What (Historical Person’s Name) Can Teach Us About treponema pallidum dark field microscopy | dark field microscope,dark field microscope manufacturer.
dark field microscopy,dark field microscope,darkfield microscope,darkfield microscopy
We are dark field microscopy,dark field microscope manufacturer.Welcome OEM.

What (Historical Person’s Name) Can Teach Us About treponema pallidum dark field microscopy

treponema pallidum dark field microscopy

What treponema pallidum dark field microscopy for point-of-care syphilis diagnosis?

What treponema pallidum dark field microscopy for point-of-care syphilis diagnosis?

Syphilis is a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum subspecies pallidum. Globally, an estimated 12 million cases of syphilis occur annually. In the United States, 13,997 cases of primary and secondary (infectious) syphilis were reported to the Centers for Disease Control and Prevention (CDC) in 2009, a 3.7% increase from 2008 and a 134% increase from 2000, when a post-war low of 5,979 primary and secondary syphilis cases was reported. Men who have sex with men (MSM) — especially those who are HIV infected — and blacks are disproportionately affected by syphilis. Geographically, urban areas and the Southeastern region of the United States have the highest rates.

Syphilis is most commonly transmitted by skin-to-skin (or mucous membrane) contact. Following exposure, the infection passes through the following stages:

Primary syphilis, characterized by a painless ulcer, called a chancre, usually develops three weeks after exposure (range 10 days to 90 days) at the site of inoculation. The chancre heals spontaneously after several weeks.

Secondary syphilis is most often characterized by a generalized rash that also resolves without treatment. Rash on the palms and soles can also occur, as can systemic manifestations such as fever, malaise, and lymphadenopathy. Given the widely variable nature of the rash and other manifestations of the disease, syphilis has acquired the moniker “The Great Imitator.”

Early (one year) latent syphilis, defined by the absence of signs or symptoms of disease and diagnosed by serologic evidence of infection.

Tertiary syphilis, which affects about a third of untreated patients and manifests with cutaneous, cardiovascular, or neurologic disease.

Syphilis can also be acquired in utero at any stage of pregnancy and lead to congenital syphilis. Routine syphilis screening and treatment in pregnant women has made congenital syphilis rare in the United States.

Approaches to syphilis diagnosis

Because T pallidum is too fragile an organism to be cultured in the clinical setting, diagnostic testing relies on two approaches: direct detection of the organism and indirect evidence of infection.
Syphilis – Treponema pallidum on darkfield.

Direct methods include darkfield microscopy, molecular assays to detect T pallidum DNA, and histopathologic examination of biopsies of skin or mucous membranes (which can also provide indirect evidence of infection, on the basis of patterns of inflammation in the tissue). Direct methods have the advantage, in some cases, of detecting infection before a patient has mounted a measurable antibody response that results in a reactive serologic test result.

treponema pallidum dark field microscopy allows visualization of live treponemes obtained from a variety of cutaneous or mucous membrane lesions, as follows.

In primary syphilis, the chancre teems with treponemes that can be seen with darkfield microscopy. The sensitivity of darkfield microscopy for the diagnosis of primary syphilis is approximately 80%. Darkfield sensitivity declines over time and can also decrease if the patient has applied topical antibiotics to the lesion(s). Of note, the mouth harbors normal non-pathogenic treponemes that are indistinguishable microscopically from T pallidum. Therefore, oral specimens cannot be used for darkfield microscopy because of the possibility of false-positive test results.

In secondary syphilis, mucous patches (as long as not oral) and condyloma lata (found in moist areas between body folds) are appropriate specimens for darkfield microscopy. Dry skin lesions usually do not contain sufficient organisms for darkfield testing.

In congenital syphilis, moist discharge from the nose (snuffles) and vesiculobullous lesions of the skin are high-yield specimen sources for darkfield testing.

Indirect methods of diagnosis include serologic testing of blood or cerebrospinal fluid (CSF) and detection of CSF abnormalities (elevated white blood cell count or protein) consistent with neurosyphilis. Serologic testing of blood involves demonstration of host antibody to either endogenous antigens (non-treponemal tests) or to antigens of T pallidum (treponemal tests). Non-treponemal tests, including the rapid plasma reagin test and the venereal disease research laboratory test, have historically been used as the initial screening tests for the serologic diagnosis of syphilis. If a patient’s non-treponemal test is reactive, confirmatory testing with a treponemal test is performed, using either the T pallidum particle agglutination test, the fluorescent treponemal antibody-absorbed test, or another treponemal test. A reactive treponemal test confirms the diagnosis of a new or previously treated case of syphilis. If the treponemal test is non-reactive, the positive non-treponemal test result is considered a biologic false-positive that is not diagnostic of syphilis. A newer algorithm that is gaining in popularity begins with a treponemal enzyme immunoassay as the initial test, followed by a non-treponemal test, and if necessary, a “tie-breaker” third test, using a different treponemal test.

treponema pallidum dark field microscopy

In 1830, J.J. Lister (the father of Joseph Lister) invented the darkfield microscope, in which the standard brightfield (Abbe) condenser is replaced with a single- or double-reflecting darkfield condenser. The use of indirect light allows visualization of organisms too small to be seen under direct-light microscopy. In 1906 in Vienna, Karl Landsteiner and Viktor Mucha were the first to use darkfield microscopy to visualize T pallidum from syphilis lesions. Since then, darkfield microscopy has served a vital role in the diagnosis of infectious syphilis.

Clinicians and laboratorians should use universal precautions in collecting, transporting, and handling specimens for darkfield examination. Acquisition of syphilis through occupational exposures, including contact with specimens collected for darkfield microscopy, has been reported.

Proper specimen collection and handling is critical for optimizing the sensitivity of darkfield testing. The clinician should gently cleanse and abrade the lesion with moist gauze, while trying not to cause bleeding. The goal is to obtain serous exudate, while minimizing contamination by blood or pus caused by secondary infection. The clinician might need to apply pressure at the margins of the lesion to express adequate serous fluid. The clinician transfers the serous fluid to a glass slide, either by direct application of the slide to the lesion, or by transferring the fluid with a bacteriologic loop or the edge of a cover slip. If necessary to prevent drying of the specimen, a drop of non-bacteriostatic normal saline may be placed on the slide; however, the saline might dilute the specimen and reduce test sensitivity. The clinician places a cover slip on top of the specimen. A trained microscopist then examines the specimen as soon as possible, no greater than 20 minutes after specimen collection. Placing the slide in a closed container such as a Petri dish during transport to the microscope might reduce evaporative drying.

Definitive identification of T pallidum depends on visualizing not only its typical morphology but also its typical motility. T pallidum is a delicate, tightly spiraled, corkscrew-shaped organism that rotates as it slowly moves backwards and forwards (translational movement); these movements are sometimes accompanied by a slight side-to-side oscillation. T pallidum will occasionally flex or bend sharply in the middle when obstructed by cellular elements or debris in the field but then spring back to its usual linear shape. In the genital region, Treponema refringens, which is part of the normal genital flora, can be distinguished from T pallidum by T refringens’ more coarsely wound spirals, greater flexibility, and rapid translational movement across the slide. In addition, the less experienced observer must guard against misidentifying Brownian movement of fibers or other linear debris as T pallidum.

After a methodical scanning of the entire specimen field of each slide, results are reported as one of the following:

Positive darkfield: Organisms with the characteristic morphology and motility of T pallidum observed

Negative darkfield: Either no treponemes found or spiral organisms seen but without the characteristics of T pallidum.

Unsatisfactory darkfield: The specimen could not be interpreted either due to drying or the presence of too many refractile elements, such as blood cells or fibers.

Syphilis is a legally reportable disease in all health jurisdictions in the United States. A positive darkfield examination should trigger a case report, regardless of clinical presentation or serologic results.

Because up to 25% of patients with primary syphilis have non-reactive serologic test results for syphilis, darkfield microscopy provides a critical complementary role in the identification of infectious syphilis. treponema pallidum dark field microscopy requires, however, a special microscope and a trained microscopist in close proximity to where patients are examined, and few clinical facilities other than STD clinics and some hospitals have the capacity to perform darkfield microscopy. Given the resurgence of syphilis in the United States, the development and maintenance of facilities and skills to perform darkfield microscopy are essential to syphilis prevention and control.

treponema pallidum dark field microscopy

How to Make a treponema pallidum dark field microscopy

How to Make a treponema pallidum dark field microscopy

You don’t need to buy a huge expensive set-up to experiment with dark field illumination.

To create a dark field, an opaque circle called a patchstop is placed in the condenser of the microscope. The patchstop prevents direct light from reaching the objective lens, and the only light that does reach the lens is reflected or refracted by the specimen. Easy enough, right?

If you want to make a dark field microscope you’ll first need a regular light microscope. Below is your full list of “ingredients”:

Dark field microscopeMicroscope
Hole punch
Black construction paper
Transparency film
Glue
Scissors
Pen

Now use the following steps to make your patchstop:

Set up your microscope and choose the lowest-power objective lens.

Set the eyepiece aside somewhere safe.

Open the diaphragm as wide as possible. Then slowly close it until is just encroaches on the circle of visible light.

Now bend over and take a look at the diaphragm from below. See that opening? It’s only slightly smaller than the finished patchstop you’ll create.

Punch a few circles in the black construction paper with the hole punch. Measure one against the diaphragm opening. If it’s more than 10% larger, cut it down to about that size (10% larger than the diaphragm opening). If it’s smaller, cut out a larger circle.

Cut a 5 cm square of transparency paper.

Glue the black circle onto the transparency film, about 2 cm from the corner of the square. In that free 2 cm of paper, write the correct magnification power of your objective.

Mark the patchstop with the correct magnification power.

Repeat the above steps for all the objective powers except the oil immersion lenses.

Now use your patchstop to turn a light field unit into a dark field microscope:

Select the correct patchstop for the objective power to be used.

Slip the patchstop between the filter holder and condenser. If your microscope has no filter, hold it manually below the condenser.

Remove the eyepiece.

Open the diaphragm and move the patchstop until the light is blocked entirely. Use tape to secure it if there is no condenser on your microscope.

Replace the eyepiece and examine the sample.

Thanks to Windtrader for this original guide. You can read it here on Ebay.

As you can see, a dark field microscope can let users see specimens in a whole new way, bringing those into focus that don’t stand out under intense light. Using dark field illumination can open up a whole new view of microscopy.

treponema pallidum dark field microscopy

What is treponema pallidum dark field microscopy?

Dark Field microscopy is a microscope illumination technique used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background.

When light hits an object, rays are scattered in all directions. The design of the dark field microscope is such that it removes the dispersed light so that only the scattered beams hit the sample.

The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object.

The result is a “cone of light” where rays are diffracted, reflected and/or refracted off the object, ultimately, allowing you to view a specimen in dark field.

A dark field microscope is ideal for viewing objects that are unstained, transparent and absorb little or no light.

These specimens often have similar refractive indices as their surroundings, making them hard to distinguish with other illumination techniques.

Dark field can be used to study marine organisms such as algae and plankton, diatoms, insects, fibres, hairs, yeast, live bacterium, protozoa as well as cells and tissues and is ideal for live blood analysis enabling the practitioner to see much more than is possible with other lighting methods.

treponema pallidum dark field microscopy

What is treponema pallidum dark field microscopy?

dark field microscopy of sugar crystals
Dark Field illumination is a technique used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background.

Pictured right: Highly magnified image of sugar crystals using darkfield microscopy technique

When light hits an object, rays are scattered in all azimuths or directions. The design of the dark field microscope is such that it removes the dispersed light, or zeroth order, so that only the scattered beams hit the sample.

The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object.

The result is a “cone of light” where rays are diffracted, reflected and/or refracted off the object, ultimately, allowing you to view a specimen in dark field.

treponema pallidum dark field microscopy

How treponema pallidum dark field microscopy work?

 

Microscopes are used to magnify objects. Through magnification, an image is made to appear larger than the original object. The magnification of an object can be calculated roughly by multiplying the magnification of the objective lens times the magnification of the ocular lens. Objects are magnified to be able to see small details. There is no limit to the magnification that can be achieved; however, there is a magnification beyond which detail does not become clearer. The result is called empty magnification when objects are made bigger but their details do not become clearer. Therefore, not only magnification but resolution is important to the quality of the information in an image.

The resolving power of the microscope is defined as the ability to distinguish two points apart from each other. The resolution of a microscope is dependent on a number of factors in its construction. There is also an inherent theoretical limit to resolution imposed by the wavelength of visible light (400-600nm). The theoretical limit of resolution (the smallest distance able to be seen between two points) is calculated as:

Resolution = 0.61 l/N.A.

where l represents the wavelength of light used and N.A.is the numerical aperture. The student-grade microscopes generally have much lower resolution than the theoretical limit because of lower quality lenses and illumination systems.

Standard brightfield microscopy relies upon light from the lamp source being gathered by the substage condenser and shaped into a cone whose apex is focused at the plane of the specimen. Specimens are seen because of their ability to change the speed and the path of the light passing through them. This ability is dependent upon the refractive index and the opacity of the specimen. To see a specimen in a brightfield microscope, the light rays passing through it must be changed sufficiently to be able to interfere with each other which produces contrast (differences in light intensities) and, thereby, build an image. If the specimen has a refractive index too similar to the surrounding medium between the microscope stage and the objective lens, it will not be seen. To visualize biological materials well, the materials must have this inherent contrast caused by the proper refractive indices or be artificially stained. These limitations require instructors to find naturally high contrast materials or to enhance contrast by staining them which often requires killing them. Adequately visualizing transparent living materials or thin unstained specimens is not possible with a brightfield microscope.

Darkfield microscopy relies on a different illumination system. Rather than illuminating the sample with a filled cone of light, the condenser is designed to form a hollow cone of light. The light at the apex of the cone is focused at the plane of the specimen; as this light moves past the specimen plane it spreads again into a hollow cone. The objective lens sits in the dark hollow of this cone; although the light travels around and past the objective lens, no rays enter it (Fig. 1). The entire field appears dark when there is no sample on the microscope stage; thus the name treponema pallidum dark field microscopy. When a sample is on the stage, the light at the apex of the cone strikes it. The image is made only by those rays scattered by the sample and captured in the objective lens (note the rays scattered by the specimen in Figure 1). The image appears bright against the dark background. This situation can be compared to the glittery appearance of dust particles in a dark room illuminated by strong shafts of light coming in through a side window. The dust particles are very small, but are easily seen when they scatter the light rays. This is the working principle of treponema pallidum dark field microscopy and explains how the image of low contrast material is created: an object will be seen against a dark background if it scatters light which is captured with the proper device such as an objective lens.

The highest quality darkfield microscopes are equipped with specialized costly condensers constructed only for darkfield application. This darkfield effect can be achieved in a brightfield microscope, however, by the addition of a simple “stop”. The stop is a piece of opaque material placed below the substage condenser; it blocks out the center of the beam of light coming from the base of the microscope and forms the hollow cone of light needed for darkfield illumination.

treponema pallidum dark field microscopy

Have any question, Please enter the form below and click the submit button.


*
*
*
*
5 + 3 = ?
Please enter the answer to the sum & Click Submit to verify your registration.

Related Items