Accepted Manuscript
                          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
                          Author: Pious Thomas Aparna C. Sekhar Reshmi Upreti
                          MohammadM.MujawarSadiqS.Pasha
                          PII:                 S2215-017X(15)00045-4
                          DOI:                 http://dx.doi.org/doi:10.1016/j.btre.2015.08.003
                          Reference:           BTRE109
                          Toappear in:
                          Received date:       27-4-2015
                          Revised date:        28-7-2015
                          Accepted date:       5-8-2015
                          Pleasecitethisarticleas:PiousThomas,AparnaC.Sekhar,ReshmiUpreti,Mohammad
                          M.Mujawar, Sadiq S.Pasha, 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, Biotechnology Reports
                          http://dx.doi.org/10.1016/j.btre.2015.08.003
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            Ms. Ref. No.:  BTRE-D-15-00056 Manuscript: FINAL –dt 280715  
            Section: Agricultural and food biotechnology / Environmental biotechnology 
             
            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 
             
                                                                           1
            Pious  Thomas*, Aparna C. Sekhar, Reshmi Upreti, Mohammad M. Mujawar  and Sadiq S. 
            Pasha 
            Division  of  Biotechnology,  Indian  Institute  of  Horticultural  Research,  Hessaraghatta  Lake, 
            Bangalore-560089, India 
            *Correspondence:  Pious  Thomas,  Division  of  Biotechnology,  Indian  Institute  of  Horticultural 
            Research, Hessaraghatta Lake, Bangalore, India – 560 089 
            Tel: 91-80-28466420 (4 lines) Ext.410; Mob: 91-9480514820; Fax: 91-80-28466291  
            E-mail: pioust@iihr.ernet.in; pioust@gmail.com 
             
            1
             Present address: Department of Neurochemistry, NIMHANS, Bangalore- 560029, India 
            Graphical Abstract 
             
            Highlights: 
                      •   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 
                  
                 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 
                                                                              0
                 the prime recommendation of sample anchoring (10  stocks). For pure cultures, serial dilutions 
                                                       0                                                       1       6
                 were prepared from 0.1 OD (10 ) stock and 20 µl aliquots of six dilutions (10  to 10 ) were 
                 applied as 10-15 micro-drops in six sectors over agar-gelled medium in 9-cm plates. For liquid 
                 samples 100 to 105 dilutions, and for colloidal suspensions and solid samples (10% w/v), 10¹ to 
                    6
                 10  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. 
                 Abbreviations:  cfu,  colony  forming  units;  CNA,  cetrimide-  nalixic  acid-  agar;  OD,  optical 
                 density; NA, nutrient agar; NB, nutrient broth; PDA, potato dextrose agar; PP, polypropylene 
                 bag; PS, peptone-salt; SATS, spotting- and- tilt- spreading; SP-SDS, single plate - serial dilution 
                 spotting; tmtc, too many to count 
       Key  words:  agricultural  biotechnology;  cfu  estimation;  environmental  biotechnology;  food 
       microbiology; pour-plating; spread-plating 
       1.  Introduction 
       Estimation of colony forming units (cfu) through serial dilution plating on a nutrient medium 
       forms the most widely accepted method for monitoring cultivable bacteria and yeasts in different 
       spheres  of  microbiology  (Messer  and  Johnson,  2000;  Wise,  2006;  ISO  2007,  2013a,  b). 
       Cultivation-based  methods  being  simple  to  practice,  command  enormous  significance  and 
       applications in bacteriology. This holds good in spite of the emergence of molecular techniques 
       such as fluorescent in situ hybridization, real-time quantitative PCR, flow cytometry, etc., which 
       although  provide  a  precise  account  of  metabolically  active  cells  (Davis,  2014;  Sohier  et  al., 
       2014) demand much expertise and resources. Further, cfu-based techniques provide information 
       on the most abundant populations among the cultivable community (Epstein, 2013; Prakash et 
       al., 2013). Viable colony counts also form essential tools in biotechnology such as gene cloning, 
       surveillance  of  genetically  modified  organisms,  bioremediation  effects,  testing  novel  anti-
       microbials, etc. besides serving as standards during molecular investigations.  
          Spread-plating and pour-plating form the standard approaches for bacterial and yeast cfu 
       estimations (ICMSF, 1978, 1986; Messer and Johnson, 2000; Messer et al., 2000; ISO 2013a, b). 
       Spread-plating offers several advantages over pour-plating such as more flexibility in handling, 
       less interfering effects on temperature sensitive organisms, the avoidance of aerobic organisms 
       getting trapped inside agar medium, the surface enumeration of cfu and the easy selection of 
       distinct colony types (Hoben and  Somasegaran, 1982; Messer et al., 2000; Wise, 2006). Here, 
       the bacterial sample is applied over agar-gelled nutrient medium with the help of a glass, plastic