FEMS MicrobiologyLetters 19 (1983) 7-9
Published by Elsevier
7
Statistical consideration of using colony diameter as a measure of
mycoplasmal growth
K. K i h a r a , S. Ishida, M. Shintani a n d T. Sasaki
Department of General Biologics Control, National Institute of Health, 2 - 10 -35, Kamiosaki, Shinagawa -ku, Tokyo 141, Japan
Received 19 January 1983
Accepted 15 February 1983
1. I N T R O D U C T I O N
The mean value of colony diameter has often
been used as a measure of mycoplasmal growth
[1-3] but there have been few reports, if any,
showing that the use of colony diameter (CD) can
be valid in statistical analysis.
In this paper we report our results showing that
C D is applicable in statistical analyses based on
the hypothesis of normal distribution. A relationship between mean CD and the number of colonies per inoculum is also presented.
2. M A T E R I A L S A N D M E T H O D S
2.1. Strains
Mycoplasma arthritidis 07, M. arthritidis YK,
M. fermentans, M. gallinarum, M. gallisepticum,
M. hominis YK, M. iners, M. mycoides subsp.
mycoides, M. neurolyticum, M. orale N - l , M.
pulmonis, M. primatum, M. pneumoniae Mac, M.
salivarium and A choleplasma laidlawii Laidlaw were
maintained for many years in our laboratory. M.
arginini G230, M. hominis PG21 K-l, M. orale C H
19299 and .4. laidlawii A PG8 were supplied by
courtesy of Prof. M. Nakamura, Medical School of
The Kurume University.
Pure Chemical Industries, Ltd., Osaka) and 10%
unheated equine serum (National Institute of
Health, Tokyo).
2.3. Inoculation of the organisms and measurement
of the CD
The surface of an agar plate, approx. 2.6 m m
thick, was divided into 6 sections of equal size,
0.01-ml samples of serial dilutions of the inoculum
to be tested were placed in each of the sections,
and the plate was incubated aerobically or
anaerobically in a humidified incubator at 36°C
for 5 days. The diameter of well-isolated colonies
was microscopically measured with a micrometer.
The measurement was carried out on colonies so
selected as to avoid inclination to any locality
within a spot. One division of the micrometer
equals 188.2/~m. For anaerobic culture, Gas Pak
jar (BBL) was used.
The probability level for all significance tests
was fixed at P = 0.05.
3. RESULTS A N D D I S C U S S I O N
3.1. Test for normality of frequency distribution of
CD value
2.2. Medium
Agar medium contained 2.1% Bacto PPLO
broth, 0.8% Bacto yeast extract, 0.8% agar (Wako
Test for normality of frequency distribution of
C D values was carried out on colonies formed
under either the aerobic or the anaerobic condi-
0378-1097/83/0000-0000/$03.00 © 1983 Federation of European MicrobiologicalSocieties
8
Table 1
Test for normality of frequency distribution of CD of M.
arthritidis 07
Actual
CD
Actual
frequency
(Fo)
2.0
3.0
4.0
5.0
6.0
7.0
3
18
28
34
20
6
Sum
109
Theoretical
frequency
(Fc)
(Fo - Fc) 2
4.19
14.74
31.24
33.47
18.89
5.58
0.336
0.721
0.336
0.008
0.065
0.032
Fc
X2 =
1.498
Mean 4.623; SD, 1.200.
tion. As an example, the results with M. arthritidis
07, cultured aerobically, are shown in T a b l e 1. A
total of approx. 100 C D was m e a s u r e d in 5 spots
each c o n t a i n i n g 26 to 40 colonies. As there were
n o significant differences in the m e a n C D ( F =
3.404) a n d variances (X 2 = 0.099, Bartlett's m e t h o d
[4]) a m o n g 5 spots, the d a t a from 5 spots can be
safely c o m b i n e d . Theoretical frequency was calculated a c c o r d i n g to an e q u a t i o n of n o r m a l distribution b y using m e a n value a n d variance o b t a i n e d
from the actual data. T o e x a m i n e differences between actual frequencies a n d theoretical ones, a
goodness-of-fit test for x 2 - d i s t r i b u t i o n was practised. As the m e a n value a n d the variance of the
s a m p l e s were used for the calculation of the theoretical frequency, the degree of freedom was 3.
T h e calculated X 2 value, 1.498, was smaller than
the theoretical one, X 2 = 7.815. Therefore, the norm a l i t y of frequency d i s t r i b u t i o n was not denied.
W i t h ca. 100 C D m e a s u r e d in one spot containing ca. 250 colonies of M. arthritidis 07 cultured
either aerobically or anaerobically, the n o r m a l i t y
of frequency d i s t r i b u t i o n of C D was also confirmed.
The n o r m a l i t y of frequency d i s t r i b u t i o n of C D
was true also for M. arthritidis Y K , M. neurolyticure, M. orale N-1 a n d A. laidlawii L a i d l a w with
a p p r o x . 100 C D m e a s u r e d in one spot or in comb i n e d spots.
In conclusion, the n o r m a l i t y of d i s t r i b u t i o n of
C D was usually c o n f i r m e d irrespective of cultural
conditions.
3.2. Test for variance homoscedasticity of CD
T h e d i a m e t e r of 10 colonies was m e a s u r e d in a
s p o t c o n t a i n i n g similar n u m b e r of colonies f o r m e d
either a e r o b i c a l l y or anaerobically. T h e n u m b e r of
colonies in a spot ranged from 20 to 60 for all the
test strains. W h e n i n d i v i d u a l species grown u n d e r
b o t h aerobic a n d a n a e r o b i c c o n d i t i o n s were subj e c t e d to the test for variance, the variance ratios,
t h o u g h d i s t r i b u t e d from 1.032 to 2.882, were not
significant for all the test strains (details not
shown).
In conclusion, the significance test for the difference in m e a n C D of each strain between the
two culture c o n d i t i o n s is statistically valid.
3,0
2.0
1.0
t___l
1
I
2
1~
3
Log n u m b e r o f c o l o n i e s p e r s p o t
Fig. 1. Relation between mean CD and log number of colonies
of M. arthritidis 07 cultured aerobically. Ordinate, mean CD;
abscissa, log number of colonies per spot; vertical bar, 95%
confidential interval calculated by the use of common variance;
linearity, F = 0.068.
3.3. The relationship between mean CD and number
of colonies per spot
As an example showing the relationship between mean CD and number of colonies per spot,
a result with M. arthritidis 07 cultured aerobically
is given. In each of the spots containing 53, 70 or
300 colonies, 10 CD were measured. In the case of
a spot containing from 3 to 5 colonies (mean 4), or
from 6 to 7 (mean 6.5), 4 or 2 spots were combined
to make 10 CD in total, respectively. An hypothesis of variance homoscedasticity among each CD
was accepted. Then 5 values of mean CD were
plotted against log number of colonies (Fig. 1). A
linear relationship between the mean CD and log
number of colonies with negative regression coefficient was demonstrated.
Similar results were obtained with other test
strains irrespective of cultural conditions. So, the
comparison of the size of C D was valid only when
colonies were sampled from the spots containing a
similar number of colonies.
From these analyses, it was concluded that a
statistical method based on a hypothesis of normal
distribution can be applied to statistical analyses
of CD. However, the comparison of mean C D was
valid only when the number of colonies per inoculum were similar to each other.
ACKNOWLEDGEMENTS
We thank Dr. M. Kurokawa for his statistical
examination and Dr. J. Yasuda for his revision of
the manuscript. We are also grateful to Dr. K.
Akama, Director of this Department, for his revision.
REFERENCES
[1] Smith, P.F. (1956) Appl. Microbiol. 4, 254-259.
[2] Razin, S., Masover, G.K., Palant, M. and Hayrick, L.
(1977) J. Bacteriol. 130, 464-471.
[3] Leland, D.S., Lapworth, M.A., Jones, R.B. and French,
M.V. (1982) J. Clin. Microbiol. 16, 709-714.
[4] Bartlett, J.C. and Smith, D.M. (1960) Canad. J. Chem. 38,
2057-2065.