Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples

• SP-SDS forms a simple tool for bacterial cfu estimation for samples with unknown cfu.
• Prime recommendation of anchoring specimens to fixed initial OD or a standard base.
• Six serial dilutions of 20 μl each applied per 9-cm plate followed by manual counting.
• Suits pure and mixed bacterial stocks, spores, yeasts and composite samples.
• Superior to alternate techniques like track-dilution, drop-plating or drop-spotting.

We propose a simple technique for bacterial and yeast cfu estimations from diverse samples with no prior idea of viable counts, designated as single plate-serial dilution spotting (SP-SDS) with the prime recommendation of sample anchoring (100 stocks). For pure cultures, serial dilutions were prepared from 0.1 OD (100) stock and 20 μl aliquots of six dilutions (101–106) were applied as 10–15 micro-drops in six sectors over agar-gelled medium in 9-cm plates. For liquid samples 100–105 dilutions, and for colloidal suspensions and solid samples (10% w/v), 101–106 dilutions were used. Following incubation, at least one dilution level yielded 6–60 cfu per sector comparable to the standard method involving 100 μl samples. Tested on diverse bacteria, composite samples and Saccharomyces cerevisiae, SP-SDS offered wider applicability over alternative methods like drop-plating and track-dilution for cfu estimation, single colony isolation and culture purity testing, particularly suiting low resource settings.

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