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5 Questions to Ask Before You dark field and bright field microscopy

dark field and bright field microscopy

How to Make a dark field and bright 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.
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
The first picture of the plankton was taken by Uwe Kils and is from Wikipedia under the GNU Free Documentation License.

dark field and bright field microscopy

When to Use a Dark Field Microscope?

dark field and bright field microscopy are used in a number of different ways to view a variety of specimens that are hard to see in a light field unit. Live bacteria, for example, are best viewed with this type of microscope, as these organisms are very transparent when unstained.

There are multitudes of other ways to use dark field illumination, often when the specimen is clear or translucent. Some examples:

Dark field illumination of caffeine crystalsLiving or lightly stained transparent specimens
Single-celled organisms
Live blood samples
Aquatic environment samples (from seawater to pond water)
Living bacteria
Hay or soil samples
Pollen samples
Certain molecules such as caffeine crystals (right)
dark field and bright field microscopy makes many invisible specimens appear visible. Most of the time the specimens invisible to bright field illumination are living, so you can see how important it is to bring them into view!

dark field and bright field microscopy

What dark field and bright field microscopy for point-of-care syphilis diagnosis?

What dark field and bright 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.

dark field and bright 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.

dark field and bright 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. dark field and bright 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.

dark field and bright field microscopy

What Disadvantages of dark field and bright field microscopy

What Disadvantages of dark field and bright field microscopy

A dark field microscope can result in beautiful and amazing images; this technique also comes with a number of disadvantages.

First, dark field images are prone to degradation, distortion and inaccuracies.
A specimen that is not thin enough or its density differs across the slide, may appear to have artifacts throughout the image.
The preparation and quality of the slides can grossly affect the contrast and accuracy of a dark field image.
You need to take special care that the slide, stage, nose and light source are free from small particles such as dust, as these will appear as part of the image.
Similarly, if you need to use oil or water on the condenser and/or slide, it is almost impossible to avoid all air bubbles.
These liquid bubbles will cause images degradation, flare and distortion and even decrease the contrast and details of the specimen.
Dark field needs an intense amount of light to work. This, coupled with the fact that it relies exclusively on scattered light rays, can cause glare and distortion.
It is not a reliable tool to obtain accurate measurements of specimens.
Finally, numerous problems can arise when adapting and using a dark field microscope. The amount and intensity of light, the position, size and placement of the condenser and stop need to be correct to avoid any aberrations.
Dark field has many applications and is a wonderful observation tool, especially when used in conjunction with other techniques.

However, when employing this technique as part of a research study, you need to take into consideration the limitations and knowledge of possible unwanted artifacts.

dark field and bright field microscopy

dark field and bright field microscopy advantages

 

No one system is perfect, and dark field and bright field microscopy may or may not appeal to you depending on your needs.

Some advantages of using a dark field microscope are:

Extremely simple to use

Inexpensive to set up (instructions on how to make your own dark field microscope are below)

Very effective in showing the details of live and unstained samples

dark field and bright field microscopy

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