Centrifugation of bacterial cells on slide surface improves the spatial distribution, cell recovery and reduces the time of enumeration for fluorescence in situ hybridization.
Fluorescence in situ hybridization (FISH), which is used for the enumeration of bacteria in various ecosystems including the human intestinal tract, has several limitations. One of the major problems encountered is the uneven distribution of bacterial cells on the slide surface, which increases the coefficient of variation between repetitions and thereby increase the time required for enumeration. In order to improve the spatial distribution, we designed a centrifugation device, which allows the direct centrifugation of bacterial cells onto the slide surface. Another problem is the loss of bacterial cells during the hybridization procedure. This leads to an underestimation of the true cell numbers in the sample. To overcome this problem we tested the use of silanized or chrome gelatine coated slides. Our study indicates that the use of the centrifugation device in conjunction with chrome gelatine coated slides highly improves the quality of enumeration data obtained by manual and automated microscopic counting and shortens the time of analysis.
Namsolleck
Pawel
P
Department of Gastrointestinal Microbiology, German Institute of Human Nutrition Potsdam-Rehbrücke, Arthur-Scheunert Allee 114-116, 14558 Nuthetal, Germany.
Mohan
Ruchika
R
Koebnick
Corinna
C
Blaut
Michael
M
D000704:Analysis of Variance; D002498:Centrifugation; D015169:Colony Count, Microbial / Q000295:instrumentation / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D015203:Reproducibility of Results