The antibodies produced by bacteria combine with antigen and form antigen-antibody complex. This complex can be observed after fluorescing it against a dark background with UV light by using a fluorochromes i.e. fluorescent dye.
- Spirit lamp
- Glass slide
- Cover slip
- Petri dish
- Whatman No. 1 filter paper
- Lint-free paper
- Fluorescent microscope
- Phosphate buffer saline (pH 7.4 -7.6)
- Specific conjugate
- Distilled water
- Low-fluorescence immersion oil
- Mounting fluid (9 ml glycerine plus 1 ml of PBS, pH 9.0)
- Fluorochrome (such as rhodamine, fluoroscein isothyocyanate, acridine orange, auramine, etc).
- Inoculate the bacterial colonies into a few drops of PBS having pH 7.4 – 7.6 and make suspension.
- Clean a slide and put a drop of suspension on the test area. Prepare smear, air dry and heat fix the bacterial cells on glass slide by gently warming. Do not make the slide hot over the flame of the spirit lamp.
- Put a drop of bacterial conjugate over the smear and distribute it evenly over the smear. Care should be taken not to dry the conjugate otherwise it will give false result.
- Place the slide in a Petri dish containing a moist filter paper and incubate at 37 C for one hour.
- Rinse the slide in a jar containing PBS for 10 minutes. Finally, rinse the slide with distilled water.
- Gently, blot dry the slide using lint-free paper. For each test use separate slide.
- Place a drop of mounting fluid and apply the cover slip.
- Clean the objective lenses and condenser lenses of the microscope using lens paper.
- Place 1-2 drops of low-fluorescence immersion oil on the condenser after lowering it.
- Place the slide on the stage of the microscope and put a drop of immersion oil on the cover slip. Adjust the microscope to view the required area.
- Examine the field under low power and concentrate the light in a small area by adjusting the condenser.
- Now, switch over to oil immersion objective and observe under the microscope using U.V. light.
The bacterial cells that produce specific antibodies will show fluorescence.