Journal of Applied Bacterioioqy 1983, 54, 313-327 1066/11/81

Quality assurance of selective culture media

for bacteria, moulds and yeasts : an attempt at
standardization at the international level

D . A . A . MOSSEL,TREES M.G. B O N A N T S- V A N L A A R H O V E N ,
A N N I E KM.TH. L I G T E N B E R G - M E R K&U M
S A R I AE.B. WERDLER
Laboratory of Microbiology, Department of the Science of Food of Animal Origin,
Faculty of Veterinary Medicine, Uniuersity of Utrecht, Utrecht, The Netherlands

Received 23 November 1981 and accepted 3 June 1982

MOSSEL, D.A.A., BONANTS-VAN LAARHOVEN, TREES M.G.,

LIGTENBERG-MERKUS, ANNIEKM.TH. & WERDLER,MARIA E.B. 1983.
Quality assurance of selective culture media for bacteria, moulds and yeasts: an
attempt at standardization at the international level. Journal of Bacteriology 54,
31 3-327.
To facilitate monitoring of culture media, a simple quantitative streaking technique,
implying ever-decreasing numbers of colony-forming units per surface area, as in
spiral plating, was developed. The procedure evaluates, in quantitative terns, the
ability of media (1) to support the formation of colonies by organisms that it was
designed to grow and (2) to resist colonization by organisms that it is expected to
suppress. The procedure was therefore termed ecometric evaluation. The ecometric
results appeared to agree well with observations made on productivity and selecti-
vity of the media studied during routine examination of specimens.
These encouraging results prompted further, rigorous standardization of eco-
metry. A template was developed to standardize inoculation and the depth of the
agar was controlled to within *lo”/;,. Finally the attributes of the inocula used
were accurately defined.
The standardized ecometric technique has been found useful for the following
purposes: ( 1 ) to assess the practical significance of the inhibitory effect of gentamicin
observed in some moulds and yeasts (this was solved by replacing poorer basal
media by one particular richer modification, viz. yeast morphology agar); (2) the
development of a blood-free selective enumeration medium for Campylobacter
jejuni, i.e. sulphide iron motility agar plus the combination of antibiotics suggested
by Skirrow (1977); and (3) verification of the absence of antimicrobial activity of
enzyme preparations, e.g. catalase, used in culture media to remedy sublethal
damage in certain groups of bacteria.
Ecometric evaluation can now be recommended for (1) routine monitoring of
consignments of dehydrated or ready-to-use, purchased media; and (2) in-house
checking of the functioning of medium preparation departments. Only occasionally
is it necessary to use conventional counting techniques to confirm the results.

Quality assurance of bacteriological procedures A typical example of the consequences of this

is routine now in clinical bacteriology (Stokes
1979; Stokes & Ridgway 1980; Vera & Power
1980). However, very unfortunately this is not
universal practice for media used in the micro-
biological monitoring of foods.
0021-8847/82/06O0-0313 $02.00
@ 1983 The Society for Applied Bacteriology
lack of quality assurance experienced by the
senior author is reported. About 1962 it was
found useful to modify existing media for the
detection of Enterobacteriaceae in foods pro-
cessed for safety. This was done by replacing
lactose, as currently used in brilliant green bile
broth (BGB), with glucose (Mossel et al. 1963);
the modified medium was named Entero-
314 D. A . A . Mossel et al.
bacteriaceae enrichment, or EE broth. When
applying the commercially available dried prep-
aration in MPN counts on dietetic products
lower values were found in EE than when using
BGB. This was most surprising because (1)
many more Enterobacteriaceae utilize glucose
than lactose, and (2) in both instances any sub-
lethally stressed cells occurring in the dietetic
product had been resuscitated (Allen et al. 1952)
prior to making the dilutions in the selective
enrichment broths. Similar results were obtained
independently by Busse (1968) and Moussa et
al. (1973). These erratic data were subsequently
shown to result from the effect of the intrinsic
antimicrobial properties of the EE preparation
used on completely unimpaired populations of
Enterobacteriaceae. Similar observations con-
tinued to be made, e.g. with violet red bile glu-
cose agar, until as recently as 1979 (Mossel et al.
1979a).
It is clear that to achieve the delicate equi-
librium required between the productive and
selective properties of culture media is not
always an easy task. It calls for regular quality
monitoring both by the industry and by the
practising bacteriologist. This should be ex-
tended to some non-selective media as well,
since these too often show various deficiencies
(Abdou & Stoeckel 1980; Mossel et al. 1980b;
Pflug et al. 1981; Reimer & Reller 1981).
Many techniques, the majority of which rely
on colony counting, have been suggested for this
purpose (Lang 1954; Gillissen & Birkenhoven
1958; Schwarz et al. 1961, 1969; Vera 1971;
Prier et al. 1975; Brinkley & Huber 1978;
Taylor & Barrow 1981; Corry 1982a; Richard
1982; Baird & van Doorne 1982; Terplan et al.
1982; Corry 1982b). In such examinations a
spectrum of organisms of varying robustness
which are expected to grow on or in the
medium should always be included. When
monitoring selective media it is essential to use
a similar range of test strains whose growth
should be suppressed. Consequently medium
monitoring is rather complex. This often acts as
a deterrent to its systematic application.
This prompted us to develop a simplified,
semi-quantitative technique for this purpose
(Mossel 1971). The method relies on inoculating
standardized cultures on solid or solidified
liquid media with a carefully standardized, se-
quential streaking technique leading to ever-
decreasing numbers of colonies, as in spiral
plating (Corry 1982b). After suitable incubation
the final streak that leads to substantial colony
formation is assessed. The readings are ex-
pressed as absolute growth index.(AGI) which is
defined in Table 1. AGIs are generally con-
sidered in relation to the extent of colonization
on a suitable control medium similarly ex-
pressed. The data may then be converted to a
relative growth index (RGI), defined as the pro-
portion of the AGIs on the test medium and on
the reference medium. Selective media in par-
ticular, but also non-selective media for more
fastidious organisms, give a series of RGIs
which satisfactorily describe their performance.
This technique is called ecometric evaluation
because it assesses the ability to support colo-
nization of media by the organisms sought, and
also their resistance to colonization by the mic-
robial types the media were designed to
suppress.
When applied to some twenty media current-
ly used in public health microbiology, ecometric
results agreed well with observations made
during routine examination of specimens. The
results also substantiated those of more detailed
studies on media, where the fate of inoculated
test organisms was studied as a function of time,
temperature and interactions with other or-
ganisms which often interfere with the fun-
ctioning of media (Mossel et al. 1980b). This
justified experimental studies on further rigor-

Table 1. Assessment of absolute growth index: AGI

Almost all streaks in quadrant numbers n - 1 and n show (cl. Fig. 1) full growth, while there is virtually no
growth on any of the streaks in quadrants numbers n + 1 and n + 2, etc. . . . . . . . . . . . . . . . . . . . . . . . .AGI = n
Approximately half of the streaks of quadrant number n show growth, quadrant number n + 1 displays virtually
no growth and quadrant number n - 1 full, or almost full, growth . . . . . . . . . . . . . . . . . . . . . . . . .AGI = n - 4
Where the expected sequential growth pattern is interrupted, plates should be reincubated and checked after a
further 16 h. If no improvement is apparent this is indicative of a delicate balance between growth and
inhibition in this particular instance. The AGI assigned in these circumstances corresponds to the highest-
numbered sector where growth is observed.
 Quality assurance of selective culture media 315
ous standardization of the ecometric procedure, Table 2. Test species used since 1971*
particularly as a tint step in its possible use at
Group 1. Gram negative bacteria
the international level. The latter is highly desir- Acetobacter aceti
able, especially to determine acceptance or re- Campylobacter jejuni
jection of large consignments of culture media Citrobacter freundii
when these are tendered to institutes of public Escherichia coli
Klehsiella sp.
health, academic institutions or hospitals. K luyvera sp.
This paper describes the results of these stu- Proteus morganii
dies and its use in some additional areas of ana- Pseudomonas aeruginosa
lytical microbiology. Pseudomonasjluorescens
Salmonella enteritidis
Salmonella hadar
Methods and Media Salmonella pullorum
Salmonella typhimurium
INOCULA Serratia liquefaciens
Shigella sonnei
The strains used for assessing performance of Vibrio parahaemolyticus
various media and their selection are listed in Yersinia enterocolitica, serotype 0 : 3
Table 2.
Group 2. Gram positive bacteria
Bacillus cereus
Bacteria Erochothrix thermosphacta
Lactobacillus acidophilus
Cultures of strains to be used in any particular Lactobacillus fructivorans
instance are generally made in brain heart in- Micrococcus sp.
fusion broth (BHI). The BHI preparation used is Staphylococcus aureus
Streptococcus durans
previously examined for productivity by the Streptococcus equinus
dilution-to-extinction technique (Mossel et al. Streptococcus faecalis
1956; Baird & van Doorne 1982; Richard 1982)
using particularly fastidious test strains, inclu- Group 3. Yeasts
Candida curuata
ding Lactobacillus fructivorans and Streplo- Candidafamata
coccus equinus. The temperature of incubation is Candida lberica
generally 30-32"C, where virtually all test Candida sake
strains grow well (Mossel & van Diepen 1956). Candida zeylanoides
It is well established that the stage of growth Cryptococcus laurentii
Debaryomyces hansenii
of microbial populations markedly influences Leucosporidium scottii
their response to various stresses (Sherman & Rhodotorula graminis
Cameron 1934; Hershey 1939; Fry & Greaves Saccharomyces cerevisiae
1951; Strange et al. 1961; Mathison 1968; Sporobolomyces puniceus
Torulopsis norvegica
Palumbo & Alford 1970; Zyskind & Pattee Trichosporon beigelii
1971; Granai & Sjogren 1981; Sackin 1981). Trichosporon pullulans
Therefore, both log phase cultures ( i t . incubated
for about 4 h, the exact value depending on the Group 4. Moulds
length of the lag phase under optimal AspergillusJIavus
Aspergillus uersicolor
conditions) and stationary phase (overnight) cul- Eyssochlamys fulua
tures are used to test the performance of media. Cladosporium herbarum
However, in many instances only inocula in the Fusarium sporotrichioides
stationary phase need to be used because this Mucor mucedo
Neurospora sitophila
corresponds to the practical situation where the Penicillium citrinum
medium is applied. Penicillium islandicum
Rhizomucor pusillus
Yeasts and moulds Rhizoous stolonifer
~~

Yeasts and moulds were cultured in yeast ex-

* It is recommended to use both subcultures of
strains purchased from reputable collections and as
tract glucose broth. The incubation temperature many fresh isolates as is feasible in each particular
of mesophilic and psychrotrophic strains was instance.
316 D . A . A . Mossel et al.
23 f 3°C for one to three days, depending on
the growth rate of the particular yeasts and
moulds under study. This once more corre-
sponds to the practical situation (Dijkmann et
al. 1979; Mossel et al. 1980a). For psychrophilic
organisms 17°C was used (Mossel & Ratto
1973).
When monitoring media to be used for the
enumeration of mould propagules, challenge in-
ocula consisting mainly of spores are recom-
mended. This will greatly improve accuracy
which is low in the presence of fragments of
mycelium. Spore inocula can be obtained by sc-
raping off the growth of test strains on, e.g., malt
agar slopes, and suspending this in ca 0.5 ml
Tween-peptone saline, containing (g/l): peptone
1.0; NaCl 8.5; Tween 80 1.0. After homogeniza-
tion of this suspension on a ‘vortex’ eccentric
shaker, it is filtered, under aseptic precautions,
through an approximately 5 cm diameter funnel
containing a closed mat of carefully folded glass
wool. To avoid spore germination, the filtrate
obtained is inoculated without delay onto test
plates as described below.
DETAILS O F INOCULATION
To achieve the attempted continuous release
and thereby dilution of the inoculum, similar to
the situation in spiral plating (Corry 1982b) it is
essential to observe the following precautions.
Fig, 1. Network, serving to guide inocu-
lation during ecometric evaluation of
media, as imprinted by template of Fig. 2.
Inoculation is by 1 microlitre plastic dispos-
able loops. It is essential that perfectly straight
loops are used exclusively. A fresh loop must be
used for each plate inoculated.
Only the loop itself and not the stem should
be immersed in the test culture. Care should be
taken that it is completely charged with inocu-
lum. Excess fluid should be removed by care-
fully pressing the flat side of the loop three times
against the side of the tube.
During the inoculation procedure the loop
must be held at a shallow angle flat against the
surface of the medium. It should not be lifted
vertically. Turning, or even tilting, should be
avoided. The original position of the hand on
the bench should be retained during the whole
operation, using the fingers only to transmit
movement.
PROCEDURE
The cultures are homogenized with the aid of a
vortex mixer immediately before each inoculat-
ion. Streaking is done on 9 cm diameter plates.
previously marked with a template, as shown in
Fig. 2. The template is a standard type rubber
die with a wooden handle. It can be ordered
from an office supplier. The ink should be suit-
able for marking plastic. The four quadrants are
inoculated in succession with series of five paral-
lel lines. the first one at the perimeter. Finally
 Quality assurance of selective culture media
Fig. 2. Template used to mark bottom of plates to be tested by the ecometric procedure.
one line is drawn passing through the centre
(Fig. 1). Plates are prepared by pouring 25 1
ml of the medium tempered at 49 f 1°C. This is
done to standardize the intrinsic physico-
chemical situation at the surface which can be
markedly influenced by diffusion phenomena
and hence by the depth of the agar layer.
Similarly the water activity (a,) at the test
surfaces is carefully standardized. This is
achieved by pouring the agar plates while the
medium temperature is 49 & 1°C and by drying
the poured plates upside down with lids closed
for 19 & 1 h at 37°C in stacks of no more than
five plates with the stacks not less than 2 cm
apart. In this way 9&160 plates can be dried in
an incubator of approximately 100 litres. Pro-
vided the temperature of the agar is controlled
when poured, this results in adequately stan-
dardized surface moisture conditions. Further-
more, this procedure also serves to test the
plates for sterility (Mossel & van de Moosdijk
1964).
INTERPRETATION A N D ACCURACY
OF R E S U L T S
In all instances the results are expressed in AGIs
according to the definition in Table 1. This, ob-
viously leads to a maximum AGI = 5. As in-
dicated previously (Mossel et al. 1980b) the
precision of ecometric readings is +0.2 units.
This entails that any AGI below 4.6 has a con-
fidence interval of f0.6 units.
When control plates showed ‘maximal’ AGI
317
values, i.e. comprised between roughly 4 and 5 ,
all data are reported as AGIs. However, when
using mould spores, for example, as the chal-
lenge, the propagule count of the inoculum is
often low, leading to AGIs of the order 2. In
these instances all AGIs are, in addition, recal-
culated to RGIs. These, clearly, vary from 1.0 to
0.0.
MEDIA
Ecometric testing was first applied to 13 media
in current use in our laboratory. These com-
prised : buffered tryptone soya peptone glucose
agar (TSB); TSB agar with crystal violet added
(CVA); lactose lauryl sulphate agar (LLA);
violet red bile glucose agar (VRBG); Mac-
Conkey agar (MCA); xylose lysine deoxycholate
novobiocin agar (XLDN); lysine iron bile salts
novobiocin agar (LIBN); cetrimide nalidixic
acid agar (PSM); TSB agar with phenyl ethanol
added (PhA); mannitol egg yolk polymyxin agar
(MYP); Rogosa et d ’ s acid acetate agar (ROG);
Baird-Parker’s egg yolk pyruvate tellurite gly-
cine agar (BPM); and oxytetracycline gentami-
cin agar (ODYA). Media challenged with Vibrio
parahaemolyticus always contained 3% NaCI.
The composition and preparation of most of the
bacteriological media have been fully described
by Mossel et a!. (1980b) and Van Netten &
Mossel (1980). Cetrimide nalidixic acid agar
(Brown & Lowbury 1965; Goto & Enomoto
1970) was purchased in dried form as Pseudo-
monas Selective Medium (Oxoid) and heated to
318 D. A. A . Mossel et al.
dissolve the ingredients only instead of steriliza-
tion. Mannitol egg yolk polymyxin agar was
prepared according to Mossel et al. (1967).
The mycological media used are dextrose
yeast extract agar (DYA) of the formula elabo-
rated by Mossel et al. 1980a, dextrose yeast
extract agar with 10 8/1 of peptone added
(DPYA); oxytetracycline dextrose yeast extract
agar (ODYA) with concentrations of gentamicin
varying from 0 to 50 mg/l, Malt Agar (MA,
Oxoid), Yeast Morphology Agar (YMA Difco),
Mycophil Agar (Myc, BBL).
Some new media were tested by the same pro-
cedure. These included selective and indicative
media used for the isolation, enumeration and
identification of Campylobacter jejuni. One non-
selective medium was found extremely useful, i.e.
sulphide iron motility (SIM)medium (Blazevic
1968) with the agar content increased to a final
concentration of 15 g/l. The growth-promoting
properties for bacteria of the Campylobacter
group result in all probability from its ferrous
sulphate content (George et al. 1978). This
medium was tested with and without a combin-
ation of antibiotics, as recommended by Lau-
wers et al. (1978), Skirrow (1977), Blaser et al.
(1980) and Patton et al. (1981). Incubation was
carried out in candle jars two-thirds full of Petri
dishes, at 42°C (Skirrow & Benjamin 1980) for
48 h.
All media were tested in freshly prepared
form. Clearly the technique is also applicable to
media stored for customary periods of time, as a
rule no more than 5 d at 7"C, and after an un-
realistically long period (more than 14 d) of
chilled storage, to assess their durability.
Results and Discussion
MEDIA M A D E SELECTIVE FOR C E R T A I N
G R O U P S OF B A C T E R I A
The results of the ecometric testing of 1 1 selec-
tive bacteriological media in current use in our
laboratory are presented in Table 3. The data
obtained with five strains of Campylobacter
jejuni using basal SIM agar and with the usual
antibiotics added are summarized in Table 4.
The experimental data in Tables 3 and 4 sub-
stantiate our earlier observations that the eco-
metric technique allows reliable performance
testing of selective and, in essence, non-selective
culture media. The precision of the ecometric
data obtained after the technique had been fur-
ther standardized was found considerably im-
proved by comparison with the previous
method of Mossel et al. (1980b). In spite of this,
as demonstrated by the data in Table 4, eco-
metric results continue to show an accuracy
below that of conventional colony-count tech-
niques. However, there is often no need for the
highest precision in performance testing.
When solid media used for the isolation of
enteric pathogens following selective enrichment
are monitored, ecometric data are quite sum-
cient as they represent the practical situation.
When low RGI values, e.g. for wanted Entero-
bacteriaceae, are obtained and/or the 'unwan-
ted' test strains give relatively high RGI values,
the medium is of only limited practical worth.
This is because the detection technique then
relies too heavily on the preceding enrichment
step (Vassiliadis et al. 1981). However, it should
be noted that this failing is often not caused by
a fault in any particular batch or brand of
medium, but is a deficiency in the original for-
mulation, e.g. brilliant green agar.
In many instances the ecometric technique
can also be used to monitor colony-count
media. When the recovery of test strains, for
whose enumeration the medium was designed, is
low in comparison with the AGI obtained on
the control medium, the performance of the
medium tested can confidently be reported as
insufficient. When RGIs are satisfactory, eco-
metric testing can be extended by using one or
two additional decimal dilutions of the standard
inocula to assess the functioning for the very
low numbers of cells (asymptotically approach-
ing one) which must develop into visible colo-
nies for enumeration.
M Y C O L O G I C A L MEDIA R E L Y I N G ON T H E
USE O F G E N T A M I C I N
The ecometric technique has also proved valu-
able in assessing the practical significance of the
recently established mycostatic properties of
gentamicin (Janes & Tilbury 1981; Corry
1982b).
Concentrations of this antibiotic from 1 to 50
pg/ml were tested in the standard medium oxy-
tetracycline gentamicin agar (Mossel et al.
1980a). Twenty-five strains of common food
yeasts and fifteen of similar mould species were
examined for colonization on these media. It
became evident that when applied to moulds,
 Table 3. Application of the rigorously standardized ecometnc technique to 11 selectivemedia and one control medium in general use

Absolute growth index (AGI) assessed on medium

Temp. Reading
Strain (“C) (days) TSBA CVA LLA VRBG MCA XLDN LIBN PSM PhA MYP ROG BPM
Gram negative bacteria
Escherichia coli 30 1 5-0 nd nd 5-0 5.0 2.5 nd QO 5*0* 0.0 nd nd
Pseudomonas aeruginosa 30 1 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0* 0.0 0.0 0.0
Salmonella typhimurium 30 1 5.0 5.0 5.0 5.0 5.0 5-0 5.0 0.0 0.0 0.0 nd nd
Shigella sonnei 30 1 5.0 5.0 5.0 5.0 5.0 5.0 nd 0.0 0.0 0.0 nd nd
Vibrio parahaemolyticus 30 1 5.0 nd nd nd nd 0.0 nd 0.0 0.0 5.0 nd nd
Yersinia enterocolitica 30 1 5-0 5.0 nd 5-0 5.0 5.0 5-0 0.0 0.0 0.0 0.0 0-0
Gram positive bacteria
Bacillus cereus 30 1 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 5.0 0.0 0.0
Lactobacillus acidophilus 30 2 nd 0.0 nd 0-0 nd nd 0.0 nd nd nd 5.0 0-0
Staphylococcus aureus 30 1 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 5.0 0.0 5.0
Streptococcus dwans 30 1 5-0 nd nd nd nd 0.0 nd 0.0 5.0 5.0 nd nd
Streptococcus faecalis 30 1 5.0 5.0 0.0 0.0 0.0 0.5* 0.0 0.0 5.0 5.0 0.0 0.0
Yeasts
Candida curvata 23 f 3 5 5.0 0.0 0.0 0.0 0.0 1.5 0-0 0.0 0-0 5-0 0.0 0-0
Debaryomyces hansenii 23 f 3 5 437 nd nd nd nd 5.0 nd 0.0 0.0 5.0 nd nd
Saccharomyces cerevisiae 23 f 3 5 4-5 nd nd nd nd 4.5 nd 0.0 0.5 0.5 nd nd
7 4.5 nd nd nd nd 4.5 nd 0.0 4.5 4.0 nd nd
Trichosporon beigelii 23 f 3 5 3.5 nd nd nd nd 0.0 nd 0.0 0.0 0.0 nd nd
Moulds
Aspergillus Javus 23 f 3 5 4.5 nd nd nd nd 5.0 nd 0.0 0.0 5.0 nd nd
Fusarium sporotrichioides 23 f 3 5 5.0 nd nd nd nd 0.5 nd 0.0 0.0 5.0 nd nd
Penicilliwn citrinum 23 - 3
+ 5 1-5 nd nd nd nd 2.0 nd 00 0.0 5-0 nd nd

TSBA, buffered tryptone soya peptone glucose agar CVA, TSB agar with crystal violet
LLA, lactose lauryl sulphate agar VRBG, violet red bile glucose agar
MCA, MacConkey agar XLDN, xylose lysine deoxycholate novobiocin agar
LIBN, lysine iron bile salts novobiocin agar PSM, Pseudomonas aeruginosa selective medium
PhA, TSB agar with phenyl ethanol MYP, mannitol egg yolk polymyxin agar
ROG, Rogosa et al.’s acid acetate agar BPM, Baird-Parker’s egg yolk pyruvate tellurite glycine agar
* Pin points.
t Irregular growth.
nd, not done.
W
c
W
320 D . A . A . Mossel et al.
Table 4. The recovery of 5 strains of Campylobacter jejuni on SIM agar with and without Skirrow's
antibiotics (sk). A comparison of colony counts and ecometric measurement
SIM SIMsk
'Temp. Reading
Strain ("C) (days) AGI cfu/ml AGI cfu/ml
Campylobacter jejuni H42 42 2 5.0 5.9 x 10" 5.0 6.0 x 10"
F157 42 2 5.0 4.3 x lo7 5.0 3.1 x 10'
K18 42 2 5.0 5.8 x 10" 5.0 5.2 x 10"
Klll 42 2 5.0 1.7 x 10" 4.0 1.4 x 10'
C186 42 2 5.0 6.7 x 10' 5.0 6.0 x 10"

ecometric results had to be expressed by a
second parameter, in addition to AGI, viz. the
degree of aerial mycelium formation (AMF). As
shown in Fig. 3 this was often markedly reduced
in the presence of gentamicin, even when the
AGI was almost unaffected. The full results thus
obtained are reported in Tables 5 and 6. Early
aerial mycelium formation did not make it im-
possible to assess AGIs because these could be
estimated by reading the bottom of the plates.
Among the yeasts tested, two of the five Can-
dida species examined showed concentration-
related sensitivity to gentamicin at levels 5-10
pg/ml. One Rhodotorula and Trichosporon were
sensitive to 50 pg/ml, but barely to lower con-
centrations. The other 19 types were grossly
gentamicin-resistant. Slight sensitivity to genta-
micin, particularly expressed in growth velocity
but not in AGI, was observed in 2 out of 15
mould species tested, i.e. Aspergillus versicolor
and, to a lesser extent, Rhizomucor pusillus.
These findings substantiate our earlier experi-
ence with gentamicin containing media when
examining food samples (Mossel et al. 1975).
Janes & Tilbury (1981) have recently stressed
the influence of the composition of the basal
medium on the recovery of yeasts in the pre-
sence of given antibacterial antibiotics. This

Fig. 3. Aerial mycelium formation as a parameter in the ecometric monitoring of media made selective for yeasts
and moulds. Right: Mould on a control plate, without antibacterial antibiotics. Left: Ecogram made in exactly
the same way, but on medium with oxytetracyclineand gentamicin added.
 Table 5. Influence of mntamicin on the growth of yeasts as assessed by the ecometric technique at 30 or 17°C as indicated
Absolute erowth index (AGI)
ODYA + gentamicin (mg/l)
Temp. Reading
Strain (“c) (days) MA ODYA
DYA DPYA - 5 10 20 50
Candida curvara, strain can 30 3 nd nd nd 5.0 5-0 4.0 4.0 5.0
curvata, strain C 30 3 nd nd nd 5.0 5.0 5.0* 5.0 5.0
curvata, strain F 30 6 nd nd nd 5.0 3.5 4.0 5.0 5.0
curvata, strain H 30 6 nd nd nd 5-0 4-0 5.0 5.0 5.0
famata 30 6 nd nd nd 4.0 4.0 4-0 4.0 3.5
ibmica 30 6 nd nd nd 5.0 5.0 1.5 0.5 0.0
sake 17 3 5.0 5.0 5.0 5.0 5.0* 5.0 5.0 5.0
zeylanoydes 30 6 nd nd nd 5.0 2.5 0.5 0.0 0.0
Cryptococcus hurentii 30 6 nd nd nd 2.5 4.0 5.0 3-0 3.5
Debaryomyces hansenii, strain Deb 30 6 nd nd nd 2-5 4.0 4.0 3-5 4.0
hansenii, strain I 30 6 nd nd nd 4.5 5.0 4.0* 2.5 5*0*
hansenii, strain 0 30 6 nd nd nd 5.0* 5.0 5.0 5.0* 5.0
Leucosporidium scottii 17 5 3.5 4.0 4.0 5.0 2.0 2.5 5.0 4.0.
Rhodotorula I 30 6 nd nd nd 3-5 5.0 5.0 4.0* 2.0
m
111 30 6 nd nd nd 4.0 5-0 5.0 3.5 5.0 2
graminis, strain D 17 5 4.0* 4.0 3.5 5.0* 2.5 4.0 4.5* 4*0*
w
graminis, strain G 17 5 2.0 3.5 5.0 5.0 50*t 5.0t 4.0*t 5o*t 2.
Tr. I 17 5 5.0 5.0 5.0 3.0 5.0 3.5* 5.0 5.0 C
m
Tr. I1 30 6 nd nd nd 4.0 5.0 5-0 3.5 3-0 c,
Saccharomyces cerevisiae 30 6 nd nd nd 3-5 2.5 4.0 2.5 4.0 E
Sporobolomyces puniceus 17 5 4.5 2.5* 4.0 5.0* nd 5.0* 4.5 4.5
Trichosporon beigelii, strain E 30 6 nd nd nd 3.5 4.0 1.5 3.5* 2.5 $-
beigelii, strain S 30 6 nd nd nd 1.5 3.0 5.0 2.0 2.0
(2.0)$§ (3.3)$§ (1.3)#§ (1-3)#§
beigelii, strain Trich. 30 4 4-0 2.5 0.5 4.0* 3-5* 1.5 2.5* 1.5*
pullulans 17 3 4.0 5.0 4.0* 4.0 5.0 5.0 5.0 5.0*
Weighted average 4.0 4.7 4.1 4.0 3.7 3.7
MA, malt agar DYA, dextrose yeast extract agar
DPYA, dextrose peptone yeast extract agar ODYA, oxytetracyclinedextrose yeast extract agar
* Irregular growth in sectx 4.
t Small colonies.
$ RGI.
5 In this instance the antibiotic seems to stimulate growth, as an RGI > 1.0 is obtained. w
nd, not done.
!2
322 D. A . A . Mossel et al.
Table 6. Effect of gentamicin on the growth of moulds as assessed by the ecometric technique at
20 f 3°C
Absolute growth index (AGI)
Aerial mycelium formation (AMF)
ODYA + gentamicin (mg/l)
Reading
Strain (days) MA DYA DPYA ODYA 5 10 20 50
Absidia* 2 nd 4.0 4.0 3.0 4.0 3.5 3.5t 3.0
Aspergillusflavus 5 4.0 nd nd 5.0 4.0 4.0 4.0 4.0
uersicolor 2 1.0 nd nd 1.0 1.0(1*0)$1.0(1.0)$1.0(1.0)$03§(0*5)$
4 AMF nd nd AMF AMF AMF AMF 4.011
Byssochlamysfulua 5 1.0 nd nd 1.0 l.a(l.O)$1.0(1.0)$1.0(1.0)f 1.0(1.0)$
CIadosporium herbarum 5 0.5 nd nd 0.5(1.0)$ 0-5(1.0)$ 1~5(3-0)$~~0~5(1~0)$
0 3 1 .O)$
Fusarium sporotrichioides 3 4.0 nd nd 4.0 4.0 2.5 3.0 4.0
Neurospora sitophila 3 1.0 nd nd 1.0 1.0(1.0)$19(1.0)$ 1.0(1.0)$ 1.0(1.0)#
Penicillium citrinum 5 2.5 nd nd 4.0t 4.0 3.0 23 4.0
I* 6 nd 5.0 4.0 4.0 5.0t 5.0 4.0 5.0
11* 6 nd 4.0 4.0 4.0 5.0t 3.5t 5.0 4.0
III* 6 nd 5.0 4.0 4.0 4.07 4.0t 4.0t 5.0
IV* 2 nd 5.0 3.5 3.0 4.0 4.0 35 3.q
Rhizomucor pusillus 13 1.0 nd nd 0.5 0.0(0.0)$O.o(O.O)$ 0.0(0.0)$ 0.0(0*0)$
Rhizopus stolonveer 2 1.0 nd nd 1.0 1.0(1.0)$ l.O(l.O)$I.O(l.O)$ 1.0(1.0)$
Weighted average 1.9 4.6 3.9 2.1 3.0 2.8 2.1 3.1
. -

MA, malt agar DYA, dextrose yeast extract agar

DPYA, dextrose peptone yeast extract agar ODYA, oxytetracycline dextrose yeast extract agar
* Isolated from air.
t Irregular growth.
$ RGI.
5 Small colonies.
1) Antibiotic stimulating growth.
nd, not done.

prompted us to repeat part of the experiments and/or oxytetracycline appear to stimulate

reported in Table 5, using various basal media.
These included yeast morphology agar as re-
commended by Janes & Tilbury (1981), Moir &
Russell's (1939) glucose yeast extract agar, soya
peptone glucose agar and malt agar. The results
are presented in Table 7. They show that the
inhibitory properties of gentamicin towards all
moulds and yeasts that have been found suscep-
tible can indeed-to a great extent-be over-
come by the use of one particular, richer basal
medium, viz. yeast morphology agar. However,
although this medium markedly reduces the in-
hibitory effect of gentamicin, there still seems to
be a residual growth-retarding activity, i.e. with
Rhizomucor and, only with respect to colony
size, also for Candida zeylanoides. In some tests
Cladosporium herbarum and Fusarium sporotri-
chioides grew to an AGI of about 1 to 2 only,
due to low levels of spores in the particular ino-
cula.
It is quite striking that chloramphenicol
growth in the case of Rhodotorula strain Tr. I
and Fusariutn sporotrichioides in Table 7 ,
though not so conspicuously in any other case.
In more general terms the response of yeasts
and moulds to antibacterial antibiotics in the
media was not very consistent. This is illustrated
by a comparison of the AGIs for gentamicin of
Tables 5, 6 and 8 with those of Table 7. This is,
however, a rather commonly observed effect
with marginal values of inhibitory factors. These
data indicate that, at any rate, rigorous stan-
dardization of the nutrient composition of my-
cological media containing antibacterial
antibiotics is required. First and foremost the
attributes of the yeast extract and peptone used,
particularly their contents of the various essen-
tial B-vitamins, should be carefully standardized
and monitored. It seems prudent to check, in
addition, the complete final medium, e.g. by the
ecometric procedure used in this investigation.
Finally, it remained to be checked whether
Table 7. The effect on the recovery of yeasts and moulds of oxytetracycline, oxytetracycline with gentamicin and chloramphenicol in media of varying basal composition at
23 f 3°C
Absolute growth index (AGI)

Reading MA MA MA YMA YMA YMA Myc Myc Myc DYA DYA DYA
Strains (days) TSBA MA ch ox ox g YMA ch ox ox g Myc ch ox oxg DYA ch ox oxg

Yeasts
Candlda curuata
strain can 5 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 4.5 5.0 5.0 5-0 43 5.0 5.0 5.0 0.0
iberiea 5 nd 5.0 nd nd 0.5 5-0 nd nd 5.0 5.0 od nd 0.0 5.0 nd nd 00
zeylanoides 5 nd 4.0 nd nd 0.0 5.0 nd nd 4.5. 5.0 nd nd 0.0 4.0 nd nd 0.0
Debmyomyces hansenii
strain Deb. 5 5.0 4.5 50 5.0 4.5 4.0 5.0 5.0 4.5 5.0 5.0 5.0 4.5 5-0 5.0 5.0 0.0
Rhodotomla
strain 1 5 5.0 5.0 4.0 5-0 5.0 5-0 5.0 3.5 5.0 5.0 5-0 5.0 5.0 5-0 5.0 5.0 00
strain 4 7 5.0 5.0 5-0 5-@f 5.0 5-0 5.0 4.0 5-0 5.01 3.5 3-5 3.5 4-0 5.0 5.0 05
strain 5 5 5.0 5.0 5.0 3.5 0.0 4.0 5.0 5.0 3.5 5.0 5.0 5.0 1 .o 3.0 5.0 5.0 0.0
strain 6 5 5.0 5.0 5.0 5.0 0.0 3.5 5.0 5.0 5.0 5.0 5.0 5.0 5.0'7 5.0 5.0 5.0 0.0
strain G 7 5.w 4.0 50 40 0.0 5.0 4.0 5.0 5.0t 5.0 5.0 3.5 4,ot 4.0 5.0 5.0 0.0
strain HI 7 2.0 5.0 5.0 5.0 0.5 5.0 5.0 5.0 5.0 5.0 5.0 5-0t 1.0' 5.0 4.0 5.0t 0.0
strain Tr. 1 I 5.0 2.5 5.0 5.0 0.0 2-5 5.0 4.0' 2-5t 5.0 5.0 5.0 0.0 4.5 4.0 5.0 00
strain Tr. 11 5 5.0 4.5 5.0 3-57 0.0 5.0 5.0 5-0 5-0 5.0 5.0 50 0.5 5-0 50 5.0 00
Sacchpromyces cereuisiae
strain sac 5 3.5 5.0 5.0 5.0 5.0 5.0 5.0 5.0 4.0 5.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0
Trichosporon beigrlii 7 5.0 5 .O 5.0 5.0 5.0 5.0 5.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 3.0
strain Trich.
Moulds
Aspersillus flavus 5 5.0 5.0 5-0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5-0 4.0 5.0 5.0 5.0 5.0
uersicolor 5 nd 5.0 nd nd 3.5 nd nd nd nd 43 nd nd 4.0 4.5 nd nd 5.0
Cladosporium 1 0.5 0.5 05 0.5 0.0 0.5 0.5 0.5 0.0 I .o 1.0 0.5 0.5 0.5 0.5 0.5 0.0
herbarum (1.01t (1W (0.0): (19t (1.0)$ (OW (IW (03f (0.51f (1QH (1.0): (OW
FUSWiWVi 5 1-0 1.0 1.0 1.o 00 1.0 I .o I
.o I .o AMF 2.w 3.w 1.05 1.0 1.0 4.0 00
spaorrichioides (1-0): (1.0)t (Wt (1.0)f (IW (1-0): (IW (4.0): II (om
Rhizomucor
pusillus 5 5.0 5.0 5.0 5.0 0.0 4.0 AMF AMF 1.o AMF AMF AMF 0.0 5.0 2.5 4.0 0.0

t ~ ~ MA,malt agar
TSBA. b u f f e d t ~ y psoya YMA. yeast morphology agar Myc. mycophil agar DYA, dextrose yeast extract agar
peptone glucose agar ch. chioramphenicol50 nq?,l ox, oxyletracycline LOO I@ g gentamian 50 rndl

Colonies markedly smaller than on control. 1RGI. I1 In this instance the antibiotic again seems to stimulate growth.
t Irregular growth. 5 RGI could not be calculated bccause of AMF in control. nd, not done.
w
h,
w
324 D. A . A . Mossel et al.
Table 8. The extent of development of 16 test bacteria and one yeast on media of two
different basal compositions made selective with oxytetracyclineand gentamicin
Absolute growth index (AGI)
Reading
Strain (days) TSBA YMA ox g Mycox g APT
-
Gram negative bacteria
Escherichia coli 1 5.0 0.0 0.0
5 0.0 0.0
Klehsiella sp. 1 5.0 0.0 0.0
5 0.0 0.0
Kluyvera sp. 1 5.0 0.0 0.0
5 0.0 0.0
Pseudomonas aeruginosa 1 5.0 0.0 0.0
5 0.0 0.0
Salmonella enteritidis 1 5.0 0.0 0.0
5 0.0 0.0
hadar 1 5.0 0.0 0.0
5 0.0 0.0
t yphimurium 1 5.0 0.0 0.0
5 0.0 0.0
Shigella sonnei 1 5.0 0.0 0.0
5 0.0 0.0
Vibrio parahaemolyticus 1 5.0 0.0 0-0
5 0.0 0.0
Yersinia enterocolitica 0 : 3 2 5.0 0.0 0.0
5 0.0 0.0
Gram positive bacteria
Bacillus cereus 1 4.0 0.0 0.0 -
5 4.0 0.0 0.0
Lactobacillus acidophilus 2 0.0 0.0 0.0 5.0
5 0-0 0.0 0.0
Micrococcus sp. 1 3.0* 0.0 0.0 -
2 4.5 0.0 0.0
5 5.0 0.0 0.0
Staphylococcus aureus 1 5.0 0.0 0.0 -
5 0.0 0.0
Streptococcus durans 1 5.0 0.0 0.0 -
5 0.0 0.0
faecalis 1 5.0 0.0 0.0 -
5 0.0 0.0
Yeast
Rhodotorula Tr. I 5 5.0*
7 5.0
TSBA, buffered tryptone soya peptone glucose agar YMA, yeast morphology agar
ox, oxytetracycline 100 mg/l g, gentamicin 50 mg/l
Myc, mycophil agar
Temperature 30°C except Rhodotorula Tr. I, 20°C
* Pin points.

the richer mycological media maintained their
bacteriostatic properties. As the data in Table 8
show, this was indeed found to be the case.
MEDIA C O N T A I N I N G C A T A L A S E
TO REPAIR SUBLETHAL LESIONS
In some culture media enzyme preparations, e.g.
superoxide dismutase (Bucker & Martin 1981),
and particularly catalase are used to overcome
inhibitory properties to stressed microbial popu-
lations (Mossel & Corry 1977). These include
catalase positive organisms deprived of this
enzyme by thermal or freezing injury (Martin et
al. 1976; Raccach & Juven 1976; Flowers et al.
1977; Rayman et al. 1978; Andrews & Martin
1979) or essentially catalase negative taxa
(Holman 1955; Harmon & Kautter 1976,1977).
We observed that an occasional batch of ca-
talase exerted a marked inhibitory effect on
 Quality assurance of selective culture media
some inocula at the required concentration of
about 100 u/ml. Guided by ecometric monitor-
ing, inhibitory batches of catalase could be
eliminated and satisfactorily functioning prep-
arations could be identified. In confirmatory
assays, preparations which had been shown to
be non-inhibitory by ecometry were invariably
found to function well in recovery media.
Conclusion
It thus appears that the further standardized
technique for ecometric monitoring can be rec-
ommended for the routine performance testing
of batches of purchased dehydrated or ready-to-
use culture media. The latter include culture
media layered on so-called agar immersion pla-
ting and contact (‘Dip’) slides, as demonstrated
earlier by Mossel et al. (1979b). Ecometric per-
formance evaluation can, clearly, also be used
for checking the functioning of in-house culture
medium preparation units.
Nonetheless, it may remain necessary to
apply conventional colony-count techniques to
validate results obtained by ecometric monitor-
ing. Our experience since 1970 and in part re-
ported here demonstrates, however, that this is
only exceptionally required.
The authors wish to tender their gratitude to
Miss Lianne Klerkx and Mr Jan-Karel van
Goch, for their support in this investigation.
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