Identification of Bacteria: Molecular vs. Standard Techniques

This essay delves into the identification of bacteria, primarily contrasting molecular and standard culture techniques. It uses Proteobacteria, specifically Salmonella typhi, as a case study to illustrate the application of various methods. The essay begins by highlighting the limitations of traditional methods and the importance of accurate identification for diagnosis, treatment, and disease outbreak tracing. It then details molecular techniques, including DNA extraction and massively parallel sequencing, emphasizing their sensitivity and ability to reveal bacterial diversity. The essay also discusses the role of electron microscopy, serology, and antigen detection. In contrast, it explores the use of direct detection in smears, axenic media, and detection in tissue sections. The essay concludes by acknowledging the advancements of molecular techniques while recognizing the continued relevance of cultural methods, especially given the cost and simplicity considerations.

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IDENTIFICATION OF BACTERIA
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Abstract
Bacteria are microorganisms which cannot be seen just by naked eyes, so some special
apparatus or machines like electron microscope are employed to help view these organisms
(Acton, 2014). And there must be some techniques used to help do the identification of these
types of the organism. When doing the identification, the scientist must be able to look for some
distinctive features in that bacteria by the use of the electron microscope. After that, he or she
will be able to categorize that kind of the bacteria in the phylum and finally of its species and
genus. There are no many other taxa for the organisms in kingdom Monera where all the
bacteria are classified. Taxa like class, family among others are not available for the kingdom
Monera (Bavoil, 2010). Identification of bacteria should be undertaken keenly and accurately,
this entails bacterial identification and detection of pathogens to enable the scientist correctly
diagnoses the bacterial disease. Identification of the bacteria will also help in the treatment of
infection and trace back to the outbreak of disease-related to microbial infection. In some case, it
used in criminal investigations, environmental studies and microbial forensics (Chang Lu, 2015).
Introduction
For this essay, we will use the molecular versus standard culture techniques to help
identify Proteobacteria as an example of bacteria. This includes several bacteria but we will take
Salmonella typhi as a specific bacteria for this essay (Chang Lu, 2015). This type of bacteria is a
pathogen which is a causative agent for typhoid. This bacteria in most case lives in dirty water
which when drunk then affect a human being. Identification of Salmonella typhi basically results
from a series of occasions encompassing serology, molecular tools, microscopy, and culture.

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Serology will deliver evidence for causality (Debnath, 2014). Culture entails exceptional merits
for the study and identification of this type of bacteria since it permits the antigenic studies,
experimental models, susceptibility testing. Identifying any bacteria help the nurse for easy
diagnosis of the disease for which the pathogen ( bacteria) is a causative agent for.
Molecular skills have made the scientist to evaluate Salmonella typhi bacteria more
keenly and very sensitive more than the cultural skills. Molecular technology can disclose order
of greatness of bacterial variety in human body fluid. Scientist employs the molecular bacterial
diagnostic details to come up with individualized medicine approaches to help treat the disease
which is caused by this type of bacteria (Quinn, 2011). It is highly promising that with a
molecular technique for the microbial diagnostic test creates an enabling environment for both
the nurse and the patient as it improves the healthcare of the patient and it makes the diagnosis of
the nurse very easy.
Molecular identification of bacteria
This technique of identification of bacteria involves preparation of samples of body fluids and
carefully analyzing them. Molecular identification entails the following;
DNA Extraction
The debridement samples were centrifuged then the sample was left to rest in about
600μL buffer A sterile of about 5 mm was applied ( steel bead) and 600 μL sterile of 0.1 mm
glass beads were fully put for the bacteria lysis. And this was done in a Qiagen Lyser Tissue. It
was then left to run for about 5 minutes at a frequency of 50 Hz. The sample was then whirled
for a short time, then about 100 μL of hundred percent ethanol was fully added to the 100 μL
aliquot of the supernatant sample (Liu, 2011). To the DNA spin column, this mixture which had

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been made was added and there was a recovery protocol for the DNA. Starting at the tissue
protocols, there was elusion of DNA with about 30 μL of water then the dilution of the sample
was done correctly up to an ultimate concentration of 20ng/ μL was attained (Leonard, 2010).
Massively Parallel b TEFAP Titration
The bacteria encoded tag FLX- Titanium pyrosequencing was done as designated
hitherto. In the making of FLX titanium sequencing, a model of a two- stranded DNA was joined
with capture beads of DNAs. Then the amplification was done by emulsion PCR. Afterwards
bead enrichment and bead recovery, there was denaturing of the bead attached to DNA by the
use of the Sodium Hydroxide (NaOH) then the sequencing primer was hardened. A 454
sequencing run was done on a GS pico TiterPlate by the help of Sequencer Genome FLX system
(Stratton, 2011).
Amplification and sequencing
With the application of universal primers which help to diagnose conventional loci like
16rDNA gene for encoding, the specific sequence can be directly amplified from the diseased
host body tissues (say human being for our case). It is then compared with a sequential database
to help deduce relationship of the phylogeny (Acton, 2014). Due to the amazing merits of this
technique (molecular), it can easily be applied to identify the origin of the bacteria (Salmonella
typhi) on the basis of the clinical response and histologic studies. Moreover, culture and isolation
were attained at the same time as the molecular identification. These scenarios propose that
molecular skills are basically important for taxonomic learning and identification of the
Salmonella typhi (Debnath, 2014).

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Electron microscopy (EM)
Negative staining is a swift EM technology which is very vital to a doctor or a nurse to
diagnose a patient who is not able to explain about his or her illness (unexplained illness)
(Leonard, 2010). Moreover, its sensitivity and specificity can be improved by the application of
immune-capture assay. Hence, for the patient suffering from typhoid, Electron Microscopy gave
the initial identification of Salmonella typhi in the bloodstream (Acton, 2014). EM is also vital
for resolving information several hundred times smaller can be seen through when compared to
the light microscope. Therefore the causative agent of typhoid can be recognized as Salmonella
typhi are available in the bloodstream. However, EM faces challenges like its cost, need for
qualified operator and availability, it is also time-consuming.
Serology and antigen Detection
Serology can be employed to provide evidence between the newly identified bacteria
(Salmonella typhi) and the disease (typhoid). Serology is beneficial to evaluate the engrossment
of in human disease of the microorganism which has been discovered in the environment due to
its effect but has not been fully identified (Quinn, 2011). For instance, when the disease typhoid
is with us, we need to fully identify that particular pathogen then we start on how to eradicate it.
Where the eradication will require a full and accurate identification of the bacteria and know its
features and where the bacteria can survive and in which environment it cannot survive in. For
the Salmonella typhi, the organism is not able to survive in a hot environment and that is why it
can be eradicated when the drinking water is boiled to enable the killing of the bacteria (Stratton,
2011).

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Serology is also a tool for discovering typhoid spectrum of the Salmonella typhi.
Therefore, serology technique contributes to the cognition of Salmonella typhi as the main and
the only agent of typhoid. In addition, the impact of serologic studies to the identification of
Salmonella typhi should be treated with a lot of care (Stratton, 2011). Serologic cross-reaction
are similar in the member of a given bacterial genus. Engrossment of the Salmonella typhi in the
human body tissue was proposed by detection of particular antibody in the patient’s body fluid.
This reliable analysis of serologic cross-reaction was possible due to the evidence of
intraleukocytic morulae (Liu, 2011).
Antigen Detection
This generates particular antibodies in studies done with animals as a model during the
experiment, this will provide immunochemical identification skills. We can employ the use of
direct immunofluorescence staining which can be smeared patient’s respiratory fluid (patient
with pneumonia) (Stratton, 2011). Immunohistochemistry is very vital in the demonstration of a
disease causation since this offers proof for in connotation of situ between the histologic
structure and the microorganism (Salmonella typhi ). For this technique, Salmonella typhi was
noticed in the patient’s body fluid (blood) which was identified with vomits, fever, and loss of
body weight and loss of appetite
Cultural techniques.
These techniques involve the traditional and cultural ways of identifying a bacteria, the below
are some of these techniques elaborated in details;
Direct Detection in Smears

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Traditionally, morphologic approaches play a vital role in identifying Salmonella typhi
and they are still being employed for diagnosing infections which are caused by agents
(Salmonella typhi ) but not periodically cultured. Since the microscopic diagnosis of smear stains
from body tissues imprints is usually easy and quicker, it has been done to patients who are not
able to explain their condition since they are dumb, though its elucidation is highly subjective
and its specificity and sensitivity are basically low (Debnath, 2014). The first substantiation for
the accountability of Salmonella species in a man with a severe febrile illness was detected by
diagnosing the smears of blood stains in conjunction with a Romanowsky stain. Evaluation of
smear is very vital when several organisms are cultured from a nostril site (Stratton, 2011).
Axenic Media
Broad spectrum media permits formerly unrecognized positive bacteria like for our case
salmonella typhi. For salmonella species, a selective antibiotic having media could be
satisfactorily substituted by non-selective blood stains, so long as the stool specimen had been
fully filtered with what is known as a cellulose acetate. The helpfulness of the wide spectrum
media must not obscure the fact that some bacteria especially salmonella typhi would have been
isolated without a specific media. Basically, conjoining diverse types of the medium by the help
of both solid and liquid specific media upsurges the efficiency of cultural techniques. For
instance, in our case study salmonella typhi can be detected in the bloodstream of an individual
who has these types of bacteria (Stratton, 2011).
Detection in Tissue section
Even though specific bacteria basically are not identified in hematoxylin and eosin stain
body tissues fragments, exceptions in many scenarios occurs. Clusters of minute particulate

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basophil material can easily be identified (seen) in the stain fragments of a blood sample which
indicate serious typhoid, curved bacteria with many Salmonella typhi may be visible in clots of
the blood which was used to do the diagnosis (Bavoil, 2010). In addition, as histopathogenic
harm and casual microorganism always have a long-recognized connotation, microscopic
evaluation of stain tissue segments during the diagnosis of unexplained illness (the illness can be
unexplained due to the status of the patients) may result to thesis on the condition of the etiologic
agent of this Salmonella typhi (Bavoil, 2010). Morphologic skills, in fact, are not allowing exact
identification of the noticed bacteria. Notwithstanding this bottleneck, the method constituting
the identification of contagious injury which is not obvious but can sometimes appear in the
patient's mouth and agents by application of histologic inspection and cytologic appeared
sometimes to be valued than molecular techniques (Stratton, 2011).
Conclusion
An in-depth study of the histories of bacterial infections gives a new vision into a very
vital role played by the different identification skills used like the molecular and cultural
techniques. Due to the spectacular progress of the molecular techniques, cultural methods have
been highly treated as obsolescent. Even if molecular faces some bottlenecks like cost, buts it is
still better than the cultural method. Cultural techniques are very cheap and simple to use
sometimes but they are not as accurate as for the molecular methods (Chang Lu, 2015). Cultural
is still the exceptional method of identifying a bacteria even if it faces some challenges. The
history of contagious illness indicates that no man’s bacterial illness is unproductive so far
(Bavoil, 2010). The real scenario seems to show whether a scientist is capable to decide the
ecological conditions obligatory by the prokaryotic agent for development or not. Cultural

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methods for identifying bacteria was predominantly applied in the traditional world when the
modern technologies were not available. Molecular methods are basically modern techniques
employed for identification of bacteria (Acton, 2014).

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