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Summary BMW Practical Medical Microbiology Subtest
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A summary from Labbuddy for the practical part test of medical microbiology
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Labbuddy questionsBacterial resistance
You determine the sensitivity of the E. coli strain(s) using the diffusion method, in which the bacteria are
plated on medium and a disc containing antibiotics is placed on the medium. You do this to confirm that
the strains are indeed sensitive and resistant to the antibiotics as indicated.
Determine antibiotic sensitivity
Kanamycine, tetracycline, streptomycine is/are an aminoglycoside antibiotic(s) and as such a protein synthesis
inhibitor. Ampicilline is/are a β-lactam antibiotic(s), and as such acts by inhibiting the synthesis of bacterial cell
wall. Kanamycine, tetracycline and streptomycine are broad-spectrum aminoglycoside antibiotics extracted from
bacteria and are powerful protein synthesis inhibitors. Aminoglycosides work by binding to the bacterial ribosomal
subunits, causing misreading of t-RNA, leaving the bacterium unable to synthesize proteins vital to its growth. In
contrast, ampicilline is a broad-spectrum β-lactam antibiotic, which works on the active replicating stage of
bacteria, inhibiting the synthesis of bacterial cell wall. The loss of cell wall leads to formation of a spheroplast,
which is extremely vulnerable and leads to highly decreased cell viability.
The temperature of 37 °C is similar to the natural conditions at for which E.coli bacteria evolved: human bodies.
From this result (with the antibiotic amounts used in this experiment) you can conclude that this bacterium is the
most sensitive to FOX & TE and the least sensitive to P. If there is a large inhibition zone, the bacterium is
sensitive to that antibiotic. When there is a small inhibition zone, the bacterium can grow in close proximity to the
antibiotic and is not very sensitive to it.
In this experiment you will investigate the transfer of plasmid DNA, namely R-factor DNA, from a donor
strain to an acceptor strain of Escherichia coli, as is done between E. coli and pathogens like Salmonella.
The research question is:
Can transfer of R-factor DNA take place from an antibiotic resistant E. coli strain to an antibiotic sensitive
E. coli strain? (PLASMID TRANSFER)
Prepare conjugation culture
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact. Where
usually a donor cells transfer its DNA to a recipient E. coli cell. It is a mechanism of horizontal gene transfer as
are transformation and transduction although these two other mechanisms do not involve cell-to-cell contact.
Prepare pure culture
You have correctly identified the Lac+ (here E. coli Lactose+ PC2348RP4+ ) bacteria. The pink coloration occurs
when an intact E. coli lac operon breaks down the lactose into glucose/galactose, which then gets broken into
acidic end products that drop the pH below 6.8 of the surrounding medium. MacConkey agar contains Neutral
Red which turns a bright pink/red at acidic pH. In contrast, Lac- E. coli, such as Lactose- PC1568, won't digest the
lactose and will eventually start to make the pH somewhat alkaline (which turns Neutral Red yellow) by breaking
down peptides.
It is of importance that the previous streak is crossed. This type of smear makes for a dilution so that individual
colonies grow. This type of smear of the cell suspension creates a dilution. One very important method in
microbiology is to isolate a single type of bacteria from a source that contains many. The most effective way to do
,this is to use this streak plate method, which dilutes the individual cells by spreading them over the surface of an
agar plate. The cells are diluted with each stroke to ensure that you get individual colonies.
PC2348RP4 is resistant to tetracycline and susceptible to streptomycine. Because it can metabolize lactose it will
convert the medium to a pink color and the colonies exhibit a pink color. PC1568 is resistant to streptomycine but
not to tetracycline and can therefore not grow on a medium that also contains this compound. PC1568 is not able
to metabolize lactose. Therefore the McConkey medium will not change color and the colonies will be white.
If a strain is now able to grow on a medium that it was previously resistant to, probably that strain is the acceptor
strain. A diffusion test should be done to confirm these observations.
Single cells reproduce and create millions of clones, which all pile up on top of the original cell. The piles of
bacterial cells observed after an incubation period are called colonies. Each colony represents the descendants of
a single bacterial cell, and therefore, all of the cells in the colonies are clones. Therefore, when you transfer a
single colony from the streak plate to new media, you have achieved a pure culture with only one type of bacteria.
Resultaten
- donor: Lac+
- acceptor: Lac-
Because LAC- got the tetracycline-resistant genes
Determine antibiotic sensitivity
Lac- is resistent for kanamycine, tetracycline, ampicilline and streptomycine.
, Isolating DNA and send sequencing facility
During DNA isolation, the bacterial cell walls en membranes are broken down to release the cell contents.
Proteins like DNases are digested by an enzyme that is added to the bacteria.
Determine plasmid transfer
Taking into account all data, can transfer of R-factor DNA take place from a antibiotic resistant E. coli strain to an
antibiotic sensitive E. coli strain?
- Yes, the transfer of R-factor DNA, carrying antibiotic resistance genes, can occur from an antibiotic-
resistant E. coli strain to an antibiotic-sensitive E. coli strain through processes like conjugation,
transformation, or transduction. This horizontal gene transfer can lead to the acquisition of antibiotic
resistance by initially sensitive strains, contributing to the spread of antibiotic resistance.
- Conjugation involves the direct transfer of genetic material, including plasmids (which may carry
antibiotic resistance genes), from one bacterium to another.
o In the case of antibiotic resistance, the resistant strain (donor cell) may transfer a plasmid
carrying resistance genes to the sensitive strain (recipient cell).
- Transformation is the uptake of naked DNA from the environment by a bacterial cell. This DNA can be in
the form of plasmids carrying antibiotic resistance genes.
o If the resistant strain releases DNA into its environment, the sensitive strain might take up this
DNA and incorporate it into its own genome.
- Transduction involves the transfer of genetic material from one bacterium to another by bacteriophages
(viruses that infect bacteria).
o If a bacteriophage infects an antibiotic-resistant E. coli strain, it may pick up resistance genes
and then transfer them to an antibiotic-sensitive strain during subsequent infections.
The goal of this experiment is to test if the bacteria that have obtained resistance due to plasmid transfer
maintain this property when there is no more selective pressure by the environment. The research
question is:
Is antibiotic resistance maintained in absence of selective pressure? (MAINTAINING RESISTANCE)
Serial passenge technique
For a quick and cheap inhome test the antibiotic diffusion test can be done. To obtain detailed information about
the insert sequence, sequencing can be done. For serial passaging, bacteria are used that have obtained
resistance by plasmid transfer. If these bacteria are passaged in medium with antibiotics, it is expected that they
maintain their resistance due to the continuous selective pressure. If these bacteria are passaged in medium
without antibiotics, it is expected that they lose their resistance due to the energy cost of resistance. The
passaging is done with antibiotics as a control. These bacteria are still under selective pressure and will not lose
their resistance. Whether the bacteria lose their resistance when there is no more selective pressure, is what you
will find out based on the results. You might reason that resistance is maintained when the gene responsible has
transposed into the chromosome of the baceteria. You might reason the opposite that resistance is lost because it
costs energy.
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