Hihihihi! This is the 3rd week of SIP and its my turn to post! Hope all of you guys are enjoying your SIP!
Well I’m attached to the Microbiology department for 4 weeks. It has 2 laboratories, Microbiology itself and Molecular microbiology. One easy and interesting method we learnt back in school to detect virus/bacteria is immunofluourescence. I had the opportunity to practice it daily in Molecular microbiology lab.

In the lab I was attached to, tests were mostly done to detect respiratory viruses in children and infants. Such viruses include Respiratory Syncytial Virus, commonly known as RSV, Adenovirus, Influenza and Parainfluenza. Among these, RSV is considered the most common virus affecting children, especially below the age of 6. It rarely affects adults. RSV infects the lungs and the breathing passages causing wheezing, whooping cough and other breathing difficulties in children.

Usually when the doctor diagnoses pneumonia, bronchitis, bronchiolitis or other respiratory illnesses, they would send for tests to detect what virus has affected the child.
Samples sent to the lab are nasopharyngeal swabs or aspirates. Most common ones are swabs.
This is how it is being processed:

1.Firstly, samples should be checked with patient’s test request form to confirm that the identity is similar to the sample.
2.About 0.5-1.0ml of PBS should be added into a clear tube and the swab is placed into the tube.
3.A wire cuter is being used to snip the swab into the tube and capped. It is then vortexes until the suspension becomes turbid enough to be placed on the slide.
4.The slide is contains small wells and 2 wells are allocated for 1 sample. 1 drop in being placed on each well. This step is quite crucial as the size of the drop depends on the turbidity of the cell suspension. Have to make sure the size of the drop is as similar as possible on each well.
5.The slides should be air dried and fixed in cold acetone for 10 mines. It should be air dried after the slide is taken out from acetone.
6.10 µl of respiratory screen reagent should be added to one well. The respiratory reagent contains the seasonal reagent to detect the respiratory viruses.( RSV, influenza, Para influenza and adenovirus)
7.10 µl of RSV reagent should be added to the other well to detect for RSV as it is the most common virus affecting the children.
8.Slides should be incubated at 35-37 degree Celsius for 15-30 mins. After incubation slides should be rinsed with PBS for 5 mins to wash off the screening dyes.
9.They should be air dried and mounting fluid should be added followed by cover slips to be read under microscope.
10.If respiratory screen turns out positive and not the seasonal viruses, then the procedure is repeated again.

How to read the slides and recognize the viruses?

It is quite beautiful to see the slides under microscope. In fact, they are quite glaring when they turn out to be positive. Positive results mean that the cells fluoresce, which indicates that the patient is infected by that virus. Usually if the screening reagent and the RSV turn out to be positive, it proves that that patient is infected by RSV. However, if RSV turns out to be negative and screening reagent turns out to be positive, it means that the patient is infected by other viruses. It could be predicted by the morphology of the cells.

Parainfluenza would have a granular cytoplasm and the cells would look a little ‘bloated’.
Influenza virus would have a green cytoplasm.
RSV would have a crescent like shape at the edge of the nucleus.
These are the typical characteristics of the cells. However, this is not the case in all situations. Sometimes, it very difficult to identify them unless you are very used to looking at them
Hence, most of the time confirmatory screening has to be done using the specific screening reagents (like influenza, parainfluenza or adenovirus). If Influenza or parainfluenza is being identified, they are being sub grouped into influenza A or B, Parainfluenza 1,2 or 3.

So this is how immunofluourescence is done in Molecular microbiology! I know it’s quite lengthy, but sorry, I have to add all the details. Have fun!

BY NEELA
TG 02

References

http://kidshealth.org/parent/infections/lung/rsv.html