History of Food Microbiology (A Brief)
CS Custer, USDA FSIS, Bethesda, MD, USA
! 2014 Elsevier Ltd. All rights reserved.
This article is a revision of the previous edition article by N.D. Cowell, volume 2, pp. 1066–1071, ! 1999, Elsevier Ltd.
Food microbiology evolved as tools and techniques revealed
problems and solutions. When Antonie van Leeuwenhoek first
observed ‘animalcules’ in 1676 with his microscope it was
a tool that revealed there was microscopic life around us. When
Louis Pasteur demonstrated that fermentation was caused
by yeasts, not spontaneous germination, and later that heat
could inactivate microbes, he was building on the work of
earlier scientists, such as Girolamo Fracastoro, Agostino Bassi,
Friedrich Henle, and others. But, Pasteur understood the
mechanism that heat killed bacteria and publicized it. Simi-
larly, Robert Koch built on the work of others, developed
a cadre of microbiologists, and provided tools for future food
microbiologists.
Before Pasteur and Koch, people had practical knowledge
about food spoilage and fermentations from experience.
Brewers and breadmakers knew that successful ferments could
be transferred to new batches. The effect of heat on spoilage was
known years before Nicholas Apert won his 12 000 Franc prize,
but he developed a reliable method for food preservation.
Centuries prior, humans had discovered that salting or drying
would prevent food spoilage; however, food microbiologists
began understanding the mechanisms of how these methods
worked and developed more successful and reliable methods.
Methods
Plating
One of the major tools for food microbiologist is the ability to
identify and characterize foodborne microbes. Selective and
differential agar media were and are still a principal tool.
In 1881, to isolate bacterial and yeast colonies, Robert Koch
built on Bartolomeo Bizio’s 1819 technique using polenta and
Schroeter’s 1872 techniques using solid media, such as potato,
coagulated egg white, starch paste, and meat. Koch used gelatin
mixed with meat extracts. According to Hitchens and Leikind’s
1939 paper, “With the aid of this medium and his plating and
dilution method, Koch revolutionized bacteriological tech-
niques. Isolating pure cultures was, in comparison with the
older techniques, enormously simplified.”
Gelatin is a liquid at 37 ! C and many bacteria digest it.
When the gelatin dissolved during Walther Hesse’s experiments
isolating airborne bacteria, his wife, Frau Fannie Eilshemius
Hesse (a housewife from New Jersey) recommended agar agar.
It worked and Hesse communicated the development to his
mentor Robert Koch. In 1882, Koch made the first printed
reference to the use of agar in his preliminary note on the
tubercle bacillus. He also mentioned the cultivation of bacteria
in agar agar in The Etiology of Tuberculosis.
Two years later, Fredrick Loeffler added peptone and salt to
Koch’s basic meat extract for a better bacterial growth. In 1887,
another of Koch’s workers, Julius Richard Petri, modified
a plate with an overhanging lid, thus eliminating the flat plate
and bell jar.
Encyclopedia of Food Microbiology, Volume 2 http://dx.doi.org/10.1016/B978-0-12-384730-0.00165-8
Microbiologists next began developing selective and differ-
ential media. In 1905, MacConkey published a solid medium
using bile salts to inhibit Gram-negative bacteria plus lactose
and neutral red to identify lactose fermenters. In his 1905 paper,
he outlined some of the earlier history of selective and differ-
ential agar media:
A consideration of the fermentation-reactions of the various organ-
isms shows that by the use of certain substances, either alone or in
combination, we can separate organisms by means of colour reac-
tions. Thus, if lactose alone be added to the agar, only the lactose-
fractors will produce acid and show pink colonies; if dulcite alone
only the dulcite fermenters; if sorbite alone only the sorbite
fermenters, and so on. This is the idea underlying all such differential
media as Wurtz litmus-lactose agar, or the litmus-lactose-nutrose
agar of Drigalski and Conradi. . The addition of sorbite gives an
agar upon which the Bacillus typhosus will produce coloured colonies.
In addition to testing human and animal feces (fed different
diets), MacConkey tested milk. His conclusion number 9 of 14
will elicit a smile from current microbiologists: “(9) At present
there is no means of differentiating the lactose fermenting
organisms of human from those of animal origin; or those of
normal dejecta from those found in enteritis.”
Microbiologists quickly investigated additional inhibitors
and indicators. Kessler (1927) and Fischer (1947) presented
excellent summaries of the early development of dyes and
inhibitors. For instance in 1912, Churchman showed that
derivatives of triphenylmethane, such as gentian violet and
brilliant green dyes, were inhibitory to bacteria, particularly
Gram positives, and crystal violet causes some inhibition of
fungi.
With suitable plating media, the science of isolating and
identifying bacterial cultures with selective and differential
plating medium leaped. Although in 1895, the American
Public Health Association began formulating methods for
conducting bacteriological analyses of water, analyses of food
(milk) would take another 15 years. The first edition of Stan-
dard Methods for the Bacteriological Examination of Milk was
published in 1910. According to Mudge (1927), as imple-
mented, those were hardly ‘standards.’ The fifth edition, Stan-
dard Methods of Milk Analysis, was published in 1927. In 1934,
the sixth edition stated, “In view of the extensive alterations
and additions, it is urged. . that they discard the fifth edition
published in 1927 in favor of the new.”
The exact origin of violet red bile (VRB) agar, one of the
standard tools of food microbiology, is elusive. McCrady and
Langevin (1932) employed gentian violet bile and the 2%
brilliant green bile. A number of researchers evaluated VRB:
MacCrady in 1932 for the Committee of Standard Methods of
Milk Analysis of the American Public Health Association,
Bartram and Black for the isolation of coliform bacteria in
raw and pasteurized milk, and Miller and Prickett concerning
213
214 History of Food Microbiology (A Brief)
the recontamination of milk. All found the medium satis-
factory because results were obtained within 24 h of incuba-
tion. Yale, in 1937, evaluated several solid media and
concluded, “The best of the experimental agars proved to be
one now on the market under the name of ‘Bacto-violet red
bile agar.’ This medium permitted the direct plating of 1 ml
quantities of milk and gave results as good as the desoxy-
cholate agar.”
Later, David Mossel tweaked VRB by adding 1% mannitol
(1957) and then glucose (Mossel et al., 1962) so all Enter-
obacteriaceae could be counted.
Sublethal Injury Repair
Selective agar media are great tools for food microbiologists.
Their shortcomings became apparent, however, when food
microbiologists noted that some selective agents would not
recover sublethally injured bacteria that otherwise might
recover in the food. Frank Bustaand Jezeski, in 1963, testing the
thermal death times of Staphylococcus aureus noted that S-110
agar gave lower thermal death times than plate count agar or S-
110 with reduced NaCl. He wrote
The apparently lower thermal death times were found to be related
to the NaCl content of the S-110 medium, because use of S-110 agar
containing lesser concentrations of NaCl resulted in growth of larger
numbers of heat-shocked S. aureus 196E. fewer heat-shocked
S. aureus 196E were detected with S-110 than with PCA. Therefore, it
was felt that heat treatment may have altered the ability of this
organism to grow on the S-110 medium.
A few years later, Scheusner et al., working with Gram-
negative bacteria, investigated which selective agents were the
most lethal, writing
The objective of this study was to identify the specific agents in
selective media that impair the growth of injured Escherichia coli. This
knowledge would be useful in designing new and improved selective
media.
The bile salts mixture alone in the medium prevented as many
injured cells from growing as did any combination of the selective
agents and inhibited as many injured bacteria as were inhibited by
Violet Red Bile Agar itself.
Sodium deoxycholate was the most inhibitory of the bile salts in the
bile mixture.
That information led one of the most innovative food
microbiologists, Paul Hartman, to devise a two-layer plate. The
bottom layer was VRB and the top layer was a nonselective
basal agar. Thus, spread-plated bacteria had a period to recover
before the selective agents diffused to the top. In 1975, the
authors wrote, “If a period of recovery is afforded the injured
cells before exposure to a secondary insult, however, many of
the injured cells become refractive to one or more secondary
stresses.”
In 2003, Busta et al. eloquently summed up the issue of
sublethal injury and the necessity of resuscitation for analyzing
foods:
Microbiological criteria are extremely dependent upon accurate
and precise analyses of the microorganisms present in the sample
of product under test. It is critical that the analytical method
detects injured cells because pathogens existing in the food in
a similar injured state could remain virulent, or could be resusci-
tated later to cause a foodborne infection upon ingestion. Some
contemporary analytical techniques may not suffer from the
same problems if they do not depend on an amplification step
that requires growth in a selective or restricted growth system. With
many methods that utilize selective agents, a nonselective growth
step is needed to allow the opportunity for resuscitation of injured
cells.
Rapid Methods
Early on, food microbiologists sought to develop methods that
were faster, more selective, and cheaper. An old engineering
adage is “There is strong, light, and cheap; pick two, all three
are impossible.” Microbiologists sought speed, sensitivity,
selectivity, and cost.
As early as 1899, scientists had measured physical differ-
ences in the media or food in which bacteria were growing.
Thus, dyes that changed with microbial multiplication were
quickly used in milk. The methylene blue or resazurin reduc-
tion tests were fast and cheap. Thorton and Hastings in a 1930
paper, concluded, “The methylene blue reduction test is as
accurate a measure of the keeping quality of milk as any
method yet available. . It is inexpensive and as nearly fool-
proof as any method for this purpose available to the dairy
bacteriologist.” Five years later, Ramsdell et al. (1935) praised
the advantages of resazurin with, “Only 1 h is required to
complete the resazurin test as prescribed in the text, while the
methylene blue test requires over 5 h.” Other scientists adapted
dye reduction, including tetrazolium reduction, to develop
rapid methods for evaluating the bacteriological quality of
other foods.
Another rapid method for microbiological food quality
was developed by James Jay for beef. The extract release volume
test required 20 min and was based on beef ’s reduction of
water-holding capacity with increasing bacterial levels. Others
adopted Jay’s method for rapid quality test for other products,
including pork and shrimp.
The next step toward achieving ‘Speed, sensitivity, selec-
tivity, and cost’ for detecting pathogens was the development of
nucleic acid probes. Serological tests had been used success-
fully, especially with enzyme-linked immunosorbent assay
(ELISA). Monoclonal antibody development had reduced
cross-reaction problems. But the sensitivity and selectivity
offered by DNA/RNA won out.
In 1981, Walt Hill, building on earlier work, published
a paper on detecting enterotoxigenic E. coli isolates containing
the plasmid for enterotoxin. Hill used a radio-labeled DNA
fragment to hybridize plasmid fragments. Isolation and
detection of the fragments was done with Southern blot elec-
trophoresis. Two years later, Renee Fitts et al. published
a similar method for detecting Salmonella spp. Her team used

restriction enzymes to build libraries of Salmonella DNA frag-
ments and selected those common to most serotypes. These
methods offered sensitivity and selectivity, but speed and low
cost were lacking.
The next step was developing a better method for detecting
those DNA fragments. Luckily in 1983, ‘Kary Mullis conceived
the idea for the polymerase chain reaction.’
The polymerase chain reaction (PCR) is a method of
faithfully duplicating DNA strands to easily identifiable levels
(Mullis received the Nobel Prize for the discovery of PCR in
1993). PCR enabled a leap in detecting foodborne pathogens.
The BAX method for E. coli O157:H7, developed by Dupont
Qualicon in the mid-1990s uses automated PCR. With
enrichment options of 8 h and a processing time of 60 min, the
AOAC certified method offered speed along with selectivity and
sensitivity.
In a 1998 paper, Johnson et al. wrote, “The BAX for
Screening/E. coli O157:H7 assay outperformed the other
methods, with a detection rate of 96.5%, compared to 39% for
the best cultural method and 71.5% for the immunodiffusion
method.”
Speed is important when dealing with perishable food
products, such as produce and raw meats. Beef packers initiated
a procedure in which they sampled combo bins of beef trim.
The sample went to the laboratory, and the bins were shipped
under seal to the customer. When the test result was negative,
the bins were released. If the results were presumptive, the
bins remained under control until the presumptive results
were confirmed negative or positive. Confirmed positive bins
remained under seal and shipped to a cooking facility. By 2001,
BAX was proven to be so selective that many packers no longer
waited for confirmation. Presumptive positive lots were sent for
cooking.
To keep up with the development of rapid methods, one of
Paul Hartman’s students, Daniel Fung, founded the Rapid
Methods Workshop in 1980 (Now the ‘International Rapid
Methods and Automation in Microbiology Workshop and
Symposium’). Two years earlier, P.C. Vasavada founded the
annual ‘Current Concepts in Foodborne Pathogens and Rapid
Methods in Food Microbiology’ at the University of Wiscon-
sin–River Falls, Food Microbiology Symposium. These have
been annual events for many food microbiologists seeking the
latest development in rapid methods.
Viruses
PCR enabled easier and more sensitive detection of viruses in
food. Before nucleic acid assays, virus assays were accom-
plished by separating the virus from the food by filtration or
flocculation. The filtrate was then placed on an appropriate
tissue culture and examined for plaques. Kostenbader and
Cliver described innovations in those methods.
In 1990, Gouvea et al. published the first use of PCR for
detecting and typing rotaviruses from stools. They used the
1987 PCR method published by Mullis and Faloona. More
viruses and techniques quickly followed in clinical samples,
not food. In 1996, Jaykus et al. published the first method for
detecting enteric viruses in oysters using reverse-transcription
PCR. More foods and more viruses quickly followed, and the
methods became routine.
History of Food Microbiology (A Brief) 215
Toxins
Bioassays have been critical for detecting toxins in food.
Potentates used ‘tasters’ to prevent poisoning, but enlighten-
ment and the end of slavery encouraged the use of other
animals. Speed is always desired and toxicologists strove to
develop faster, more sensitive, and specific assays.
Staphylococcal enterotoxin lent itself to more practical
assays. When in 1936, Dolman et al. published the inter-
abdominal kitten test, other in vivo assays (e.g., monkeys) were
abandoned. The Dolman assay offered a tool that led to the
1941 immunological studies by Hammon. That in turn
enabled Berdoll and Casman to further characterize the
chemistry and serology of two enterotoxins in the mid-1950s.
Thus, within another decade, Casman and Bennett, adopting
methods by Ouchterlony and Wadsworth, developed the
Casman–Bennett Staphylococcal Enterotoxin assay. The assay,
because of its flexibility, prevailed for decades.
Greater speed, simplicity, and sensitivity were still desired,
however. In a 1973 international meeting on staphylococcal
enterotoxins at Pennsylvania State, C.E. Park illustrated that
desire. Holding his thumb and finger a fraction of an inch
apart, he said, “Reggie Bennett can detect this amount of
staphylococcal enterotoxin with the Casman-Bennett assay.”
Opening his finger and thumb farther apart, he exclaimed,
“I can only detect this amount.” Closing his thumb and finger
close again, he stated, “But with radioimmunoassay I can detect
this amount.”
Radioimmunoassay had been developed and published in
1960 by Yalow and Berson, federal government workers at the
Veterans Administration Hospital in New York. Yalow and
Berson later received the Nobel Prize for Medicine in 1977 for
a peptide hormone assay. The sensitivity and speed of radio-
immunoassay lent itself to being adopted for many assays; they
offered spectacular sensitivity over immunoassays, but the
equipment and protocols were expensive. That was to soon
change with ELISA.
In 1971, Van Weemen and Schuurs, in the Netherlands, and
Engvall and Perlmann, in Sweden, separately developed and
published enzyme-labeled assays. The simplicity and porta-
bility of the method caught on. In 1976, George Saunders
working at Los Alamos Laboratories under a US Department of
Agriculture (USDA) grant demonstrated the method to two
USDA scientists. In a moment of inspiration, he applied
a double sandwich procedure to a staphylococcal enterotoxin
sample and had a positive in 15 min. With additional devel-
opment, that method was published in 1977. In 2005, Lequin
published an excellent review of the history of radioimmuno-
assay and enzyme-labeled assays.
Botulinal toxin is the most lethal foodborne toxin. The
mouse bioassay was an early development and, despite
advances in immunoassays, it remains the most common
means of detecting the deadly neurotoxin. There are five known
serotypes and a mouse reacts with all giving the typical pinched
waist and labored breathing symptoms of a neurotoxin.
Still, there have been attempts for in vitro assays. In 1966,
Food and Drug Administration (FDA) scientists Johnson et al.
attempted to develop an in vitro serological assay for serotypes
A, B, and E using hemagglutination and bentonite flocculation.
They were partially successful stating, “The antitoxin-sensitized
216 History of Food Microbiology (A Brief)
SRBC technique is extremely sensitive, detecting toxins at levels
of less than one to several LD50 per 0.5 ml.”
Two years later, other FDA scientists, Vermilyea et al.
(1968), published their efforts to adopt the Casman–Bennett
immunodiffusion assay for staphylococcal enterotoxin to
botulinal toxin. They concluded, “Perhaps lower concentra-
tions of toxin can be detected by the gel-diffusion techniques, if
antitoxin with higher biological activity is used.”
Improved production methods yielded antiserums with
higher specificity. That, together with the development of the
ELISA method, led to Notermans et al. publishing their assay
for botulinal toxin A in 1978. That was soon followed by assays
for botulinal toxins E and G. Two excellent reviews on botu-
!
linal toxin assays are Capek and Dickerson (2010) and Sharma
and Whiting (2005).
Microbiological Control
In two papers written in 1989 and 1993, David Mossel
described ‘ Wilson’s Triad’ as an early system for controlling
food safety. As described by both Mossel and Wilson, there
were five components to the triad: (1) heat treatment or
pasteurization to kill pathogens, (2) posttreatment sanitary
handling to prevent contamination, (3) cooling to stabilize
the treated product and prevent outgrowth of heat-resistant
microbes, (4) employing testing to select sound ingredients, and
(5) controls (e.g., temperature) for the first four components.
Treatments
Heat
The most famous early application of heat is Pasteur’s heating
wine to prevent souring. Although a century earlier scalding of
cream had been advocated to increase the shelf life of butter,
the mechanism of destroying bacteria was not understood.
Louis Pasteur and his colleague Claude Bernard completed
their first test of heating liquids to kill bacteria and molds on 20
April 1862.
Milk
On the heels of (1882) Koch’s germ theory came the connec-
tion of tuberculosis with tubercular cows. According to Wiki-
pedia, “Pasteurization of milk was suggested by Franz von
Soxhlet in 1886.” In the United States, pasteurization was first
used in the 1890s. The New York City Board of Health issued
an order requiring the pasteurization of milk in 1910. In 1924,
the US Public Health Service developed the Standard Milk
Ordinance, today known as the Pasteurized Milk Ordinance.
Canning
Nicolas Appert had established a food preservation plant in
1804, decades before Pasteur. In January 1810, Appert won
a 12 000 Franc prize for his technique of preserving food in
sealed bottles and boiling them for hours. Appert’s nephew,
Raymond Chevalier-Appert, adapted Denis Papin’s 1679
‘steam digester,’ a pressure cooker, to his uncle’s process and
patented it in 1851. In 1860, Isaac Solomon a Baltimore
tomato canner, added calcium chloride to the water, thus
raising the boiling point and reducing the process time from
between 5 and 6 h to less than 1 h.
But there were still swollen cans. Wanucha (2009) reported,
that in 1897 William Underwood sought help from MIT. He
was passed to Samuel Cate Prescott, a chemist. Prescott found
that the swollen cans of Underwood Potted Meat were full of
bacteria; bacteria that could survive hours of boiling. Addi-
tional experiments showed that 10 min at 120 ! F would
destroy the bacteria.
That heat treatment was fine for those small cans but not for
larger containers. Later, C.O. Ball’s 1923 and 1927 papers,
pioneering thermal death time research for Clostridium botu-
linum and establishing Fo and Z values, was improved on by
others in ensuing decades – e.g., Stumbo and Longley in 1966
and Stoforos in 2010. Additional work over the following
decades improved the required treatments and also character-
ized the conditions that affected heat resistance. In a 1936
paper, Williams wrote,
A great deal of work on heat resistance has therefore been done, and
it has been found that the resistance of any particular species is not
fixed but varies with several factors. The conditions under which
the spores are produced as well as their age and previous treatment
apparently are important. The concentration of spores, pH of the
substrate in which they are heated, and the presence or absence of
protective colloids or salts are also factors.
Clostridium botulinum was not the most heat-resistant bacte-
rium. Fortunately, the more heat-resistant bacteria, such as
Clostridium thermosaccharolyticum (now Thermoanaerobacterium
thermosaccharolyticum), are not toxigenic or pathogenic and do
not ordinarily multiply at room temperature. McClung charac-
terized and named this bacterium from cultures isolated from
swollen cans and soils. As a colleague once quipped, the sound
of cans of chili exploding in the warehouse during the Texas
summer underlined that C. thermosaccharolyticum was not a rare
bug. Thus, canned foods destined for ‘tropical service’ require
much higher heat processes than needed to destroy C. botulinum.
Surrogates
Because noninfective or nontoxigenic strains are safer to use,
especially in food plants, food microbiologists found surro-
gates to substitute for the pathogen in validating interventions.
For instance, the target organism for canning was C. botulinum;
however, a nontoxigenic surrogate soon became the preferred
organism to use. According to Bradbury et al., the surrogate –
a strain of Clostridium sporogenes designated Putrefactive
Anaerobe (PA) 3679 (ATCC 7955, NCTC 8594) – originally
was isolated from spoiled canned corn in 1927.
With the advent of hazard analysis and critical control
points (HACCP) and the importance of validating and veri-
fying processes, the importance of surrogates has increased. In
2010 the National Advisory Committee on Microbiological
Criteria For Foods (NACMCF) published a paper on protocols
for validating processes including using surrogates. A copy
is available at http://www.fsis.usda.gov/ophs/nacmcf/2004/
nacmcf_pasteurization_082704.pdf.

Subsequently, Marshall et al. (2005), Liu and Schaffner
(2007), Niebuhr et al. (2008), and Sinclair et al. (2012) pub-
lished papers on the use of and criteria for surrogates.
Meat
Except for milk and canning, little research appeared to have
been done on inactivating infective bacteria in meats. Trichinae
in pork, however, were a major concern. Control of the
nematode was achieved primarily by microscopic examination
of diaphragm tissue to certify that the carcass was trichinae free.
In the early part of the twentieth century, USDA scientist
Brayton Ransom published a series of papers on destroying the
nematode by heat, freezing, or curing. His research had effects
on later developments in bacterial meat safety.
In Ransom and Schwartz’s (1919) paper on heat, the
authors wrote, “The vitality of the larvae of Trichinella spiralis is
quickly destroyed by exposure of the parasites to a temperature
of 55 ! C (130 ! F), gradually attained.” The key phrase was
‘gradually attained.’ In the body of the paper, the authors rec-
ommended 137 ! F/58.3 ! C as the endpoint temperature, add-
ing that only large pieces of pork are cooked. That temperature
remained the standard until the mid-1980s when Food Safety
and Inspection Service (FSIS) amended the regulation, 9 Code
of Federal Regulations (CFR) 318.10(c), based on research by
Agricultural Research Service (ARS) scientist Tony Kotula. The
amendment replaced the single 137 ! F/58.3 ! C with a time
temperature table ending at 144 ! F/62.2 ! C, which was close to
the prescribed 7D Salmonella kill.
Poultry
In the early 1960s the USDA ARS research group recom-
mended cooking poultry to 160 ! F/17.1 ! C based on research
in poultry rolls. That temperature was promulgated into
the 9 CFR 381.160 and remained the standard until FSIS
replaced it with a Salmonella Performance Standard a half
century later.
Thus, milk, pork, and poultry – but not beef – had prescribed
minimum heat treatments. Following a series of New Jersey
salmonellosis outbreaks in beef in 1977, USDA Food Safety
Quality Service (FSQS now FSIS) published an emergency rule
prescribing a minimum temperature of 145 ! F/62.8 ! C based
on previous work by Angelotti who had just become the FSQS
administrator. The beef industry complained that the 145 ! F/
62.8 ! C requirement would prohibit rare roast beef. They also
responded with Goodfellow and Brown’s research validating
a series of times and temperatures ranging from 120 to 145 ! F
that would yield a 7D Salmonella kill.
The new times and temperatures ranged from 130 ! F
(121 min) to 145 ! F (instant) and were promulgated as 9 CFR
318.17. Goodfellow and Brown’s validation also showed that
the salmonellae on the surface of dry-roasted beef rounds were
more heat resistant than those in the interior. That surface heat
resistance was substantiated by USDA ARS Roy Blankenship in
two additional papers.
Those times, temperatures, and conditions were published
in 9 CFR 318.17 until replaced by Performance Standards in
1999. The times, temperatures, and conditions as ‘safe harbors’
were copied into FSIS’ Appendix A: Compliance Guidelines for
History of Food Microbiology (A Brief) 217
Meeting Lethality Performance Standards for Certain Meat and
Poultry Products, which are available at http://www.fsis.usda.
gov/oa/fr/95033f-a.htm.
In the antiregulation period of the 1980s, only products that
had been linked to an outbreak were covered by the rule. Thus,
while corned beef was regulated, pastrami was not. Similarly,
ground beef was not covered until 1993 after three outbreaks
culminating in the 1993 Jack-in-the-Box outbreak. Using data
from Line et al., FSIS promulgated times and temperatures for
hamburgers into 9 CFR 318.23.
Heat Resistance and Solute Concentration
That the salmonellae on the surface of dry-roasted beef rounds
were more heat-resistant than those in the interior was
surprising to a few but not to most food microbiologists.
Decades earlier, it was known that dry heat was less lethal than
wet heat. Thus, while dry glassware required 250 ! F for 2 h to
sterilize it, wet heat required only 121 ! C for 15 min. Blan-
kenship (1978) wrote, “The enhancement of heat resistance by
reduced water activity among microorganisms is well docu-
mented.” And Goodfellow and Brown postulated, “that the
unexpected survival of Salmonella inoculated onto the surface
of beef rounds was due to rapid dehydration of the inoculated
organism which in turn resulted in increased heat resistance.”
Williams (1936) noted that factors affecting the heat resis-
tance of spore-forming bacteria included ‘pH of the substrate in
which they are heated, and the presence or absence of protec-
tive colloids or salts are also factors.’ Others began doc-
umenting the effects on non-spore-forming bacteria. In 1966,
Calhoun et al. published the effects of salt or glucose on the
heat resistance of Salmonella, Pseudomonas fluorescens, and S.
aureus. That paper was followed in 1970 by Baird-Parker et al.
and Goepfert et al. publishing separate papers on the effect of
water activity (aw) on heat resistance of salmonellae and other
Gram-negative bacteria. Goepfert et al.'s paper showed sugar
was particularly protective. In sucrose, at aw 0.96 other Salmo-
nella serotypes approached the heat resistance of Salmonella
senftenberg 775W, and at aw 0.93, some exceeded its heat
resistance. These results were similar to Goepfert and Biggie's
(1968) paper in which Salmonella typhimurium was more heat
resistant in chocolate than S. senftenberg 775W. For a more
recent review, see Doyle and Mazzotta (2000).
The strain, S. senftenberg 775W, would become included in
inoculated cocktails for heat-resistance studies, including
Goodfellow and Brown. Winter et al. first reported the
extraordinary heat resistance of a strain of S. senftenberg in
1946. It survived almost 5 min of heating at 60 ! C in liquid
egg. Solowey et al., in 1948, designated the strain as 775W. In
1969, USDA ARS scientist, Henry Ng characterized the heat
resistance of 296 strains representing 75 Salmonella serotypes,
including S. senftenberg 775W. He noted, “.S. blockley 2004
was 5 times more heat-resistant and S. senftenberg 775W was
30 times more heat-resistant than S. typhimurium Tm-1, the
reference strain in this study.” Ng also characterized the effect
of growth temperature, growth phase, and substrate on the
heat resistance of those salmonellae. The next year, Baird-
Parker et al. reported finding two isolates, an S. senftenberg and
a strain of S. Bedford, in the United Kingdom with similar heat
resistance to 775W.
218 History of Food Microbiology (A Brief)
Fermented Sausage
The treatment prescribed for inactivating trichina in pork led to
procedures for inactivating E. coli O157:H7 in fermented
sausages. In the early 1980s, FSIS questioned the adequacy of
validation used for proprietary trichina treatments by Swift &
Co. Earlier, ARS scientists had shown that salmonellae could
survive some fermented sausages processes. Swift scientists
responded positively and validated a new procedure. That
procedure was later promulgated as 9 CFR 318.10(c)(3)
Method No. 7. The new Method No. 7 added a low temperature
(125 ! F/51.7 ! C) heat treatment that was claimed also to
inactivate any salmonellae in the raw pork.
When the November 1994 E. coli O157:H7 outbreak from
fermented dry salami occurred, industry associations looked
for remedies. The Blue Ribbon Task Force of the National
Cattlemen’s Beef Association funded the Food Research Insti-
tute to conduct validations for safe processes. John Luchansky
at the Food Research Institute chose 9 CFR 318.10(c)(3)
Method No. 7 as a starting point. The result was a 1996
booklet, by Nickelson et al., listing time, fermentation
temperature, and pH that would result in a 5 log kill for E. coli
O157:H7 using the low-heat Method No. 7. Those results were
later published in peer-reviewed journals by Calicioglu et al. as
well as Kasper and Luchansky.
7D vs 6.5D
In 1977, when FSQS (now FSIS) amended the roast beef rule
(9 CFR 318.17) to include the time and temperatures validated
by Goodfellow and Brown, they prescribed the 7D kill suggested
by Angelotti in 1961. The basis of that standard was reputedly
the highest level of salmonellae encountered in a ground beef
product plus a 2D safety margin. Similarly, the 5D standard
for E. coli O157:H7 in beef was based on the highest level found
in beef during an outbreak in New Jersey plus 2D. FSIS revisited
the 7D Standard with the proposal of Salmonella Performance
Standards for the HACCP rule. FSIS used the levels of salmo-
nellae in beef or poultry from FSIS’ baseline program and
nationwide surveys. FSIS concluded, “Once the number of
organisms in raw product is determined, it is possible to esti-
mate the probabilities of the number of surviving organisms
for a given x-log10 lethality reduction process.” Those data
resulted in a prescribed 7D kill for poultry products and a 6.5D
kill for beef products. The report, “Lethality and Stabilization
Performance Standards for Certain Meat and Poultry Products:
Technical Paper,” FSIS 1998, is available at http://www.fsis.usda.
gov/OPPDE/rdad/FRPubs/95-033F/95-033F_tech_paper.pdf.
Alternate Methods to Inactivate Microbes
In addition to heat, other means to inactivate bacteria in foods
have been researched over the past century, including irradia-
tion (gamma, X-ray, electron), high pressure, pulsed electric
fields, pulsed light, ohmic and inductive heating, microwave,
oscillating magnetic fields, and ultraviolet light. Currently,
the two most successful alternate methods are irradiation and
high pressure.
According to Jay et al., B.E. Proctor in the United States was
the first to employ the use of ionizing radiation to preserve
hamburger meat in 1943. The history of using irradiation for
inactivating bacteria goes back another 39 years, however. In
1904, Samuel C. Prescott (the same who aided Underwood
with canning), described the bactericide effects of irradiation
from radium on yeast and what are now known as E. coli and
Corynebacterium diphtheriae. There were additional papers on
the destruction of pathogens by irradiation, including
Schwartz’s (1921) paper on destroying T. spiralis. Sixty-four
years later, in 1985, the FDA approved irradiation for the
control of T. spiralis.
Summary
This history of food microbiology is incomplete because food
microbiology is evolving rapidly with new tools and tech-
niques for discovering, inactivating, and preventing foodborne
diseases. Space restricted the inclusion of the history of indi-
vidual pathogens, control programs, stabilizing foods, and
controversies over issues, such as irradiation and nitrite.
A half century ago Salmonella, S. aureus, and C. botulinum
were the foodborne pathogens. Prions, Campylobacter spp., and
Shiga toxin-producing Escherichia coli (STECs) were not on the
list. Since that time, we have learned that benign organisms
such as E. coli and Citrobacter can acquire virulence factors and
become pathogenic. Perhaps, in the future, microbiologists will
assay for virulence factors, not genera and species. A century
from now, microbiologists will smile on our ignorance. Let us
be envious of their knowledge and continue to build on the
foundations of their discoveries.
See also: Clostridium : Clostridium botulinum; Clostridium :
Detection of Neurotoxins of Clostridium botulinum; Escherichia
coli: Escherichia coli; Fermented Meat Products and the Role of
Starter Cultures; Heat Treatment of Foods: Principles of
Canning; Heat Treatment of Foods – Principles of
Pasteurization; Staphylococcus: Detection of Staphylococcal
Enterotoxins; Trichinella; Virology: Detection; Indicator
Organisms; Nonthermal Processing: Irradiation; Identification
Methods: Introduction; Injured and Stressed Cells.
Further Reading
Older original papers published by the American Society for Microbiology are free of
charge as are those with a URL. Other papers are available by membership,
libraries with subscriptions, or purchase from the publisher.
Angelotti, R., Foter, M.J., Lewis, K.H., 1960. Time-temperature effects on Salmonellae
and Staphylococci. In: Robert, A. (Ed.), Foods. II. Behavior at Warm Holding
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Sanitary Engineering Center, Cincinnati, Ohio.
Angelotti, R., Foter, M.J., Lewis, K.H., 1961. Time-temperature effects on Salmonellae
and Staphylococci in foods. III. Thermal death time studies. Applied Microbiology 9,
308–315.
Baird-Parker, A.C., Boothroyd, M., Jones, E., 1970. The effect of water activity on the
heat resistance of heat sensitive and heat resistant strains of Salmonellae. Journal
Applied Bacteriology 33, 515–522.
Ball, C.O., 1923. Thermal process time for canned food. Part I, No. 37. Bulletin of the
National Research Council 7.
Ball, C.O., 1927. Theory and practice in processing. The Canner 64 (5), 27. (Cited by:
Stumbo. C.R., Longley, R.E., 1966. New parameters for process calculation. Food
Technology 20 (3), 341–345.)

Bartram, M.T., Black, L.A., 1936. Detection and significance of the coliform group in
milk. Journal of Food Science 1, 551–563.
Bergdoll, M.S., 1956. The chemistry of staphylococcal enterotoxin. Annuals New York
Academy of Science 65, 139–143.
Blankenship, L.C., June 1978. Survival of a Salmonella typhimurium experimental
contaminant during cooking of beef roasts. Applied and Environmental Microbiology
35 (6), 1160–1165.
Blankenship, L.C., Davis, C.E., Magner, G.J., 1980. Cooking methods for elimination of
Salmonella typhimurium experimental surface contaminant from rare dry-roasted
beef roasts. Journal of Food Science 45, 270–273.
Bradbury, M., Greenfield, P., Midgley, D., Li, D., Tran-Dinh, N., Vriesekoop, F.,
Brown, J.L., 2012. Draft genome sequence of Clostridium sporogenes PA 3679,
the common nontoxigenic surrogate for proteolytic Clostridium botulinum. Journal
Bacteriology 194, 1631–1632.
Busta, F.F., Jezeski, J.J., 1963. Effect of sodium chloride concentration in an agar
medium on growth of heat-shocked Staphylococcus aureus. Applied Microbiology
11, 404–407.
Busta, F.F., Suslow, T.V., Parish, M.E., Beuchat, L.R., Farber, J.N., Garrett, E.H.,
Harris, L.J., 2003. The use of indicators and surrogate microorganisms for the
evaluation of pathogens in fresh and fresh-cut produce. Comprehensive Reviews in
Food Science and Food Safety 2 (Suppl. 1), 79–185.
Calhoun, C.L., Frazier, W.C., Wisconsin, U., 1966. Effect of available water on thermal
resistance or three nonsporeforming species of bacteria. Applied Microbiology 14,
416–420.
Calicioglu, M., Faith, N.G., Buege, D.R., Luchansky, J.B., 1997. Viability of Escherichia
coli 0157:H7 in fermented semidry low-temperature-cooked beef summer
sausage. Journal of Food Protection 60 (10), 1158–1162.
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!
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