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Biotechnology Reports 8 (2015) 45–55
Contents lists available at ScienceDirect
Biotechnology Reports
journal homepage: www.else vie r.com/locat e/btre
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 ,
Sadiq S. Pasha
Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake, Bangalore 560089, India
A R T I C L E I N F O A B S T R A C T
Article history: We propose a simple technique for bacterial and yeast cfu estimations from diverse samples with no prior
Received 27 April 2015 idea of viable counts, designated as single plate-serial dilution spotting (SP-SDS) with the prime
Received in revised form 28 July 2015 recommendation of sample anchoring (100stocks). For pure cultures, serial dilutions were prepared from
Accepted 5 August 2015 0 1 6
Available online 20 August 2015 0.1 OD (10 ) stock and 20 ml aliquots of six dilutions (10 –10 ) 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), 101–106dilutions were used. Following incubation, at least one
Keywords: dilution level yielded 6–60 cfu per sector comparable to the standard method involving 100 ml samples.
Agricultural biotechnology Tested on diverse bacteria, composite samples and Saccharomyces cerevisiae, SP-SDS offered wider
cfu Estimation applicability over alternative methods like drop-plating and track-dilution for cfu estimation, single
Environmental biotechnology
Food microbiology colony isolation and culture purity testing, particularly suiting low resource settings.
Pour-plating ã 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
Spread-plating (http://creativecommons.org/licenses/by/4.0/).
1. Introduction bioremediation effects, testing novel anti-microbials, etc. besides
serving as standards during molecular investigations.
Estimation of colony forming units (cfu) through serial dilution Spread-plating and pour-plating form the standard approaches
plating on a nutrient medium forms the most widely accepted for bacterial and yeast cfu estimations [8,9,14,15]. Spread-plating
method for monitoring cultivable bacteria and yeasts in different offers several advantages over pour-plating such as more flexibility
spheres of microbiology [14,27]. Cultivation-based methods being in handling, less interfering effects on temperature sensitive
simple to practice, command enormous significance and applica- organisms, the avoidance of aerobic organisms getting trapped
tions in bacteriology. This holds good in spite of the emergence of inside agar medium, the surface enumeration of cfu and the easy
molecular techniques such as fluorescent in situ hybridization, selection of distinct colony types [7,15,27]. Here, the bacterial
real-time quantitative PCR, flow cytometry, etc., which although sample is applied over agar-gelled nutrient medium with the help
provide a precise account of metabolically active cells [3,20] of a glass, plastic or steel spreader where the spreader is generally
demand much expertise and resources. Further, cfu-based considered a mere tool to distribute the inoculum over the agar
techniques provide information on the most abundant populations surface [5,15,28]. We have documented that the inoculum-
among the cultivable community [4,17]. Viable colony counts also spreader employed during standard spread-plating could impart
form essential tools in biotechnology such as gene cloning, significant injury to bacterial cells and affect the cfu depending on
surveillance of genetically modified organisms, assessing the extent of its usage on the agar surface [23]. This was
demonstrated in comparison with the alternate approach that
did not involve the use of spreader, namely, spotting- and- tilt-
spreading (SATS). Any spreader movement on agar surface
Abbreviations: cfu, colony forming units; CNA, cetrimide- nalixic acid- agar; OD, subsequent to the exhaustion of free moisture proved detrimental
optical density; NA, nutrient agar; NB, nutrient broth; PDA, potato dextrose agar; PP, to the bacterial cells further influenced by the operator practices
polypropylene bag; PS, peptone-salt; SATS, spotting- and- tilt- spreading; SP-SDS,
single plate-serial dilution spotting; tmtc, too many to count. and moisture levels in the medium. The physical impaction effects
* Corresponding author. Fax: +91 80 28466291. on vegetative cells varied between different organisms governed
E-mail addresses: pioust@iihr.ernet.in, pioust@gmail.com (P. Thomas). by the cell characteristics of the bacterium with Gram-negative
1 Present address: Department of Neurochemistry, NIMHANS, Bangalore 560029,
India. organisms being more vulnerable than Gram-positive bacteria,
http://dx.doi.org/10.1016/j.btre.2015.08.003
2215-017X/ã 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
46 P. Thomas et al. / Biotechnology Reports 8 (2015) 45–55
cocci less susceptible than rods and more risk to larger cells than 2. Materials and methods
smaller cells [25]. The physical impaction effect also applied to the
supposedly hardy spores of Bacillus spp. which seemed comparable 2.1. Bacterial and yeast cultures and composite samples
to glass globules that crumble under physical pressure [26]. Thus,
the spreader-independent SATS approach proved to be a simpler Pure cultures of bacteria belonging to different phylogenetic
and safer alternative to spread-plating for bacterial cfu estimations groups varying in Gram reaction, cell characteristics and sporula-
with several other advantages [23,25,26]. tion potential were used towards optimizing the single plate-serial
Generally 25–250 or 30–300 colonies per agar plate (100 ml dilution spotting (SP-SDS) technique employing spotting- and- tilt-
sample) are prescribed as the acceptable cfu for accurate counting spreading (SATS) [23,25], as the standard procedure. The
[14,23,25,27]. When there is no clear indication of the dilution level organisms included Enterobacter cloacae, Escherichia coli, Acineto-
that yields this cfu range, several plates representing different bacter junii (Proteobacteria), Bacillus pumilus, Bacillus subtilis,
dilutions and replications need to be employed leading to Bacillus thuringiensis, Staphylococcus epidermidis, Staphylococcus
considerable wastage of time, manpower and material resources haemolyticus (Firmicutes) and Microbacterium esteraromaticum
[2,6,13]. This applies invariably to pure bacterial cultures, water, (Actinobacterium) described elsewhere [25]. E. cloacae was used as
food, soil and various environmental and biotechnological speci- the primary candidate for protocol optimization followed by B.
mens. As we found that inoculum-spreader was wholly dispens- pumilus. One strain of ascosporogeneous wine yeast (Saccharomy-
able, accommodating multiple dilutions in a plate was considered. ces cerevisiae) was used in this study employing potato dextrose
Similar attempts in the past included drop-plating [2,6,16], track- agar (PDA). Different composite samples representing public
dilution [13] and drop-spotting with digital imaging [19], but these health, food, environmental, agricultural, clinical and biotechno-
studies used pure bacterial cultures that yielded confined colony logical settings described below were also tested for bacterial or
growths. The situation is different when the samples involve fast yeast cfu. Additionally, an endophytic bacterial strain of Pseudo-
growing organisms, mixture of different bacteria varying in growth monas aeruginosa from banana [18] that could be monitored
rates or colony characteristics, and with food and environmental distinctly from other organisms on cetrimide- nalidixic acid- agar
samples. The present studies were undertaken to optimize a (CNA) selective medium [22] was used as a representative of
simple and resource saving method for bacterial cfu estimations clinical specimens and genetically modified organisms. Unless
that allows the accommodation of multiple dilutions in a plate and mentioned differently, overnight nutrient agar (NA)/nutrient broth
to test the feasibility of the technique across diverse samples (NB) derived (18–24 h) cultures were used in all studies involving
including pure bacterial and yeast cultures and composite samples. pure bacterial cultures except for spores.
Fig. 1. An illustration of single plate-serial dilution spotting (SP-SDS) technique for pure bacterial / yeast cultures and composite samples.
P. Thomas et al. / Biotechnology Reports 8 (2015) 45–55 47
applied 1 0
2.2. Nutrient media area). The cfu ml of the 10 stock was arrived at as the
product of n 5 10d+1 (d = dilution level yielding the countable
Nutrient agar sourced from M/s HiMedia Biosciences (Mumbai, colonies).
India) formed the standard bacteriological medium while the other
media formulations mentioned later were employed for specific 2.4. Preliminary SP-SDS trials
organisms/samples and also to test the applicability across
different media. Unless mentioned differently, NA/fresh PDA An initial SP-SDS trial was set up employing serial dilutions of E.
prepared in pre-sterilized disposable Petri-dishes on the same cloacae and with irrigation grade open-tank water using six
day about 2 h post-pouring (referred to as fresh plates) or that decimal dilutions from the 100 stock with four replications. An
prepared on the previous day and incubated overnight at 37C assessment of the need for sample vortexing to disperse the
after sealing in polypropylene (PP) bags were used in all trials. The bacterial cells during serial dilutions was undertaken using E. coli
nutrient plates used in a specific trial belonged to the same batch of and E. cloacae practicing vortexing for 10 s spans during decimal
preparation unless mentioned differently. serial dilutions.
2.3. SP-SDS and SATS procedures 2.5. Assessing intra- and inter-plate variations in SP-SDS employing E.
cloacae and mix inoculum
For pure bacterial and yeast cultures, a uniform cell suspension
was prepared by dispersing the overnight colony growths from agar To get an estimate of the possible sector to sector variations in a
plates, orNB culture afterone spin-washinsterilewaterinthecase of plate or inter-plate variations during SP-SDS, E. cloacae serial
Bacillus spp. After allowing any cell clumps to settle down, the clear dilutions of 104 and 105 were spotted (20 ml) in three sectors each
upper part was transferred to a fresh tube. The optical density (OD) in ten fresh NA plates of which 104 yielded tmtc and 105 countable
was determined at 600 nm employing a 1:10 diluted stock in a uv/ colonies. The cfu per sector (average of three sectors at 105),
vis spectrophotometer (Genesis 10 UV, Thermo Scientific, MA, USA) standard deviation (SD) and coefficient of variation (CV) were
based on which the ‘anchored stock’ of 0.1 OD (100) was prepared. worked out for each plate individually. A similar experiment was
1 6 7
Decimal serial dilutions (100–1000 ml) of 10 –10 or 10 were undertaken employing the mixed inoculum of five organisms (E.
prepared from the 100 stock in 1.5 ml tubes with 4–5 repeated cloacae, B. pumilus, B. thuringiensis, S. epidermidis and M.
flushing esteraromaticum)
and changing of tips (see movie: https://youtu.be/ which were pooled in equal proportions employ-
LEqmWmBVIpA). For preparing the stock and serial dilutions, ing the dilution levels that yielded the acceptable cfu (30–300 per
filter-sterilized distilled water (FDW) aliquoted and stored at 20C 100 ml).
was preferred unless the water was freshly autoclaved. This was
essential to avoid the chances of any hardy autoclaving defying 2.6. Assessing the number of replications needed for comparable cfu
spores multiplying during the post-autoclaving storage [21]. Spore estimates in SP-SDS and SATS
preparations and dilutions were made from 7-day old NA plates in
50% ethanol as described elsewhere to avoid their germination [24]. SP-SDS was undertaken in comparison with SATS using E.
For water and clear liquid specimens, the direct sample formed the cloacae 105 dilution. SATS involved 100 ml sample applied in 12 NA
100 anchored stocks. Thick and colloidal suspensions such as milk plates while in SP-SDS, 105 dilution was applied in six sectors in 12
and fruit juice were used directly or after adjusting OD600 nm to 1.0 plates. Colony counts were made adopting one sector per plate
or 10 while for solid specimens (food, soil) a suspension prepared in sequentially representing the six sectors across 12 plates in SP-SDS
water at 1.0 g sample per 10 ml formed the 100 stock. In this study, and the cfu/100 ml was recorded in both methods. The mean, SD
our emphasis was on cfu enumeration technique rather than and CV were worked out sequentially for 2–12 replications.
sampling methods for which the accepted standard procedures Further, the data were tested for significance through single factor
prescribed were to be adhered (e.g. [8–14]). ANOVA considering two to 12 replications sequentially. ANOVA
To execute SP-SDS, the reverse of the 9-cm Petri-dishes between SATS and SP-SDS was also done considering the average
containing surface dry agar media were drawn to six sectors with cfu counts from the six sectors in the 12 SP-SDS plates. The
the marking of first and last dilution sectors for clear identification. experiment was repeated employing a composite sample com-
Using a calibrated micropipette, 20 ml aliquots from selected six prising of E. cloacae, B. pumilus, B. thuringiensis, S. epidermidis and
dilutions were applied as 10–12 micro-drops in these demarcated M. esteraromaticum prepared as above but in irrigation grade tank
areas (Fig. 1). During sample spotting, the same tip was used water with a prior SP-SDS assessment of cfu to fix the appropriate
starting with the lowest dilution. Care was exercised to avoid tip dilution level.
marks on the medium during sample application not to mistake
them for cfu. The sterility of the diluent was ensured by spotting 2.7. Testing SP-SDS versus SATS on additional pure cultures and
20 ml at the bottom part of the plate. The plates were exposed in composite samples
the laminar air-flow (LAF) cabinet for the droplets to dry off (8–
10 min for fresh plates and 3–4 min for pre-prepared surface-dry The applicability of SP-SDS was tested employing pure cultures
plates), sealed in polypropylene (PP) covers and incubated inverted of different bacteria. This included E. coli, E. cloacae, P. aeruginosa, A.
at 28–37C as required for specific organisms. For SATS, 100 ml of junii, B. pumilus, B. subtilis, B. thuringiensis, S. epidermidis, S.
different dilutions were applied as 20–25 micro-drops per plate haemolyticus and M. esteraromaticum employing NA at 30C except
and spread on agar surface by mere tilting or gentle twirling of for E. coli for which trypticase soy agar (TSA; 37C) was used.
0
plate followed by surface drying (5–6 min) in the LAF [23,25]. Cfu Employing d1–d2 source cultures, 0.1 OD (10 ) stocks were
enumeration was done after 18–48 h with the marking of colonies prepared in FDW followed by the preparation and usage of six
on the reverse of the plate. decimal serial dilutions. A similar experiment was undertaken
The colony development pattern at different dilutions in SP-SDS with the yeast strain on PDA. The composite samples included
was recorded as spot growth, too many to count (tmtc) or irrigation-grade tank water, milk, ground mixed-vegetables and a
countable/acceptable (6–60 range). After recording the dilution soil sample. For pure bacterial cultures, the tested dilutions
1 6 0 5
level yielding acceptable colonies and the cfu per sector, cfu per included 10 –10 , for clear water 10 –10 , for milk, ground
100 ml was worked out as n 5 (n = colonies in 20 ml sample vegetables and soil, 101–106 avoiding the particulate 100. Cfu
48
P. Thomas et al. / Biotechnology Reports 8 (2015) 45–55
enumeration was done manually after 18–48 h and beyond as serial dilutions adopting 40–400 ml or 50–500 ml decimal dilutions
1 6
needed depending on the organism/sample. (10 –10 ). As controls 100–1000 ml and 40–400 ml dilutions in
Based on the information of the appropriate decimal dilution 1.5 ml microfuge tubes were employed. Additionally, ELISA plates
that yielded 30–300 cfu per 100 ml sample, SP-SDS was undertaken (Greiner Bio-One GmbH, Germany) were tried which accommo-
in comparison with SATS employing four replicate plates for SATS dated 200 ml sample per well employing 20–200 ml dilution series.
and adopting cfu from first four of the six sectors in an SP-SDS plate
for statistical analyses. Two independent serial dilutions were 2.11. Testing of SP-SDS methodology for microbiological and
prepared each applied in duplicate SATS plates or three SP-SDS biotechnological samples
1 0
sectors each. The cfu counts were translated to cfu ml of 10
Different
stock and analyzed for significance employing single factor ANOVA samples representing biotechnology, agriculture, med-
(Microsoft Excel 2010) after logarithmic transformation. icine, food microbiology, environmental microbiology and applied
microbiology where there was no clear idea about the prevalent
2.8. Comparison of SP-SDS with alternate resource saving approaches bacterial or yeast cfu in the sample were tested through the SP-SDS
approach. The preferred dilutions from the anchored stocks included
0 5 1 6 1 6
A comparison of SP-SDS with alternate resource saving 10 –10 or 10 –10 for liquid samples, and 10 –10 for solid samples
0
techniques was undertaken employing pure cultures of E. cloacae, avoiding the particulate 10 . Further, SP-SDS was tried for parallel
B. pumilus, S. epidermidis and irrigation grade water. This included testingof two ormultiple samples in a plate. Thisincludedtesting the
6 6 drop-plating as per [2] and track-dilution as per [13]. For effect due to different diluents on E. cloacae where the 100 stock in
1
track dilution, 12 12 cm plates from M/s. HiMedia BioSciences, FDW was taken through serial dilution in saline (NaCl 9 g l ),
Mumbai were employed. phosphate buffered saline (PBS), peptone–water (10 g l1 peptone
1 1
and 5 g l NaCl; pH 7.2) [1], peptone-salt (1 g l each peptone and
2.9. Testing SP-SDS approach across other media and NA plates of NaCl; pH 7.0; [23]) or nutrient broth (pH 7.4) employing FDW as
different batches control. In another trial, E. cloacae dilutions prepared in FDW and
peptone salt was monitored with SP–SDS after static incubation
SP-SDS approach was tested across other media including Luria over 5 h at 20 min intervals during the initial one hour and hourly
3 8
Bertani agar for E. coli, plate count agar, brain heart infusion agar, thereafter employing the decimal dilutions 10 –10 . Further
Muller experimental
Hinton agar and MacConkey agar for E. cloacae and details are provided under Results and Discussion.
irrigation grade water. CNA medium was tested employing P.
aeruginosa. The media formulations were sourced from M/s 2.12. Statistical analysis
HiMedia Biosciences, Mumbai.
Based on the earlier documentations that the quantity of For direct comparisons within SP-SDS trials, the mean colony
medium per plate, the age of plates after the preparation and the counts per sector in a plate and for comparisons between SP-SDS
pre-treatments given to the plates did not alter the cfu estimates in and SATS techniques, cfu per 100 ml samples were used for
SATS [23], fresh plates with 15, 20 or 30 ml NA were tested in SP- statistical analysis. The mean, SD and CV were employed for direct
SDS for the time needed for droplet drying and the cfu after comparisons estimated with the S function in Microsoft Excel
applying the 105 dilutions of E. cloacae and B. pumilus. Further, 2010. In the trial comparing SP-SDS versus SATS, the significance
20 ml NA plates prepared on the same day or that prepared 1–7 was tested through single factor ANOVA or Student’s t-test using
days before and the plates given a 37C pre-warming treatment the Data Analysis Tool of Microsoft Excel 2010 after logarithmic
were tried in SP-SDS wherein fresh 20 ml NA plates served as transformation of cfu for the 100 stocks. Unless mentioned
control. The experiments were repeated wherein B. pumilus culture differently, four replications were employed for comparing SP-
4
was employed at a non-decimal dilution (1:3 of 10 ) to get more SDS versus SATS.
acceptable cfu range (>100 per 100 ml) as in the earlier study [25].
3.
Results and discussion
2.10. Testing SP-SDS methodology employing multi-well plates
3.1. Preliminary SP-SDS trials
SP-SDS methodology was tested with E. cloacae and B. pumilus
using 96 cavity (500 ml) autoclavable polypropylene assay plates In the initial trial employing E. cloacae, the first three serial
1 3 4
(Cat. No. P.96-450R-C; Genaxy Scientific Pvt., Ltd., Solan, India) for dilutions (10 –10 ) showed spot growth, 10 displayed tmtc and
Fig. 2. SP-SDS with Enterobacter cloacae involving 101–106dilutions showing acceptable cfu at 105(A); SP-SDS with static water sample from an open tank at 100–105dilutions
displaying countable colonies at 103 dilution (B).
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