International Biodeterioration & Biodegradation 32 (1993) 55-66

Spoilage of Bread by Bacillus

Jaekie M. Thompson, Christine E. R. Dodd & Will M. Waites

Department of Applied Biochemistryand Food Science, Faculty of Agricultural and Food
Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough,
Leicestershire UK, LE12 5RD

ABSTRACT

Bacteria belonging to the genus Bacillus are capable of causing the food
spoilage condition in bread known as rope. This involves mainly Bacillus
subtilis, but also B. licheniformis, B. megaterium and B. cereus. Rope is
characterized by a sweet fruity odour, often described as resembling over-
ripe pineapples, together with patchy discolouration and softening of the
loaf crumb. Differentiation of microorganisms can be carried out in a
variety of ways. These include classical methods, for example the Gram
stain, commercially available kits, such as the Analytical Profile Index
(API) strip, and modern molecular methods, such as ribotyping. The
various methods used to differentiate the strains are an important aid in
highlighting the areas within the bakery processing line that are involved in
contributing to the food spoilage condition known as ropey bread.

INTRODUCTION

Bacteria belonging to the genus Bacillus are capable of causing the food
spoilage condition in bread k n o w n as rope (Voysey & Kaur, 1987;
Kirschner & Von Holy, 1989; Von Holy & Allan, 1990). Bacillus
mesentericus was initially described as the causative agent. This was later
referred to as B. subtilis (Barton-Wright, 1943). Other rope-inducing
species include B. licheniformis, B. megaterium and B. cereus (Collins
et al., 1991).
55
International Biodeterioration & Biodegradation 0964-8305/94J$07-00 © 1994 Elsevier
Science Limited, England. Printed in Great Britain.
56 Jackie M. Thompson, Christine E.R. Dodd, Will M. Waites

Spores of Bacillus species are numerous in the environment (Collins

et al., 1991). It is thought that they attach to the ears of wheat during
cultivation (Kirschner & Von Holy, 1989), and subsequently pass from the
wheat into the flour during milling (Seiler, 1986; Kirschner & Von Holy,
1989; Volavsek et al., 1992). Brown breads, which have a high bran
content, appear to be more susceptible to rope (Kirschner & Von Holy,
1989). Other sources of Bacillus contamination include raw materials such
as baker's yeast and improvers, as well as the bakery atmosphere and the
surfaces of processing equipment (Collins et al., 1991; Volavsek et al.,
1992). Spores are able to withstand the high temperatures reached during
the baking process and can then multiply under favourable conditions
(Kirschner & Von Holy, 1989; Volavsek et al., 1992). Close stacking of the
loaves after baking, for example, provides warmer, more humid condi-
tions because of slower cooling of the loaves, and thus favours spore
germination and subsequent growth of the vegetative cells produced
(Voysey & Kaur, 1987; Kirschner & Von Holy, 1989). Underbaking of the
loaves also provides favourable conditions for rope development. This not
only allows a greater number of spores to survive, but also provides a
moister crumb (Seiler, 1986).
Baked loaves can become ropey within 12 h (Von Holy & Allan, 1990).
Rope is characterized by a sweet fruity odour, often described as resem-
bling overripe pineapples. Patchy discolouration can then be seen in the
central portions of the loaf. The crumb becomes very soft, and eventually
web-like strands can be pulled out from the loaf, so giving the condition
its name (Voysey & Kaur, 1987; Kirschner & Von Holy, 1989; Von Holy
& Allan, 1990). It is thought that the encapsulation of the bacterial cells,
together with the digestion of the starch granules by bacterial amylases
and proteases to glutinous extracellular polysaccharide material, contri-
butes to the production of rope (Kirschner & Von Holy, 1989; Von Holy
& Allan, 1990).
As a result of the application of good manufacturing practices,
advanced process control and high hygiene standards, the problem of rope
spoilage is not common in the Western world (Volavsek et al., 1992). The
addition of preservatives, such as 0.3% (w/v) calcium acetate or 0.2% (w/v)
calcium propionate (based on flour weight), to the bread, also helps to
prevent rope spoilage (Kirschner & Von Holy, 1989). The use of preser-
vatives in bread has decreased in recent years owing to increased con-
sumer demands for a reduction in the use of chemical additives in foods
(Jackel, 1980; Von Holy & Allan, 1990). The use of vinegar, which is
classed as a natural preservative, has therefore increased. Concentrations
of 0.1% (w/v) acetic acid (based on flour weight) are used (Kirschner &
Von Holy, 1989), but according to the U K Flour Milling and Baking
 Spoilage of bread by Bacillus 57

Research Association there has been an increase in reports of rope

outbreaks, especially during warmer weather (Von Holy & Allan, 1990).
There have also been cases recorded of food poisoning caused by the
consumption of ropey bread. These include household outbreaks invol-
ving two people in 1984, and three people in 1987 (Sockett, 1991; Sockett,
P.N., pers. comm., 1992), as well as an outbreak in the Isle of Man in 1988
(Anon., 1988).
The incidence of rope is much greater in South Africa, where consumer
preference demands warm, underbaked, brown loaves, and the climate is
warm and moist and manufacturing standards are lower than in the U K
(Von Holy & Allan, 1990; Volavsek et al., 1992). The risk of food
poisoning to the consumer, as well as monetary losses to the baker, are
therefore much higher (Collins et al., 1991; Volavsek et al., 1992).

THE GENUS BACILLUS

The genus Bacillus was first described by Ferdinand Cohn in 1872 (Collins
et al., 1991). Smith et al. (1946) carried out the first comprehensive study
on the taxonomy of Bacillus. Researchers have tried unsuccessfully to
divide the genus (Sneath, 1986). Recent studies using D N A base compo-
sition and D N A base reassociation have highlighted the broad nature of
the genus (Priest, 1981; Sneath, 1986). Small subunit (16S) ribosomal
RNA studies by Ash et al. (1991) have divided the genus into five distinct
clusters which Ash et al. suggested actually represent separate genera.
The use of classical tests to identify isolates is hampered by the need for
stringently controlled conditions. Colony characteristics of B. subtilis and
related species are known to vary considerably as a result of environ-
mental factors, such as agar depth and medium composition. For example,
colony variation can be influenced by the type of peptone used in the
medium. The degree of sporulation within the colony can also affect the
colony colour (Sneath, 1986). The use of the Analytical Profile Index
(API) 50CHB identification strips (BioMrrieux) have been shown to give
more reproducible results than classical methods (Logan & Berkeley,
1981, 1984; Collins et al., 1991). These strips examine the utilization of 49
carbohydrates, which is demonstrated by a colour change.

D I F F E R E N T I A T I O N OF B A C I L L U S ISOLATES

Collins et al. (1991) attempted to characterize 102 Bacillus isolates

obtained from a bakery environment, including 45 from ropey bread,
58 Jackie M. Thompson, Christine E.R. Dodd, Will M. Waites

as well as 14 reference Bacillus strains. They carried out a variety of

tests including catalase production, the Voges Proskauer test, growth
on anaerobic agar, growth at 50°C, growth in 7% NaCI and hydro-
lysis of starch. From these results, they differentiated their isolates,
using a dichotomous key, as B. subtilis (63-7%), B. licheniformis
(24.5%), B. pumilus (8.8%), B. cereus (2%), and B. firmus (1%). In
comparison, using API 50CHB, they grouped their isolates as B.
subtilis including B. amyloliquefaciens (58.2%), B. pumilus (20.4%), B.
licheniformis (17.4%), B. cereus (3%) and B. circulans (1%), with the
remainder giving unacceptable profiles (4%). They compared the iden-
tifications that they obtained from the key and API 50CHB, and
found that of the reference strains only eight were correctly identified
by one or both methods. For example, American Type Culture
Collection (ATCC) 14577 (B. sphaericus) was identified as B. firmus
and B. cereus by the key and API profiles, respectively. Similar
discrepancies were also seen when the bread isolate identifications were
compared (Collins et al., 1991).
Similar discrepancies to those obtained by Collins et al. (1991) were
seen when 170 Bacillus isolates obtained from swabs of bakery equipment,
raw ingredients, and British and Maltese loaves of bread, were examined
(Thompson, J.M., unpublished, 1992). The classical tests used were Gram
staining, catalase production, the Voges Proskauer test, D-xylose utiliza-
tion, hydrolysis of starch, growth at 55°C and growth on B. cereus selective
agar (Oxoid). From the results of the classical tests, only 65% of the
isolates could be identified to the species level. This was achieved using a
differential characteristic table for the expected Bacillus species, obtained
from Sneath (1986) and Collins et al. (1989). The results showed that
the majority of identified strains were B. pumilus (17-1%), followed
by B. licheniformis/B, coagulans (indistinguishable profiles) (12.9%),
B. subtilis (9-4%) and B. cereus (6.5%) (see Fig.l(a)).
The main problem when attempting to identify Bacillus using a limited
number of tests is that species cannot readily be distinguished. In parti-
cular, a single character may be used to distinguish between two species.
This is highlighted by B. pumilus and B. subtilis which, based on the above
identification system, are only distinguished by the hydrolysis of starch.
An error in the interpretation of this test can therefore totally alter the
identification of an isolate. Differentiation is also further complicated by
the fact that some Bacillus species are extremely variable in their char-
acteristics. For example, 11-89% of B. coagulans can be positive in their
test result for D-xylose utilization and citrate utilization. Positive results
can therefore make the profile indistinguishable from that for B. licheni-
formis.
 Spoilage of bread by Bacillus 59

Ca) B.licheniformisl
B.eoagulans/ B .li¢heniformis/
B B..subfili~ B.pumilus 17.1% / B.eoagulans/
.purer|us u.o'~~ / B.subtilis 0.6%
~--~.-a------~ / ~,B .stearothermophilu s/

Non-identifiable _ / ~ ~ ~ / B.circulans/
3,7% ~ A , , \ \ \ \ \ \ \ \ " ~ ~ ~ 8.ma0~...24%
~,,,~~"~ ~ _ ~--'---'- B.stearothermophilu s/
~N,~'~,~'N~ ~ B.megaterium/

.
B.stearothermoph|lusl j~'///j,~ ~ I ~ B.sphaerieus/
-13 ......
B.cireulan,/~- ~ ~ ~ l~.orevts/3.,'r.
B.maeerans 3.5% / ~ -- ~ n ';,h,,i
B.licheniformis/
B.subtilis 9.4% B.eereus 6.5% B.eoagulansl 12.9%

(b)
B.lieheniformis 28.2%
~fll I I I I ] I I t/r'~,\,,,,,,'B-megaterium3.6%
~Ji"r' ~ 11'
] I I l lll
II I ) / ~ ' Y " " B ' p o i y m y x a 3.5%
. L1~B.macerans 3.0%

B.pumilus 1 6 . 0 % ~ ( i n e l u d i n g B.myeoides) 2.4%

Y/////////////il " U n ,e ~ ep~,b le
B.stearotberrnophilus
0.6%~ Y//'~Z/~,,/~ Pr°file4.2%
B.circulans 4 . 1 % ~
B. amyloliquefaeiem2.4% t -,~ B.subtilis 32.3%

Fig. 1. Percentage distribution of 170 isolates of Bacillus using (a) classical methods and (b)
API 50CHB.

The isolates were identified by API 50CHB as B. subtilis (including

B, amyloliquefaciens) (34.7%), B. licheniformis (28-2%), B. pumilus
(16.0%), B. circulans (4.1%), B. megaterium (3-6%), B. polymyxa (3-5%),
B. macerans (3.0%), B. cereus (including B. mycoides) (2-4%) and
B. stearothermophilus (0.6%), with 4-2% giving unacceptable profiles (see
Fig. l(b)). The distribution within the samples of these identified isolates
is shown in Table 1. As in the study by Collins et al. (1991), discrepancies
were seen between the two identification methods. This is highlighted in
Fig. 1. It must also be noted that identical profiles are not always obtained
if the tests using API 50CHBs are repeated. For example, one isolate was
initially identified as B. pumilus. On repetition of the API, the profile gave
B. subtilis. It is apparent that neither the use of classical tests with an
identification key nor the use of API 50CHB can be considered as
completely reliable methods in the identification o f Bacillus species.
60 Jack& M. Thompson, Christine E.R. Dodd, Will M. Waites

TABLE 1
Distribution of 170 Bakery Bacillus Isolates Identified Using API 50CHB

Bread ingredients Swabs of Cooked loaves

and uncooked processing and
dough (%) line (%) breadcrumbs (%)
B. subtilis 22.9 4.1 5.3
B. amyloliquefaciens 1.2 1.2 0
B. licheniformis 18.2 5.9 4.1
B. pumilus 11.2 2-4 2-4
B. circulans 2-9 0.6 0.6
B. megaterium 1-8 1.2 0.6
B. polymyxa 2.9 0 0-6
B. macerans 2.4 0 0.6
B. cereus (including B. mycoides) 0.6 1.8 0
B. stearothermophilus 0 0 0.6
Unacceptable profile 0.6 2.4 1.2

PROBLEMS WITH B A C I L L U S A N D M O L E C U L A R M E T H O D S

Molecular techniques such as plasmid profiling can indicate whether

strains have come from the same or different environments, and therefore
may help to identify the source of a particular strain. This can therefore be
used to detect 'critical control points' within the production line (Dodd,
1990). In our experience, however, it is difficult to obtain plasmid
deoxyribonucleic acid (DNA) from wild type Bacillus strains with any
consistency. Other workers have also experienced this problem with
B. subtilis (Bron, 1990). This is particularly true if trying to use a uniform
method for a variety of Bacillus species. Although lysis of the cells can
often be achieved, this is not the gentle controlled lysis required for
successful plasmid DNA isolation.
One reason that has been suggested is that Bacillus are Gram positive
and therefore have a thick peptidoglycan cell wall layer with covalently
attached polyanions (teichoic acid and teichuronic acid) (Bron, 1990).
This has been suggested to decrease the sensitivity of the cells to enzymes
such as lysozyme, which hydrolyses repetitive N-acetyl-glucosamine
/~-l---4-N-acetylmuramic acid bonds in the cell wall (Chassy & Guiffrida,
1980). Furthermore, it has been reported that some B. cereus isolates are
resistant to lysozyme, as a result of the occurrence of glucosamine residues
with free amino groups in the cell wall peptidoglycan (Araki et al., 1972).
These N-unacetylated glucosamine residues have also been demonstrated
in the cell walls of B. megaterium and B. subtilis (Hayashi et al., 1973). A
 Spoilage of bread by Bacillus 6l

particular problem when working with Bacillus isolates capable of pro-

ducing rope is that the action of lysozyme may be further inhibited. This is
because they are known to secrete an extracellular polysaccharide which
has been shown to surround the cells (Kirschner et al., 1990).
Bacillus species are also known to secrete various proteins into the
medium during post-exponential growth. These include cell-associated
and extracellular nucleases, and special care is therefore required to limit
their presence. To overcome this problem, exponentially growing cultures
can be used. Also, the inclusion of ethylenediaminetetraacetic acid
(EDTA) can be used to chelate any divalent cofactor cations (Bron, 1990).

RIBOTYPING

The recently developed technique of ribotyping provides a method for

simplifying complex chromosomal D N A fragment patterns (Grimont &
Grimont, 1986). It is based on restriction fragment length polymorphisms
(RFLP) in the chromosomal DNA. D N A contains the operons encoding
the ribosomal ribonucleic acid (ribosomal RNA) (Baloga & Harlander,
1991). The ribosomal R N A molecules are ubiquitous and their sequences
extremely conserved. The ribosomal R N A operons are present in several
copies within the chromosome and comprise the 16S, 23S, 5S and some
transfer RNA genes. The number of operon copies and their location on
the chromosome are known to vary between Bacillus species (Grimont &
Grimont, 1986). Comparison of restriction endonuclease digests of chro-
mosomal DNA, after hybridization with a DNA copy of Escherichia coli
ribosomal RNA, will therefore provide taxonomic information.
Ribotyping has been used successfully with a variety of bacteria. For
example, Stull et ai. (1988) differentiated between nine E. coli isolates,
seven out of eight Pseudomonas cepacia isolates, and nine out of 10
Haemophilus influenzae isolates. Serratia proteamaculans and Serratia
liquefaciens, which are difficult species to differentiate using biochemical
tests, have been separated using ribosomal DNA patterns (Grimont &
Grimont, 1986). Isolates of Pasteurella multocida were ribotyped by
Snipes et al. (1989). Baloger & Harlander (1991) differentiated between
Listeria monocytogenes, L. innocua, L. ivanovii and L. welshimeri and also
further subdivided 28 strains of L. monocytogenes using banding patterns.
Such work showed that strains of the same (or different) serotype can be
differentiated using ribotyping (Snipes et al., 1989).
Ribotyping has therefore highlighted differences both between and
within species (Baloga & Harlander, 1991). This has also been shown
when 17 ATCC, National Collection Type Cultures (NCTC) and
62 Jackie M. Thompson, Christine E.R. Dodd, Will M. Waites

National Collections of Industrial and Marine Bacteria (NCIMB) Bacillus

reference strains were ribotyped (Thompson, J.M., unpublished).
Differences were seen in the patterns between the species, whereas both
similarities and differences were seen within species (Fig. 2). The strains of
B. subtilis (NCTC 3610 and NCTC 6276) which showed some common
bands, and B. subtilis var. niger (NCTC 7861) and B. subtilis var. globigii
(NCTC 10072) all showed different ribotypes. Bacillus subtilis var. niger
has now been placed in a separate species, B. atrophaeus sp. nov. (Nakamura,
1989). This may explain why NCTC 7861 has a different ribotype from
the other B. subtilis strains. When B. amyloliquefaciens (NCTC 10785) was
observed, this appeared to have the same banding pattern as NCTC 6276,
which is an isolate from ropey bread. This species has always been closely
linked with B. subtilis (see Priest et al., 1987). The major difference
between B. subtilis and B. amyloliquefaciens is that the latter produces
large amounts of extracellular enzymes, such as a-amylase and protease
(Priest et al., 1987). Priest et al. (1987) proposed that B. amyloliquefaciens
is distinct from other named species of Bacillus, but because of the
phenotypic similarities between B. subtilis and B. amyloliquefaciens
differentiation using classical tests alone may be difficult (Priest et al.,
1987). Previous evidence suggests less than 25% homology between the
two DNAs (see Priest et al., 1987). This is probably dependent on the
strain of B. subtilis being used, because, from the ribotype patterns, one
would expect more similarity between the D N A of B. subtilis (NCTC
6276) and B. amyloliquefaciens (NCTC 10785) than if B. subtilis (NCTC
3610) was used.
The B. licheniformis strains examined seemed less variable, in that similar
bands were seen within all four isolates. All were ropey bread isolates,
except NCTC 10341, and were originally classified as B. mesentericus. All
the remaining isolates, including B. cereus (NCTC 2599) and B. mycoides
(NCTC 926), showed different banding patterns, except for B. megaterium
(ATCC 9885), which unexpectedly had the same banding pattern as
B. subtilis var. globigii (NCTC 10073). Although B. subtilis and B. mega-
terium were placed within the same groups by 16S rRNA sequencing,
B. megaterium was positioned on a distinct line when the data were
represented as a phylogenetic tree, implying no specific relationship with
the remaining species in the cluster, including B. subtilis (Ash et al., 1991).
Ribotyping has also been applied to Bacillus isolates capable of causing
rope (Thompson, J.M., unpublished, 1992). Some isolates that had similar
API 50CHB profiles also gave the same ribotype. For example, 9-4% of
the isolates identified as B. subtilis by the API strips all had abnormal
results for inulin and lactose utilization. Ribotyping of these isolates
provided identical or very similar ribotypes, but not identical to the ribo-
 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
..... , o

12.2 K B
7.1 K B
--6.1 KB
~ 5.0 K B
--4.0 KB
°~ --3.0 KB
r~

....... - - 2 . 0 KB
~ 1.6 K B

g
Fig. 2. Ribotyping of B~lcillus species. (DNA digested with Cla 1). Lane 1:1 kb ladder; Lane 2: NCTC 3610 (B. subtilis); Lane 3: NCTC 6276
(ropey bread isolate) (B. subtilis); Lane 4: NCTC 7861 (B. suhtilis var. niger): Lane 5: NCTC 10073 (B. subtili~s var. globigii); Lane 6: NCIMB
10785 (B. amyloliqu~fociens); Lane 7: NCTC 10341 (B. liehen(formis); Lane 8: NCTC 962 (ropey bread isolate) (B. lieheni[brmis (B. mesen-
tericus)): Lane 9: NCTC 1024 (ropey bread isolate) (B. licheni/brmis (B. mesenterieus)); Lane 10: NCTC 1025 (ropey bread isolate) (B. liehe-
n([brmis (B. mesentericus)): Lane I1: NCTC 2599 (B. cereus): Lane 12: NCTC 926 (B. cereus var. mycoides); Lane 13: NCTC 2610
(B. circulans): Lane 14: NCTC 10337 (B. pumilus); Lane 15: NCTC 10335 (B.firmus); Lane 16: NCTC 10343 (B. polymyxa); Lane 17: NCTC
6355 (B. macerans); Lane 18: ATCC 9885 (B. megaterium); Lanc 19: I kb ladder.
64 Jackie M. Thompson, Christine E.R. Dodd, Will M. Waites

type patterns for B. subtilis N C T C 3610 and NCTC 6276. In some cases,
strains with the same API identifications were seen to have different
ribotypes. Two strains with similar patterns of carbohydrate utilization,
both identified as B. pumilus, gave different patterns when ribotyped, and
neither was identical to the banding obtained for B. pumilus NCTC 10337.
However, ribotypes identical or very similar to those shown by some of
the reference Bacillus strains were seen. For example, one isolate gave an
identical ribotype to that shown by NCTC 10341 (B. licheniformis).

CONCLUSIONS

Bacillus isolates that are capable of causing the spoilage of bread and
possibly food poisoning are important to both the manufacturer and the
consumer. Their differentiation by classical and molecular methods can
highlight the areas within the bakery processing line which need to be
controlled to prevent spoilage occurring.

ACKNOWLEDGEMENTS

The authors acknowledge the award of a Ministry of Agriculture, Fisheries

and Food studentship to J.M. Thompson.

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