Mc Jason Laurete 08 March 2018

ChE 40

Direct Measurement of Microbial Growth

There are many different ways to measure the growth of microbial populations. Most methods of counting are based on
indirect or direct counts of tiny samples. After, calculations are used to determine the size. Usually the procedure is done
indirectly with a series of dilutions, making it possible to estimate the number of bacteria in the original sample.

Plate Counts: this is the most frequently used method. Measures the number of variable cells, although it takes a large
portion of time for colonies to form. This is a problem in cases in which it is impossible for the lot to be held for long times.
It is assumed, with this method, that each grows and divides to produce a single colony, but is not always the case. Often
plate counts are known as CFU or colony-forming units. It is important that only a select amount of colonies develop in the
plate or there becomes a risk that the overcrowding will stop development. They use serial dilution to help with this problem.

Serial Dilutions: continued dilution of a substance in solution. The factor is always constant in the process so that it is a
geometric progression and is logarithmic.

Pour Plates and Spread Plates: Pour plate method introduces the bacterial suspension into the petri dish, nutrient agar is
poured over it and then mixed in, then finally it is incubated. In this method, colonies grow in the agar as well on the surface.
Often the Spread plate method is used though. This process includes adding inoculum to the surface of agar and spreading
it around uniformly on the surface with a special rod. This helps to avoid contact with the cells with melted agar.

Filtration: Bacteria can be counted this way when it is small enough in lakes or pure streams. This occurs through the
bacteria being filtered out and retained on the filter surface, which then gets transferred to a petri dish of nutrient medium
where colonies will rise off the surface.

Most Probable number method: The idea of this method is, the greater the number of bacteria in a sample the more dilution
is needed to reduce density so that no bacteria is left to grow in the series tubes. Useful in cases in which the microbes cannot
grow on solid media or when they do grow in a liquid differential medium

Direct Microscopic Count: a measured volume of bacterial suspension is placed in a defined area on a slide. Motile bacteria
are hard to count using this method and also it is very common, when being counted, for the dead cells to be just as likely
to be counted as the living, and finally a large number is needed in order to be able to be counted. Benefis, however, include,
no incubation time needed, and instruments can often be used to help count in this method.

Indirect Measurement of Microbial Growth

In situations where determining the number of microorganisms is difficult or undesirable for other reasons, the use of indirect
methods can be an excellent alternative. These methods measure some quantifiable cell property that increases as a direct
result of microbial growth.

The simplest technique of this sort is to measure the weight of cells in a sample. Portions of a culture can be taken at
particular intervals and centrifuged at high speed to sediment bacterial cells to the bottom of a vessel. The sedimented cells
(called a cell pellet) are then washed to remove contaminating salt, and dried in an oven at 100-105 °C to remove all water,
leaving only the mass of components that make up the population of cells. An increase in the dry weight of the cells correlates
closely with cell growth. However, this method will count dead as well as living cells. There might also be conditions where
the dry weight per cell changes over time or under different conditions. For example, some bacteria that excrete
polysaccharides will have a much higher dry weight per cell when growing on high sugar levels (when polysaccharides are
produced) than on low. If the species under study forms large clumps of cells such as those that grow filamentously, dry
weight is a better measurement of the cell population than is a viable plate count.

It is also possible to follow the change in the amount of a cellular component instead of the entire mass of the cell. This
method may be chosen because determining dry weights is difficult or when the total weight of the cell is not giving an
accurate picture of the number of individuals in a population. In this case, only one component of the cell is followed such
as total protein or total DNA. This has some of the same advantages and disadvantages listed above for dry weight.
Additionally, the measurement of a cellular component is more labor-intensive than previously mentioned methods since
the component of interest has to be partially purified and then subjected to an analysis designed to measure the desired
molecule. The assumption in choosing a single component such as DNA is that that component will be relatively constant
per cell. This assumption has a problem when growth rates are different because cells growing at high rates actually have
more DNA per cell because of multiple initiations of replication.

Turbidity. A final widely used method for the determination of cell number is a turbidometric measurement or light
scattering. This technique depends on the fact that as the number of cells in a solution increases, the solution becomes
increasingly turbid (cloudy). The solution looks turbid because light passing through it is scattered by the microorganisms
present and the turbidity is proportional to the number of microorganisms in the solution. The turbidity of a culture can be
measured using a photometer or a spectrophotometer. The difference between these instruments is the type of light they pass
through the sample. Photometers, such as the Klett-Summerson device, use a red, green or blue filter providing a broad
spectrum of light. Spectrophotometers use prisms or diffraction gratings supplying a narrow band of wavelengths to the
sample. Both instruments measure the amount of transmitted light, the light that makes it from the light source through the
sample to the detector.

Measuring the turbidity of a culture. When measuring light scattering it is important to consider the wavelength of light
used a bacterial culture. Microorganisms may contain numerous macromolecules that will absorb light, including DNA (254
nm), proteins (280 nm), cytochromes (400-500 nm), and possible cell pigments. When measuring bacteria by light scattering
it is best to pick a wavelength where absorption is at a minimum and for most bacterial cultures wavelengths around 600 nm
are a good choice. However, the exact wavelength chosen is species specific.

Due to the nature of light scattering, transmittance decreases geometrically as the cell numbers increase. It is more intuitive
to think of the units increasing as growth increases and for most bacterial analysis.

Absorbance increases in a linear fashion as the cell number increases. When measuring growth of a culture the term optical
density (OD) is normally used to more correctly represent the light scattering that is occurring; under optimal conditions,
little light is actually absorbed by the culture so the term absorbance is misleading. For most unicellular organisms changes
in OD are proportional to changes in cell number (within certain limits) and therefore can be used as a method to follow cell
growth. If a precise cell number for a given OD is desired, a standard curve can be generated, where viable plate count or
cell mass is plotted as a function of OD. It also wise to develop a standard curve to verify that the OD is actually an accurate
portrayal of cell growth. After the standard curve is made, it is then possible to simply measure the OD of the culture and
read the cell number from the curve.

The turbidity of a culture is dependent upon the shape and internal light-absorbing components of the microorganism and
therefore turbidity readings are species-specific and cannot be compared between different microbes or even between
different strains of the same species. As above, there are microbes that change cell size or shape at different stages of growth,
which introduces some inaccuracy to this method of cell counting. Also both living and dead cells scatter light and are
therefore counted. However, the method is very rapid and simple to perform and provides reliable results when used with
care, so it is an extremely common method of real time analysis of prokaryotic populations. In fact, it is one of the methods
we will use for measuring cell number in the experiment on bacterial growth. Turbidometric measurements also do not
destroy the sample.

Mechanisms of Mutation

Any heritable change in the genome is commonly called a mutation. Biochemically, it is a chemical change or alteration in
a nitrogen base of a DNA sequence resulting in the production of a defective protein or a truncated protein, which is not
functional. These altered proteins can cause serious problems in metabolism leading to changes in the morphology and
physiology of the organism. Mutations, in most cases, are spontaneous and may not be dangerous.

Even though the natural mutations are spontaneous and rare, biologists can induce mutations using different methods, which
in most cases are not desirable and precise. But, now molecular biologists can alter any amino acid of a protein by changing
the corresponding bases in its gene very precisely and accurately resulting in desirable mutations. It is possible to alter
properties such as increased stability, temperature resistance, product inhibition, substrate specificity, etc. of any enzyme.
The accurate induction of one or more point mutations on selected regions of a gene resulting in amino acid substitutions or
deletion or addition is known as site directed mutagenesis. It can be defined as the controlled alteration of selected regions
of a DNA molecule. The principle of site-directed mutagenesis is that a mismatched oligonucleotide primer is extended,
incorporating the ‘mutation’ into a strand of DNA that can be cloned. This technique of creating desired molecular mutations
in a gene has contributed greatly to the basic understanding of functions of genes, DNA-protein interactions, gene
regulations, the role of amino acids in the structure and functions of proteins, role of active centres in the enzyme-substrate
interactions, etc. A single base change in a gene permits the evaluation of the role of specific amino acids in the function
and structure of a protein. This technique also allows one to create or destroy a restriction site at specific locations within a
DNA sequence or gene.

Site-directed mutagenesis is actually one of the applications of PCR. The gene, which has to be mutated, should be made
into a single-stranded DNA by cloning into a M13 plasmid vector. By following modern PCR methods, it is possible to carry
out the site directed mutation without the participation of M13 vector. The designing and chemical synthesis of the primer
is the key factor in this technique. The part of the DNA where the mutation has to be introduced should be synthesized as
an oligonucleotide primer, which is complementary to the respective region of the DNA except for the nucleotide that has
to be changed. In short, the mutation is introduced to the gene in the form of a primer and the primer is extended with a
polymerase reaction.

The site-directed mutagenesis is a multistep process that begins with the cloning of the gene in a bacteriophage like M13 to
generate single stranded DNA. M13 is a filamentous bacteriophage that specifically infects e. coli that expresses sex pili
encoded by a plasmid F factor. M13 bacteriophage contains DNA in a single-stranded or replicative form, which is replicated
to double-stranded DNA within a bacterial cell. The primer is designed and synthesized, which is an oligonucleotide
complementary to the region of the DNA to be mutated except for the nucleotide to be changed. This oligonucleotide with
the mismatched base or bases hybridize to the single-stranded DNA and serve as the primer to start synthesizing the
complementary DNA strand with the help of a suitable DNA polymerase such as T4 DNA polymerase or Taq polymerase.
The resulting double stranded DNA will be a hybrid of the wild type parent strand and the mutated newly synthesized DNA
strand. This DNA molecule can be transformed into an e. coli cell, where the mutated DNA strand serves as a template to
replicate new strands carrying the mutation along with the wild strands. The bacteriophage plaques containing the mutated
DNA can be screened by hybridizing with the labeled probe of the original mutated oligonucleotide. By adjusting the
hybridization time and wash temperature of the hybridized probe, only the perfectly matched hybrid will remain and all
other mismatched hybrids
will dissociate. The
presence of the desired
mutation in the gene can be
checked and confirmed after
isolating the plasmid DNA
from the single positive
plaques and sequencing it.
A single base can be
mutated in recombinant
DNA plasmids with a
process called inverse PCR.
Two primers are
synthesized with their
antiparallel 5’ ends
complementary to the
adjacent bases on the two
strands of DNA. One of the
two primers carries a
specific mismatched base
that is faithfully copied
during the PCR
amplification resulting in a recombinant plasmid with a single mutated base.
Induced Mutation. Virtually any agent that directly damages DNA, alters its chemistry, or in some way interferes with its
functioning will induce mutations. Mutagens can be conveniently classified according to their mode of action. Three
common types of chemical mutagens are base analogs, DNA-modifying agents, and intercalating agents. A number of
physical agents (e.g., radiation) damage DNA and also are mutagens.

Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into the growing polynucleotide
chain during replication. Once in place, these compounds typically exhibit base pairing properties different from the bases
they replace and can eventually cause a stable mutation. A widely used base analog is 5-bromouracil (5-BU), an analog of
thymine. It undergoes a tautomeric shift from the normal keto form to an enol much more frequently than does a normal
base.

The enol tautomer forms hydrogen bonds like cytosine and directs the incorporation of guanine rather than adenine. The
mechanism of action of other base analogs is similar to that of 5-bromouracil.

DNA-modifying agents change a base’s structure and therefore alter its base pairing characteristics. Some mutagens in this
category are fairly selective; they preferentially react with some bases and produce a specific kind of DNA damage. An
example of this type of mutagen is methyl-nitrosoguanidine, an alkylating agent that adds methyl groups to guanine, causing
it to mispair with thymine. A subsequent round of replication could then result in a GC-AT transition. Hydroxylamine is
another example of a DNA-modifying agent. It hydroxylates the C-4 nitrogen of cytosine, causing it to base pair like
thymine. There are many other DNA modifying agents that can cause mispairing.

Intercalating agents distort DNA to induce single

nucleotide pair insertions and deletions. These mutagens
are planar and insert themselves (intercalate) between the
stacked bases of the helix. This results in a mutation,
possibly through the formation of a loop in DNA.
Intercalating agents include acridines such as proflavin and
acridine orange.

Many mutagens, and indeed many carcinogens, directly

damage bases so severely that hydrogen bonding between
base pairs is impaired or prevented and the damaged DNA
can no longer act

as a template for replication. For instance, UV radiation

generates cyclobutane type dimers, usually thymine
dimers, between adjacent pyrimidines. Other examples are
ionizing radiation and carcinogens such as the fungal toxin
aflatoxin B1 and other benzo(a)pyrene derivatives.
Retention of proper base pairing is essential in the
prevention of mutations. Cells have developed extensive
repair mechanisms. Often the damage can be repaired
before a mutation is permanently established. If a complete
DNA replication cycle takes place before the initial lesion
is repaired, the mutation frequently becomes stable and
inheritable.

References:

[1] A. J. Nair. Introduction to Biotechnology and Genetic Engineering. Infinity Science Press LLC, 2008.

[2] https://microbialgrowth101.weebly.com/direct-methods-of-measuring.html

[3] https://instruction.bact.wisc.edu/instr/book/displayarticle/107

[4] J. Willey; L. Sherwood; C Woolverton. Microbiology, 7th ed. McGraw-Hill, 2008.