A “spiral test” applied to bacterial mutagenesis assays

213

Mutation Research, 82 (1981) 213--227 Elsevier/North-Holland Biomedical Press

A "SPIRAL T E S T " APPLIED TO BACTERIAL MUTAGENESIS ASSAYS

SILVIO de FLORA Institute of Hygiene, School of Medicine, University of Genoa, 16132 Genoa (Italy) (Received 12 September 1980) (Revision received 4 February 1981) (Accepted 6 February 1981)

Summary A new procedure (the spiral test) has been set up and validated for the distribution of chemicals in bacterial mutagenesis assays. This m e t h o d involves the use of a special instrument (spiral plater), which dispenses, along a spiral track, decreasing volumes of liquid samples, from the near centre to the periphery o f a rotating agar plate. A gradient of concentration of a c o m p o u n d up to about 1500 : 1 is thus formed on a single plate. The activity of 18 mutagens of various potencies and chemical classes was checked in the SalmoneUa/microsome test by dispensing their solutions either on the surface of top agar (method A) or of the minimal-glucose agar medium, before the addition of molten top agar incorporating bacteria and eventually $9 mix (method B). Compared with the spot test, the gradient of concentration of a c o m p o u n d produced by the spiral diluter was much wider and more gradual. Even nondiffusible chemicals (e.g. b e n z o [ a ] p y r e n e and benz[a]anthracene) were efficiently detected in the spiral test, as well as very weak (e.g. mebanazine and trimethylphosphate) or borderline (e.g. perylene, 1,1-dimethylhydrazine and procarbazine) mutagens, which were negative in the spot test. Method B was at least as sensitive as the plate-incorporation test, such a goal being achieved in a single plate instead of in serial plates. Technical problems made m e t h o d A less sensitive, but it was more efficient in detecting unstable mutagens (e.g. ~-propiolactone). Like the plate test, the spiral test appeared to be suitable for a semi-quantitative assessment of mutagenicity data, and was efficient in demonstrating both the activation of promutagens and the deactivation of some directly acting mutagens. Preliminary assays were also carried out with repairproficient (WP2) or
The possibility of obtaining a gradient of concentration of chemicals on a single agar plate affords considerable advantages, in term of simplicity, in bac0 027-5107/81/0000--0000/$02.50 © Elsevier/North-HollandBiomedical Press

214 terial mutagenesis assays. This task is commonly achieved by spontaneous diffusion of compounds in the well-known spot test, which is considered to be a convenient m e t h o d for a qualitative pre-screening of mutagens (Ames et al., 1975). However, the spot test has 2 major limitations, consisting in its lower sensitivity, compared with the plate-incorporation test, and in its dependence on the degree of diffusibility of compounds in the agar. Accordingly, important categories of mutagens, such as most polycyclic hydrocarbons and other waterinsoluble chemicals, cannot be identified by this procedure (Ames et al., 1975). Another method, suitable for preparing a narrow-concentration gradient (1 : 10 per plate) simply by varying the thickness of the agar layer incorporating the compounds, has been proposed and validated by testing a large number of chemicals (McMahon et al., 1979). An instrument automatically dispensing a continuously decreasing volume of liquid samples is commercially available. Distribution of samples along a spiral track results in a concentration range up to about 1500 : 1 from the near centre to the periphery of a single petri plate. This instrument has been designed to determine the density of bacterial suspensions and has been successfully applied to microbiological analyses of food, water, pharmaceutical and cosmetic products, as well as of clinical samples (Gflchrist et al., 1973; Jarvis et al., 1977; Briner et al., 1978; Kramer et al., 1979). I present here a new application of the spiral-plating system, consisting in the distribution of chemical solutions, instead of bacterial suspensions, for the assessment of their mutagenicity in bacterial test systems. This method, referred to as the spiral test, has been preliminarily validated in the Ames reversion test with his- strains of S. typhimurium. A number of assays has been carried out with repair-deficient or -proficient trp- strains of E. coll. Materials and methods

Chemicals The following 18 chemicals were tested: aflatoxin B1 (AFB1) (Makor Chemicals, Jerusalem, Israel), acriflavine and trimethylphosphate (BDH, Poole, Dorset, Great Britain), 2-aminofluorene (2-AF) and benzo[a]pyrene (BP) (EgaChemie KG, Steinheim/Albuch, West-Germany), dibenz[de, kl]anthracene or perylene (Sigma Chemical, St. Louis, MO, U.S.A.), benz[a]anthracene (BA) (K and K Laboratories, Plainview~ NY, U.S.A.), fi-propiolactone {Sigma Chemical, St. Louis, MO, U.S.A.), 2-methoxy~¢hloro-9-(3-(ethyl-2¢hloroethyl)aminopropylamino)acridine • 2HC1 (ICR-170) and 2-methoxy-6-chloro-9-(3-(2chloroethyl)aminopropylamino)acridine. 2HC1 (ICR-191) (Polysciences, Warrington, PA, U.S.A.), 1,1-dimethylhydrazine (Fluka, Buchs, Switzerland), N-isopropyl-~-(2-methylhydrazino)-p-toluamide. HC1 or procarbazine or natulan (Hoffmann--La Roche, Basel, Switzerland), a-phenylethylhydrazine oxalate or mebanazine (Imperial Chemical Industries, Cheshire, Great Britain), fi-phenylethylhydrazine.H2SO4 or phenelzine (Warner, Eastleigh, Great Britain), nialamide (Pfizer Italiana, Roma, Italy), sodium azide, sodium dichromate and methyl methanesulphonate (MMS) (Merck-Schuchardt, Munich, WestGermany). These compounds were dissolved and diluted either in bi-distilled water or in dimethyl sulphoxide (DMSO).

215

Bacterial strains 5 h/s- strains of S. typhimurium (TA1535, TA100, TA1537, TA1538 and TA98) were kindly supplied by Dr. Bruce N. Ames (Department of Biochemistry, University of California, U.S.A.). Their genetic features have been described by Ames et al. (1975). 3 strains of E. coli, i.e. WP2 (trp-), CM871 (trp-/lexA-/uvrA-/recA-)and TM1080 (trp-/lexA-/polA-/R391), were kindly supplied by Dr. Carlo MontiBragadin (Institute of Microbiology, University of Trieste, Italy). Their efficiency, compared with other repair-deficient E. coli strains, has been demonstrated with a variety of DNA-modifying agents (Venturini and Monti-Bragadin, 1978). Spiral plater The spiral plater instrument is manufactured by Spiral Systems Marketing Ltd. (Bethesda, MD, U.S.A.). 2 models (B250 and C), having similar features, were made available by their distributors in Italy (Pool Bioanalysis Italiana, Milano, Italy), during 2 separate periods. The dispensing stylus of the spiral plater moves from the near centre towards the outside of a rotating plate, depositing the liquid sample in an archimedes spiral. This instrument can be regulated for distribution of liquid samples on the surface of either 90/100- or 140/150-mm plates. The dispensing time in these 2 kinds of plates is 23 and 40 sec, respectively. The cumulative volumes of sample, obtained by regulating the spiral diluter in 2 different positions, are 36.5 /~1 (90/100-mm plates) or 39 #1 (140/150-mm plates) and 92 or 100 gl, respectively. According to the principle of the spiral plater, the volume of sample dispensed at each point of the spiral track can be calculated with the aid of suitable equations and graphs supplied by the manufacturers. Table 1 gives some examples of volumes dispensed per unit area ( g l / m m 2) in the initial and terminal parts of the spiral track, as related to the cumulative volume dispensed and to the size of plates. The corresponding theoretical ranges of concentration of chemicals from the starting point to the end of the spiral track are also shown. TABLE 1 V O L U M E S O F C H E M I C A L S O L U T I O N S D I S T R I B U T E D BY T H E S P I R A L P L A T E R ( M O D E L S B250/C) AND RANGE OF CONCENTRATION OF CHEMICALS FROM START TO LIFT-OFF POSITIONS OF THE DISPENSING STYLUS Cumulative volume dispensed (~1)

V o l u m e d i s p e n s e d per u n i t a r e a ( / J l / m m 2 ) Start position rffi12mm

Lift-off position r = 40ram (90/lO0-mm plates)

rf64mm ( 1 4 0 / 1 5 0 - m m plates)

36.5--89

8.0 X 10 -2 (1)

1.4 X 10 -3 (1 : 57)

8.6 X 10 -$ (1 : 930)

92--100

2.8 × 10 - t (1)

0.3 X 10 -3 (1 : 88)

1.9 X 10 -4 (1 : 1515)

r, r a d i a l p o s i t i o n , as m e a s u r e d f r o m the centre o f t h e t u r n t a b l e .

216 Mutagenicity assays The spiral test differs from the standard procedures of the Salmonella/microsome test (Ames et al., 1975) only because chemicals are distributed by the spiral diluter instead of being incorporated into the top agar (plate-incorporation test) or placed at the centre of agar plates (spot test). The volumes of media and reagents were doubled in 140-mm plates, compared with 100-mm plates. $9 mix, containing liver $9 fractions (adjusted to a protein concentration of 30 mg/ml) from rats treated i.p. with Aroclor 1254, was used as metabolic system. Solutions of compounds were dispensed either on the surface of top agar (method A) or of the minimal-glucose agar medium, before the addition of molten top agar embedding bacteria and eventually $9 mix (method B) (see Results and Discussion). The evaluation of results was usually carried out after 48 h of incubation at 37°C. Assays with repair-deficient or -proficient E. coli DNA-repair assays were carried out in parallel with strains WP2, CM871 and TM1080 of E. coli, by distributing compounds with the aid of the spiral plater (method A), as described for mutagenicity assays with Salmonella strains. The only difference was that tryptophan (20 pg/ml), instead of traces of histidine-biotin, was added to the top agar. The diameter of the zone of inhibition of bacterial growth was measured after 24--48 h at 37°C. Results and discussion

Comparison with the spot test Mutagens that failed to be detected in the spot test, owing to their lack of diffusibility, such as benz [a ] anthracene and benzo[a]pyrene, were conversely clearly positive in the spiral test, with both methods. This provides evidence that, although these compounds are not incorporated into the top agar, they can easily interact with activating enzymes and with bacteria embedded in the thin layer of soft agar. Fig. 2 gives an example of the appearance of the mutagenic response induced by benzo[a]pyrene in the spiral test, compared with controls of the same bacterial strain (Fig. 1). Considerable advantages over the spot test were also recorded with all the diffusible chemicals tested. For example, the gradient of concentrations produced by the spiral diluter is much wider and more gradual, and the active concentrations of mutagens are distributed over a larger area of the agar plate. This results in a better and progressive distinction between toxic and mutagenic effects and in a greater sensitivity of the spiral test. Other examples of positive results obtained with well-known mutagens are presented in Fig. 3, showing the very intense mutagenic response to 2-aminofluorene (cumulative dose of 40 ~g per plate) and in Fig. 4, giving an idea of the tremendous mutagenic potency of aflatoxin B1 (0.4 ~g). The spiral test also detected very weak mutagens, such as mebanazine, as well as borderline mutagens, such as perylene, 1,1-dimethylhydrazine and procarbazine (see under Interpretation of results). Fig. 5 refers to mebanazine, which had been previously shown in this laboratory to be negative in the spot

217

Fig. 1. A 140-mm control plate. A cumulative volume o f 1 0 0 p~l DMSO was dispensed b y t h e spiral di l ut e r o n t o t h e s u r f a c e o f t h e minimal-glucose agar m e d i u m ( m e t h o d B).

test and weakly positive in the plate test (Parodi et al., 1981). Trimethyl phosphate, a liquid compound positive in the plate test only when undiluted or diluted 1 : 2 (i.e. 120 and 60 mg per plate, respectively), with an extremely low potency (0.0003 revertants per ~mole) (De Flora, 1981) and which produced a poor cluster of revertants in the spot test, was convincingly positive in the spiral test.

Comparison with the plate-incorporation test Several assays were carried out to compare the efficiency and the sensitivity of the spiral test (methods A and B) with the plate-incorporation test. With this aim, serial 2-fold or 4-fold d ~ t i o n s of known mutagens were assayed in parallel with these methods and t~le resulting dose--response curves were drawn by relating the amounts of compound to the total number of revertants scored in the corresponding plate. 2 examples are reported in Fig. 6: a water-soluble, diffusible and directly acting mutagen (sodium azide) and a water-insoluble,

218

Fig. 2. Mutagenic r e s p o n s e i n d u c e d by b e n z o [ a ] p y r e n e (4 #g in a cumulative volume of 100/~1 DMSO) i n the spiral t e s t , u n d e r t h e s a m e c o n d i t i o n s i n d i c a t e d in t h e l e g e n d t o Fig. 1.

non
219

Fig. 3. M u t a g e n i c r e s p o n s e i n d u c e d b y 2-aminofluorene (40 #g in a cumulative volume of I 0 0 #I DMSO) in the spiral t e s t , u n d e r t h e s a m e c o n d i t i o n s as i n d i c a t e d in t h e l e g e n d to Fig. 1.

od B) and the plate test (Fig. 6) provided evidence that the former procedure was clearly the more efficient when high concentrations of mutagens were tested. This presumably depended on the circumstance that, in the case o f toxic compounds (e.g. azide), killing of bacteria affects only the internal area of the plate (unless at extremely high doses), instead of affecting the whole culture area. The same explanation might also apply to mutagens reaching a plateau in the plate test (e.g. benzo[a]pyrene), probably depending on shortage of activating enzymes at high doses. In general, the plate test gave a slightly higher n u m b e r of revertants at intermediate doses, while the spiral test (method B) was somewhat more sensitive at low doses of mutagens (Fig. 6). The different distribution of compounds over the dish seems to justify the above profiles of dose--response curves. In fact, although the cumulative doses of compounds dispensed were equal in the plate test and in the spiral test, in the latter test their concentrations were greater in

220

F i g . 4. M u t a g e n i c r e s p o n s e i n d u c e d b y a f l a t o x i n B1 ( 0 . 4 ~ g in a c u m u l a t i v e v o l u m e o f 1 0 0 ~1 D M S O ) i n t h e s p i r a l t e s t , u n d e r t h e s a m e c o n d i t i o n s as i n d i c a t e d i n the legend to Fig. 1.

the central areas of plates. For instance, incorporation of I mg of a c o m p o u n d into the t o p agar (plate test) resulted in a mean concentration of 65 X 10 -3 ~g/ mm 2 in a 140-ram dish. By distributing the same solution with the aid of the spiral distributor with the 100-gl dispensing mode (i.e. 10 ~g c o m p o u n d per #l solution), its concentration was 2.8/~g/mm 2 at the starting position and 1.9 X 10 -3 ~g/mm: at the end of the spiral track, as calculated from the data presented in Table 1. By using the 39-#1 dispensing m o d e (i.e. 25.6 gg c o m p o u n d per #l solution), the corresponding concentrations were 2.0 and 2.2 X 10 -3 /~g/mm 2, respectively. Obviously, it is likely that diffusion of chemicals in the spaces between adjacent tracks tends to decrease the above concentration values per unit area. Moreover, diffusible c o m p o u n d s tend to spread from the inner track to the centre of plates and from the outer track (lift-off position of the dispensing s t y l u s ) t o the periphery of the culture area. Even SQ, the gradient

Fig. 5. M u t a g e n i c r e s p o n s e i n d u c e d b y m e b a n a z i n e (1 m g i n a c u m u l a t i v e v o l u m e o f 9 2 #I D M S O ) i n t h e s p i r a l t e s t ( m e t h o d A ) , p e r f o r m e d i n 1 0 0 - r a m p l a t e s . T h e p l a t e is d i v i d e d i n t o 4 c o n c e n t r i c a r e a s ( A - - D ) , as d e s c r i b e d i n t h e t e x t .

~000r

9000I 80001 Na azide

/\

TA1535 $9"

~

7000I

Benzo(a)pl/rene TAI00 $9+

i ~500[

/

/

/O~o

6000~

[ i 5000

i

1~'~&~A

/ /

/o--~

O15°°h5ool l OO ":Ii'°,',"~'~

300( 2000

1000 0

1

4

16

64

01

.

.

o 256 Amount per plate (~g)

.

.

I

.

.

.

.

4

.

.

16

64

,

,

255

Fig. 6. D o s e - - r e s p o n s e c u r v e s o b t a i n e d w i t h s o d i u m a z i d e a n d b e n z o [ a ] p y r e n e ( 1 0 0 #I o f s o l u t i o n s p e r p l a t e ) i n t h e p l a t e - i n c o r p o r a t i o n t e s t (~) o r i n t h e s p i r a l t e s t , p e r f o r m e d w i t h e i t h e r m e t h o d A (o) o r B ( e ) . All t h e a s s a y s w e r e c a r r i e d o u t i n d u p l i c a t e in 1 4 0 - r a m p l a t e s . C o n f i d e n c e l i m i t s a r e n o t r e p r e s e n t e d , for the sake of graphic clarity.

222 of concentrations per plate is still more pronounced than expected on the basis of the values presented in Table 1. D i s t r i b u t i o n o f r e v e r t a n t s in the spiral t e s t

The distribution patterns of revertants in the spiral test can be checked by expressing the mutagenic response as number of revertants per unit area of the plate (e.g. per sq. cm) rather than of total revertants per plate. With this aim, 4 concentric areas (A--D) were considered in 90/100-ram plates (see for instance Fig. 5) and 5 areas (A--E) in 140/150-mm plates (Figs. 7 and 8). The inner area (A) has a radius of 12 mm, i.e. it corresponds to the starting position of the dispensing stylus. Similarly, the concentric annular areas have a thickness of 12 mm, with the exception of the external area, whose thickness depends on the size of plates. Fig. 7 shows the relationship between the amounts of sodium azide or benzo[a]pyrene dispensed by the spiral diluter and the density of revertants in the concentric areas A--E. The maximal density of revertants tended to shift from the centre to the periphery of plates by increasing the cumulative amounts of both these compounds. Some differences between azide and benzo-

6O

Na

88

/t" //~,~\ \ \

azide

it, \' \

TA 153S $ 9 -

8O 45

Ben zo(a)pi;rene

,/ ,, ,,\ 7 ,, !;

4O

//

E t~

$9÷

^

i/

/

i

/ ,/ //

28

..," ii /"/ ; /I i

/

!I/ , i / / / i '1 /

,

/

/

/

15

/

,-' I

/ / /

// / /

/...

I ##

5

/

\

.i/

/

/ /i' / /"

10

/ !

1 i I

i

/

/•

/

/

/

I

I

/

I

I

I

/

i

I

i I

/ /

"~

;

/ / ,! '

,/ \.~\ / ,' - ~--i...~

/

i

i

I

/

/

/

/

/

;

.

.I

/

/

/

I /"

",,

/

,.,\ / \ \ , '\ ~ '~ \\ ,I ", \

./ // I/ i / / /

..,' !

I/,"/

i/

/

//\\ /-.

30

@ > @

TA ZOO

38

U"

~"

7!

~C" i /'// ',,"--~U---':/

•/

........ /

i

/ ~

'

i

'

4

'

~

'

~

' ~

Amount

o

per

1

4

16

64

256

plate (~,g)

Fig. 7. D e n s i t y o f r e v e r t a n t s ( m e a n o f d u p l i c a t e s ) in 5 c o n c e n t l d c areas ( A - - E ) o f 1 4 0 - m m plates. S o d i u m a z i d e a n d b e n z o [ a ] p y r e n e w e r e a s s a y e d at various c o n c e n t r a t i o n s in t h e spiral t e s t ( m e t h o d A, 100-#1 c u m u l a t i v e v o l u m e s o f s a m p l e s ) . C o n f i d e n c e l i m i t s are n o t r e p r e s e n t e d , for t h e s a k e of-graphic clarity.

223

Fig. 8. M u t a g e n i c r e s p o n s e i n d u c e d b y s o d i u m d i c h r o m a t e ( 3 0 # g i n a c u m u l a t i v e v o l u m e o f 3 9 #1 w a t e r ) i n t h e s p i r a l t e s t , u n d e r t h e s a m e c o n d i t i o n s as i n d i c a t e d in the legend to Fig. 1, e i t h e r i n t h e absence ( S g - - ) o r t h e presence (S9+) o f S9 m i x . P l a t e s ( 1 4 0 r a m ) w e r e d i v i d e d i n t o 5 c o n c e n t r i c axeas ( A - - E ) , as described in the t e x t .

[a]pyrene can be ascribed to their distinctive mutagenicity profiles (Fig. 6) and mainly to their different diffusibility patterns. For instance, the density of revertants in the central area (A), where no sample was directly distributed b y the spiral diluter, was elevated in the case of azide and n o t significantly increased over controls in the case of b e n z o [ a ] p y r e n e . Testing of c o m p o u n d s in duplicate or triplicate plates provided evidence for a quite satisfactory homogeneity of results.

Interpretation of results The spiral test is suitable for both a qualitative and a semiquantitative evaluation of results. In the great majority of plates the visual appearance left no d o u b t a b o u t the positivity or negativity of results. Therefore, as in the spot test, an evident visual increase of revertants over controls and the patterns of their distribution in plates is sufficient to provide a qualitative appraisal of the activity of test compounds. For a semiquantitative assessment of results, the total number of revertants can be counted over the whole plate and be compared with controls. Criteria for positivity are those suggested for the plate test (Ames et al., 1975; de Serres and Shelby, 1979), being the dose--response effects appreciated in a single plate.

224 For extremely weak mutagens, a further increase of sensitivity in the evaluation of results can be achieved by separately counting the number of revertants in concentric areas, as specified in the previous paragraph, and by comparing their density with the corresponding areas in control plates. Such a procedure allowed the detection, in the spiral test, of mutagens which in this laboratory had been found to be borderline positive in the plate test, such as perylene, 1,1dimethylhydrazine and procarbazine (De Flora, 1981; Parodi et al., 1981). Counting of colonies can be done either manually, with a specially designed viewer grid with radial lines and concentric circles supplied with the spiral plater, or by an automatic laser colony counter, which is commercially available. Because each area marked on the viewer grid corresponds to a known volume of sample, which can be inferred from suitable graphs supplied with the instrument, it is even possible, at least in method A, to determine the amount of chemical deposited in the area with the greatest density of revertants. Obviously, this is a rather sophisticated approach, which can be faced for a more complete evaluation of mutagenicity data, e.g. for the assessment of the mutagenic potency.

Evaluation of metabolic effects The procedure of dispensing chemicals by the spiral diluter appears to be quite compatible with their activation by metabolic systems, such as $9 mix, as shown with a number of test compounds (e.g. aflatoxin B1, benzo[a]pyrene, benz[a]anthracene and perylene). Furthermore, the spiral test was also efficient in detecting the decrease of mutagenicity afforded by liver homogenates with some direct-acting mutagens included in the experimental protocol, such as sodium azide, the ICR compounds and sodium dichromate, that we have previously demonstrated to be deactivable by $9 mix in the plate test (De Flora, 1978, 1981; Petrilli and De Flora, 1978; De Flora et al., 1979; De Flora and De Flora, 1981). An example concerning sodium dichromate is shown in Fig. 8. In the absence of $9 mix (Fig. 8A), a central zone of bacterial inhibition was surrounded by a ring of induced revertants, whereas in the presence of $9 mix (Fig. 8B) no toxic effect was detectable, and clustering of revertants, compared with controls, occurred only at the centre of plates. General evaluation of methods for the performance of the spiral test The results obtained and the experience acquired in the use of the spiral diluter for the performance of the Ames test indicate that both methods A and B have some advantages and disadvantages, which should be taken into account. Method A has the advantage that chemicals distributed on the surface of freshly solidified top agar can readily interact with bacteria and metabolic systems, thus keeping the accuracy of the spiral gradients. For the same reason, heatJabfle or unstable compounds can be successfully tested. The problem of avoiding contamination among plates is solved by simple mechanical rinsing of the dispensing stylus, which is done by aspirating DMSO and sterile water. This is the regular procedure for sterilizing the stylus in the original application of the spiral plater, i.e. when bacterial suspensions are distributed. The major disadvantage of method A, as already discussed, is t h a t a more

225 concentrated top agar is needed to prevent formation of grooves and scratching of the agar surface by the dispensing stylus. This change involved some technical difficulties, especially over-heating of the molten top agar before incorporation of bacteria and S9 mix, which was presumably responsible for the lower sensitivity of method A compared both with method B and with the plateincorporation test. In method B the dispensing stylus lies on a sterile medium, thus avoiding problems of sterilizing the stylus when the same chemical solution is distributed over several test plates. The main advantage is that this method is at least as sensitive as the plate-incorporation test, such a goal being achieved by using a single plate instead of serial plates. For instance, in 140/150-mm plates the spiral track covers a range of concentration corresponding to about 11 serial 2-fold dilutions in separate plates. A disadvantage of method B is that several chemicals tend to diffuse during the drying of the spiral track, before addition of top agar, which affects the accuracy of spiral gradients. However, such an inconvenience does not appear to be important for routine purposes, i.e.unless information on the exact concentration of chemicals in each point of the spiral track is needed. Moreover, diffusion of chemicals mainly occurs at the centre and at the periphery of plates, i.e. inside and outside the spiral track, while in the remaining areas of plates diffusion is likely to be limited to the spaces between adjacent tracks. Another limitation of method A is that labile compounds tend to be inactivated before the application of the bacteria. A n example of such a problem was provided by ~-propiolactone, whose activity was greatly reduced after overnight drying of the spiral track at room temperature. Conversely, the same compound was almost as active as in the plate-incorporation test when assayed with method A. Therefore, attention should be paid when unstable chemicals are tested with this method. Assays with E. coli strains A number of assays were carried out, with repair-proficient (WP2) or -deficient (CM871 and TM1080) strains of E. coli, on MMS, sodium dichromate, phenelzine and niaiamide as test compounds, in the absence of $9 mix. All 4 compounds were positive in repair tests, though with a variable efficiency and a different spectrum of sensitivity of bacterial tester strains. In virtue of the wider and more regular formation of the concentration gradient obtained with the spiral test, the differences in the size of inhibition zones between WP2, CM871 and TM1080 plates were considerably more pronounced than in parallel assays with the conventional spot test. An example of the sharp difference of the toxic activity of MMS towards 2 tester strains is reported in Fig. 9. On the other hand, application of compounds with the spiral diluter sometimes uncovered some problems in the evaluation of results. For example, owing to the more progressive shift from non-toxic to toxic concentrations, the edge of the inhibition zone was less clear,cut than in the spot test. Therefore, additional work seems to be desirable to ascertain the convenience of such an application of the spiral diluter. In a limited number of experiments, encouraging results (Table 2) were obtained by equipping the instrument with an additional cam, which had been designed by the manufacturers to

226

Fig. 9. Z o n e o f i n h i b i t i o n g r o w t h d e t e r m i n e d b y m e t h y l m e t h a n e s u l p h o n a t e ( 2 0 m g in a c u m u l a t i v e volu m e o f 3 9 ~l) d i s p e n s e d b y t h e spiral d i l u t e r o n t h e t o p agar o f 1 4 0 - m m p l a t e s i n c o r p o r a t i n g strains WP2 o r T M 1 0 8 0 o f E. c o i l

distribute a constant rather than a variable volume of sample. In this w a y bacterial suspensions can be homogeneously distributed along a spiral track just before test chemicals are dispensed by the "variable volume" cam. Therefore, no top agar is needed and, because bacteria grow in concentric tracks, the inhibition zone is more clearly defined. Furthermore, although no specific experiment was carried out in this laboratory, it is likely that, following its original application, the spiral diluter might be successfully used in bacterial D N A damage assays, as well as in any other short-term microbial system involving measurements of the density of test organisms (e.g. survivors or revertants), without the preparation of serial dilutions of test samples or multiple plates.

TABLE 2 D I A M E T E R ( m m ) O F T H E Z O N E O F I N H I B I T I O N ( m + 2s O F T R I P L I C A T E S ) O F B A C T E R I A L G R O W T H ( S T R A I N S WP2, C M 8 7 1 A N D T M 1 0 8 0 O F E. coli) I N D U C E D BY 4 C H E M I C A L S A S S A Y E D , I N E Q U A L A M O U N T S , E I T H E R BY T H E S P I R A L T E S T O R T H E S P O T T E S T I N 1 0 0 - m m P L A T E S Compound (cumulative amount)

Spiral t e s t a

Spot test b

WP2

CM871

TMI080

WP2

CM871

TM1080

Methyl methanes u l p h o n a t e ( 1 . 5 rag)

24.2 + 0.6

8 0 . 8 + 1.5

85.2 + 2.5

19.3 + 0.6

37.8 + 1.5

43.0 ± 2.0

Sodium dichzomate ( 1 0 ~g)

2 7 . 0 ± 2.0

58.3 + 2.5

6 4 . 3 + 4.2

2 0 . 7 + 1.1

34.2 + 0.6

3 8 . 8 + 1.1

P h e n e l z i n e (1 m g )

2 5 . 5 + 1.7

45.2 + 4.5

3 7 . 8 + 1.5

1 0 . 0 + 2.0

1 8 . 2 + 1.5

1 4 . 8 + 1.5

N i a i a m i d e (8 m g )

24.3 + 3.0

5 1 . 2 + 3.5

2 4 . 2 ± 1.5

1 4 . 7 + 1.1

2 0 . 8 + 2.5

1 4 . 0 + 2.0

a P e r f o r m e d b y distributing b a c t e r i a w i t h t h e u n i f o r m c a m and c h e m i c a l s w i t h t h e variable c a m o f t h e spiral diluter. b P e r f o r m e d b y p o u r i n g c h e m i c a l s in w e l l s at t h e c e n t r e o f agar plates c o n t a i n i n g bacteria.

227

Note added in proof Studies on the application of the spiral plater to the Ames test are also in progress at the University of Denver. Drs. N. Couse and J. King presented a poster on this subject at the Annual Meeting of the American Environmental Mutagen Society (Nashville, TN, 16--20 March 1980).

Acknowledgements This investigation was supported by Italian CNR (Progetto Finalizzato Controllo della Crescita Neoplastica). I thank Drs. R. Ligugnana and S. Centenaro (Pool Bioanalysis Italiana) for making available the spiral-plating instrument throughout the periods of experiments. The valuable assistance of Drs. C. BenniceUi, P. Zanacchi and A. Camoirano is gratefully acknowledged.

References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for de t e c t i ng carcinogens and mut a ge ns w i t h the S a l m o n e l i a / m a m m a l l a n - m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 3 1 , 3 4 7 - - 3 6 4 . Briner, W.W., J,A. Wunder, D.W. Blair, J.J. Parran, T.L. Blaney and W.E. J o r d a n (1978) Use of spixal bacterial plating and laser colony counting t e c h n i q u e s in studies of the microbial ecology of man, in: A.N. Sharpe and D.S. Claxk (Eds.), Mechanizing Microbiology, Thomas, Springfield, IL, Publ. No. 1010, American Lecture Series, pp. 154--169. De Flora, S. (1978) Metabolic deactivation of mutagens in t he Salmonella-microsome test, Nature (London), 2 7 1 , 4 5 5 - - 4 5 6 . De Flora S. (1981) S t u d y of 106 organic and inorganic c o m p o u n d s in the S a l m o n e l l a / m i c r o s o m e test, Carcinogenesis, 2, No. 3. De Flora, S., and A. De Flora (1981) Variation of the t~ameshift activity of a m u t a g e n (ICR-191) following n i t r o s a t i o n in h u m a n gastric juice, Cancer Lett., 1 1 , 1 8 5 - - 1 8 9 . De Flora, S., R. Coppola, P. Zanacchi and C. Bennicelli (1979) Reversal of s odi um azide m u t a g e n i c i t y by liver preparations and by gastric juice, M u t a t i o n Res., 61, 389--394. De Serres, F.J., and M.D. Shelby (1979) The Salmonella m u t a g e n i c i t y assay: r e c o m m e n d a t i o n s , Science, 203, 563--565. Gilchrist J.E., J.E. Campbell, C.B, Donnelly, J.T. Peeler and J.M. Delaney (1973) Spiral plate m e t h o d for bacterial d e t e r m i n a t i o n , Appl. Microbiol., 2 5 , 2 4 4 - - 2 5 2 . Jarvis, B., V.H. Lach and J.M. Wood (1977) Evaluation of t h e spiral plate m a k e r for the e n u m e r a t i o n of microorganisms in foods, J. Appl. Bacteriol., 43, 149--157. Kramer, J.M., M. Kendall and R.J. Gilbert (1979) Evaluati on of the spiral plate and laser c ount i ng technique for the e n u m e r a t i o n of bacteria in foods, Eux. J. AppL Microbiol. Bioteclmol., 6, 289--299. McMahon, R.E., J.C. Cline and C.Z. T h o m p s o n (1979) Assay of 855 test chemicals in t e n tester strains using a new m o d i f i c a t i o n of the Ames test for bacterial mutagens, Cancer Res., 39, 682--693. Parodi, S., S. de Flora, M. Cavanna, A. Pino, L. Robbiano, C. Bermicelli and G. Brambilla (1981) DNA damaging activity in vivo and bacterial m u t a g e n i c i t y of sixteen hydra z i ne derivatives as related quantitatively to th eir carcinogenicity, Cancer ltes., 41, No. 4. PetrUli, F.L., and S. De Flora (1978) Metabolic deactivation of he xa va l e nt c h r o m i u m mut a ge ni c i t y, Mut a t i o n Res., 54, 139--147. Venturini, S., and C. Monti-Bragadin (1978) I m p r o v e m e n t in the sensitivity of DNA polymerase I-deficient Escherichia coli for detecting mutagens and carcinogens, Appl. Environ. Microbiol., 36, 794-797.