Journal of Microbiological Methods 172 (2020) 105891

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Journal of Microbiological Methods

journal homepage: www.elsevier.com/locate/jmicmeth

Culture medium brand choice impacts colony swarming behavior among T

industrial Bacillus isolates and the accuracy of aerobic plate counts
John P. Gorsuch , Daniel LeSaint, Zachary Jones, Korey Bielecki, Peyton Woodruff
⁎

BiOWiSH Technologies, 2717 Erie Avenue, Cincinnati, OH 45208, USA

ARTICLE INFO ABSTRACT

Keywords: Aerobic plate count assays are an industry standard method for the enumeration of microbial products. Colony
Bacillus swarming among industrial Bacillus isolates on solid medium can impact a counting technician's interpretation of
Aerobic plate count colony count, promote inter-technician variance and reduce the agreement of plate counts with growth-in-
Tryptic soy agar dependent enumeration methods. In the present study, we examined swarming behavior among four industrial
Bacillus species as a function of culture medium brand choice. Colony diameter for three Bacillus species was
found to be influenced by culture medium brand, as was colony count interpretation among three out of four
plate counting technicians. Estimations of Bacillus endospore concentration were likewise influenced by culture
medium brand, leading to an increased incidence of QC failure for replicate samples of a Bacillus-based microbial
product as a function of brand choice. Results suggest that culture medium brand choice may be an additional
source of variance when plate counting is used for the enumeration of Bacillus-based microbial products. We
recommend that plating medium brand availability in testing laboratories be considered by microbial product
manufacturers when considering sources of variance in customer and regulatory laboratories.

1. Introduction
Due to their metabolic dormancy and resistance to environmental
and chemical stressors, Bacillus endospores are popular inclusions in
microbial products (Cutting, 2011). Global labeling and regulatory
mandates require the verification of label claims for such products, and
thus commercial Bacillus formulations are routinely subjected to enu-
meration assays. Despite the availability of growth-independent enu-
meration methods (Davis, 2014; Gorsuch et al., 2019a) the aerobic
plate count (APC) assay is among the most common methods used to
enumerate microbial products. A staple of benchtop microbiology for
over a century (Breed and Dotterrer, 1916; Fisher et al., 1922), the APC
assay's accuracy is limited by numerous sources of variance which re-
sult in a tendency towards the underestimation of microbial con-
centration in tested samples (Sutton, 2012; Davis, 2014). Among these
sources of variance is the subjectivity inherent in data collection, as the
APC assay relies heavily upon the counting technician's subjective in-
terpretation of colony count. Multiple colonies that have merged to-
gether may be interpreted by the counting technician as a single colony.
This can be especially problematic when the APC assay is used for the
enumeration of species which produce “swarming” or “spreading” co-
lonies on solid medium. Swarming behavior among industrial Bacillus
colonies has been shown to exacerbate the APC assay's tendency to-
wards the underestimation of microbial concentration and to reduce the
method's upper limit of quantification (LOQ), limitations which can be
mitigated through the formulation of a swarming inhibitor into plating
medium (Gorsuch et al., 2019b). However, the growth media approved
for use in standardized plate counting methods – such as those de-
scribed in the FDA's Bacterial Analytical Manual (BAM) (Maturin and
Peeler, 2001) and by the American Society for Testing Materials (ASTM
D5465-16, 2016) – are not formulated to mitigate swarming in Bacillus
colonies, nor are their upper LOQ appropriate for swarming colonies;
thus, these methods risk underestimating Bacillus endospore con-
centrations in tested samples (Gorsuch et al., 2019b).
As Bacillus endospores become more common in microbial product
formulations, mitigating the impact of colony swarming on the accu-
racy of APC counts will become a topic of greater interest among the
producers and consumers of Bacillus-based products. Observations by

Abbreviations: APC, Aerobic Plate Count; BAM, Bacteriological Analytical Manual; BA, Bacillus amyloliquefaciens; BS, Bacillus subtilis; BL, Bacillus licheniformis.; BP,
Bacillus pumilus; CFU, colony forming unit; FDA, Food and Drug Administration; ISO, International Organization for Standardization; NPK, Nitrogen, Phosphorous
and Potassium; PBS, Phosphate Buffered Saline; TSA, Tryptic Soy Agar; LOD, limit of detection; LOQ, limit of quantification; QC, quality control
⁎
Corresponding author.
E-mail addresses: jgorsuch@biowishtech.com (J.P. Gorsuch), dlesaint@biowishtech.com (D. LeSaint), zjones@biowishtech.com (Z. Jones),
kbielecki@biowishtech.com (K. Bielecki).

https://doi.org/10.1016/j.mimet.2020.105891
Received 10 March 2020; Received in revised form 11 March 2020; Accepted 11 March 2020
Available online 12 March 2020
0167-7012/ © 2020 Elsevier B.V. All rights reserved.
J.P. Gorsuch, et al.
Table 1
Composition of Tryptic Soy Agar (TSA) brands used in plating exercises. The
product label for Brand #1 makes no mention of acceptability for standard
methods, while the product label for Brand #2 claims acceptability for use with
EP, USP, ISO, JP and FDA-BAM standard plating methods.
Agar brand #1 Agar brand #2
Enzymatic digest of Casein (15 g/L) Pancreatic digest of Casein (15 g/L)
Soy peptone (5 g/L) Papaic digest of soybean (5 g/L)
Sodium chloride (5 g/L) Sodium chloride (5 g/L)
Agar (15 g/L) Agar (15 g/L)
pH 7.3 ± 0.2 pH 7.3 ± 0.2
plate counting technicians familiar with the enumeration of Bacillus-
based products suggest that some brands of a given agar formulation
may promote increased colony swarming relative to other brands of the
same agar type. Given the impact of Bacillus colony swarming upon the
accuracy of plate counts, such observations may implicate agar brand
choice as another source of variance in plate counting data. In the
present study, we investigated the impact of agar brand choice on the
swarming behavior of four industrial Bacillus strains by culturing
colony-forming units (CFUs) of each strain on two different brands of
Tryptic Soy Agar (TSA). We also conducted a plate counting exercise
using both brands of TSA to examine the impact of agar brand on en-
dospore concentration estimations produced by APC assays. If APC
assays are to be used for the regulation and quality control of the
emerging class of Bacillus-based microbial products, a thorough un-
derstanding of their limitations and potential mitigation strategies is
essential.
2. Materials and methods
2.1. Preparation of plating media
Two brands of Tryptic Soy Agar (TSA) were selected for use in this
study and will be referred to here as Brand #1 and Brand #2, respec-
tively. The compositions of each brand of TSA are presented in Table 1.
The product label for Brand #1 did not claim that the formulation was
approved for use in standardized plate counting methods, while the
product label for Brand #2 claims acceptability for use with EP, USP,
ISO, JP and FDA-BAM plate counting methods. Product lots of each
brand used in this experiment were within their claimed shelf life, with
Brand #1 expiring in September 2022 and Brand #2 expiring in August
2024.
Agar was prepared according to manufacturer's instructions by
adding 40 g of dry medium to 1.0 L of DI water, heating to a boil while
mixing on a magnetic stir plate, and then sterilizing in an autoclave for
15 min at 121 °C, 15 psi for 15 min. Flasks of molten agar were allowed
to equilibrate on the benchtop until comfortable to the touch, and
medium was dispensed aseptically in 20 mL aliquots into sterile, dis-
posable Petri dishes. Plates were allowed to solidify and cure benchtop
for 24 h under ambient conditions before use. Because agar formula-
tions were not visibly distinct, plates were labeled on their bases and
lids with color-coded labels to mark them as plates of Brand #1 or
Brand #2, respectively.
2
Journal of Microbiological Methods 172 (2020) 105891
2.2. Impact of agar brand on Bacillus colony swarming
Axenic endospore suspensions of four industrial Bacillus isolates:
Bacillus amyloliquefaciens (BA), Bacillus subtilis (BS), Bacillus pumilus
(BP) and Bacillus licheniformis (BL) were obtained from an industrial
fermentation company (produced by the supplier using proprietary
methods that were not made available to the authors) and subjected to
serial dilution as described in previous work (Gorsuch et al., 2019b).
Dilution schemes were devised to render both a “countable” plate as
well as a lower-concentration plate on which isolated colonies could be
measured independently of the effects of colony crowding should ex-
cessive swarming take place. The manufacturer claimed
5.0 × 1010 CFU/mL for each suspension, thus the “countable” plate was
a 10−9 dilution, achieved by collecting a 100 μL sample from a 10−8
dilution of the product ( ± 500 CFU per 1.0 mL in the 10−8 dilution
bottle = ± 50 CFU per 100 μL plating aliquot) and the “reduced
crowding” plate was an 80% dilution of a 10−8 dilution bottle (20 mL
of solution from the 10−8 dilution bottle into 80 mL peptone blank to
yield ± 100 CFU per 1.0 mL = ± 10 CFU per 100 μL plating aliquot) to
render ± 10 spores per 100 μL plating aliquot. Ten replicate plates per
TSA brand were prepared from each serial dilution, rendering an ex-
pected sample size of ± 100 colonies per species per TSA brand. Plates
were incubated at 37 °C for 24 h. After incubation, colony swarming
was assessed by measuring colony diameter with a pair of Vernier ca-
lipers. Only colonies that were completely separate from other colonies
on the plate were measured (n = 30 colonies per species per agar
brand). Mean colony diameters for each species on each TSA brand
were compared using a one-way ANOVA (McDonald, 2014). After
measurements were taken, plates were counted and the evenness of
interpreted colony distribution across replicate plates was compared to
a theoretical Poisson distribution using a Chi-Square analysis
(McDonald, 2014) to rule out differences in the growth potential of the
selected Bacillus isolates between the two agar brands.
2.3. Impact of agar brand on colony count interpretation
Fifteen replicate samples of a commercially available NPK fertilizer
coated with a mixed-species assemblage of the BA, BS, BP and BL strains
described in Section 1.2 above were subjected to homogenization, serial
dilution, plating and incubation as described in previous work (Gorsuch
et al., 2019a). For each replicate, 100 μL aliquots of the targeted dilu-
tion bottle were spread onto triplicate plates of TSA Brand #1 and TSA
Brand #2, respectively. This resulted in fifteen, triplicate countable
plate series per agar brand. Each series of plates was counted in-
dependently by four separate counting technicians, with experience in
Bacillus plate counting ranging from 2 years to 11 years, and each
technician's counts were recorded separately. To determine the impact
of agar brand on each technician's interpretation of colony count, mean
colony counts for plates of Brand #1 and Brand #2 TSA were compared
for each counting technician using a one-way ANOVA (McDonald,
2014).
2.4. Impact of agar brand on APC endospore concentration estimations
The plates produced in Section 1.3 above were also used to examine
the impact of agar brand upon the agreement of APC results with
J.P. Gorsuch, et al. Journal of Microbiological Methods 172 (2020) 105891

Fig. 1. Colonies of BA, BS, BP and BL grown on TSA Brand #1 (frames a-d, respectively) and Brand 2 (frames e-h, respectively). Colonies of BA, BS and BL were
appreciably larger on plates of TSA Brand #2 than on TSA Brand #1. Colonies of BP compared favorably on both plating media.

Table 2 was the only metric assessed here.

Impact of TSA brand choice on colony diameter. Average colony diameters
(n = 30 colonies) were larger on Brand #2 than on Brand #1 (statistically
significant values denoted with a *) for species BA, BS and BL. BP colony dia- 3. Results
meters compared favorably on Brand #1 and Brand #2.
BA BS BP BL 3.1. Impact of agar brand on Bacillus colony swarming

Average colony diameter TSA 2.97 0.9 0.97 6.17

Qualitative differences in BA, BS, BP and BL colony size after 24 h of
brand #1 (mm)
Average colony diameter TSA 4.78 2.95 1.02 7.88 incubation at 37 °C are presented in Fig. 1. Upon visual inspection,
brand #2 (mm) swarming behavior of BA, BS and BL colonies appeared more vigorous
σ (Brand #1) 0.32 0.21 0.18 0.91 on TSA Brand #2 than on TSA Brand #1. Colonies of BP appeared to
σ (Brand #2) 0.36 0.24 0.25 0.92
compare favorably between the two brands of agar.
p (one-way ANOVA) < 0.001* < 0.001* 0.06 < 0.001*
A quantitative consideration of average colony size as a function of
TSA brand is presented in Table 2. Average diameters of BA, BS and BL
product label claims. The plate counting exercise produced fifteen sets colonies were significantly larger on TSA Brand #2 than on TSA Brand
of countable plates (n = 3 plates per set) per agar brand. Each set of #1 (Table 2). Average diameters of BP colonies compared favorably on
plates was counted independently by four separate counting techni- both brands of TSA. No significant difference in the distribution of
cians, with experience in Bacillus plate counting ranging from 2 years to colonies across replicate plates was detected between the two brands of
11 years, and each technician's counts were recorded separately. Counts TSA (Chi-Square test, p = .76).
from each technician were averaged (n = 12 plates per replicate pro-
duct sample) to produce consensus estimations of microbial activity for
each of the 15 replicate product samples. The industry standard for APC 3.2. Impact of agar brand on colony count interpretation
assays is to report the average of three countable plates as the microbial
concentration of a tested sample (Maturin and Peeler, 2001). For this Trends in colony size for a mixed BA/BS/BP/BL assemblage diluted
exercise, we reported the average of three countable plates as inter- and plated on TSA Brand #1 and Brand #2, respectively, are displayed
preted by four different counting technicians as the microbial con- in Fig. 2. Bacillus colonies were larger on plates of Brand #2 relative to
centration of the tested sample. This resulted in two consensus enu- Brand #1, an observation which compares favorably with the results of
meration values for each replicate product sample: one produced from colony diameter experiments (Table 2).
plates of TSA Brand #1, the other produced from TSA Brand #2. These A comparison of colony count interpretation among four separate
consensus enumeration values were then compared with the Bacillus- counting technicians as a function of TSA brand is presented in Table 3.
coated NPK product's label claim of 5.0 × 108 CFU/g. No statistical Average colony counts for each technician were calculated from 42
consideration of APC data is prescribed by standard methods, and replicate plates per agar type. Three out of four technicians interpreted
product samples are either passed or failed based on the numerical a significantly higher number of colonies on plates of TSA Brand #1
agreement of consensus counts with the product's label claim. Thus, the than on plates of TSA Brand #2 (one-way ANOVA, α = 0.05).
numerical agreement of consensus counts with the product's label claim

3
J.P. Gorsuch, et al. Journal of Microbiological Methods 172 (2020) 105891

Fig. 2. Serial dilutions of a Bacillus-coated NPK fertilizer spread on plates of TSA Brand #1 (a) and TSA Brand #2 (b). Plate curing time and incubation time was
identical for both brands.

Table 3 microbial concentration estimations as interpreted by four different

Impact of TSA brand choice on colony count interpretation by four different counting technicians met or exceeded the product label claim in 93% of
counting technicians. Replicate dilution aliquots from the same bottle were tested samples when TSA Brand #1 was used, while counts met or
spread on plates of TSA Brand #1 and Brand #2, respectively. All technicians exceeded product label claim in 53% or tested samples when TSA Brand
interpreted numerically higher colony counts on plates of Brand #1 than on #2 was used.
Brand #2 (n = 42 plates per agar type). For three of the four counting tech-
nicians, counts from plates of TSA Brand #1 were significantly higher than
counts from plates of Brand #2 (one-way ANOVA, significant differences de- 4. Discussion
noted with an *).
Average σ (Brand Average σ (Brand p (one-way Quality control and regulatory enumeration assays are vital safe-
count (Brand #1) count (Brand #2) ANOVA) guards for the producers and consumers of microbial products. The
#1) #2)
standardized assays used to carry out these enumerations, such as those
Technician 1 61 10.27 47 9.16 0.0001* put forth by the FDA (Maturin and Peeler, 2001) and the ASTM (ASTM
Technician 2 56 10.75 48 9.63 0.001* D5465–93 16) are widely recognized and accepted by both consumers
Technician 3 61 11.81 57 10.63 0.12 and regulators around the world; however, recent research has shown
Technician 4 53 8.96 46 8.28 0.0003* that these methods may be limited in their accuracy for the enumera-
tion of products formulated with industrial Bacillus assemblages
(Gorsuch et al., 2019b). This presents challenges for the manufacturers
3.3. Impact of agar brand on APC endospore concentration estimations
of Bacillus-based products, who must choose how best to address the
limitations of standard methods. Manufacturers must either undertake
The results of APC enumeration assays conducted using TSA Brand
the development and validation of Bacillus-appropriate standard
#1 and Brand #2, respectively, are displayed in Fig. 3. Consensus
methods and promote their adoption by regulators in the relevant

Fig. 3. Impact of TSA brand on agreement of APC counts and product label claim. Consensus Total APC counts were calculated for each replicate product sample
(n = 15 samples) using plate counts collected by four separate technicians (Table 3). Use of TSA Brand #1 resulted in passing values for 93% of product samples;
however, use of TSA Brand #2 resulted in passing values for 53% of product samples.

4
J.P. Gorsuch, et al.
markets, or they must thoroughly characterize the limitations of stan-
dard methods for the enumeration of their products and set con-
servative product label claims which take these limitations into ac-
count.
The merging of multiple colonies into a single unit is a documented
source of variance that can drive underestimation of microbial con-
centration in plate-counting assays, as it makes interpretation of colony
count difficult for the counting technician (Sutton, 2012). The
swarming behavior exhibited by industrial Bacillus colonies on solid
medium has been shown to exacerbate this tendency towards under-
counting, suggesting that standard plate-counting methods may be even
less accurate for the enumeration of Bacillus-based products than for
other microbial products (Gorsuch et al., 2019b). Colony swarming and
its impact on the accuracy of the microbial concentration estimations
produced during APC assays are thus a topic of interest. In the present
study, we examined the impact of culture medium brand choice
(Table 1) on swarming behavior among colonies formed on solid
medium by industrial BA, BS, BP and BL CFUs. Average colony diameter
was significantly larger among colonies of BA, BS and BL cultured on
TSA Brand #2 than among colonies cultured on TSA Brand #1 (Fig. 1,
Table 2). Additionally, we examined the impact of culture medium
brand on the number of Bacillus colonies interpreted by counting
technicians on replicate plates produced from serial dilutions (n = 15)
of a Bacillus-based microbial product. Three out of four counting
technicians interpreted significantly fewer colonies on plates of TSA
Brand #2 relative to identically inoculated plates of TSA Brand #1
(Table 3). This lower interpretation of colony count led to an increased
incidence of consensus estimations of microbial concentration which
fell below product label claim for TSA Brand #2 when compared to TSA
Brand #1 (Fig. 3).
These data support the hypothesis that culture medium brand
choice influences the degree of swarming behavior among colonies of
three industrial Bacillus isolates. Previous work has demonstrated a
negative relationship between swarming behavior and the accuracy
APC enumerations (Gorsuch et al., 2019a); thus, these data suggest that
culture medium brand choice may be an additional source of variance
when APC assays are used to enumerate products formulated with
Bacillus species which form swarming colonies on solid medium. The
colony count data presented in Table 3 and the enumeration data
presented in Fig. 3 further support this relationship, as TSA Brand #2 –
which produced larger Bacillus colonies per unit time – produced lower
interpreted counts among three out of four counting technicians
(Table 3) and lower interpreted consensus enumeration values (Fig. 3)
than did TSA Brand #1. Though both tested culture media are sold as
Tryptic Soy Agar, differences in composition are noted upon compar-
ison of the product labels (Table 1). We hypothesize that the differences
observed in swarming behavior among these Bacillus colonies on plates
of these TSA brands may be attributable to these compositional dif-
ferences. It should be noted that TSA Brand #2, which promoted colony
swarming behavior and produced lower interpretations of colony count
among three out of four counting technicians, is listed as acceptable for
use with EP, USP, ISO, JP and FDA-BAM standard plating methods.
Conversely the composition of TSA Brand #1, which produced smaller
colonies and higher interpretations of colony count among three out of
four counting technicians, is not listed as acceptable for use with these
standard methods.
Journal of Microbiological Methods 172 (2020) 105891
Whether the manufacturers of Bacillus-based products intend to
address the limitations of standard methods for the enumeration of
Bacillus assemblages methodologically or through the adoption of more
conservative label claims, their decisions will be informed by a thor-
ough understanding of the sources of variance which impact the ac-
curacy of APC enumerations. It has been the authors' experience that
the brand of culture medium available to plate counting technicians
often varies geographically, as well as among testing laboratories de-
pending on their approved suppliers. Therefore, if standard plate-
counting methods are to be used by customers or regulators for QC
enumeration of Bacillus-based products, it may be advantageous for
manufacturers to consider setting label claims that can be supported by
the specific brand of culture medium available to the end user.
Funding source declaration
All authors are BiOWiSH employees. Funds for this research were
entirely from BiOWiSH Technologies, Inc.
Credit author statement
John Gorsuch: Conceptualization, Methodology, Validation, Formal
Analysis, Data Curation, Investigation, Resources, Writing (Original
Draft, Reviewing & Editing).
Daniel Le Saint: Data collection, data interpretation, writing (re-
viewing and editing).
Zachary Jones: Data interpretation, writing (reviewing and editing).
Korey Bielecki: Data interpretation, writing (reviewing and editing).
Peyton Woodruff: Laboratory support, writing (reviewing and
editing).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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