol
filooontous torus, oppoors to bo tho group which inoroosos most
rspidly. Only o for of thoso giro tho oonploto rooctions or '
thosillni.(Iz2isnsitznlsoziso

Poroontsgos of tho organisms Iron.oooh sorios which
goyo positivo results with tho vsrious physiological tosts hsvo
boon oomputod ond oro rocordod in Toblo 1119

Table III.
Physiologiosl Roootions of tho Organisms Isolotod
tron Prosh ond from Putrofying loot. A Sunory of
Toblos I ond II.

Orgonisns from fresh Orgsnisms from putro-

-Honbur or Stout tying Hamburgor Stout
" posit ivo % positivo
'fi;5§3§3£'§£iiii'"""""""'Ei""'"""""§i"""""
'ZESSSII'""""""'"'"""'5Z'"""""""85"""""
5i$ii’i§3£'éi£§3£§" """ ""'"55""""'"""S§"""""
'éii'i;3;'6i£5333"""""""'i2""'"""""ii"""""
7i3€£§i;£'£i;;5i.i£$3£"""""Ei’""""""“;5"“"""
’SI§;;£33'ESEJJJE'"""""""55"""""""'35"""""
';;;;;""'"""""""“"""a""'""“"";a"""""'

Two groups or orgonions sppoor in tho sorioo tron
putrofying'lost which do not oppoor, ot loust to any notiooublo
oxtont in thot fron.frosh moot. Thoso sro tho groups dosoribod

 

.- A.
- c—

H - . 1' ,H ,-
r
.- .- — o- »- v - - -
. - H - H H _ H
H .H . , 7, ,
- - .. .. .. H. . - .
Ha- - — H H ‘ - H
H H ~ .— ,- -. a H .
_. ,o , . -. V ,
- H «A r. — H o H -
o

- .-
O
- H - ‘
-- c-
- Hi - 4.
,H H .H ,

-- H - o H- - H- .— H—
- — - — ¢ - u- '— uv H
_ H — H- , - H . H. H

 

, . H - H - H H H -

. .
.H - H o. H- H . o
H. H. H — a H H. .H ,H H. .
H. .— »« H H. — H - - .H (I-

.

H- H - — co - on! .4 H
<0 — as H- - 4 H

H

 

-23-.

as short rods, of which thoro woro l3, ond us protons-liko
organisms, of which thoro woro 32, in a total of 62 oulturos.
rho charsctoristics of thoso groups aro rocordod in Toblo IV.

TOblB IV.

Short rods Protons-liko organisms
% positivo 7% positivo

Hydrogon sultid 98 100
'TZiiSEI;""""'"""""""'ZE""""'""'55"""""
'71333'E:3§'5i£5332'""""'""3£ """"" "’i56 """" "'
"é£;'§;;;'5i33333""’ """" ""5' """"""" i6"""""
"i;§£;i;;’£i£3'§25£535""5""'ES""""""”3§"""""
'7§I€;;£3;5§Si;525"' """" ""'E£"""""""E;"”"""
"i555i"""""""""""""i5'""""""'ZZ""'""'

It is or particular noto that a largor porcontsgo
of tho crganimms isolotod from.trosh hamburgor stoak roduood
‘mothylon.bluo, nitrotos, and producod ammonia, than of tho
organismo isclstod from.thot in tho process or putrotoction.
.dotion on glucoso soomod to bo common to both groups. I! thoso
roots bo acooptod so conclusivo thon any tost bosod on thoso
charactoristios doponds ontiroly upon tho incrosso in numboro
of organisms found in tho moot and not upon.spooi£icity. This
loans that any such.tost would bo lioblo to most of tho
criticisms of tho bootorisl count and would probably bo ot’no

moro vsluo.
no organimms in tho first sorios produced indcl.

In tho sooond 28% producod it. Oomporsblo with this is tho

H-

- .— H. _
H— a- -
H- H. .H
H— - H _

-I—DO-Io‘

-. H. — Hv .-
.H H- H. H
— H H— H— —

.—
H— _ -H a» — H.
.- o-H '— c-H- H. - _ .-
H— .H - _ . _

H- - w —- — .H ' -

 

w— — — -
_ .. H- .
.H a. - -
~ — H— sl-

.-
— H
.1

.-
H- H .H 7
H. p _ - .— — — - .- - -
- — .- — - .- - — —- ' ‘1
’ - H- .H - _ .- — .— — —
I
. - .H — — H- - - — —
n
A .. _ _. .— — .1 c— _ _ —

.H—HH--,-_—__HH—

—_——¢—

--—-—-—

”—3--—

~i'_

—p.‘-.—

0-.--—

 

-24.

increeee in hydrogen eulfid producere. fhie increaee ie only
pertielly ehown by the percentege of 81 ee coepered to 51.

loet of the hydrogen eulfid producing beoterie found in the
firet eeriee were coccue forne, producing a eufficient enount
to be obeerved firet, et the end of two to six deye. In the
eeoond eeriee noet of the hydrogen eulfid producere were rode,
producing it ebundently in one dey. Thie would indicete thet
eny teet beeed on hydrogen eulfid production ehould ehow e euoh
nore ebundent evolution of thie gee during the etegee where
putrefeotion ie commencing, It ie worthy of note thet 92% of the
ehort rode end 100% of the proteue-like orgenieee produced
hydrogen eulfid.

Su-ery

Of 51 orgenieee ieoleted fro- freeh heeburger eteek
88% were coccue foree . 0f 63 ieoleted froe einiler eeet in the
etegee of beginning putrefection only 21% were cocci. Thie
increeee in red foree wee due to the eppeerenoe of two groupe
of becterie not found in the freeh eeet.13 were deeoribed ee
ehcrt rode end 38 ee proteue-like orgenine.

Deepering the two eeriee of culturee, 'it wee noted
thet e lerger percentege of the orgenieee from freeh eeet
produced monie end reduced methylen blue end nitretee, then of
the orgeniene froe putrefying meet. There wee no epprcieble
difference in the ection of the two greupe on glucoee.

Judging free theee reeulte there ie e decided
increeee in the umber of indol end hydrogen eulfid producere
during the etege of incipient putrefection of meet. It wee not

detereined whether thie would hold true with meat stored at e

-45-

different “temperature ‘tlw .140".

P." IIe

Hydrogen Bulfid feete for the Detection of Incipient
Putrefection of fleet.

In.Pert I it wee noted thet indol producing
orgenieee eede their eppeerence during the etegee in which
putrefecticn wee commencing..dleng with thie wee e very eerhed
increeee in the nuebere which ectively evolve hydrogen eulfid.
dn.etteept hee been nede to develope e teet or teete for the
etege of incipient putrefhcticn end for the eeount of effective
contenineticn, beeed on the preeenoe of theee groupe of orgenieue.
By the eeount of effective ccnteeineticn ie meet the eecunt of
ccnteeinetion.with thoee orgenieee which will be ective in
decoepceing, or ecre properly, putrefying the meet. In other
worde it eeeeed deeireeble to heve e teet which would predict
the length of tine during which e given.eeet product would be in
condition for coneueption. Eeeburger eteek wee egein.choeen for
the inveetigetione.

ror'the detection of the preeenoe end ectivity of
indol producing crgeniene the cotton wool plug teet for indol ee
developed by Gore (41) wee ueed. Thie ie elnodificetion of
Ehrlich'e method in which the reegente ere pleced on the cotton
plug end the indol voletilieed by heating the culture in e weter
beth. It ie e very eetiefectcry method where the color of the
medium would meek the color of the reecticn. One gran eenplee
were weighed into tubee conteining 10 cc of meet infueion broth

end the tubee incubeted et 37’. These tebee were removed free

-27-

the incubator and teeted for indol at hourly intervele. It wee
found that regardleee of whether the hamburger eteak wee putrid
or freeh, the time of the first appearance of indol was between
12 end 24 houre. The amount of indol formed with putrid meet

did not appear to be greatly in excewe of the amount formed with
comparatively fresh meet..ie these preliminary teete did not
appear to give favorable resulte the method wae not investigated
further.

A eimider method has been developed to test for the
presence end activity of hydrogen eulfid producing organieme. One
gram portions of hamburger eteak ere weighed into tubee containing
exactly 10 cc of etandard infueion broth, adJueted to ph 7.
Stripe of filter paper ecaked in lend acetate eolution ere
euepended beeide the plug ee indicatcre of the production of

hydrogen eulfid. The tubee are incubated at 37' end hourly
obeerveticne made for blackening of the etripe of lead acetate
paper. The time of the first appearance of blackening ie ueed
as the index.

Semplee were collected from various meat markete
and tested by this method. Duplicate tubes were run aerobically
end elec in the partial vacuum induced in e Rcvy Jar by a water
pmnp. Before the completion of the experiemnte a vacuum pump
wee purchased. The pump was found to produce almost identically
the same degree of vacuum as the water pump used with the firet
eanplee. In the first experiments the samples were divided and
'etored in deep culture dishes at 20° and at ice box temperature

which varied around 5'. They were tested at intervals of from

one to three days and particular attention was payed to the
results at the time when the odor of putrefacticn was first
noticeable.

Tables V and VI give the results obtained from the
examination of two samples. The sample recorded in Table V was
purchased from.market.d. The meat from which it was obtained had
been ground and stored in a pen in the refrigerator at the market.
It wee frozen solid at the time it was bought. It was not tested
until the day following the one on which it wee collected. It
appeared to be in good condition and free from excessive
contamination. Table VI gives the results of a sample from
market 3. All hamburger steak from this market was ground at the
time of selling..dnaerobic spore tests were made on both samples
according to the method of Weinsirl (40). Positive results were
obtained.

Sample 3 (Table V) after 24 hours incubation at 5°
contained enough active hydrogen eulfid producers to give a
positive test in 8 hours both aerobically and anaerobically.

.dt the end of nine days when a putrid odor was first noticed

a poeitive'test was obtained in 10 hours aerobically and in 5

hours anaerobically..dfter storing at 20° for one day the odor

was described as very sour and there was a very slight indication
of beginning putrefacticn..d positive test was obtained in 6 hours
aerobically and in 5 hours anaerobically. At the end of two

days, when the meat was decidedly putrid, a positive test was
obtained in 3 hours aerobically and in 2 hours anaerobically.

Sample 0 (Table VI), on immediate testing gave a
positive result aerobically in 8 hours and anaerobically in

10 hours. After one day at 20‘ it gave off a slightly putrid

-29-

Tflbl. Ve

Hydrogen Sulfid Production by Organisms from Hamburger

Steak. Sample B. Recorded in Hours Necessary to Obtain
a.Poeitive Test. Incubated 37'.

0-9'---§---COO-Oocoooouccococfiocuu ----------------------- .

Age u Stored at 5‘ e Stored at 20° :

1n 'CO---.-*-.-.--.*.-...---- ooooooo i ...... fl oooooooo 0-. .

Days :aerobicganaerob{ condition aerobic anaer.;conditicn ;

Q. .QC‘OOOCOOC‘CCCOC ...... OO-oafinocuuoofiuunoucoq-vocccoouooq
: not : run. .good . not : run : good :

.- -CO}.------+---.D-OJOOO--OCCOJ-O-CQO-J-OOOOOOJ cccccccc «mo-d.

u 8 a 8 : good : 6 3 5 : ecur .

vrvr-----'-v---v---v-v------- -----'-4-------{-----------4

do On NI 0-H 0|

r--‘--v--'--‘--fi---'-----‘

: 7 g 1 : good : 3 g 8 : putrid :

t 3 n 8 . fair , : g :

-- .-.}--.--..*---C---1.--------J. ..... --"--.-.--J-----------‘.
: 8 : 6 I sour 0 I I ' n

9 , 10 , 6 ,put. odor: : g :

Occuuobbaco-o-‘QO-----§------¢--’--co---,u-o---u,.o-oo--o----

Table VI.

Hydrogen Sulfid Production by Organisms from.Hamburger
Steak. Sample 0. Recorded in Hours leoeseary to Obtain
a Positive Test. Incubated 37‘.

O-----'----- ---------------- c ...... c ------------------- ---fi .

Age : Stored at 5‘ ' Stored at 20' 0

Days :aerobiczanaer. Jonditicn .aercbic.aneer. dondition :

O ' 8 - 10 : good : 8 : lO : good i

------:r-------',-- ..... '---------'------- C-----.*----------
1 . : 3 good : 6 g 6 put. odor :

convucr-OUC-C-tocucuwe-t-Do---nus-:occunun'C-UOCOO' ..... uuucu‘
e . s . 5 .put. odor. : 3 ' 2

...... L.CC----t---.---‘---------‘-O-----‘-------‘----------‘

 

—D.ller‘

"ta—”—

‘0.-

”

—--

‘el-l‘ “-

odor and the hydrogen eulfid test was positive in 6 hours

aerobically and in 5 hours anaerobically..After 5 days at 5°

the "‘E.E§9 reached a stage comparable to that reached in one
day at 20'. A positive test was obtained aerobically in 8 hours
and anaerobically in 6 hours.

Aerobic hydrogen eulfid producing organisms do not
appear to develop« at 5'. The test completed under aerobic con-
ditions seems to give comparable results with that run under
anaerobic conditions where the meat has been stored at the higher

temperature. One sample gave a positive test in 10 hours
anaerobically and the other in 8 hours, at the time of first
testing. It is worthy of note that all examinations made at the
time of the first orgencleptic indications of putrefacticn
showed a production of hydrogen.sulfid in 5 hours anaerobically.
This was independent of the temperature ofstorege and seemed
to indicate that the anaerobee which produce hydrogen eulfid
were the organisms chiefly concerned in active putrefacticn.
Deter experiments show similar results. This agrees with the
findings of recent investigators concerning the causal
organieme found in true putrefactimn.

Table VII gives the results obtained by examining
36 other samples of hamburger steak, using the same test..dll of
these samples were tested immediately after collection and again
at intervals of from one to three days until? the meat was shown
to be putrid according to crganoleptic tests. In all cases the
meat was stored in deep culture dishes at approximately 5'. no
covers were placed on the dishes and an attempt was made to

approximate the conditions under which it would be stored in

-31-

Table VIIe
Hydrogen Sulfid Production by Organisms from
. Hamburger Steak. Recorded in Hours Necessary to

Obtain a Positive Test. Incubated 37‘.

3 I
"t time °f “1371318 until odor ears
[arket }«u-n-r-o --------------- 322-1 Condition

1
I
3
percbic Ieneero : aerobic :anaer. I 3
'3
I

: .1 g s g s : 10 g 5 ; good

‘C.----.--------‘------*--------h--O-----‘-- ......... . oooooo q
3 B 3 s 3 lo 3 a : 5 : good :
*COQCOCFOCC-C-C-{-OQ-OQJ-C-------%------CCJ'-------.-‘--------d'
I B : '7 I 5 I 7 I 4 I fair I

I

I

: ‘ g 7 g a g .. g - :bad-cdor as bought'

‘Dav-noficoncououOuaoucnd-uCO-.-CQhO-QOCOUOQOQon-cccuucooccucoq
3 s 3 12 3 9 3 a 3 6 3 800d 3
*‘O..--*--..----*-C---CJ-D-CQCCQQBOCO0.0--J..-.---.--Q--. ..... a].
g d g 5 I 4 I - : - :bad-cdcr as boughtI
.'C-coo-rocouwe-0-I-oocwe-1-a-----curse-nee---o‘coouououocouccox--fi
. B , 8 , 8 , '7 6 , good :

I
‘COOC-..-------.‘C-.---‘---------L----.---‘---O-.COC-O-C-----q
i D 3- 5 3 4 3 5 3 8 3 poor 3

nun--- coo-o--- C-CCO- sec-no---- onus-es-- coco-ounoqcoouucss-J
I 3 3 a 3 8 3 7 3 4 3 fair 3

:r:7:7:7;2; poor :

fi--canofi-nccoo-c‘OCOQCCflOOCCCoacofi-o-Q----,----.-Q-------UDID-q
3 15 - 3 13 3 7 3 6 3 3004 3

5 I e 3 5 3 5 3 poor 3

""""7""" """"’P“""" """"""'°""*

8 g ‘7 g '7 g 5 : good :

.-------.------*--------*--‘-----’---C-------C-----.
3 3 2 3 - 3 - 3bad-cdor as bought 3

--------‘------J ...... --+.-------‘---------- -------- a
I 4 I I 8 3 poor 3

: 7 : : 4 : 6°°4 '

.-------:------‘--------T-C------"-.---------------q
-c------‘------J---------L---Dunc-‘C-uc-oc-ucuunocucud

I 2 I 3 - 3bad-odcr as bought' .

--------I------I--------++--------I---------~--------I

: 6 : nogt run : good :

-------0.0-----d--u-----*~-------'---------con-n---u‘
3 5 3 mil: run 3 poor :

I 6 I I 3 3 good 3

IUI

I

I

I
"C‘---

3
IN!
I
I

--+-3-3--3-3-3-s3-

WI

p— we
I
~— .-

a-—a-o-p-—

.-

\—

_ ..;.-

I
I
e

I
I
i
I
I

.-

-L-
3

 

 

Table VII. Gout.
Hydrogen fillfid Production by Organisms from
Hamburger Steak. Recorded ,in Hours Necessary to

Obtain a Positive Test. Inoubated 37’.

."*""I""""‘"""" ufiei'iidiifié'l"z""""""""’"

t time of buying
Isrket 3.--.-----------JEEHfifiEflBBSE? condition

3 3aerobic3 anaer. 3aerobic 3anaer. I
3------3-------3--------3--------3--------3------------------

3A3 353 I33 good

.D-----p-C-----*--.--.--'O--DQOCC‘Q-CO--O.’------------.-----fi

In; 353 353 fair 3

‘-C---Of-‘O----J-C-CCCCO‘--.-----‘.C.‘O-.-:-------------OOD-O‘
d I 3 d 3 3 - 3bad-cdcr as bought3

I I 5 3 I - Ibad-odor as bought 3

I I 2 I I #- 3bad-cdcr as bought I

.- -‘ooo.-Q-J-ouuococ‘c.OCD.--‘----¢--o.----no-----...-.--d
3 7 I 3 d 3 good

3 7 3 3 5 3 good 3

sees-ownsoJ-O-ccuaso‘ooq-I-o-o‘-.-o-u-nb-Qco-uasocuq-cacao-Ia...-
3 a 3 3 6 3 500d 3

I I 3 I I - » Ibad-odor as bought3

I I I I I
I I I I I
I I I I I
I I
I I
I I

-.-

I I
IUI

. .QC'..-..--‘-----.-C'--------‘.------COr---- ....... .O....
3 3 8 3 3 . 3bad-odcr as bought I
D.coo-‘roconc‘ocooocqo‘.ooouqvw-‘ww-c-uoocfioucooqoooc9C.-.q-oq

3 w: 3'i .2
I I? I mctrun3 good I

00"---o..-;-.-..9-0"COCO-OQ;CQOCOOQQ'.--caccCCQQQQCCO-n

3 3 7 3 not run 3 good 3

.C-‘CCOO-UO‘COOCQQ--‘.--Q..OQ‘O0--C.--.---------.--------q
3 3 4 3 3 - 3bad-odcr as bought, '

wIP

v .- 4
.- — — a .I
.— .- - g
—- .—
—— ._ v _.
_ 7— .—
— o - _
.- —« — .—
- _ 4'
_ .- - _.

.‘F’—

I W —
I
. .- .—
3
- _, _
A
I
a...‘ _ .-
5. .-
—. '- -
_l _. ._

 

 

-35-

the average market . For the sake of brevity only'the results
obtained by testing immediately after collection.end those
obtained by testing when the first faint odor of putrefacticn
became evident are given. The meat was graded according to the
appearance, odor, condition of the shop, etc. into four classes;
good, fair, peer, and bad. Only meat which gave a slight odor
of putrefacticn at the time at which it was collected was
recorded as bad. These ratings do not give a good method for
determining the quality of the meat or the stage of incipient
putrefacticn but they do furnish es gced a method as was
obtainable with which to compare the results of the hydrogen
eulfid test. The test was run aerobically only on the first
fiftean samples, as it did not appear to give good results on
meat stored at 5'.

The results agree very well with those given in Tables
V and.VI. They show conclusively that the aerobic hydrogen
eulfid producing bacteria do not increase in activity appreciably
during the first stages of putrefacticn at 5‘. On the other
hand the anaerobic hydrogen eulfid producing group appears
to increase in activity decidedly. a summary is given in Table
VIII showing the number of samples rated as good, fair, poor,
and bad according to the appearance of the meat and the
average number of hours required for the production of hydrogen
eulfid under the conditions of the test, applied anaerobically.
These averages are computed for the immediate test and for the
test applied when the meat began to evolve odors of

putrefacticn

Table VIIIe
Hydrogen Sulfid Iroduction by Organisms from Hamburger
Steak. Recorded in Hours necessary to Obtain a Positive
Test. a Sumary of Table VII. Incubat ion at 37' under
Anaerobic Conditions.

.--..--.----.--.---...C..-------...---.---.--C--------------.

Orgmoleptio Tested when:
'0. 0: .mp1... ---.----.---....-‘--OODOOCOOCQD
Rating Purchased Putrescent"
a”! a 1;. .u-.-;:;o..---.Qcocc-o;:g..o--o-
Eh;- anoocoonoogauocon-o-.-.;:5----oa---.---.§:;anus---
’00! 6 ‘.5 -- Im C..- 3:8 0- em .
ICC-”531C odor -11 - - ----5e3--- - ---- -- w-------

.------------.------.---.---.-.---.-.-----..---.-.----------.

* when putrid odor first became evident.

According to Table VIII 16 samples rated as good
gave a positive test in an average of 7.5 hours. At the time
when noticeable putrefacticn appeared these same samples
gave positive test at the end of 4.5 hours. Three samples
3 rated as fair gave positive test at the end of 6 hours and at
the end of 3.7 hours after active putrefacticn became apparent.
Six samples rated as poor gave positive tests at the end of
4.5 hours and at the end of 2.8 hours at the time of the first
appearance of a foul odor. This would indicate that the test
shows two conditions; first, the stage of incipient putrefacticn,
second, the amount of effective contamination. The shortening of
the length of time required to obtain a positive test, as the
meat ages, shows that the test is indicative of the activity

of a group of organisms which increases in numbers or activity

u. h - l
— — 7 h
~ - m— ‘-

—-.t'-vv

rah—p-

V .— 9 .— o v .- —
0

. g. - 3.- p .- — — -

- .— - .. — — .- —

-e—el-~—-~‘rw--.

.— —— a — - a n» a—
- v .a ’ . _ ,. - ~
-— - u— m— an— .a- - — ‘—

._-'————.-

_-... I...
r“-..-
— —-—
-A..-

0-...

.--—0.

~.——--..——_

a”.~—_~—.‘-————

.. a— - — ‘
v -- -— — .-
__ - _.. .. .

—---—-'_

oa—v—‘~--‘-

.- - ‘- au- ' a:— - .—
— — — - .Im .- .-
— n - - .-. ~ a .-

'I‘

.-

-——.~‘q—.-—a--—.—

~w—————--—-

—-a———-pu-—-

.I-ue-b-a-o-e—g-a-

—e———-——

—-e——-—‘

----—m-w

—a--v—e-—

"~-—-

 

-36-

or both during the first stages of putrefacticn, before the
meat would be condemned by crgenoleptic tests. It is highly
probable that the samples rated as poor by'their appearance
were more highly contaminated than those recorded as good.

The ”good" samples gave positive tests at the end of 4.5
hours and the "poor" samples at the end of 2.2 hours, when
tested at that stage at which they began to evolve odors of
putrefacticn. This difference is probably due to the increased
amount of effective contemineticn.in the poor samples.

The average length of thee elapsing before the meat
rated as good appeared putrid was five to seven days. That
rated as fair remained in good condition for approximately
three days. That rated as poor usually commenced to evolve
foul odors after one day. In general the length of time
elapsing before noticeable putrefacticn became evident agreed
well with the condition of the meat as indicated by the
hydrogen eulfid test.

Pour samples which were collected from a market,
later discovered to be using sulfites as preservatives, were
tested. These samples gave positive tests in from 8 to 12
hours at the time of collection. oh storage at 5° the time
required to obtain a positive test increased until at the time
at which the meat became noticeably bad only a slight test
was obtained in 24 hours. If the definition of ”putrefacticn”
given by Rettgsr I35) and other investigators be accepted, vis.,
that it is marked by the evolution of a decidedly foul odor,
then no true ”putrefacticn" took place in these samples of

meat. The spoilage appeared to be more closely comparable to

the "decomposition" described by Hunter (55-58) and by other-I

.36-

which has been ascribed to aerobic bacteria, molds, etc. A
species of mold found growing on these samples was isolated
and identified as W Midhis meet remained in
apparently good condition for from 10 to 20 days. The fact

that the preservative prevents the development cf’the organisms
responsible for a positive anaerobic hydrogen eulfid test and
at the same time prevents the development of the organisms
responsible for the putrefacticn of the meat would serve as '
evidence that both types of organisms may be the same.

lhether the test as developed for haeburger eteak
would be applicable for any other class of meat cannot be stated
without further investigation. The condition of hamburger steak
as sold in the average market is such that some method for
determining the amount of effective contamination and the length
of time during which it will remain useable after selling, I
should certainly be applied. Prom.the results obtained on the
III-plea tested and recorded in Tables v, u and III it appears
that this method would be preferable at least to plate counts
and to chemical methods in that it is quicker and easier to
perform, that it is more specific for the organisms developing
during incipient putrefacticn and that it will show incipient
rather than advanced putrefacticn, along with the amount of
effective contamination.

Any definite standard set for the condemnation of
unfit hamburger steak must depend upon the quality offimeat
which is considered salable. Hamburger steak which will not
give a positive hydrogen eulfid test, using the method
employed in this investigation, in 6 hours may safely be

predicted to keep at least three days in.a good refrigerator,

-37-

before putrefacticn becomes evident. This does not appear to
be too high a standard to set, although it would condemn at
least half of the hamburger steak which is now being sold in
the markets.

Some samples were tested by the hydrogen eulfid
production test and also by the methylen blue reduction method
of Tillmans, Strchecker, and Schutse 331). By this method 5 gas
of well mixed meat is placed in a 60 co flask, which is filled
with water heated to about 40'. One cc of a methylen blue
solution is added. The flask is tightly sealed and placed in a
water bath at 45'. The value of the meet is Judged by the
length of time required for the reduction of the methylen blue.
Tillmans and his associates concluded that meat was decomposed
and unfit for use as human food when the methylen blue was
reduced in less than one houre

Table 11 gives the results obtained by the testing
of eight samples by both methods. Hone of these were of very
good quality. All of them gave positive tests for hydrogen
eulfid producing organisms in less than 7 hours. a period of
from 4 to 8 hours was required for the reduction of the
methylen blue. All of the samples were tested again at the time
when putrefacticn became evident by the odor. Portions were
taken from the surface, from the center, and from a mixture of
the two, for the final test on the last six samples. The results
show that the methylen blue was reduced, within the period of
one hour set by the German investigators, after the meat became
noticeably putrid. There was a wide variation in the length of
time which the reduction required depending upon the spot of

Curling. For example in the testing of Sample III, using

-38-

Table II.
A Comparison of the Hydrogen Sulfid Production and
lethylen Blue Reduction Tests. Recorded in Hours
necessary to Obtain a Positive Test.

3..P1. Is

Ige in days, I Hydrogen Sulfid 3 leth.Blue RedI3Condition meet

0 3 c 3 c , : fair
3 3 3 5 3 poor

3 d 3 2 3 1/2 ‘ 3putrid odor

‘---.------.‘-.-------------.-C---------------L---------C---.

-- B--

SIIPIO II.

------.-.-----------.--------- --------------. -------.---.-

dge in days, I Hydrogen Sulfid 3 Ieth.Blue Red.3 Condition mes;

‘OCOCOO-COCCiCOOOCOOOCCCOCD-C-.-‘.‘--O.------..'--------.O..-"
I O 3 2 3 d 3 poor 3

3. s I l I l 3putrid odor 3

.0-0.-.--.-.--..-.--.-..-.---- ----------.---. -.--.-.----..J

Sample IIIe

xE-oacceocut t--..---.----------.----.$---coca-o---con-o-o-nooc

s in daymI Hydrogen Sulfid Test 3leth. Blue Reduction 3

".--..--..-'--------.---- ..... ------ ----- ........ -..-------
3 o 3 c 3 s 3
..... .---.'.---...----.------.-----"-----.-------.--'-.----‘

3Surface 3Center 3Hixed 3Surface 'Center 3llixed 3

*UC.CO..-‘--.----;.--U---:----..-. ---00--:----¢m-d
4 3 e I c I e I 1/2 3 s 31 1/23

'-.----'

M1. I'e

-.-------- .----.-.--------.-------‘-------------.---C--.--.

in days3r Hydrogen Sulfid Test 3lleth. Blue Reduction 3

I--------.-3--------------~~--------I-------- ----- ----------t

3 0 3 5 3 I I

3Surface 3Center 3Hixed 3Surface 3Centcr 3Hixed 3

. COO.--- Q--.-em-‘-o--coo‘oou-----‘-------.------‘-

d 3 1 3 1 3 1 35am.mhm.nhmn

1 - f -
A '- .n — u. .I _
< -— u— c—
— .-. - ...
_. _ _ .. .m .— U
s
on '— . 'e v
I

—’--

I
I
_._-_.—-._d

IL...—

-
h
—'-a-’
I
.- .
._I...

—-
H-v-
.-.—_.-
-..

-m’-—.—
A o
- .-

.a‘v-Jaa—
I

a. g..--

_ _ - - —
_ — _ I. _ fl
- — - .— v ..
i
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—3—sw——

—.

v

-39-

Table IX. Cont.
a Comparison of the Hydrogen Sulfid Production and
Iethylen Blue Reduction Tests. Recorded in Hours
necessary to Obtain a Positive Test.

Sample 7 e

...--.-.....r-...... ..... - ....... ---- ------- ----- -----------

Ige in days 3 Hydrogen Sulfid Test 3 Heth. Blue Reduction 3

I O I 6 3 6 3

3Surface ICenter Illixed 3Surface 3Center 3Hixed 3

I-------- ------- ------- --------I---.I--I------

5 3 d 3 5 3 5 3 .l 3 1/2 3 1/2 3

.-0.0-..---‘O-.---..‘-.---.-‘--.C---’---.C------O-.C- ccccccc .

'----

Sample VI.

'---------.-'------.------.-.-------- ' --------------.------.-

Me in days 3 Hydrogen Sulfid Iest 3 Heth. Blue Reduction 3

‘OCOO-QC“.-0‘-.-‘OCCOOOOCOOOCOCCCCCCQ'OOCOOCOOO ..... -C-.-----q
3 0 3 6 3 8 3
3Surface ICenter Illixed ISurface ICenter ilixed I
r.....-.. U...... ..... C ..... . OOOOOOOO Q- -‘C-.-

5 343434325/351/e353

‘0‘...--..*..........-.-...’.......'...--..-..........-...-.

'O---.

Sample VII.

--.------.-*---..-.--.-----.-‘-.---. ‘ --.--------------------

figs in days 3 Hydrogen Sulfid Test 3lleth. Blue Reduction ‘3

'5.9-o-u-coucr-qQC-cuccccncGnu-Q.--coop-o---nconnoccuouoonoo-Q‘
I 0 I 5 3 8 3
*o....-.-.- .0.....- wees--cocccnocccokccucocouccocoon-onccuoofl

3Surface ICenter 3llixed 3Surface 3Center 3Hixed 3

, ----- --- ------- ------- -- ------ I-------I------

I 3 8 3 3 3 3 37min. 35min. 35min.3

cnun-ooouocd.a.--c---...-----'-..----'.-use—ocuccssooccouooucouo

3M1. VIIIe

OO----.----*------.-----.----D----O ococuuooocImC-c-I-u-sesecm--.
age in daysi Hydrogen Sulfid Tea 3Ieth. Blue Reduction '3
coco-0---- coo-uuocoocunws-oc ..... capo-ooocucncocococcuoocsm-’
o 3 e : 5 ' 3
Ooucoccvo-iocwe-c- --------------- 5---..-cccooccouooouuoou:

3Surface 3 Center 3Hixed 3Surface 3Center 3Hixed I

t 3 l 3 l 3 1 312min.3lOmin.3llm

ode-oouocchC-occncofio-oocoor ------ .F uuuuuuuuuuuuuu .o.......

r---.-‘.--

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neat fro-.the surface the methylen blue was reduced in one half
hour while if neat from the center was used it required two
hours for the reduction to be complete. The place of sampling
appeared to have little effect on the hydrogen sulfid test.

The hydrogen sulfid production test appears to be
superior to the methylen blue reduction test in that it furnishes
a nethod by which the condition of the seat say be indicated
before it,reaches the stage of evident putrefacticn, and in that
the place of’sanpling does not seen to influence the result to
as great an extent. The uethylen blue reduction test would be
superior if a test for advanced putrefacticn was desired because
it is sore rapid and is slightly easier to run.

rurther investigations were nade to see how much the
results obtained in the use of the test for a routine method
night be influenced by slight errors in technio and to see if
the technio employed in the test night be improved so as to lake
the test more delicate or acre accurate. Bight samples were
tested using different amounts of neat. Along with the tubes
centaining l gn other tubes were used containing 1/8 and 8 ga
portions. Results showed that within this range the difference
in the length of tine required to obtain a.pesitive test was
insignificant. At no tine was there a difference in results
between the tubes containing 1 gn.of neat and those containing
8 ga. Occasionly 1 hour longer was required to obtain a positive
test in the tube where only 1/8 gs was used. Fro- these
investigations it was concluded that in the routine use of the
test extremely accurate weighings would not be necessary. One

gran appears to be the logical aaount of seat to use.

-41..

The posibility of substituting peptone solution
for infusion broth was investigated. The results were very un-
satisfactory. The length of time required to obtain a positive
test was increased and the reaction was not as well defined.
It was found that neat extract broth gave identical results
with infusion broth. Broth was tried which contained different
amounts of peptone- standard broth, one half strength broth
and double strength broth. The reaction given in half strength
broth was ordinarily as quick but not as strong. lo difference
sas noted between the results obtained with standard and with
double strength.

‘ To investigate the effect of the reaction of the
medium used standard broth was adjusted to the following phs;
6.5; 6.753 7.0; 7.25; and 7.5. Between 6.75 and 7.86 there was
litt1e_differenoe in the results obtained with the six samples
tested. Beyond this range a longer period of time was required
to obtain a positive test and the reaction was not as well
defined. Broth adjusted to ph 6.8 or 7.0 seems to be at the
optinum reaction for the production of hydrogen eulfid through
the action of the anaerobic organisms concerned in the

putrefacticn of meat.

Bun-ary’

in.attempt to apply a test for incipient putrefacticn

of neat based on the appearance of indol forming organisms was

unsuccessful.

Thirty six samples of hamburger steak were investi-
gated by a test designed to show the increase in activity of

hydrogen eulfid producing organisms during the period,before

putrefacticn can be demonstrated by crgenoleptic methods. The
method of testing was as follows; Place 1 gm of meat in a tube
containing exactly 10 cc of standard infusion or extract broth.
Suspend a strip of lead acetate paper beside the cotton plug.
Incubate at 37' in an anaerobic jar under partial vacuum. Examine
and record the number of hours necessary to obtain blackening
of the acetate paper due to the production of hydrogen eulfid.

Positive tests were obtained from comparatively good
hamburger steak in 7-10 hours. it the time when putrefacticn
‘ became evident by the production of a foul odor, positive tests
were obtained in 8-5 hours. Hamburger steak which does not give
a positive test in a hour. will keep at least 3 days in a good
refrigerator. This is suggested as a tentative standard.

Based on results obtained the test is recommended
as being efficient in showing not only the first steps in
putrefacticn but also the amount of effective contamination.
The test is not extremely sensitive to errors in the preparation
of the sample of meat used, method of sampling, or in the
reaction of the broth employed as a medium.

The hydrogen eulfid test was found to be of’more
value than the methylen blue reduction test because it shows

incipient rather than advanced putrefacticn.

-43-

In concluding I wish to acknowledge
my indebtedness to Dr. lard Giltner and to the
other members of the Bacteriology Department for
suggestions and assistance received during this

investigation.

III

(2)

(3)

I4)

(5)

I6)

”I

I8)

I9)

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.p—_———-

a
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