Very good work, please see my feedback below.
Points: 44/50
Dr. Singh
Food Microbiology
30 September 2021
Assignment 1
General Microbiology and Historical Perspectives
Any discussion on microbiology must begin with an understanding of microorganisms,
which fall into two main categories: prokaryotes and eukaryotes. These organisms have survived
on Earth for more than three billion years; the prokaryotes are classified as unicellular organisms
that lack a nucleus or membrane-bound organelles. On the other hand, Eukaryotes are
multicellular with a true nucleus, and their organelles are, in fact, membrane-bound.
The sub-categories of microbes come in the form of bacteria, fungi, algae, viruses,
protozoa, and helminths. These microbes have a myriad of uses to us as humans; many microbes
are harmful. However, various microbes benefit us in different ways. They are a double-edged
sword; on one side, they are used to produce some foods like yogurt or alcohol, on the other side,
they can cause diseases such as botulism or AIDS.
One of the most notable structures of bacteria in relation to microbiology, in general, is
the cell wall. There are two types of cell walls in bacteria that lead to two classifications known
as gram-positive or gram-negative. At first, this distinction refers to the difference in appearance
between the two types of bacteria after Gram staining. Gram-positive bacteria will appear purple
under a microscope due to the thick peptidoglycan wall with teichoic acids embedded in the wall.
Gram-negative bacteria appear pink or red due to their relatively complex wall with a thin
peptidoglycan layer and outer lipopolysaccharide filter. Staining is carried out since bacteria
have a similar refractive index as water and are near impossible to see under a microscope alone.
It allows researchers to more clearly see the organelles and assist in ruling out many other
possible microbes.
It can be said that microbiology may have begun with food microbiology as the first
practice applications were used in food. Alcohol, cheese, and bread fermentation techniques are
some of the original, ancient crafts employed thousands of years ago, around 6000 BC. I will
further discuss the history of food microbiology in specific at a near point in this write-up.
However, the significant beginning of microbiology can be attributed to Louis Pasteur. He
presented the idea that fermentation does not spontaneously create organisms through chemical
reactions but that there were organisms, to begin with.
Fast-forwarding microbiology to today, the Food and Drug Administration began with
the 1906 Pure Food and Drugs Act which brought together many bills over a long period to halt
the abuse of the consumer marketplace. This act and the FDA’s formation can be attributed to
Harvey Washington Wiley’s efforts towards public health protection “In response to the public
outrage at the shockingly unhygienic conditions in the Chicago stockyards that were described in
Upton Sinclair’s book ‘The Jungle.’” (FDA, 2018). The Meat Inspection Act was also passed
that day after awareness of abysmal and unsanitary meat handling, dangerous preservatives and
dyes, and baseless medications were shed to light. The FDA brought a federal standing to the
establishment of public health.
Historical Highlights in Food Microbiology
To expand upon the milestones of general microbiology listed above, 10 significant discoveries
in the field of food microbiology are listed below. All of these accomplishments have increased
the understanding of microbial growth and control in foods and helped define food safety
regulations.
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4000 B.C.E. +: Early food preservation methods were recorded, including drying,
pickling, and fermenting. Although these methods were generally effective for short term
food storage, they were not regarded as safe for long-term storage.
1800: Nicolas Appert invented canning during the French Revolutionary War as a
response to the needs to preserve food from spoilage. His discovery offered a new way to
preserve food and increase storage life, by heating it and cooling it under vacuum, but the
process was not well understood until many decades later.
1850: Nicolas Appert and Raymond Chevallier-Appert improved the canning process
efficiency and quality of canned food using steam sterilization.
1880: A. A. Gartner isolated Salmonella enterica serovar Enteridis from a man who
became ill from eating contaminated meat. This was one of the first documented
instances of Salmonella food poisoning.
1890: Milk was pasteurized for the first time.
1900: Upton Sinclair exposed the meat industry for its harsh labor conditions and
corruption, and the Food and Drug Act (FDA) was passed in response.
1930: Refrigeration became available as a household commodity and replaced cellars and
iceboxes as primary ways of keeping food cold. The FDA also began to enforce
regulations over food production and processing.
1980: E. coli O157:H7 was identified as a food pathogen, and the first genetic probe
using PCR was developed for Salmonella.
1990: Irradiation was developed and approved as a new method to control pathogenic
bacteria in the meat and poultry industries.
2000: Many improvements in food safety regulation occurred during this decade
including irradiation of other food types (vegetables and eggs) and high pressure
processing (USDA). The Food Safety Modernization Act was also established.
Microbial Growth, Survival, and Enumeration, History of Food Microbiology
Beginning with growth, referring to the growth of a population of microbial cells rather
than the growth in size. Prokaryotic and bacterial microbes mainly replicate through the process
of binary fission, where a single DNA molecule replicates and attaches to different sides of the
cell as it splits apart into two genetically identical components. This binary fission is known as
reproductive doubling, where proliferation occurs in doubles as fission continues approximately
every twenty minutes.
Microbial growth can be described by a growth curve that comes in stages. Initially, the
lag phase is the point at which a bacterium is exposed to a new environment and must adapt to it
in order to survive. All living organisms have conditions that must be meant for their life to
continue, and microbes are no different. Once the bacteria have adapted to their environment,
they will move into the log, or exponential, phase. This is known as the log phase because the
population experiences exponential, or logarithmic, growth in a short amount of time. Following
the log phase, the stationary phase shows no change in population size as the bacteria uses up an
abundance of nutrients and growth enablers. Eventually, the death phase shows a decrease in the
bacterial population as there are no more nutrients available and the accumulation of toxins and
waste from the exponential growth earlier.
It is important to note that the number of cells at any phase results from the growth rate
against the death rate. The log phase sees exponential proliferation because, at that time, the
growth rate far surpasses the death rate. The stationary phase sees no change in growth because
the growth rate and death rate are equal. Logically, then, the death phase is the result of the death
rate outperforming the growth rate (adopted from Ref: Singh et al., 2021).
For food microbiologists, the goal is to extend the lag phase as long as possible to
ultimately stop the spoilage of food and the presence of foodborne illness. If it is difficult for a
bacterium to adapt to its environment, it will not enter the log phase and proliferate to damage
the food eventually. Depending on the microbe itself, there are different mechanisms for survival
based on the change in conditions. I will speak further on intrinsic and extrinsic factors that
affect growth. However, one survival strategy involves the acid tolerance response of Salmonella
in the stationary phase after experiencing acid shock. The sigma factor encoded by the rpoS
locus “enhances the ability of stationary-phase cells to survive under hostile environmental
conditions” by helping RNA polymerase to transcribe genes encoding for starvation proteins
(Montville et al., p. 213). Another example of a survival strategy in response to a stimulus would
be how cold shock proteins act as chaperone proteins to reduce mRNA folding in colder
temperatures that would otherwise be difficult for proliferation.
Successful survival and proliferation would lead to enumeration wherein a microbiologist
identifies the number of viable microbes in a sample. There are multiple methods available with
various levels of accuracy, time efficiency, and cost. A direct microscopic count (DMC) assay
involves uniformly spreading a sample of food or liquid onto a microscope slide. The
microbiologist counts the cells in a specific precinct of the field. The average number of cells per
field is then used to calculate the number of cells in the entire sample with high specificity. This
method is notably quicker than other enumeration techniques as it does not require an incubation
period for the cells to proliferate (Montville et al., 213).
As mentioned earlier, the history of food microbiology can be understood to be started by
Louis Pasteur. His swan-necked flask experiment proved that contamination of nutrient-rich
broth results from microbes in the air trapped in the neck of the flask. This discovery disproved
the idea of spontaneous generation facilitated by air with enzymes and chemicals. It promoted
the thought that life is required to bring about life, known as the Theory of Biogenesis
(Worcester Medical Museum). He is also responsible for the Germ Theory of Disease and
showing that certain microorganisms are responsible for fermentation, leading to his discovery
that heat application can reduce the number of bacteria in the aptly named pasteurization process.
Joseph Lister is known as the Father of Modern Surgery as he found that requiring his
surgeons to wash their hands in carbolic acid drastically reduced the number of gangrenous
wounds that were so fatal in surgeries at the time (Bacover, 2019). Robert Koch expanded on
much of Pasteur’s work, presenting his postulates such as “The specific disease must be
reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host” and
that “bacteria must be present in every case of the disease,” which provided a benchmark to
understanding the relationships between bacteria and clinical diseases (Stoppler, 2021).
Paul Ehrlich’s theory that therapeutic agents are dependent on their side chains was that
backing for his synthesizing the first drug to treat infections, what he would call a ‘Magic
Bullet.’ This bullet was known as Salvarsan, a synthetic derivative of arsenic that effectively
treated syphilis patients until the invention of antibiotics (Cook & Naglak, 2020). Another
microbiologist, Edward Jenner, is renowned “For his innovative contribution to immunization
and the ultimate eradication of smallpox” by developing vaccines that inoculated patients
initially through cowpox (Riedel, 2005). Preceding many of these microbiologists, Nicolas
Appert invented the concept of canning foods as a method of preservation. He was unsure
exactly why this method worked, Pasteur later proving it with his Theory of Biogenesis, but
Appert reasoned that it worked with wine. Therefore it should work with food – first boiling it in
a bath then sealing tightly to prevent air contact (Famous Inventors).
Microbial Growth:
The bacterial growth curve consists of the below 4 distinct phases, where the microbes reproduce rapidly
under favorable conditions at an exponential rate for population growth. The four phases are as follows:

Lag phase: During this initial phase, the bacteria are adapting to their environment and preparing
for replication and increase in size and cell number. The cells cannot divide during this stage;
therefore, they keep maturing, synthesizing new components and preparing for the next phase.
During this time, the “duration of the lag phase is determined by many factors, including the
species and genetic make-up of the cells, the composition of the medium, and the size of the
original inoculum” (Anonymous, n.d.).

Log (exponential) phase: The cells are dividing rapidly by binary fission. This phase is important
because the growth rate is maximal and constant. Furthermore, the “population is most uniform in
terms of chemical and physical properties” and the cells display balanced growth (Singh, 2021).
The population also doubles every generation because the conditions are favorable. Microbes are
the most active in their middle and late exponential phase because they have the most primary
and secondary metabolite production.

Stationary phase: At this point, the cells are still metabolically active but the population starts to
die as there are a number of unfavorable conditions, such as nutrient limitation, endospore
formation, toxic waste accumulation and limited oxygen availability (Singh, 2021). The rate of
the bacterial cell growth equals the cell death. This is the stage where cells are on survival mode
and are trying to figure out different ways to stay longer since there are not enough nutrients and
space. Additional changes that the cells experience are nucleotide condensation, protoplast
shrinkage, increase of starvation proteins as well as decrease in size.

Death phase: The population starts to die in greater numbers. Waste products increase, the
nutrients are less available and the living cells decline exponentially in this final phase.
Figure 1. The growth curve is represented by this logarithm of living bacterial cells over a period of time.
The 4 distinct phases of a bacterial growth curve (lag, log, stationary and death phases) are shown in this
figure.
Survival and Enumeration:
Enumeration refers to the number of organisms that are growing in a sample, which involves small
dilutions and large number of cells. The growth of microorganisms is determined by their number under
various conditions. In some enumeration methods, the sample, which represents a 100 dilution, is diluted
in a 10-fold dilutions before enumeration and “to enumerate the bacteria, 0.1 ml from each dilution tube is
plated on an agar plate of purpose medium” (Montville et al., 2012, p.16). The colonies are counted after
incubation time and the number is multiplied by the dilution factor. The Most Probable Number (MPN)
and Colony Forming Units (CFU) are used to determine the number of viable microorganisms in the
sample tested. MPN is used for liquid broth and CFU is used for solid agar samples. MPN differs from
CFU because it is an estimation method, less precise, estimates viable numbers of microorganisms in 10fold dilutions as well as works “only when low levels (<30 CFU/ml) are present (Montville et al., 2012,
p.16). On the other hand, CFUs are more precise and the results are based on the number of colonies. The
plate count method identified the number of organisms that are able to grow on a given medium after
incubation.
Understanding the different intrinsic and extrinsic factors that are affecting the microbial growth in
various foods helps food scientist and microbiologists reduce food contamination as well as the survival
chances of microorganisms, such as yeasts, mold and bacteria. The growth of microorganisms is
influenced by factors like temperature, oxygen, moisture and pH; therefore, controlling these factors
reduces food spoilage, transmission of diseases and unwanted microbial contamination.
Intrinsic and Extrinsic Factors Affecting Microbial Growth in Varying Food
Various factors affect a microbe’s ability to grow in food. Just like how all living
organisms have specific parameters required for continuous life, microbes need specific food,
acidity, time, temperature, oxygen, and moisture levels to optimize their proliferation. The food
environment must first be ideal; starch, moisture, and protein levels are involved, such as in raw
meat. The nutrients available to the bacteria must also be accounted for as they are a source of
energy, water, vitamins, ion concentration, and minerals that perpetuate growth. The pH level is
an intrinsic factor as microbes prefer neutral to somewhat acidic pH levels from food in general
for growth. There are many bacteria and some known as acidophiles, that prefer more acidic
environments, but this is not the case for the most part. Exemplefeficatied by the fact that
fermentation is a popular method for preservation as the very acidic environment halts microbial
growth. If the pH of the environment is out of the microbe’s ability to survive, then it will not
grow; similarly, human beings experience severe problems if blood pH changes even slightly
from its regular reading.
Moisture is another intrinsic factor to food and is measured by water activity on a scale of
0 to 1 Aw, dry to pure water. Microbes usually need higher than 0.9 Aw for growth, which is
why dehydrating, freezing, and salting are all efficacious methods for preserving foods from
bacterial growth. This is why foods such as rice, dry beans, and nuts can be stored away for long
periods of time without the need for preservation methods. The extrinsic factor of time is critical
for microbial growth concerning the growth curve mentioned earlier – the goal is to prolong the
lag phase as much as possible to keep food sanitary for consumption. This concept is commonly
exemplified by expiration dates on perishable foods and “use-periods” for foodservice or
retailers (CANR, 2001).
Temperature is another extrinsic factor that, similar to pH, has many outliers that survive
in extreme heat or cold climates. However, generally speaking, 40-140° Fahrenheit is the danger
zone, and it is advised to keep temperatures below or above this zone. Cold temperatures have
been shown to decrease the doubling time of bacterial growth, which increases the shelf life,
which explains why freezing is a viable preservation technique, in conjunction with the fact that
it also reduces water activity. Some cells may undergo homeoviscous adaptation where they
synthesize double bonds in fatty acid chains preventing solidification and allowing for gene
expression of compatible solutes at cold temperatures to be used by bacteria.
The presence of oxygen is another extrinsic factor that modulates the growth of bacteria,
and it is seen in two primary forms. Some microbes are aerobic, meaning they require oxygen to
grow, and others are anaerobic, and the presence of oxygen can be fatal. There are, of course,
many sub-categories of these first two because there are many kinds of microbes. They also
differ in how much oxygen they need or whether oxygen is a preference rather than a
requirement. Oxygen sensitivity is essential for fresh produce as ozone can disinfect apples,
lettuce, berries, grapes, and more from Escherichia Coli O157:H7 as it yields toxic, reactive
oxygen species such as superoxide or hydroxide radicals (Montville et al., 481). Another
atmospheric gas that is very important to food preservation is carbon dioxide since it is used in
modified-atmosphere packaging that prevents spoilage by pathogenic microbes.
Something to keep in mind with all these factors is that they do not act in manners
exclusive to each other. Some variation in extrinsic factors may cause fluctuations in intrinsic
factors, creating compounding, deleterious effects on growth. As mentioned earlier, freezing
temperatures are a valuable method of preservation as it increases the time needed for
reproductive fission, halts the growth of thermophilic bacteria, and decreases the moisture of the
food host by turning the water into ice. Placing foods into conditions that are not favorable for
microbes to survive is the mission of increasing lag time and preserving foods.
Cell Stress, Sub-lethal Cell Damage Affecting Culturing Ability of the Cell
There are two main categories of cells that have undergone some form of stressor. Some
cells may be injured, and other cells may be viable but non-culturable (VNC). This stress was
discussed in the section just previously involving the multitude of factors that affect the growth
of a microbe. Stressing a cell involves removing its favorable growth conditions, however, not
intensely enough to cause lethal damage. Sub-lethal levels of heat, such as the difference
between cooling E. Coli to 30° Fahrenheit, would make it VNC but cooling all the way down to
4° Fahrenheit would kill it all together (Montville et al., 45). The difference between a cell being
injured and being VNC is that injured cells can still be cultured on non-selective media, and
VNC cells cannot be cultured on any media.
The degree of injury for cells is also dependent on a combination of the factors that were
mentioned before. Cell exposure to pH or temperature levels outside of its normal conditions will
have varying levels of damage based on, perhaps, the amount of time that the exposure lasted. It
is also possible that injured cells are cultured in a rich media that allows for repair if given the
proper allotment of time. Oxygen toxicity, as mentioned earlier, may also cause injury, which
can be undone with catalase or pyruvate in the absence of oxygen as they are peroxidedetoxifying agents. Cell injury can be dangerous because cells that were classified as dead but
were unknowingly injured instead may cause the scientists to overestimate the intensity of the
lethal conditions provided that were thought to kill off the microbes. Cells that were thought to
have been killed off may also repair if given enough time before consumption and cause
foodborne illness. Just as cell stress depends on the intensity of the environmental factors, the
rate of repair is also influenced by them. It is noted that L. monocytogenes that has been injured
at 55° Fahrenheit for twenty minutes will “repair immediately at 37°C” but only begins repair
after “8 to 10 days and full recovery requires 16 to 19 days” if it is kept at 4° Fahrenheit
(Montville et al., 20).
Cells may transition from a vegetative state over to VNC as a method of survival for
nonsporulating bacteria, such as Salmonella, Campylobacter, Escherichia, Shigella, and Vibrio.
There is a morphological change in which the rod-like cells shrink into spherical bodies in a
process that takes anywhere from two days to weeks for the entire population (Montville et al.,
20). Although they cannot be cultured, VNC cells are still helpful in studying structures such as
membranes using fluorescent nucleic acid stains. Detection of VNC cells with an intact cell
membrane will fluoresce green, and those with a damaged membrane will stain as a red color.
Iodonitrotetrazolium violet is a different stain reduced by respirating cells to form an insoluble
compound that is detectable by a microscope.
The culturing ability of VNC cells is not permanent as they can be brought back to a state
of culturing viability with the presentation of nutrients over an extended period of time with
specific modulations of temperature. One consistent batch of cells can go through cyclical states
of VNC and culturability without proliferation. It is possible to restore culture capabilities of E.
Coli O157 and Vibrio parahaemolyticus by the addition of catalase or sodium pyruvate, which
induces the “Rapid production of superoxide and free radicals” which, after hydrolysis and
degradation of hydrogen peroxide, yields acetic acid and carbon dioxide (Montville et al., p. 22).
Microbial Antagonism, Quorum Sensing, and Microbial Food Spoilage
Microbial antagonism can be understood as the inhibition of one bacterium by the
presence of another in a setting. Just as there is a vast array of microbes, with that comes a
complex web of relationships between these microorganisms, which can be antagonistic in
nature.
This figure introduces some of the most common forms of microbial antagonism. Protozoan
grazing on pathogenic microorganisms and viruses, phage induced lysis of pathogenic bacteria
and protozoa, “Bdellovibrio and like organisms” predation of pathogenic bacteria, and microbial,
chemical substances with antimicrobial activity such as lactic acid and violacein to kill or inhibit
its opponents (Feichtmayer et al., 2017).
Just like how there are many signal transduction pathways in our bodies that regulate
many specific actions, bacteria have their forms of signaling for communication of gene
transcription. Quorum sensing is the phenomenon in which a cellular response is dependent on
the threshold concentration of a particular signaling compound. This signaling compound is
produced by the bacterial population itself, released into the environment, and its concentration
depends on the population's growth. After the signaling compound has reached a higher
extracellular concentration, it will diffuse back into the cell, where it “Binds to an intracellular
regulator protein that affects transcription of a regulon(s) to elicit a cellular response” (Montville
et al., 23). Because the gene for the signal compound is located on the same regulon, it is deemed
auto-induced.
The auto-inducers of quorum sensing in Gram-negative bacteria are sometimes detected
by histidine kinase receptors with two components. The binding activates receptor kinase activity
wherein it autophosphorylates and gives another phosphate to a different cytoplasmic response
regulator. In Gram-positive bacteria, the quorum sensing system is somewhat different. These
bacteria use auto-inducing peptides as signaling molecules which are produced in the cell then
secreted. Once it reaches a high concentration, the process moves forward in the same way as
Gram-negative bacteria. At the end of this process, the auto-inducing peptides can be
“transported back into the cell cytoplasm where they interact with transcription factors to
modulate the transcription factor’s activity and, in turn, modulate gene expression changes”
(Rutherford & Bassler, 2012). The membrane-spanning histidine kinase receptors are involved in
signal transduction pathways where phosphorylated response regulators modulate gene
expression, enzyme activity, and other phenotypic phenomena.
Quorum sensing may be upregulating the synthesis of aggregates of cells known as
biofilms. They provide some semblance of organization and circulation among microcolonies
that need to transport nutrients and waste. The cells in biofilms are more resistant to stressors
like heat and sanitizers, seen by the fact that the “Lethal combination of sodium hypochlorite and
heat to Listeria monocytogenes is approximately 100 times lower for biofilms than for free cells”
(Montville et al., 24). This can pose a significant threat the food safety because many bacteria
like E. Coli O157:H7 and Yersinia enterocolitica can form biofilms that are only removable by
proper cleaning techniques. There are many developments in the realm of biofilm control and
degradation, aptly moving towards the field of prevention with equipment that retards the initial
absorption step.
Although it may be rational to think that quorum sensing influences food spoilage, there
has not been any evidence to prove this form of thought. A study in which meat was inoculated
with wild-type strains or N-acyl homoserine lactase synthase knockout mutants all spoiled at the
same rate. Even the introduction of quorum sensing inhibitors did not affect the spoilage of
vacuum-packed meat (Montville et al., 23).
Food spoilage itself is the culmination of everything discussed in this write-up and
beyond. Pathogenic microbes in various environments have intrinsic and extrinsic factors such as
moisture and pH that must be met to grow in population size in their environment. This means
that the log phase growth rate far surpassed the death rate and compounds further for the bacteria
to continue to proliferate. If there are stressors to the cell that alter the favorable factors to
growth, the injured cells may have enough time and resources to repair and continue to culture.
The microbial antagonism will ensure the dominance of some microbes over others in specific
environments, and quorum sensing will assist in communicating gene expression that is
beneficial for the microbe population as a whole.
Eventually, food spoilage will present itself as damage to the original food through taste,
appearance, and/or nutritional value. The spoilage will lend itself to foodborne illnesses, food
poisoning due to the toxicity attributed to the original microbe’s specifications. There have been
many endeavors to reduce food spoilage as much as possible, from tactics as old as fermentation
to modified-atmosphere packaging. Spoiled foods will have varying characteristics with different
compound causations for the spoilage, such as trimethylamine bringing a fishy characteristic to
meat, eggs, and fish. Microbial-mediated spoilage may attack foods' different qualities, such as
degradation of carbohydrates or lipids based on the food type and microorganism available.
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