European J. Appl. Microbiol.

,o,Apphed
European
J.....

Biotechnol. 9, 59--62 (1980) Microbiology and

Biotechnology
9 by S p r i n g e r - V e r l a g 9 1980

A Method for Turbidimetric Measurement of Bacterial

Growth in Liquid Cultures and Agar Plug Diffusion
Cultures, Using Standard Test Tubes

K. J. Brown

Section of Child Health Research, Department of Paediatrics, University of Auckland School of

Medicine, Auckland, New Zealand

Summary. A method has been developed for turbidimetric measurement of

bacterial growth in standard inexpensive test tubes with closures in-place.
Liquid cultures and agar plug diffusion cultures can be assayed using an un-
modified spectrophotometer. Growth curves of replicate cultures grown in
test tubes, are reproducible with respect to similarity of curve shape, onset
of logarithmic growth phase, and maximum growth.

Introduction

In the course of assessing the effects of phenylalanine analogues on bacterial growth

in a plate assay (Brown and Lines 1976, 1978), it became necessary to compare cul-
ture growth on plates to that in liquid media, and to that in agar plug diffusion cul-
tures. The diffusion cultures consist of plugs of solidified agar, in the bottom of
test tubes, which are overlayered with inoculated culture medium.
Among the methods considered for measuring growth were nephetometry and
spectrophotometry. The disadvantages of using a nephelometer were that, with the
light source located at the bottom of the detector well, agar plug diffusion cultures
could not be accurately read and compared to liquid cultures, due to disturbance of
the light path. Also, in using a nephelometer, measurements could only be read in
arbitrary units. A 'Spectronic 20' spectrophotometer (Bausch and Lomb, Rochester,
N.Y.), equipped with a scale reading in absorbance units, proved suitable for assaying
both liquid and agar plug diffusion cultures.
The standard, cap-less cuvettes for the 'Spectronic 20', though usable for end-point
experiments, did not allow for long-term monitoring of uncontaminated cultures.
Spectrophotometer-matched, side-arm flasks were expensive and impractical in terms
of required amount of media, time, and incubator space. Also, as many as 100 cul-
tures, representing replicates of both controls and a number of tested factors, needed
to be examined in a single set of experiments.
A method was therefore developed for turbidimetrically measuring growth of cul-
tures of non-chaining, non-clumping bacteria in standard test tubes, with slide-on

0171-17 41/80/0009/0059/~ 01.00

60 K.J. Brown

closures in;place, directly in an unmodified spectrophotometer. Using this procedure,

growth could be monitored at regular intervals, without disturbing cultures or risking
contamination.

Materials and Methods

Serotyped cultures of Escbericbia coli 086/B7 and Salmonella typbi, isolated from
infant pathologiczl spec{mens, were obtained from Princes~ Mary Hospital for Chil-
dren, Auckland, New Zealand. Cultures were maintained on Dorset Egg Medium
(Dorset 1902) at room temperature.
For growth assessment experiments, cultures were inoculated into 5 ml of sterile
liquid media in 12 x 100 mm test tubes (Glass Wholesales Supplies, London, U.K.),
which were then capped with autoclavable plastic slide-on closures (Downing Plastics
Industries Ltd., Masterton, New Zealand). These tubes, with closures in-place, fitted
into the detector well of a 'Spectronic 20' spectrophotometer, and allowed the well-
cover to be fully closed for making readings. Cultures were first grown in peptone
water [2% (w/v) tryptone (Oxoid, London) and 0.9% (w/v) NaC1/1]. Amounts of
0.05 ml of logarithmic phase culture were then transferred to duplicate or triplicate
tubes of Trypticase Soy Broth (BBL, Cockeysville, Maryland), or to the phenylalanine-
free defined medium, Medium VI (Brown and Lines 1978). Cultures were grown in
an incubator, without shaking, at 37~ Turbidity was assayed by direct examination
of the tubes with closures inplace at 450 nm, in a 'Spectronic 20' spectrophotometer.
This was calibrated with an uninoculated control tube. Internal controls were pro-
vided for each experiment through the use of replicate tubes, as well as the uninocu-
lated media tubes. Measurements were made at three or four hourly intervals, for 48
to 60 h.
Agar plug diffusion cultures were prepared to study delay of contact of inhibitory
substances with, and of uptake by, bacterial suspensions in the liquid medium. For
agar plug diffusion cultures, 0.5 ml of 1.5% (w/v) sterile agar medium at 46~ with
or without test substances added, was pipetted into the bottom of 12 x 100 mm
culture tubes. These were subsequently overlayered with 4.5 ml of inoculated liquid
culture medium. In some experiments, the agar plug was first overlayered with an
interface layer consisting of 0.2 ml of concentrated agar [4.5% (w/v) agar in Medium
VI] before introducing the inoculated liquid medium (4.3 ml). A tube with an agar
plug and with uninoculated liquid medium was used for calibration. A plug consisting
of even 0.7 ml o f agar medium, in the bottom of the tubes, did not interfere with
spectrophotometric readings_

Resuks

Turbidimetric readings of culture growth were reproducible when bacterial cultures

were grown in standard test tubes, and read directly in the spectrophotometer (Fig. 1).
The turbidimetric readings were previously shown to be directly proportional to the
number of colony forming units (Brown and Lines 1976). By marking each tube, the
orientation could be maintained for subsequent readings. The direct reading method
enabled measurements to be taken without disturbing the cultures (as occurs during
Growth Measurement in Test Tubes 61

1.0
0.9
0.8
0.7
0.6
0,5

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E
0

0.3

c
1 I! IJ
0.2
o

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4. 8 12 16 20 24. 28 32 36 z,O 44. /,8 52 56
Time(h)
Fig. 1. Growth curves at 37~ of replicate cultures of Escherichia coli 086/B7 (. . . . ) and
Salmonella typhi ( ) in Medium VI (Brown and Lines 1978). (A) No phenylalanine analogue
added. (B) 50 #1 0.0t M g3-2-thienylalanine(Sigma Chemical Co., St. Louis, Missouri) added/5 ml
medium

transferring to cuvettes), and permitted about 80 determinations to be made in 30

min. Although cultures were not disturbed for spectrophotometric determinations,
similarly shaped curves were found for identically inoculated cultures which were
suspended using a vortex mixer. Perkins et al. (1963) also reported no qualitative
differences between shaken and static cultures, in their procedure for recording effects
on growth or sporulation of B a c i l l u s c e r e u s by inhibitors.
In the identically inoculated replicate tubes, growth curves were reproducible with
respect to (1) similarity of shape of the curve; (2) onset of logarithmic growth phase
as measured from the first reading after the initial lag phase in which all portions of
the curve between further readings gave a positive slope, until stationary phase was
reached; and (3) maximum growth, the asymptotic portion of the curve (stationary
phase). This is shown in Fig. 1.

Discussion

In order to assay liquid media cultures, a technique has been developed for reading
culture growth in standard test tubes, directly in a spectrophotometer. This method
has a number of advantages. Transfer to sterile cuvettes is not required, results are
similar among replicately-inoculated cultures, and the tubes are inexpensive as com-
pared to cuvettes. Also, the small size of the tubes, when compared to spectrophoto-
62 K.J. Brown

meter-matched, side-arm flasks, allows about 100 cultures, including both replicate
controls and tested variables, to be incubated and assayed in one experiment. This
allows a great saving in media cost and incubator space. The method is applicable to
agar plug diffusion cultures, and should be adaptable for growth measurement of
strictly anaerobic cultures in suitable pre-reduced media, by using either rubber stop-
pered tubes, or tubes having screw caps with rubber liners.
The application of this method, in the study of inhibition of bacterial growth by
phenylalanine analogues, using both liquid cultures and agar plug diffusion cultures,
will be reported elsewhere.

Acknowledgments. This work was supported in part by grants from the National Children's Health
Research Foundation, New Zealand, and the Auckland Medical Research Foundation.

References

Brown KJ, Lines DR (1976) Can J Microbiol 22:1673-1679

Brown KJ, Lines DR (1978) Aust J Exp Biol Med Sci 56:507-511
Dorset M (1902) Am Med 5 5 5 - 5 5 6
Perkins JP, Louie DD, Aronson JN (1963) Can J Microbiol 9:791-797

Received September I 1, 1979