Comparison of machine and manual staining of direct smears for acid-fast bacilli by fluorescence microscopy.

Abstract

SYNOPSIS Comparisons were made in Lusaka and in London between manual staining and staining in an automatic machine with auramine-phenol of direct smears of sputum and other types of specimen for acid-fast bacilli. No evidence was obtained of carry-over of acid-fast bacilli from positive to negative smears during machine staining. There was improved contrast between bacilli and the background in smears prepared with the machine.The staining of direct smears for acid-fast bacilli in Coplin jars is often considered undesirable because of the risk of transferring bacilli from a positive to a negative smear (Pagel et al, 1964). Similar objections could be raised to the use of automatic staining machines. Cremer (1968) investigated the possibility by staining 200 smears prepared from two negative sputum specimens known not to contain acid-fast bacilli, together with a similar number of positive smears prepared from heat-killed cultures of Mycobacterium tuberculosis. No evidence of carryover bacilli from the positive to the negative smears was obtained. However, because of the artificiality of the method of preparation of the positive smears and the rather small samples tested, we felt that a further study was necessary before machine staining could be recommended for general use. We report here the results of two studies, one in Lusaka and the other in London, in which manual and machine staining were compared, particularly to explore the possibility of carry-over from positive to negative smears. Material and methodsSeparate studies were carried out in Lusaka and in London. In both laboratories, smears of sputum or other types of specimen were spread on slides with a loop made from thick (22 SWG) nichrome wire. A separate slide was used for each specimen. After heat

Survival of tubercle bacilli in heat-fixed sputum smears.

“…All procedures were carried out within laboratory Accepted for publication 12 In the first experiment two direct smears were prepared from each of 187 sputum specimens. One slide from each pair of smears was passed three times through the flame of a gas burner with the smear side uppermost.…”

Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda

“…When contamination occurred, the original sample was reprocessed and reinoculated. Any suspected growth was tested for morphology using auramine O staining and fluorescence microscope as previously described [25]. All manipulations of samples including processing, inoculation, and DNA extraction were performed in the biosafety level three at National Reference Laboratory (NRL), Kigali, Rwanda.…”

Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda

“…When contamination occurred, the original sample was reprocessed and reinoculated. Any suspected growth was tested for morphology using auramine O staining and fluorescence microscope as previously described (21). All manipulations of samples including processing, inoculation, and DNA extraction were performed in the biosafety level three at National Reference Laboratory (NRL), Kigali, Rwanda.…”