Intracellular Borrelia Spirochaetes Reproduce Inside Erythrocytes

Summary

The experiment adds to previous work showing borrelia spirochaete invasion of erythrocytes(1). It provides video evidence that the spirochaetes continue their development within the cell.

Materials and methods

The medium to hold blood cells stable on a microscope slide mini-culture was prepared as:

Deionised water with:-
Sodium Chloride 9 gms/litre
Sodium Citrate 3 gms/litre
Dextrose 5 gms/litre
Triton X-405 0.5 mls/litre

Gelatine granules were dissolved in the liquid as per the manufacturer's instructions (Dr Oetker Gelatine - www.oetker.co.uk) to create a soft jelly.

The slide was prepared as a normal blood drop thin-film smear on a Polysine TM (Thermo Scientific) cell adhesion microscope slide. No drying time was allowed. A drop of the prepared medium was immediately placed on the smear and a 50mm coverslip placed. After blotting, the periphery of the coverslip was sealed with microscope immersion oil.

Results

An effect of the gelatine was to cause intense rouleaux of the RBCs which did not adhere to the slide surface, creating some odd formations:



On days 9 to 11 after preparation of the culture the rouleaux had relaxed but the RBCs mostly remained in tight clumps of various sizes. Intracellular spirochaetes were observed at various stages of 'string of pearls' morphology. The horizontal field of view for maximum magnification videos is ~27 microns. All videos play at normal speed and each clip is trimmed to around 7 seconds viewing time. Videos are edited for noise and levels.




 

Still images are edited for noise, levels and sharpness:






































Extracellular spirochaetes on the same slide. These subjects showed no motility:
















Reference:
Morten M. Laane and Ivar Mysterud: A simple method for the detection of live Borrelia spirochetes in human blood using classical microscopy techniques. Biological and Biomedical Reports 01/2013; 3(1):15-28. PDF available at:

A simple method for the detection of live Borrelia spirochetes in human blood

This page created Aug 26th 2014

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