Dr.T.V.

Rao MD

Dr.T.V.Rao MD 1
 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"

Dr.T.V.Rao MD 2
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.

Dr.T.V.Rao MD 3
Differences between Conventional and
Fluorescent Microscope


 A fluorescence microscope,
The Conventional uses a much higher
microscope uses intensity light source
visible light (400-700 which excites a
fluorescent species in a
nanometers) to sample of interest. This
illuminate and fluorescent species in turn
emits a lower energy light
produce a magnified of a longer wavelength
image of a sample. that produces the
magnified image instead
of the original light
source.
Dr.T.V.Rao MD 4
 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.

Dr.T.V.Rao MD 5
 What is Fluorescence
Microscopy?


 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.

Dr.T.V.Rao MD 6
 Principle of Fluorescent
Microscopy


 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.

Dr.T.V.Rao MD 7
 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.
Dr.T.V.Rao MD 8
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.
Dr.T.V.Rao MD 9
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.

Dr.T.V.Rao MD 10
 Advantages of Fluorescent
Microscopy


 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.

Dr.T.V.Rao MD 11
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.
Dr.T.V.Rao MD 12
 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

Dr.T.V.Rao MD 13
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.
Dr.T.V.Rao MD 14
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.
Dr.T.V.Rao MD 15
How to Use a
Fluorescence
Microscope
Dr.T.V.Rao MD 16
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.

Dr.T.V.Rao MD 17
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.
Dr.T.V.Rao MD 18
 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.

Dr.T.V.Rao MD 19
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
Dr.T.V.Rao MD with fluorescein). 20
 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.
Dr.T.V.Rao MD 21
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.

Dr.T.V.Rao MD 22
 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.

Dr.T.V.Rao MD 23
Step 7


Clean off the
microscope lens
with lens paper,
or if really dirty,
use a cotton
swap and glass
cleaner.
Dr.T.V.Rao MD 24
 Direct Immunofluorescence


 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).

Dr.T.V.Rao MD 25
 Indirect Immunofluorescence


 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,

Dr.T.V.Rao MD 26
 Fluorescence Microbiology Modernises the
Diagnostic Laboratories


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.

Dr.T.V.Rao MD 27
Dr.T.V.Rao MD 28
 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.

Dr.T.V.Rao MD 29
 Reagents for staining
Auramine-phenol solution
Auramine O

25 g
Ethanol 3000 ml
Phenol 250 g
Distilled 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.
Dr.T.V.Rao MD 30
 Reagents for staining


Acid-alcohol for decolorization

Sodium chloride 20 g
Hydrochloric acid, A.R. 20 ml
Distilled water 500 ml
Ethanol 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.

Dr.T.V.Rao MD 31
 Reagents for staining


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.

Dr.T.V.Rao MD 32
 Fuchsin-stained smears require -


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 Dr.T.V.Rao MD 33
 Staining procedure


 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
Dr.T.V.Rao MD 34
 Staining procedure - Precautions
 Avoid under-decolorization. AFBs are difficult to over-decolorize
since the decolorization procedure with acid-alcohol is relatively


milder 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 loss
of 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 to
confirm findings. However, Z-N stained smears cannot be used for
FM
 Fading: Stained smears may fade on exposure to light. To be stored
wrapped in brown or black paper and kept away from light

Dr.T.V.Rao MD 35
 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)

Dr.T.V.Rao MD 36
Screen the Smear in Defined
Pattern

Dr.T.V.Rao MD 37
Identify the Acid Fast Bacilli
with Caution

Dr.T.V.Rao MD 38
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)

Dr.T.V.Rao MD 39
 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

Dr.T.V.Rao MD 40
 QUANTIFICATION OF FLUOROCHROME
SMEAR RESULTS
FM Magnification
ZN REPORT
250x 450x 630x
1000x

0 NO.AFB 0 0 0

DIVIDE
1-9/100FIELD EXACT NO by 10 by 4 by 2
10-99/FIELD 1+

1-10/FIELD 2+

>10/FIELD 3+

Dr.T.V.Rao MD 41
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

Dr.T.V.Rao MD 42
 FM Grading

No. of bacilli per HPF Grade

Less than 6 per field 1+

6-100 bacilli per field 2+

More than 100 per field or large 3+

clumps

Dr.T.V.Rao MD 43
Fluorescence Microscopy Advantages in
Acid Fast Bacilli Identification


 More than 50 smears examined/day
 Continuous availability of power
 More sensitive. where small No. of bacilli are
present
 Majority of FM +ve samples are also +ve by
culture
 Does not yield more false positive than ZN
 Doubtful smears to be re examined by ZN
Dr.T.V.Rao MD 44
 How to get better Photomicrograph for
Documentation


 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.

Dr.T.V.Rao MD 45
 QBC Malaria Test
Uses the Principles of fluorescence


The QBC Malaria
Test is a
fluorescence
microscopy-based
malaria diagnostic
test that speeds and
simplifies malaria
detection
Dr.T.V.Rao MD 46
 Quantitative Buffy Coat
(QBC) Test


 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.

Dr.T.V.Rao MD 47
 EXAMINATION OF SPECIMENS for
Fungal Diseases


 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
Dr.T.V.Rao MD 48
 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.

Dr.T.V.Rao MD 49
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

Dr.T.V.Rao MD 50
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

Dr.T.V.Rao MD 51
 Immunofluorescent antibody (IFA) procedure employing
cryptosporidium- specific polyclonal or monoclonal antibodies has been developed

•Polyclonal AB, raised against 18 and 20 kDa C. parvum

coproantigen, were used to react with C. parvum
sporozoites in an immunofluorescence assay.
•Monoclonal antibody reagents offer increased
sensitivity and an excellent alternative to conventional
staining methods.
•These reagents are helpful when screening large
numbers of patients or those with minimal symptoms.
•Elimination of the problems of false-positive and false-
negative results with routine staining methods.

Dr.T.V.Rao MD 52
 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
Dr.T.V.Rao MD 53
 Enumerable Possibilities with
Florescent Methods


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

Dr.T.V.Rao MD 54


Created by Dr.T.V.Rao MD for ‘ e ‘
learning resources for Microbiologists
in the Developing world
 Email
 doctortvrao@gmail.com

Dr.T.V.Rao MD 55