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Method - Sputum samples included in the study were direct smear positive with a minimum volume of 5 ml. Samples were thinned for 15 min by addition of an equal volume of NaOH/NALC. Samples were then divided into two 5 ml volumes. One 5 ml volume of thinned sputum was placed through the TB-Bead protocol which takes less than 5 min and does not involve centrifugation. The final eluate was adjusted to 0.5 ml with phosphate buffer and 50 µl analysed on a slide by auramine and ZN acid-fast stain. The other 5 ml of thinned sputum was made up to 50 ml with phosphate buffer and then centrifuged for 20 min. The pellet was resuspended in 20 ml phosphate buffer and the sample recentrifuged as before. Finally, the pellet was adjusted to 0.5 ml with phosphate buffer and 50 µl analysed on a slide by auramine and ZN acid-fast stain.

Results - The figures in the table show the total number of acid-fast mycobacteria counted in 20 fields using a 40x objective.



Conclusion - The results show that concentration of mycobacteria from sputum using either centrifugation or TB-Beads are comparable and that, as expected, both show a higher concentration of mycobacetria on the slide than direct smear. In both concentration methods, the mycobacteria were resuspended in a relatively large volume of liquid in order to facilitate counting. In practice, more concentrated and more sensitive smears could be performed by reducing the volume of the resuspension buffer.



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Microsens Medtech

Investigation of the concentration of mycobacteria from sputum using TB-Beads compared to centrifugation


In this issue we look at a study that compares TB-Beads with centrifugation for the concentration of mycobacteria from sputum. In the next issue we will discuss different disinfectants for TB.

Rationale - To compare the ability of centrifugation and TB-Beads to concentrate mycobacteria from sputum as determined by ZN and auramine microscopy



Consumption  May 2011/1

Sputum

Direct smear

TB-Beads

Centrifugation


ZN

Auramine

ZN

Auramine

ZN

Auramine

1

22

30

115

134

99

84

2

26

19

138

161

111

146

3

51

38

418

508

386

407

4

11

19

78

71

59

88

5

9

15

65

83

63

71