Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

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Sir George G. Stokes

� The phenomenon of fluorescence was known by the middle of the nineteenth century. British scientist Sir George G. Stokes first made the observation that the mineral fluorspar exhibits fluorescence when illuminated with ultraviolet light, and he coined the word "fluorescence"

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Discovery of Fluorescence Microbiology

� The fluorescence microscope was devised in the early part of the twentieth century by August Köhler, Carl Reichert, and Heinrich Lehmann, among others. However, the potential of this instrument was not realized for several decades, and fluorescence microscopy is now an important (and perhaps indispensable) tool in cellular biology.

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Differences between Conventional and Fluorescent Microscope

�The Conventional microscope uses visible light (400-700 nanometers) to illuminate and produce a magnified image of a sample.

� A fluorescence microscope, uses a much higher intensity light source which excites a fluorescent species in a sample of interest. This fluorescent species in turn emits a lower energy light of a longer wavelength that produces the magnified image instead of the original light source.

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What is Fluorescence?

�Fluorescence is light produced by a substance when it is stimulated by another light. Fluorescence is called "cold light" because it does not come from a hot source like an incandescent light bulb.

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�� Fluorescence microscopy is a unique way of using a

microscope to discover facts about specimens that often are not shown by standard bright field microscopy. In bright field microscopy, specimens are illuminated from outside, below or above, and dark objects are seen against a light background. In fluorescence microscopy, specimens are self-illuminated by internal light, so bright objects are seen in vivid color against a dark background. Bright objects against dark backgrounds are more easily seen. This characteristic of fluorescence microscopy makes it very sensitive and specific.

What is Fluorescence Microscopy?

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��Most cellular components are colorless and cannot be

clearly distinguished under a microscope. The basic premise of fluorescence microscopy is to stain the components with dyes. Fluorescent dyes, also known as fluorophores of fluorochromes, are molecules that absorb excitation light at a given wavelength (generally UV), and after a short delay emit light at a longer wavelength. The delay between absorption and emission is negligible, generally on the order of nanoseconds. The emission light can then be filtered from the excitation light to reveal the location of the fluorophores.

Principle of Fluorescent Microscopy

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Principle of Fluorescent Microscopy

� Fluorescence microscopy uses a much higher intensity light to illuminate the sample. This light excites fluorescence species in the sample, which then emit light of a longer wavelength. The image produced is based on the second light source or the emission wavelength of the fluorescent species -- rather than from the light originally used to illuminate, and excite, the sample.

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Works on Faster Transmission of Light

� Fluorescence, describes light emission that continues only during the absorption of the excitation light. The time interval between absorption of excitation light and emission of re-radiated light in fluorescence is of extraordinarily short duration, usually less than a millionth of a second.

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Works on Principles of Light Pathways

� Specifically, a dichroic mirror is used to separate the excitation and emission light paths. Within the objective, the excitation/emission share the same optics. In a fluorescence microscope, the dichroic mirror separates the light paths.

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�� Fluorescence microscopy is the most popular method for

studying the dynamic behavior exhibited in live cell imaging. This stems from its ability to isolate individual proteins with a high degree of specificity amidst non-fluorescing material.

�The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer.

� Different molecules can now be stained with different colors, allowing multiple types of molecule to be tracked simultaneously. These factors combine to give fluorescence microscopy a clear advantage over other optical imaging techniques, for both in vitro and in vivo imaging.

Advantages of Fluorescent Microscopy

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Fluorescence Microscope

�Fluorescence microscopy by

epi-illumination is the most commonly used method today because it is simple to do, needs relatively simple equipment, and is efficient.

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Epifluorescence Microscopy

� Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life sciences The excitatory light is passed from above (or, for inverted microscopes, from below), through the objective lens and then onto the specimen instead of passing it first through the specimen. The fluorescence in the specimen gives rise to emitted light which is focused to the detector by the same objective that is used for the excitation

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The Specimens to be Stained

�Most specimens for fluorescence microscopy must be stained. Fluorescent stains are called "fluorochromes."

Acridine orange, auramine O, and fluorescent antibody (FA) are the fluorochromes used most.

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Fluorescence Microscopy applied in Many Braches of Science and Medicine

�uses of fluorescence microscopy are many and varied. They are in medicine, public health, biological research, and environment monitoring. The most common

application is medical laboratory diagnosis.

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How to Use a Fluorescence Microscope

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How to Use a Fluorescence Microscope

� The object to be studied is marked with a molecule called a fluorophore (a dye). When the florescent light is activated, the light used for illumination is separated from the florescent molecule (the fluorophore), which is much weaker. This is done through an emission filter.

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Step 1� Locate the light switch on

the side of the microscope that turns on the light. Turn the microscope on.

�Write down the exact time you turn on the light. The florescent light is mercury-based, and a time log must be kept for exposure and use of the light.

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Step 2

�Locate the toggle switch on the right side of the microscope between the oculars and objectives. This switch controls the shutter for the mercury light to the objective lens.

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Step 3

� Select the appropriate dye for your object (this will depend entirely on what you are going to be studying). The most common dyes include I3 (for use with CTC, DTAF and fluorescein), A (for use with DAPI and f420), N21 (for use with Rhoda mine) and L3 (for use with fluorescein).Dr.T.V.Rao MD 20

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Step 4

�Put the filter (dye) into the tray operated by the silver sliding knob. To remove the tray, simply pull the silver knob out.

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Step 5

� Select the lens you would like to use. The 63x objective lens will have the highest numerical aperture. The 100x objective lens will have the highest magnitude that can be used with the mercury-based florescent light source.

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Step 6

� Turn the light off when finished, and mark the time. Wait 30 minutes before turning the light back on, or the lamp could explode. It is a good idea to keep track of how many hours the lamp is in use and replace it according to the manufacture's guidelines.

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Step 7

�Clean off the microscope lens with lens paper, or if really dirty, use a cotton swap and glass cleaner.

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�� A specific antibody is labeled by chemically attaching a

fluorophore to form what is known as a conjugate, which is then spread on a microscope slide containing the suspected presence of a particular antigen known to stimulate production of the antibody. If the antigen is present, the labeled antibody conjugate binds to the antigen and remains bound to the specimen after it is washed. The presence of the chemically attached fluorescent conjugate and antigen is demonstrated when the fluorophore is excited at its excitation peak, and the subsequent emission intensities at various wavelengths can then be observed visually or captured by a detector system (digital or traditional camera).

Direct Immunofluorescence

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�� Indirect immunofluorescence here serum possibly containing

unlabeled antibody and its related, but known, antigen are incubated together. A fluorochrome conjugated to an anti-human antibody (if the specimen being tested is human) is then placed on the slide containing the unlabeled antibody-antigen. If indeed, there has been an antigen-antibody reaction, the fluorochrome-labeled anti-human antibody attaches itself to the complex formed by the antigen and antibody. Subsequently, the labeled grouping of antigen, antibody, and fluorochrome labeled anti-human antibody is excited at the peak wavelength intensity for that fluorochrome and any resulting emission is observed. The indirect immunofluorescence technique reduces the necessity of keeping in stock large numbers of labeled antibodies, and also usually results in greater fluorescence intensity. fluorescence,

Indirect Immunofluorescence

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��Advantages of fluorescence microscopy are due to its sensitivity, specificity, rapid testing, and easy use. It is easy to set up and do, provides rapid diagnostic tests, and can be very specific. Modern technology allows conversion of most compound microscopes easily and economically into effective fluorescence microscopes.

Fluorescence Microbiology Modernises the Diagnostic Laboratories

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Fluorescent Staining in Tuberculosis

� The Auramine-rhodamine process uses a yellow fluorescent dye to visualize Mycobacterium tuberculosis under a fluorescence microscope. Potassium permanganate or acridine orange can be used as a counterstain. Under the lens, the bacterial cells will appear green.

� The Auramine-rhodamine stain is more sensitive than the Zhiel-Neelson and more cost effective.

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�Auramine-phenol solution

Auramine O 25 gEthanol 3000 mlPhenol 250 gDistilled water 5300 ml

� Suspend 25 g auramine-O in 3000 ml of ethanol in a 5-litre conical flask. Add a magnet and place on a magnetic stirrer until solution is complete.

� Dissolve 250 g phenol in 5300 ml of distilled water.� Mix the phenol solution with alcoholic auramine solution.� Store in an amber colored bottle at room temp. for up to 3 mths.

� Filter before use.

Reagents for staining

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�Acid-alcohol for decolorization

Sodium chloride 20 gHydrochloric acid, A.R. 20 mlDistilled water 500 mlEthanol 1500 ml

� Dissolve sodium chloride in distilled water

� Add conc. hydrochloric acid, mix thoroughly.

� Add alcohol

� Can be stored at room temperature for 3 months.

Reagents for staining

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�Counter stain

Potassium permanganate 1 gm

Distilled water 1000 ml

� Dissolve and store in an amber colored bottle

� Stays at room temperature for up to 3 months.

Reagents for staining

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��Use of 1000x magnification

�Use of oil immersion

�Examination of 300 microscopic fields

�About 15 minutes to examine one negative smear

�Examination by an experienced microscopist

Fuchsin-stained smears require -

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��Place the slides on a staining rack, with the smeared side facing up, the slides not touching each other

�Flood the slides with freshly filtered auramine-phenol. Let stand for 7-10 min.

�Wash well with running water

�Decolorize with acid-alcohol for 1-2 min.

�Wash as before with water and slope the slides to air dry

�Counter stain with 0.1% KMNO4 for 30 seconds

Staining procedure

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� Avoid under-decolorization. AFBs are difficult to over-decolorizesince the decolorization procedure with acid-alcohol is relativelymilder than the 25% H2SO4 used in Z-N.

� Thick smears: Interfere with decolorization, and counter stain.Mask the presence of AFB and tendency to flake, resulting in lossof smear material and possible transfer of material to other slides

� Strong counter stain: May mask the presence of AFB

� Re Staining : Smears examined by FM may be restained by Z-N toconfirm findings. However, Z-N stained smears cannot be used forFM

� Fading: Stained smears may fade on exposure to light. To be storedwrapped in brown or black paper and kept away from light

Staining procedure - Precautions

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EXAMINATION PROCEDURE

� The mercury vapor lamp : It takes about 10 min. to

reach full intensity

� Examination: Using the low power objective (100-

150x) first examine a known pos. slide to ensure

that the microscope is correctly set up

� Appearance: Bacilli appear as slender bright

yellow fluorescent rods, standing out clearly

against a dark background

� Positive: Minimum of 4 AFB in the entire smear.

� Negative: Less than 4 bacilli ( No.of bacilli to be

recorded)

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Screen the Smear in Defined Pattern

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Identify the Acid Fast Bacilli with Caution

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EXAMINATION PROCEDURE

� The mercury vapor lamp : It takes about 10

min. to reach full intensity

� Examination: Using the low power

objective (100-150x) first examine a known

pos. slide to ensure that the microscope is

correctly set up

� Appearance: Bacilli appear as slender

bright yellow fluorescent rods, standing out

clearly against a dark background

� Positive: Minimum of 4 AFB in the entire

smear.

� Negative: Less than 4 bacilli ( No.of bacilli

to be recorded)

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Grading is Essential to Determine Prognosis

� Morphological Confirmation: With a

high power objective (400-600x) - To

be done with all doubtful smears as

well as scanty positives.

� Examination: At least three horizontal

sweeps on the entire smear.

� Gradation: Grade positive smears into

three degrees of positivity using the

high power objective

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QUANTIFICATION OF FLUOROCHROME SMEAR RESULTS

ZN

1000x

REPORT

FM Magnification

250x 450x 630x

0 NO.AFB 0 0 0

1-9/100FIELDEXACT NO

DIVIDE

by 10 by 4 by 2

10-99/FIELD 1+

1-10/FIELD 2+

>10/FIELD 3+

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Examining and Reporting Acid-fast Smears

Number of AFB Observed

Report 200x,250x 400x,450x

No AFB seen 0 0

Doubtful: repeat 1-2/30F* 1-2/70F

1+ 1-9/10F 2-18/50F

2+ 1-9/F 4-36/10F

3+ 10-90/F 4-36/F

4+ >90/F >36/F

* number of acid-fast bacilli observed per microscopic field

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FM Grading

No. of bacilli per HPF Grade

Less than 6 per field 1+1+1+1+

6-100 bacilli per field 2+2+2+2+

More than 100 per field or large

clumps

3+3+3+3+

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��More than 50 smears examined/day

� Continuous availability of power

�More sensitive. where small No. of bacilli arepresent

�Majority of FM +ve samples are also +ve byculture

�Does not yield more false positive than ZN

�Doubtful smears to be re examined by ZN

Fluorescence Microscopy Advantages in Acid Fast Bacilli Identification

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�� The quality of a photomicrograph, either digital or

recorded on film, is dependent upon the quality of the microscopy. Film is a stern judge of how good the microscopy has been prior to capturing the image. It is essential that the microscope be configured using Köhler illumination, and that the field and condenser diaphragms are adjusted correctly and the condenser height is optimized. When properly adjusted, the microscope will yield images that have even illumination over the entire field of view and display the best compromise of contrast and resolution.

How to get better Photomicrograph for Documentation

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QBC Malaria TestUses the Principles of fluorescence

�The QBC Malaria Test is a fluorescence microscopy-based malaria diagnostic test that speeds and simplifies malaria detection

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�� The method is centrifugation and thereby concentration of

the red blood cells in a predictable area of the QBC tube, making detection easy and fast. Red cells containing Plasmodia are less dense than normal ones and concentrate just below the leukocytes, at the top of the erythrocyte column. The float forces all the surrounding red cells into the 40 micron space between its outside circumference and the inside of the tube. Since the parasites contain DNA which takes up the acridine orange stain, they appear as bright specks of light among the non-fluorescing red cells. Virtually all of the parasites found in the 60 microliter of blood can be visualized by rotating the tube under the microscope. A negative test can be reported within one minute and positive result within minutes.

Quantitative Buffy Coat (QBC) Test

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��MICROSCOPIC EXAMINATION

� 0.1% Calcofluor White (W/V) Solution

� (a) Use commercially available solution cellufluor 17352 (Polysciences, Washington, PA), fluorescent brightener 28.F6259 (Sigma, St. Louis, MO), 1 gm

� (b) distilled water, 100 ml

� (c) Gently heat if precipitate develops. Filter if precipitate persists. Store at 25 C in the dark.

� (3) Commercially prepared kits are

EXAMINATION OF SPECIMENS for Fungal Diseases

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Need Specific Protocols

� Microscope filter system: An epifluorescent microscope equipped with a mercury vapor lamp and either an ultraviolet (UV) or blue-violet (BV) excitation filters to achieve radiation on the slide below 412 nm should be used since the maximum absorbance of CFW is 347 nm.

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Fluorescent stains for demonstrating Cryptosporidium spp.oocysts include Auramine- rhodamine, Auramine CarbolFuschin and Acridine orange . Confirmatory staining ofsuspected oocyst by another method may be required

Casemore 1984

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Fluorescent stains Cryptosporidium

spp.

� Fluorescent stains for demonstrating Cryptosporidium spp. oocysts include Auramine-rhodamine, Auramine Carbol Fuschin and Acridine orange . Confirmatory staining of suspected oocyst by another method may be required

�Casemore 1984

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Immunofluorescent antibody (IFA) procedure employing

cryptosporidium- specific polyclonal or monoclonal antibodies has been developed

•Polyclonal AB, raised against 18 and 20 kDa C. parvumcoproantigen, were used to react with C. parvumsporozoites in an immunofluorescence assay.•Monoclonal antibody reagents offer increasedsensitivity and an excellent alternative to conventionalstaining methods.•These reagents are helpful when screening largenumbers of patients or those with minimal symptoms.•Elimination of the problems of false-positive and false-negative results with routine staining methods.

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Giardia and Cryptosporidium

�The sensitivity and specificity of the Merifluor DFA test, have been reported to be 96 to 100% and 99.8 to 100%, respectively, for both Giardia and Cryptosporidium

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��Several Immuno Diagnostic Methods are

available Using the Fluorescent Techniques, which needs specific testing material and to follow specific protocols. The Technologists should be familiar with literature available with the Kits

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Enumerable Possibilities with Florescent Methods

��Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for Microbiologists

in the Developing world � Email

� doctortvrao@gmail.com

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