CHAPTER 2: METHODS OF STUDYING BACTERIA AND CULTURE ASEPTIC TECHNIQUES Flashcards
Two types of preparations for microscopic exams:
Wet mounts and Bacterial smears
Valuable for demonstrating motility in
microorganisms
Wet mounts
– movement seen is
due to molecules of the solvent medium
bombarding with the organism’s surface;
occurs in all microscopic bodies
suspended in water
Brownian motility
– movement of a bacteria
in a given direction
True motility
in wet mount, No stain is employed since most stains kill the organisms except
vital stains
Features which may be particulate, such as spores
of fungi and ferns, and pollen grains may be best
obsserved during this technique
Wet mounts
in Normal wet mount, what is dropped in the slide
dH20
inn normal wet mount, for
greater volume of sample, or hanging
drop method preparations, use a
.
depression slide
difference of normal wet munt and hanging drop method
in normal wet mount
o Small size is okay
o It is short-term use
o uses dh20
in hanging drop mount
o Large sample size
o Long-term use
o uses vaseline or petroleum jelly
o Allows microbes to freely move around
o Allows to distinguish true motility from
Brownian motion
➢ Hanging drop method
The best smears are made from bacteria that have
grown on a solid surface such as an agar slant or
plate
The aim is to place an
appropraite concentration of cells on the slide
smear prepartion for bacterial smear
– producing a culture
Inoculation
creating the proper temperature and other
conditions to promote the growth of microbes
incubation
separating microbes from one another, grow
colonies (pure culture
isolation
observing characteristics of colonies and
cultures (color, texture, size, shape, motility)
Inspection
– main purpose is to determine the type of
microbe using biochemical, immunologic, serologic tests,
and DNA analysis
Identification
his describes the “side view” of a colony.
These are the most common
Elevation
edge of a colony (or any growth)
may be an important characteristic in identifying
organisms.
margin
provide the preparations of the antigen and the use of serum of the patients is to detect if their sample contains the antibodies specific to the antigen in the kit
Latex kits
Purpose of Serologic testing
- In vitro antigen-antibody reactions (detectability and
specificity).
o Latex kits provide the preparations of the antigen
and the use of serum of the patients is to detect if
their sample contains the antibodies specific to the
antigen in the kit. - Confirmation of identification results from other tests.
o It can detect the serotype through serologic
testing. - Determine a patient’s immune status.
o A patient is immune to a certain bacteria if the
antigen-antibody reaction is positive. (There is a
presence antibody in the patient’s serum). - Follow the course of a disease (antibody increase).
o It can be seen through serologic testing if
antibodies are increasing or decreasing in terms of
the reaction in the serological test. - Serotyping for epidemiological purposes.
t provides rapid and accurate data when it comes to dealing
with results
Molecular techniques
how bacteria evolves
Phylogenetic studies
for stain typing of
epidemiologically related organisms.
Pulsified gel electrophoresis
To separate molecular components such as DNA,
RNA and Protein in the electric field.
Pulsified gel electrophoresis
Direct detection of genes related to resistance
mechanisms such as mecA gene in Staphylococcus aureus.
Molecular techniques
– bacteria possess diverse proteins and RNA that
can sense change to their intracellular and extracellular
movement
Signaling
To detect important and unusual proteins in order
to identify bacteria and the characteristics that
were altered.
Signaling
to detect genetic
material from a specific organism such as virus, to amplify
DNA sequences
Polymerase Chain Reaction (PCR) –
– separate double-stranded DNA
Denutration
➢ – complementary sequence is attached
Annealing
– extending the gene to amplify the
genes
Extension
– specificity and quantification
of the targer organism
Quantitative PCR
is used routinely in microbiology to
separate DNA, RNA, or protein molecules using an electric
field by virtue of their size, shape, or electric charge.
Gel electrophoresis
Southern blotting, northern blotting, western blotting,
and eastern blotting are molecular techniques for
detecting the presence of microbial:
o DNA sequences (Southern)
o RNA sequences (Northern)
o Protein molecules (Western)
o Protein modifications (Eastern).
are used in microbiology as the modern
alternative to the “blotting” techniques
DNA microassays
permit the exploration of thousands
of sequences at one time.
Microassays
Used in molecular microbiology to detect the
presence of pathogens in a sample
DNA microassay
were the first to be completely analysed by DNA
Sequencing
viral genomes
was discovered as a cellular
gene regulation mechanism in 1998, but several RNAibased applications for gene silencing have already made it
into clinical trials
RNA interference (RNAi)
Leading uses for nucleic acid-based tests
- Nonculturable agents
- Fastidious, slow-growing agents
- Highly infectious agents that are dangerous to culture
- Nonculturable agents
o Human papilloma virus
o Hepatitis B virus - Fastidious, slow-growing agents
o Mycobacterium tuberculosis
o Legionella pneumophilia - Highly infectious agents that are dangerous to culture
o Francisella tularensis
To transfer living microbes from one place to another
without contamination of the culture or the sterile medium
or the surroundings
Aseptic Transfer
In order to prevent contamination of the sample, inoculating
instruments must be sterilized prior to use, immediately
before use in an incinerator or Bunser burner flame
Aseptic Transfer
- Preventing contamination of a culture with environmental
microbes
Aseptic Technique
