STUDIES ON ANAEROBIC BACTERIA
III. HISTORICAL REVIEW AND TECHNIQUE OF CULTURE
OF CERTAIN THERMOPHILIC ANAEROBES
L. S. McCLUNG*
Department of Agricultural Bacteriology, University of Wisconsin, Madison
Received for publication July 16, 1934
Although three groups of heat-resistant thermophilic bacteria
have been shown to be important in the spoilage of non-acid
canned foods, only two have been extensively studied from the
standpoint of physiology and morphology. The group of ther-
mophilic anaerobes, the suggested causal agents of the type of
spoilage known as "hard swell," has received little attention in
this respect. A cultural test (Bigelow, 1931) has been devised
for the numerical estimation of these organisms, an invention of
necessity in the solution of the practical problems arising from
contamination of sugars, etc., but difficulties in cultivation have
prevented detailed studies of the taxonomic characteristics of
the cultures in question. Paine (1931) attempted such a study
of a few cultures probably of this group, though purity of culture
was not well established and certain of the results presented were
incomplete, due to problems encountered in routine study. The
investigation now reported presents a technique of culture and
other methods essential to the preliminary studies and to the
taxonomic and biochemical data which will be presented in later
LITERATURE OF THERMOPHILIC SPOILAGE BACTERIA
Although, as early as 1895, Russell isolated bacteria from
canned foods'and later others (MacPhail, 1897; Prescott and
Underwood, 1897; Poincarr6 and Mac6, 1899; Harding and
* Now with Research Division, American Can Company, Maywood, Illinois.
174 L. S. M cCLUNG
Nicholson, 1904, and others) also incriminated bacteria as a
cause of spoilage of canned foods, it was not until 1912, with the
pioneer work of Barlow, that thermophilic spoilage organisms
received notice. The original report of Barlow is reviewed at
length by Tanner (1924). More recently the problems of isola-
tion and classification of some of these forms, together with
studies on practical tests for their detection, have been inves-
tigated. In the last five to eight years a long list of publications
has appeared on these topics, and because these are so widely
scattered, a review of the literature is felt desirable.
There have been established three major types of thermophilic
spoilage organisms in non- or semi-acid canned foods, the dis-
tinguishing characteristics of which have been reported by
Cameron, Williams and Thompson, 1928; Cameron and Williams,
1928a; Cameron and Loomis, 1930; and Bigelow and Cameron,
1932. These three groups produce spores which possess unusual
resistance to heat. They vary, however, in other respects.
The groups which have been recognized are as follows:'
1. Flat sour organisms. These are facultative aerobic bacilli,
characterized by the production of acids (lactic, Bigelow and
Cameron, 1932; and formic and acetic, Williams, 1934) but not
of gas from carbohydrates. Obligate and facultative thermo-
philic sub-groups were found by Cameron and Esty (1926), who
studied the cultural characteristics of 214 strains of this group.
The type species is B. stearothermophilus of Donk (1920). Other
descriptions of species similar or related to the "flat sour" organ-
isms may be found (Wyant and Tweed, 1923; Bitting, 1924; and
1 The grouping presented is that commonly used in the literature of this sub-
ject. It is recognized that, as the non-toxic food spoilage organisms are more
extensively studied, it will be necessary to extend or change any system of
grouping which may be proposed at the present time. It may, perhaps, be
preferable to classify the various groups according to the outward appearance
of the can of spoiled food after inoculation, followed by suitable incubation,
from a pure culture of the organism in question. Such a classification would
provide for two main divisions; those in which the can has been swelled and
those in which no outward evidence of the spoilage is observed. In this case the
"flat sour" (producing no gas) and the "sulfur stinker" (producing a soluble
gas) groups would appear as sub-groups of the latter division.
STUDIES ON ANAEROBIC BACTERIA 175
2. Gas-forming (non H2S) anaerobes. These are the so-called
"thermophilic anaerobes" or "T. A." cultures with which this
study will be chiefly concerned. By production of gases2 (carbon
dioxide and hydrogen) in large quantities from carbohydrates
these forms are responsible for the spoilage known as "hard
swell," in which the can may be bulged or the seams split due to
the pressure; a "cheesy" odor is sometimes noticed in certain
spoiled canned goods or in cultures of this type. Other than the
reports of sources from which these forms may be isolated the
only published study of this group is that of Paine (1931).
3. Sulfid or "sulfur stinker" organisms. The distinctive fea-
ture of this group is the production of hydrogen sulfid at ther-
mophilic temperatures. The causal organism was originally
classified by Werkman and Weaver (1927) and named by them
Clostridium nigrificans. ,Werkmn (1929) reported a detailed
study of the cultural characteristics. Clost8idium nigrificans is
strictly non-saccharolytic and feebly proteolytic, attacking cys-
tine with the production of hydrogen sulfid; thermophilic tem-
peratures are optimum for growth. Although spoilage due to
this species has not been common, an outbreak is serious
Efforts have been made to discover the source of the spores of
thermophilic spoilage forms in nature. Although the ultimate
source may be the soil, practical problems in the canning industry
have caused more immediate sources to be examined. Sugar
and starches have been found to be important sources of these
organisms in canneries (Cameron and Williams, 1928a and 1928b;
Cameron and Yesair, 1931; Owen, 1932; Owen and Mobley,
1932; and Clark, 1933).
Other reports have shown the widespread contamination of
sugars by thermophilic spore-forming bacteria of some one of the
above mentioned classes (Cameron, 1928; James, 1928a, 1928b
and 1928c; Williams, 1930; Cameron and Bigelow, 1931; Barnes,
1932; Owen and Mobley, 1933; and Clark, 1933). Recognition
2 According to the figures of Paine, the carbon dioxide-hydrogen ratio in the
fermentation of glucose is about 1:8, although the time at which the analyses
were made was not given.
176 L. S. MCCLUNG
of this fact led to demands for uncontaminated sugars by the
canneries. Tentative standards for thermophilic purity of sugar
were suggested in 1931 by the National Canners' Laboratory
(Bigelow, 1931). Sugar that is "certified" to meet these stand-
ards is now available. Contamination from infected bagging has
also been considered (Owen and Mobley, 1932; Owen, 1932).
In addition to the contamination of particular lots of foods a
sugar containing these organisms may present a greater danger
because of the contamination of the plant itself as, for example,
the establishment of the organisms in wooden brine lines and
tanks (Cameron, Williams and Thompson, 1927 and 1928; and
Cameron and Bigelow, 1931). Such equipment may serve as a
constant source of contamination for all products canned, and,
since the spores of these bacteria survive a more severe heat treat-
ment than the maximum possible without injury to the food
materials (Cameron, 1931; Cameron and Yesair, 1931a, 1931b;
and Townsend, 1932), the spoilage losses may be great (Bigelow
and Cameron, 1932).
That the raw product at times may be sufficiently contam-
inated to induce spoilage has been reported by Townsend (1932)
in the case of asparagus. The source of contamination here was
the soil and other reports have also shown the spoilage forms to
be present in soil, usually that which is heavily fertilized (Werk-
man and Weaver, 1927; James, 1928b and 1928c; Cameron and
Williams, 1928b; Werkman, 1929; Medaris, 1931; and Clark,
The possibility of economic loss due to these forms is not con-
fined to this country. Prickett (1932) detected thermophilic
spoilage bacteria in canned Italian shallots. The presence of the
recognized groups in Belgian, Russian, Czechoslovakian and
Dutch sugars was established by Homans (1933a and 1933b).
Although Hirst (1931-2) reported isolated cases of thermophilic
spoilage in England, no serious outbreak had occurred. He
suggested that the low temperature of storage in English ware-
houses served to minimize the danger.
Occasional reports, e.g., Duval (1931) may be found in which
the author does not agree with these generally accepted opinions
STUDIES ON ANAEROBIC BAVARIA 177
as regards thermophilic spoilage bacteria. In these reports the
absence of experimental procedures and of the exact results
eliminates from serious consideration the criticisms offered. The
validity of the conclusion that sugar may play an important r6le
in the contamination of a plant has been questioned by Ingersoll
(1930a and 1930b), who believes thermophilic contamination to be
a matter of sanitary condition of the plant. This view, however,
is not general (Bigelow, 1930), and specific answers to Ingersoll's
criticism have been given (Cameron, 1930b and Williams, 1930).
Many report the presence of viable bacteria in supposedly
sound canned foods. The pioneer work on this problem was
done by Cheyney (1919), Weinzirl (1919) and Savage (1923).
Occasional thermophilic types were encountered but descriptions
of the cultures are so incomplete as to make impossible accurate
identification of the species involved. Hanzawa and Takeda
(1931), in a study of the bacteriology of "swelled" cans of cod-
fish-cake, include a review of some of this literature.
MEDIA USED AND TECHNIQUE OF CULTURE
At the outset of this study of the thermophilic gas-producing
(non-H2S) anaerobes, it seemed evident from the literature and
communications with those studying the practical side of spoilage
problems, that special procedures would be necessary if consistent
results were to be obtained. As the work progressed this pros-
pect became a reality. As will be pointed out in the appropriate
sections, many of the procedures which have been found suc-
cessful for other anaerobes were not satisfactory for these ther-
mophilic anaerobes. Two major problems which presented un-
usual difficulty concerned growth on the surface of solid media
and the controlled production of spores in laboratory media.
Difficulty was experienced in choosing a medium for general
cultivation purposes. In a study of the enrichment media suit-
able for this group, Medaris (1931) was unable to find many that
would be useful. In addition to the liver infusion of Cameron,
Williams and Thompson (1928), potato glucose infusion and
178 L. S. MCCLUNG
certain of the infusions prepared from canned strained vegetables
were sometimes satisfactory. An extension of these experiments
has been necessary and the essential types of media tried, to-
gether with methods for their preparation, will be discussed.
Medium for enrichment and general ciUtivation purposes. Liver
infusion (liquid infusion tubed and sterilized over particles of
dried extracted tissue) has been the most extensively used for the
group, as it seems peculiarly suited to their growth requirements.
It is the medium recommended for commercial use in detecting
contamination of sugars and other materials, but in the present
study a slight modification of the original formula has been used
(McClung and McCoy, 1934). Growth is indicated by turbidity
of the infusion above the particles of ground tissue and a violent
production of gas during the early stages of growth; the liquid
also becomes lighter in color. The odor is distinctive but not
that of putrefaction.
The infusion as usually prepared is probably many times more
concentrated than is necessary; a 1:10 dilution allows growth,
although if dilute inocula are used the time for the appearance of
turbidity and gassing is lengthened. It is important that pre-
viously sterilized tubes be used for this medium, as the time of
autoclaving should not exceed forty to forty-five minutes (17
pounds pressure). Further sterilization darkens the medium and
renders it unsuitable for growth. The danger of over-steriliza-
tion of liver medium has also been observed by Jackson and
This medium, although very satisfactory for cultural purposes,
is not to be recommended for material for stained mounts. Cer-
tain Gram-positive, granular organisms, which apparently multi-
ply rapidly on the liver tissue during the drying process, appear
in certain batches. These forms are, however, characteristic
and may be recognized and disregarded.
Other media commonly used for other types of anaerobes were
found unsuitable for various reasons. The gassing and turbidity
diagnostic of growth of the thermophilic anaerobes in Robertson's
(1931) alkaline meat medium, Hall's (1920) brain medium and
others of similar type are transient, thus making theindications
STUDIES ON ANAEROBIC BACTERIA 179
of growth uncertain. Difficulty of preparation and sterilization
of these media also make them undesirable for general routine
purposes. Beef-extract peptone broth and the various peptone
solutions tested are not suitable, nor is yeast water, even with the
addition of 1 per cent glucose as an additional carbon source and
reducing substance, satisfactory. The poorly buffered excelsior
extract medium proposed by Paine (1931) for this group did not
supply the essentials for growth; addition of glucose or micro-
cosmic salt as suggested did not improve the medium. As has
been previously noted (McClung and McCoy, 1934), plain corn-
meal mash is not good for this group.
Plating medium. As was earlier demonstrated by Medaris
(1931) certain of the vegetable media have proven useful for
tube culture. Five to 10 per cent infusions of canned strained
vegetables (peas, spinach, etc.) are made, and for plating pur-
poses, 2 per cent agar is added. The inability of certain of these
vegetable infusions to support growth is probably due to the ex-
treme pH change which occurs during sterilization, as has been
previously suggested (McClung, 1932).
Pea infusion seems to undergo less change of reaction than
others and will support consistent growth; it has been almost
exclusively employed as a plating medium in this work and can
not be recommended too strongly for this purpose in culturing
this particular group. It is inexpensive and easy to prepare,
fairly well standardized, and allows spore formation. This last
point is particularly important as many of the other media which
do allow growth on solid media as, for example, liver infusion or
glucose agars are unsatisfactory in this respect.
Base medium for physiological studies. Some difficulty has
been encountered in finding a suitable base medium for the
study of the physiological characters of these cultures. Peptone
solutions (proteose, bacto and Witte) in various concentrations,
beef-extract peptone broth, yeast water and others tested are
not to be used, for even with the addition of 1 per cent of car-
bohydrate inconsistent results are often obtained, almost uni-
formly so if dilute inocula are used. Casein digest, prepared
according to the formula of Kristensen, Lester and Jurgens
180 L. B. MOCLUNG
(1925), is somewhat better in this respect but has the disad-
vantage of variation from batch to batch. The most satisfactory
base which has been found is 1 per cent tryptone broth' (in dis-
tilled or tap water with no adjustment of pH) to which the vari-
ous test substances are added. Little or no growth is obtained
in the base alone; glucose in 0.1 to 1.0 per cent concentrations
stimulates growth, the time required for the development of
turbidity of the medium or for gas production as indicators of
growth being inversely proportional to the amount of sugar
Other media. A new medium was devised for certain correlated
problems requiring large numbers of tubes, for which none of the
above described media was satisfactory. The details of prepara-
tion of this medium, containing 1 per cent dried ground liver
tissue and 5 per cent cornmeal have been described in an earlier
publication (McClung and McCoy, 1934) and need not be re-
peated. The results which have been obtained indicate that the
medium is satisfactory for routine cultivation purposes, i.e.,
serial transplant studies, sub-culture following thermal death
time tests, exarmination of sugars, Starches, soil, etc.
Other media have been prepared according to standard pro-
cedures or specific reference will be given as needed. Unless
otherwise noted all media have been sterilized by autoclaving for
thirty minutes at 17 pounds pressure. The usual practice of
exhaustion by steaming of the tubed media before use for an-
aerobic culture, has been followed except in the case of the corn-
liver medium, for which this does not seem necessary.
Technique of culture
Test tube. It has been commonly noted that consistent posi-
tive results with this group will not be obtained in tube culture
if the medium is not protected from the air. This appears to be
true even with the more reduced media like liver infusion or glu-
cose broth; the use of large inocula in part avoids this difficulty
but is often impossible or undesirable. This fact has been met
3 This medium is prepared from the desiccated product "Tryptone" of the
Difco Laboratories; formerly sold as "Tryptophane Broth."
STUDIES ON ANAEROBIC BACTERIA 181
by others working with anaerobes and the devices to circumvent
the problem have been many. Various mechanical seals, i.e.,
vaseline, oil, paraffin, etc., have been proposed but the incon-
venience of these procedures in routine culture scarcely warrants
their use. The incubation of all tubes in anaerobic jars offers
another solution but one which can not be applied if the tubes
require inspection at close intervals, nor is it convenient to use
many of the available anaerobic systems at thermophilic tem-
The addition of inert substances to certain media for pro-
moting anaerobic growth is an old method which many have
found useful. These substances are of several types and a
partial review of their uses may be found in the recent publication
of Breden and Buswell (1933). Several of these chalk, asbestos,
cotton, glass wool, pumice stone, small percentages of agar, etc.,
-were tested in various base media but without success in the
present work. The use of particles of dried liver tissue was ad-
vantageous with certain bases but the action here is probably
due as much to the soluble nutrients from the liver as to the re-
ducing action of the tissue.
Cameron, Williams and Thompson (1928) recommend in en-
richment cultivation of this group the stratification of liver in-
fusion with yeast water agar made by pouring sterile agar into
the inoculated tube, thus giving a series of physical phases-from
liquid to jel-in which growth occurs. Medaris (1931) modified
this method by first adding to the surface of the inoculated tube
a. thin layer (- inch) of sterile paraffin, which was allowed to
solidify; then sterile non-nutrient (2 per cent) agar was added to
form a 1 inch seal. This seal is effective but the paraffin feature
complicates washing of tubes, a factor to be considered if many
tubes are involved.
In some cases, as liver infusion, the paraffin layer is unnecessary
if the agar is added carefully. The plain agar seal is also possible
on most other media (vegetable infusions, milk, tryptone broth,
etc.) but only if the following precautions are observed. Cool
the inoculated tubes by immersion for several minutes in ice
water and add two or three drops of 3 per cent agar to form a
182 L. S. MCCLUNG
thin layer on the surface of the medium. After this has solidified
(two or three minutes) add the remainder of the agar as before.
Contamination of tubes prepared by this method is rare and may
be reduced to a negligible amount by the addition of gentian
violet (1: 50,000) to the agar used for sealing. The disadvantage
of the extra time required to prepare tubes by this method is
offset by the assets which it presents-ease of examination for
hourly or daily readings; ease of removing inoculum or material
for staining; elimination of the need for anaerobic jars adapted to
thermophilic temperatures. This modification of the original
method of Cameron and Williams has been used throughout
In the use of the agar sealed media it has been found useful to
transfer with capillary pipettes drawn from 7 mm. (inside diam-
eter) glass tubing in 10 cm. lengths which are thrust between
the agar and the side of the tube in obtaining culture material.
One or 2 ml. rubber bulbs are used for drawing up and expelling.
The pipettes may be cleaned and used repeatedly.
Occasionally it isdesired to incubate cultures not under seal
for periods of forty-eight to sixty hours in which case evapora-
tion at the thermophilic temperatures becomes a serious factor.
If the medium is semi-solid or of such nature that protection
from oxygen is not necessary for anaerobic growth, wrapping of
each tube in lead foil will suffice to delay evaporation. If much
longer incubation is required, all tubes may be enclosed in a jar.
Plate culture technique. Following the development of tech-
nique which made possible consistent results with various tubed
media, it seemed logical that growth on the surface of solid media
would be obtained if the same base media and inocula were used
and anaerobic incubation was provided. This, however, was
not found true. In an attempt to have as few variable factors
as possible, one standard source of inoculum was chosen for the
early experiments; this was an eighteen to twenty-four-hour
culture of the strain in question in liver-infusion broth under
agar seal. Proposed devices for anaerobic plate culture are so
numerous in the literature and the more practical ones are so
widely known that specific references are unnecessary. It is
STUDIES ON ANAEROBIC BACTERIA 183
enough to state that a large number of these were tested. Follow-
ing preliminary experiments the Novy jar (replacement with
carbon dioxide, nitrogen, or a mixture of carbon dioxide and
hydrogen), the phosphorous jar of Sellards (1904) and the vege-
table tissue system of Carroll and Hastings (1925) were chosen
for detailed study. In these were tested all the media which
were known to be favorable to growth in tube culture; in each
case the standard inoculum (active liver culture) was used. The
results were almost uniformly negative. Any positive culture
seemed a matter of chance for it could not be duplicated using,
as nearly as possible, identical conditions.
It was later shown that the inoculum was the factor responsible
for these negative results. In one experiment the use of aged
cultures in liver or vegetable infusion as the inoculum for plate
culture resulted in colony formation. Subsequent experiments
showed that colony formation could be induced in every culture
by this method; furthermore, if the plating medium used were a
vegetable-infusion agar (strained peas) spores would be produced
in the colonies formed providing an incubation time of four to
seven days were allowed. The use of old cultures or previously
prepared spore suspensions is now considered to be an absolute
essential for successful plate culture regardless of the type of
anaerobic system used. Positive cultures have thus been ob-
tained in anaerobic environments provided by various methods
whereas completely negative results have resulted with every
plate culture incubated in the same jars in which the inoculum
was from a young culture. No difficulty in plate culture has
been encountered in any instance since this method was adopted
as a routine procedure; while the complete reverse is true in any
case where this is not observed.
The vegetable tissue system (potato) of Carroll and Hastings
(1925), which has recently been shown to be effective at ther-
mophilic temperatures (McClung, McCoy and Fred, 193-), has
been used in routine culture with complete success. With this
method it is necessary to use clay tops on the petri dishes to
absorb the excess moisture formed. The use of these clay top
dishes does not over-dehydrate the agar, as might be expected
184 L. S. McCLUNG
at the high temperature of incubation, leaving a surface that is
too dry for proper growth but on the contrary the dryness aids
in the formation of discrete colonies, a distinct advantage in the
purification of cultures or in the study of colony types. This
dry surface also seems to be an essential in the production of a
high yield of spores. At least 20 ml. of agar should be used in
each dish; a smaller amount is not to be recommended since a
minimum of forty-eight hours of incubation is necessary for the
proper growth of colonies and ninety-six to one hundred twenty
hours for sporulation.
Spore formation. The best method which has been found for
the production of spores is the growth on the surface of suitable
agar, using the petri dish technique described above. The vege-
table-infusion agars (usually of strained peas) are to be used
since liver, glucose tryptone or similar agars do not allow spore
formation. If it is desired to build up a stock spore suspension,
it isbest to develop the culture by serial plating technique rather
than by using tube cultures for inoculum. Dilutions of the
growth may be prepared in tap water or physiological saline and
these serve as a stock inoculum for a number of plates; these
stock spore suspensions remain viable for considerable periods of
time. For the production of spores the petri plate technique is
preferable to growth in bottles, as the plate culture gives a better
yield of spores even in the same medium; it is probable that
moisture relations explain the difference.
Stock cultures. In any problem of this nature it is essential to
have on hand stocks from which physiologically comparable
cultures may be obtained. In the culture media which permit
the most luxuriant vegetative growth (liver infusion, etc.), it will
be found that after a certain period of incubation the cultures
of this group will become autosterilized or nearly so, and for this
reason such media are not to be recommended for the mainte-
nance of stock cultures. It has been previously mentioned that
stock suspensions of spores in tap water (or physiological saline
solution) remain viable apparently indefinitely for although
exact counts were not made, dilute water suspensions of spores
(in bottles closed by metal caps) were viable after eight months
STUDIES ON ANAEROBIC BACTERIA 185
of storage at room temperature. Another method of preserving
the stock cultures of this group is to dry spore suspensions in
tubes of sterilized garden soil. Soil stocks prepared in this
manner were viable after a period of several months with no
indication of loss in the numbers of viable spores. Similarly,
liver cultures dried upon particles of liver may serve as reference
culture sets. Although it has never been possible to demonstrate
the presence of spores in liver infusion cultures by microscopic
examination, a very low percentage of spores is probably formed.
Three major groups of thermophilic bacteria have been
established as causal organisms in certain types of spoilage of
non-acid canned foods. Of these one group, the thermophilic
gas-producing (non-H2S) anaerobes, has not previously been ex-
tensively exmned and this group has been chosen for study.
Methods are presented which are believed to be satisfactory for
use in routine study. These include the formulae of various
types of media which have been found suitable for growth as
well as especial technique necessary in test tube and plate cul-
ture. A method which has been found to be satisfactory for
the controlled production of spores in the laboratory, by surface
growth of the cultures on vegetable extract agar is discussed.
Methods for the maintenance of stock cultures are included.
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JOuBNAL OF BACTzuOLOGY, VOL. 29, No. 2