A Microliter Syringe Method for Counting Bactcria

During our study of the relationship between propagule size and reproduction capacity of microorganisms on agar media1), it was necessary to determine concentrations of various species of bacteria and actinomycetes. Since bacterial counts made in haemacytometer had been shown to be unreliable because the amount of samples counted was not precise3,4,5) and difficult due to insufficient color contrast of bacterial cells3), a method was developed to fulfill the need for such study. The microliter syringe method, also called microsyringe method2), originally developed for determining concentrations of fungal propagules was modified for making bacterial counts. Agrobacterium tumefaciens, Bacillus megaterium, Corynebacterium insidiosum, Erwinia carotovora, Pseudomonas solanacearum and Xanthomona campestris obtained from A.M. Alvarez were grown on potato dextrose agar supplemented with 0.1% yeast extract at 25C for 7 days. Six circles (ca. 1-2mm diam.) were marked on a clean glass slide with a blue wax pencil (Blaisdell, China-Marker). One loopful of bacterial cells was transferred to sterile distilled water and diluted to 10-6. The diluted bacterial suspension (0.1ƒÊl) was placed within each circle on the slide with a microliter syringe (Hamilton, 5ƒÊl). The smears were air-dried, fixed over flame and stained with 1% basic fuchsin using a disposable pipet with a fine tip made over flame. After 3min, excess stain was dissolved in about 2ƒÊl of distilled water and the solution was absorbed from the edge of the smear by a tissue paper 30sec later. Such process of removing excess stain was repeated two times. The number of bacteria per ml was calculated from the average number of bacteria per 0.1ƒÊl determined by counting the total number in the whole smear areas with an 100•~ (oil) objective. For comparison a diluted bacterial suspension was also plated on the same medium (0.1ƒÊl/plate), and the number of bacterial colonies per plate was determined after incubation at 25C for 7 days. Six plates were used for each organism. The number of bacteria in each circled area was easily counted after staining. The standard deviations and coefficients of variation were great when excess stain was removed by the usual rinse with water (Table 1). Moreover, considerable amounts of bacteria were lost during the process of washing. Such shortcomings were overcome by using a pipet to apply water and tissue paper to remove excess stain dissolved in water. For all the six species of bacteria tested the microliter