title: 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.
abstract: 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 (100stocks). 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 100-105dilutions, and for colloidal suspensions and solid samples (10% w/v), 101-106dilutions 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 andSaccharomyces 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.
authors.affiliation: Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake, Bangalore 560089, India.
authors.forename: Pious
authors.initials: P
authors.lastname: Thomas
authors.affiliation: Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake, Bangalore 560089, India.
authors.forename: Aparna C
authors.initials: AC
authors.lastname: Sekhar
authors.affiliation: Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake, Bangalore 560089, India.
authors.forename: Reshmi
authors.initials: R
authors.lastname: Upreti
authors.affiliation: Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake, Bangalore 560089, India.
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Mujawar
authors.affiliation: Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake, Bangalore 560089, India.
authors.forename: Sadiq S
authors.initials: SS
authors.lastname: Pasha
keywords: Agricultural biotechnology; CNA, cetrimide- nalixic acid- agar; Environmental biotechnology; Food microbiology; NA, nutrient agar; NB, nutrient broth; OD, optical density; PDA, potato dextrose agar; PP, polypropylene bag; PS, peptone-salt; Pour-plating; SATS, spotting- and- tilt- spreading; SP-SDS, single plate-serial dilution spotting; Spread-plating; cfu Estimation; cfu, colony forming units; tmtc, too many to count