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When samples are prepared for viewing using an SEM, they must also be dehydrated using an ethanol series. However, they must be even drier than is necessary for a TEM. Critical point drying with inert liquid carbon dioxide under pressure is used to displace the water from the specimen. After drying, the specimens are sputter-coated with metal by knocking atoms off of a palladium target, with energetic particles. Sputter-coating prevents specimens from becoming charged by the SEM’s electron beam.

  • Why is it important to dehydrate cells before examining them under an electron microscope?
  • Name the device that is used to create thin sections of specimens for electron microscopy.

Using microscopy to diagnose syphilis

The causative agent of syphilis is Treponema pallidum , a flexible, spiral cell (spirochete) that can be very thin (<0.15 μm) and match the refractive index of the medium, making it difficult to view using brightfield microscopy. Additionally, this species has not been successfully cultured in the laboratory on an artificial medium; therefore, diagnosis depends upon successful identification using microscopic techniques and serology (analysis of body fluids, often looking for antibodies to a pathogen). Since fixation and staining would kill the cells, darkfield microscopy is typically used for observing live specimens and viewing their movements. However, other approaches can also be used. For example, the cells can be thickened with silver particles (in tissue sections) and observed using a light microscope. It is also possible to use fluorescence or electron microscopy to view Treponema ( [link] ).

Micrograph a shows clear spirals on a black background. Micrograph b shows purple spirals on a clear background. Micrograph c shows three-dimensional spirals.
(a) Living, unstained Treponema pallidum spirochetes can be viewed under a darkfield microscope. (b) In this brightfield image, a modified Steiner silver stain is used to visualized T. pallidum spirochetes. Though the stain kills the cells, it increases the contrast to make them more visible. (c) While not used for standard diagnostic testing, T. pallidum can also be examined using scanning electron microscopy. (credit a: modification of work by Centers for Disease Control and Prevention; credit b: modification of work by Centers for Disease Control and Prevention; credit c: modification of work by Centers for Disease Control and Prevention)

In clinical settings, indirect immunofluorescence is often used to identify Treponema. A primary, unstained antibody attaches directly to the pathogen surface, and secondary antibodies “tagged” with a fluorescent stain attach to the primary antibody. Multiple secondary antibodies can attach to each primary antibody, amplifying the amount of stain attached to each Treponema cell, making them easier to spot ( [link] ).

Fluorochromes are attached to secondary antibodies (unstained). Two secondary antibodies are attached to a single primary antibody (unstained). The primary antibody is attached to a spiral labeled T. pallidum cell
Indirect immunofluorescence can be used to identify T. pallidum , the causative agent of syphilis, in a specimen.

Preparation and staining for other microscopes

Samples for fluorescence and confocal microscopy are prepared similarly to samples for light microscopy, except that the dyes are fluorochromes. Stains are often diluted in liquid before applying to the slide. Some dyes attach to an antibody to stain specific proteins on specific types of cells ( immunofluorescence ); others may attach to DNA molecules in a process called fluorescence in situ hybridization (FISH) , causing cells to be stained based on whether they have a specific DNA sequence.

Questions & Answers

life arises from living matter or live organism.
Swami Reply
yes
sildra
I think live matter arises from non living matter
sildra
I dont think so...can u explain with an example
Manya
living maters made by non living matters
sildra
non living matters like stones? rocks?
Manya
eno
sildra
no
sildra
then?
Manya
cells are made by C N O minerals etc
sildra
I mentioned these as non living maters
sildra
that's all
sildra
cells are made up of those things but they originate from living things..
Manya
ok
sildra
Ok..good chat:-)
Manya
where are you from
sildra
thanks
sildra
India...u?
Manya
Tamil nadu
sildra
I am from Maharashtra
Manya
what about your studies
sildra
completed bsc.. preparing for msc entrance...wbu?
Manya
same
sildra
are you microbiologist
sildra
yes i am
Manya
good
sildra
what s the scope for micro in ur state?
Manya
did you find your college to higher studies
sildra
have to give an entrance exam for every college here...so lets c
Manya
food industries, medical lab, vaccine industries ,etc
sildra
hoping for pune University...wbu?
Manya
great!
Manya
is that centeral University right
sildra
what is your namr
sildra
hello
Udhaya
Hi
Maruf
Family kindly help me with this question? 1) Shortlist the configurative measurements of the following human anatomical ranges of÷ - Blood ( haemeglobin) in both male and female - Haematocytes in both male and female - Hepatocytes in both male and female - Lymphocyte / T. Lymphocytes in both male
Gifted
My names are Gift Mwale and am a Zambian. Kindly help me with this research which goes like this... 1) Shortlist the configurative measurements of the following human anatomical ranges of ÷ - Blood ( haemeglobin) in both male and female - Hepatocytes in both male and female - Haematocytes in both
Gifted
please can anybody talk about brain tumour and its cure.
BELLO Reply
enlargement of the thyroid gland resulting in over production of hormone.
Kamal Reply
What can u say on Thyroid Cancer?
Abdulkareem
Please, talk about the thyroid cancer.
BELLO
explain the Grave's disease
John Reply
what is cell
Avi Reply
is unit of life
Kamaluddeen
Ok
mohamed
who is an industrial microbiologist
Cynthia Reply
I want to know the biochemical composition of bacteria
Josh Reply
It contains peptidoglcon, DNA nd RNA
Asiya
bacteriophage disadvantage
Momina Reply
disease due to __________ abnormalities are termed primary immunodeficiencies
Tayee Reply
Some primary immunodeficiencies are due to a defect of a single cellular or humoral component of the immune system.
Prince
Examples of primary immunodeficiencies include: chronic granulomatous disease, X-linked agammaglobulinemia, selective IgA deficiency etc
Prince
thank you
Nana
explain microbial mutation
Emerald
what is mutation
Cynthia Reply
alteration in genetic makeup
UDEME
Hi, I'm new here. I'm Bello Abdul Hakeem from Nigeria.
BELLO Reply
welcome on board
Kamaluddeen
Thanks brother I'm an undergraduate. I hope to study for MBBS.
BELLO
pls guys help me out
Linda
what is limitation of plate
Linda
limitation of plate load test . there are some that should be considered while performing load test which are given below, this test is usually performed on relatively similar plate ,usually 1 or 2 square foot area the reason is that the plate of greater are the economically not feasible
yemi
iam new here my name shafiu umar from Nigeria
Shafiu
thanks shafiu
gambo
how to differentiate Gram positive from Gram negative bacteria?
Faheem
by the cell membrane
Tim
with gram staining which has 4 steps
Freedom
Mechanism of bacterial pathogenicity
rashida Reply
Toxicity
Ibrahim
write a short note on Algea
najaatu Reply
green minutes plants organisms that are produced in turf.
Prince
Algae is a kind of a photosynthetic organism, which is usually grown in the moist areas. These are usually the simple plants that grow near to the water bodies. It contains a kind of chlorophyll pigments that act as a primary coloring agent.
Avi
they are eukaryotic and most lived in fresh water. they are photosynthetic that's, they contain chlorophyll and store starch in discrete chloroplast.
Prince
are bacteria important to man
just Reply
yes, depending on the type of bacteria .eg the normal florals, the latic acid batteries etc are important to man
Lois
I mean latic acid bacterial
Lois
I don't understand this topic
Jane Reply
what topic is that
Rose
okay. Go ahead and ask
Blessing Reply
Industrial microbiology mcq
mohamed
Okay. What's your question?
Blessing

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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