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Microbial Growth
However, during microbial growth, the cells
might actively produce biosurfactants, and/or
some of the degradation products might act as
emulsifiers, thereby influencing the particle size
distribution tremendously.
From:
Olives and Olive Oil in Health and Disease
Prevention, 2010
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Chapters and Articles
Microbial Growth
Helen Louise Brown, in
Encyclopedia of Infection and Immunity, 2022
Cell counting
Microbial growth counts provide a highly
accurate estimate of the microbial population,
which is not confounded by the presence of
debris or dead cells. One of the most accurate
ways of determining microbial numbers in
clonal population is to count a subset of the
microbial population and scale that number
up to give a concentration for the whole
population. Typically, this is done by
producing a series of dilutions (serial dilutions
are usually logarithmic, i.e., 10-fold). The
dilutions are then added to agar and
incubated in optimum growth conditions to
allow microbial growth and colony formation
(Fig. 2).
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Fig. 2. Overview of a basic method of counting the number of
colony forming units within a population.
It is assumed that each single microbe within
a dilution will grow by binary fission to
produce a clonal colony. Colony counting
methods express the concentrations as the
number of colony forming units in each mL of
the initial microbial culture or “CFU/mL.” The
term “colony forming unit” is used in
preference to a cell/mL value since although
the assumption is that each colony on the
plate is derived from a single cell, this is not
directly known. To extrapolate the total
number of cells within the starting inoculum
the following formula is used:
Many videos are available online
demonstrating colony counting methods and
how to calculate CFU/mL.
Although colony counting methods are
commonly used and considered to be one of
the best methods of estimating inoculum
concentrations, they do have some negative
aspects which should be fully considered
before use. It is important to have some
understanding of the growth dynamics and
phenotype of the species to be worked with
before counting. For example, if the species to
be worked with produces aggregates which
cannot be separated then it is very likely that
any CFU/mL values will underestimate the
actual number of viable cells. Some bacterial
species which have a mucoid or swarming
phenotype, for example Pseudomonas
aeruginosa and Proteus mirabilis respectively,
will also be difficult to count accurately since
they do not produce well defined and
separated colonies when grown on some
agars. Similarly fungal species which form
complex multicellular bodies or hyphae are
difficult to count, although this technique can
work well with species, such as yeast, which
have a unicellular, non-hyphenated growth
stage. Finally, methods for determining
CFU/mL are time consuming, requiring a
period of incubation to allow visible microbial
growth and require a large number of agar
plates. This can be mitigated slightly using
direct methods such as counting cells within a
hemocytometer, but again this is not suitable
for all species—in particular those which are
very small or motile.
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Foods, Materials, Technologies
and Risks
J.N. Sofos, in Encyclopedia of Food Safety, 2014
Inhibition of Microbial Growth
Microbial growth on meat products, as well as
other foods, is affected, not only by the type
and level of initial contamination but also by
various factors associated with the product
(intrinsic) or its environment (extrinsic).
Approaches aiming to inhibit microbial
growth are mostly based on manipulation or
changes in these factors. These include storage
at low temperatures, drying (evaporation) or
reduction by binding of water levels (salting
and sugaring) available for microbial growth
(water activity), addition of acids (low pH),
fermentation (low pH and production of
antimicrobials), packaging under modified
atmospheres such as vacuum, and use of
chemical antimicrobials. Combinations of
antimicrobial technologies, applied
individually at sublethal levels (hurdle
technology), are frequently used in many
meat products as they result in
microbiologically stable and safe products of
desirable eating quality. It is important to
properly select and apply sublethal hurdle
combinations with the objective of pathogen
control without stress adaptation or selection
of resistant pathogens.
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Growth Kinetics
Ghasem D. Najafpour, ... Ghasem Najafpour, in
Biochemical Engineering and Biotechnology, 2007
5.1 INTRODUCTION
Microbial growth is considered for the
observation of the living cell activities. It is
important to monitor cell growth and
biological and biocatalytic activities in cell
metabolism. A variety of methods are
available to predict cell growth by direct or
indirect measurements. Cell dry weight, cell
optical density, cell turbidity, cell respiration,
metabolic rate and metabolites are quite
suitable for analysing cell growth, substrate
utilisation and product formation. The rate of
cell growth is described in this chapter.
Various bioprocesses are modelled for
substrate utilisation and product formation.
Growth kinetics in batch and continuous
culture is examined in detail.
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Scientific Fundamentals of
Biotechnology
N.S. Panikov, in
Comprehensive Biotechnology (Second Edition),
2011
1.20.1 Introduction
Microbial growth dynamics is a subject of
numerous fundamental and applied research
studies in modern microbiology and
biotechnology. Usually, biotechnologists want
to know the time progress of, say, product
formation associated with cell growth,
nutrients uptake, respiration, and other
metabolic processes. We can predict growth
dynamics before setting up a real experiment
or technological line by using mathematical
simulation models of different degrees of
complexity. Such a preliminary simulation is
very useful for planning and optimizing real-
life experiments. Moreover, the verified
dynamic mathematical models provide
efficient tools to optimize the yield of target
product, minimize undesirable generation of
waste products, etc. Another important
implication of growth dynamics is that
recorded growth curves carry important
hidden information about microbial cells,
growth regulation, and interactions. Based on
growth dynamic pattern, we can distinguish
the effects of products or substrate inhibition,
identify growth-limiting substrates at various
stages, pinpoint the importance of positive or
negative interactions in a mixed or genetically
inhomogeneous culture, etc.
The scientific discipline that uses special
quantitative tools to study the development of
any processes in time – physical, chemical, or
biological – is called kinetics (from the Greek
κινετικοσ, forcing to move). Kinetic studies in
microbiology cover all dynamic
manifestations of microbial life: growth itself,
survival and death, product formation,
adaptations, mutations, cell cycles,
environmental effects, and biological
interactions. Kinetics provide a theoretical
framework for optimal design in
biotechnologies, based on fermentation and
enzyme catalysis, as well as on the
employment of outdoor activity of natural
microbial populations (wastewater treatment,
soil bioremediation, etc.).
Contrary to simple rate measurements, kinetic
studies require the perception of the
underlying basic mechanisms of studied
processes. We will define mechanistic studies
as those that interpret some complex process
as an interplay of several simpler reactions;
for example, cell growth can be explained
through the activity of enzymes and microbial
community dynamics can be interpreted
through the behavior of individual cells and
populations. Ideally, mechanistic studies infer
the coupling of experimental measurements
with analysis of simulating mathematical
models. The models formalize postulated
mechanisms, so that the comparison of
observations and the model’s predictions
allows one to discard an incorrect hypothesis.
Quantitative studies in microbiology often
involve the assessment of ‘growth
stoichiometry’. Stoichiometry (Greek
στωικηειον, element) is the quantitative
relationship between reactants and products
in a chemical reaction. In microbiology,
stoichiometry stands for a quantitative
relationship between substrates and products
of microbial processes, including biomass
formation (the consequence of complying
with mass and energy conservation laws). In
practical terms, kinetic and stoichiometry are
tightly linked to each other, but stoichiometry
mainly addresses problems of a static nature
(how much? in what proportion?), whereas
kinetics considers the dynamics questions (at
what rate? by which mechanism?).
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Proteomics for bioforensics
Eric D. Merkley, ... Karen L. Wahl, in
Microbial Forensics (Third Edition), 2020
Elucidating methods of production:
exogenous proteomic signatures of
production methods
Microbial growth environments often contain
proteins or peptides. Pathogens growing in
host tissue or laboratory medium will be
surrounded by protein-rich components.
Typical laboratory media contain digests of
proteins, which can be used by the organisms
as sources of energy, carbon, nitrogen, and
specific amino acids. Peptides from the
partially digested proteins that are a major
component of culture media can remain
associated with microbial cells, even if the
cells have been washed in buffer (Clowers
et al., 2013). Peptide components can be
determined following elution of a cell sample
with an appropriate organic solvent—without
cell lysis—and analysis by standard bottom-up
proteomic methods. We refer to these as
exogenous peptides.
Observed exogenous peptides provide
information about (1) the specific protein
from which they were derived; (2) the tissue
in which that specific protein is expressed; (3)
the organism from which that tissue was
derived; and (4) the production process
(Fig. 17.4). Thus, protein, tissue, and organism
source information can be generated from
peptides that match to predicted proteins
within the appropriate sequence databases.
This information alone may reveal whether a
microbial sample was grown in a laboratory,
particularly if components like bovine milk
casein or soybean seed storage protein
(glycinins) are identified associated with
bacterial biomass. Each of these proteins is
commonly present in tryptone and soytone
which are used in tryptic soy broth, a
commercial microbiological medium. The
signatures of commercial media
manufacturing are derived from the extraction
and breakdown of proteins to amino acids and
the process can vary between manufacturers.
These variations include whether acid or
enzymatic hydrolysis is used, which endo-
and exopeptidases are used, and the reaction
time and temperature. As a result, different
profiles of residual peptides remain in
medium components from different
processes. These peptide profiles (that is,
different sets of peptides each with a
characteristic range of relative abundance)
may represent different lengths of peptides
originating from the same or different
portions of a protein sequence. Clowers et al.
(2013) postulated that the profile of peptides
can act as a signature of a manufacturing
method that distinguishes between sources of
the same type of medium component and
possibly help to link a microbial agent to
production materials.
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Figure 17.4. Proteomic analysis of exogenous material reveals
the sources of proteins in the environment and, in the case of
manufactured media, can yield a manufacturing signature in
the form of peptide distributions.
The concept of exogenous signatures can be
extended to virus production. The culture
medium for viruses consists of a host cell and
cell culture medium, both of which are rich in
proteinaceous components. Purification of
virus proteins from host cells presents a
challenge for proteomic analysis because host
proteins can significantly outnumber viral
proteins (Mottaz-Brewer et al., 2008). The
incorporation of host cell proteins into viral
particles has been described for a number of
viruses and makes detection of host cell
proteins possible even following stringent
purification steps (Krauss et al., 2002;
Vanderplasschen et al., 1998; Varnum et al.,
2004). As a result, the number and type of
proteins associated with viral samples can
provide information on the methods used for
production.
We examined the impact of poxvirus
production systems and different purification
procedures on proteomic signatures
(Wunschel et al., 2013). In contrast to
exogenous peptides associated with the
surface of bacterial cells, which are identified
without further proteolytic digestion, the
exogenous proteome of viral samples consists
of intact proteins which must be digested
before analysis, similar to common bottom-up
proteomic protocols. Increasingly stringent
levels of purification were employed, and the
impact on the proteome revealed distinct
shifts in the number and type of host proteins
identified in each sample. Proteins with
variable peptides that differentiate between
mammalian hosts were also identified in
some samples. That is, the proteomics
measurements revealed whether the poxvirus
sample was grown in human or monkey cell
lines. These examples demonstrate the
potential to expand exogenous proteomic
signatures to viral samples to derive
information on production methods.
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Biofilms
Md Mahamudul Haque, ... Kangmin Duan, in
Methods in Microbiology, 2023
2.5 Microbial growth assay
To determine the microbial growth in liquid
cultures a growth curve was generated by
measuring OD600 over time. The cultures were
inoculated in the wells of a 96 well plate
(transparent flat bottom, Thermo Scientific™)
with a ratio of 1: 10, namely, 20 µL of culture
with OD600 of 0.5 and 180 µL of fresh medium
(TSBYE with 1% sucrose). The culture in each
well was topped with 50 µL of filter-sterilized
mineral oil (Sigma-Aldrich) to prevent the
cultures from drying out during the
experiment. This experiment was performed
in seven replicates. Microbial growth was
measured at one-hour intervals, and 3 s of
orbital shaking was performed before the
OD600 measurements in the microplate reader
(Spectramax Id5—Molecular Devices)
operated with the software SoftMax Pro
(version 7.2). A logistic growth equation was
used with nonlinear regression (curve fit) in
GraphPad Prism version® version 9 to obtain
the growth rate constant (k) values for each
culture.
Y0 is the starting population (same units as
Y).
YM is the maximum population (same
units as Y), and.
k is the rate constant (inverse units of X).
Generation time was calculated by taking
the reciprocal of the k values.
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INTERMEDIATE MOISTURE
FOODS
K. Prabhakar, in
Encyclopedia of Food Microbiology, 1999
Temperature of Storage
Low temperatures retard microbial growth
and enzymatic activity in foods and prolong
keeping quality. In temperate regions IM meat
products can be stored for long periods at
ambient temperatures, which are normally
10–20°C or less, whereas in tropical areas
shelf life is lower because ambient
temperatures are higher (30–45°C). This has
resulted in the historical development of
succulent IM foods like cured and smoked
meats in regions with cold climates. In
tropical areas, IM meat products are more dry
in nature as the demands on stability
requirements are higher. There is a
considerable energy saving if foods preserved
by deep freezing (− 18°C to − 20°C) can be
processed as IM foods which can be kept at
refrigeration temperatures (2–4°C) with the
same efficiency.
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Volume 1
Ann Shippy MD, in
Textbook of Natural Medicine (Fifth Edition), 2020
World Health Organization
• “Microbial growth may result in greater
numbers of spores, cell fragments,
allergens, mycotoxins, endotoxins, β-
glucans and volatile organic compounds in
indoor air. The causative agents of adverse
health effects have not been identified
conclusively, but an excess level of any of
these agents in the indoor environment is
a potential health hazard.”3
• Mycotoxins, or fungal toxins, are low-
relative-molecular-mass biomolecules
produced by fungi, some of which are toxic
to animals and human beings. Mycotoxins
are known to interfere with RNA synthesis
and may cause DNA damage. Some fungal
species may produce various mycotoxins,
depending on the substrate. In the case of
Penicillium, one such compound is
penicillin, a strong antibiotic. Several
mycotoxins (e.g., aflatoxin from Aspergillus
flavus and Aspergillus parasiticus) are
potent carcinogens. Many mycotoxins are
immunotoxic, but the trichothecene
mycotoxins are immunostimulating at low
doses.4 Numerous mycotoxins have been
classified by their distinct chemical
structures and reactive functional groups,
including primary and secondary amines,
hydroxyl or phenolic groups, lactams,
carboxylic acids, and amides. The
mycotoxins that have perhaps received the
most attention are the trichothecenes,
produced by Stachybotrys chartarum.
Bloom5 showed that several mycotoxins
produced by S. chartarum and Aspergillus
versicolor (i.e., macrocyclic trichothecenes,
trichodermin, sterigmatocystin, and
satratoxin G) could be present in most
samples of materials and settled dust from
buildings with current or past damage
from dampness or water damage. Charpin-
Kadouch6 compared the levels of
macrocyclic trichothecenes in samples
from 15 flooded dwellings known to be
contaminated with S. chartarum or
Chaetomium and a group of nine dwellings
without visible mold. The level of
macrocyclic trichothecenes was
significantly higher in floor dust from the
moldy houses than from the reference
dwellings; the levels in wall samples from
moldy houses were also higher.
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