Fluorescence Microscopy Products ...
 
 
 


Most confirmative Tuberculosis diagnosis test!

WHO recommended technology!

Highly sensitive due to concentration technique!

No more focusing troubles! Comes pre-packaged with "Focus it Fast" slides!

 


HOW IT WORKS?

Tuberculosis is diagnosed by finding Mycobacterium tuberculosis bacteria in a clinical specimen taken from the patient. While other investigations may strongly suggest tuberculosis, they cannot confirm it.
Wax like coating on the membrane of tuberculosis bacilli has large amount of mycolic acid. This mycolic acid has the tendency to retain the primary stain, like auramine O even after exposure to de-colourizing acid alcohol (hence; the term "AFB" (Acid Fast Bacilli)). Stained bacteria are then observed using fluorescent microscope to confirm the positive TB test.

TECHNOLOGY

The identification of myco-bacteria with auramine O (fluorescent dye) is due to the affinity of the mycolic acid in the cell wall for the fluoro-chrome.

Therefore; Fluor AFB is fluorescence microscopy based technique.

In fluorescence microscopy, light rays of shorter wavelength pass through a smear stained with fluorescent dye such as Auramine O. Auromine O has a property of absorbing light rays of shorter wavelength and emitting light rays of longer wavelength. A mercury vapour lamp or a more advanced LED based light source is used as a source of light. Either with LED of predefined wavelength or by means of suitable filter, only light rays of shorter wavelength are allowed to emerge. These rays are then used for microscopy.

 
 

FEATURES & BENEFITS

 Reccomended by WHO
Fluor AFB is unique two stage kit for tuberculosis diagnosis. Many research studies have proved that, fluorescence microscopy for TB diagnosis is far more sensitive than conventional ZN method that is performed with light microscope. WHO recommendation for the procedure is certainly a seal of authentication.

 High speed screening due to lower magnification.
The most important advantage of fluorescence microscopy is that, slides can be examined at a lower magnification; thus allowing the examination of much larger area per unit of time. An area that needs examination for 10 minutes with a light microscope can be examined in only 2 minutes using fluorescence microscopy.

 Unique concentration formula increases sensitivity.
Even though, finding the tuberculin bacilli in sputum specimen is the most confirmative TB diagnosis; many a times there is a risk of missing out on detection of the bacilli, due to very low number of bacilli present in a sputum specimen.
Fluor AFB's unique pretreatment formula helps to increase the concentration of TB bacilli in specimen by many folds thereby, immensely increasing sensitivity.

 Bright and Clear :
Mucolytic reagent provided with Fluor AFB kit frees the TB bacilli from mucus. Buffer solution provided along with the kit maintains the necessary pH balance required for optimum brightness and colour reproduction.

 Absolutely Safe to Use; No risk of contamination during centrifugation.
Fluor AFB is provided with decontaminant reagent. Active ingredient of decontaminant reagent is sodium hypochlorite, which kills the myco- bacterium, leaving behind a safe specimen for observation.

  Focus it Fast Slides:
Many research studies have proved in the past that, fluorescence microscopy for TB diagnosis is far more sensitive than conventional ZN method that is performed with light microscope. However; fluorescence microscopy could not gain popularity due to one very important limitation; in absence of any glowing bacilli, microscopic field appeared dark without giving any reference to focus the smear. This created confusion in observation. Darkness could be attributed to a negative specimen or an improperly focused smear.
"Fluor AFB comes with specially processed "Focus it Fast" slides. These slides are printed with small square grids using specially formulated ink, which glows when excited by blue UV light, thus providing the end user with a reference to focus the smear. It is now possible to focus the smear in just few seconds, making detection very easy and fast!

TRAINING

Composition of Fluor-AFB
    
The
Fluor-AFB kit contains following for approx 100 staining, contents:
 

   Reagent (A) :

  Decontaminant Solution

  (1 x 100 ml)

   Reagent (B) :

  Mucolytic

  (1 x 2.0 gms)

   Reagent (C) :

  Buffer Solution

  (2 x 100 ml)

   Reagent (1) :

  Staining Solution

  (1 x 100 ml)

   Reagent (2) :

  Decolourizing Solution

  (1 x 100 ml)

   Reagent (3) :

  Counterstaining Solution

  (1 x 100 ml)


Fluor-AFB
Grid Printed Slides                                    (2 X 50 Slides)
 
Speciman for Investigation
Sputum, pleural fluid, urine, lymph-nodes, bronchio-alveolar washing
 
STAGE 1: Pretreatment of the specimen :
 
In order to free Mycobacteria from mucus, it is recommended to treat the sputum before staining. The active ingredient dissolves the organic material by oxidation, leaving AFB unaffected. Pretreatment of specimen allows decontamination, release of bacilli by mucolytic action, concentration, and pH balancing for increased sensitivity of detection. The histological specimens need to be treated to remove paraffin & rehydrate the sections.
 
Procedure for Pretreatment :
Carry out this procedure in a clean & sterile centrifuge tube (15-20 ml) with a lid.
 
●  Take 2 ml Reagent A & add one scoop (10mg) of Reagent B & mix until dissolved

●  Add 2 ml of specimen in the above centrifuge tube & close the lid.

●  Mix well in a vortex mixer for approx 10 minutes.

●  Carefully unscrew the cap of tube & add 4 ml of reagent C & mix well.

●  CCentrifuge at 4000 rpm for 10 minutes.

●  After centrifugation unscrew the cap carefully & discard the supernatant gently in an activated 2% glutaraldehyde solution without disturbing the bottom of the tube.

●  Add 1 ml of distilled water at the bottom & re-suspend the content

●  Use this suspended material for preparation of  smear.
 
Procedure for Fixation of Smears (On focus it fast Slides)
Fixation of smear on the special Focus it Fast slides is carried out over the flame of a bunsen burner or spirit lamp 2-3 times avoiding excess of heating..
 
STAGE 2 : Procedure for Manual staining

Fill the coupling jars with approx. 50 ml reagent 1. Replace after 50 stainings. Pre-warm solution 2 & 4 to approx 40-50 degree C in water bath.

 

Stains

Time (min)

Reagent 1 (Staining Solution)

15

Rinse with Tap Water

Reagent 2 (Decolorizing)

3

Rinse with tap water

Reagent 3 (Counterstaining)

5

Rinse with Tap Water.

 
 
Dry the smears in air. Histological sections should be treated with increased concentrations of alcohol and Xylem before mounting with DPX-mountant.
 
Staining result, Morphology & Diagnosis of AFB
The slides can be examined using clear immersion oil or can be mounted with DPX mountant.
The Acid-fast Bacilli are clearly distinguished by being golden-yellow on a dark background.
Typical morphology of AFB is, slender slightly curved, acid-fast rods that are partly visible granulae; typical cord forming colonies.
The diagnosis is reported as Acid-fast Bacilli (AFB) detected or not detected.

Mycobacterium Tuberculosis: infection must be considered for any patient with repeat smear AFB positive regardless of the number of AFB seen.
Reporting as per Indian reference Institution after examination for 5 minutes covering 100 fields
 

No AFB    

  Negative

1-10 AFB                                      

  Actual Number of AFB

>10 AFB                                  

  +

Masses of AFB in several fields        

  ++

 
In order to confirm diagnosis of Tuberculosis, confirmation tests such as culture, PCR or similar methods are requested.

Note : Grid slides provided in the kit are manufactured using specially formulated hydrophobic ink. White ink provides dull green fluorescence under the blue light excitation. It is recommended to use only these slides as it helps the user in focusing the microscopic field in smear rapidly & conveniently against a dark background.
 

FAQ'S

Can I use other body fluids other than sputum with Fluor-AFB?
Yes, other body fluids like CSF, broncho-alveolar washings can be used with Fluor-AFB.
 
Can I buy additional Focus it Fast slides?
Yes, please contact your authorized Florotek dealer with your additional order request.
 
Can I buy the components of Fluor-AFB kit individually?
Yes, please contact your authorized Florotek dealer for the same.
 
Once stained how long can I wait before observing the slide?
It is recommended to observe the slide immediately upon staining. However It has been observed that staining is stable for up to 12 hours.
 

LITERATURE

Please click here to download Fluor-AFB Literature