- Email: [email protected]
A technique for the effective enrichment and isolation of Bacillus thuringiensis
FEMS Microbiology Letters 142 (1996) 173-177
A technique for the effective enrichment and isolation of Bacillus thuringiensis Clare Johnson, Alistair H. Bishop * School
ofChemical and Life Sciences. University of Greenwich, Wellingron Street, London SE18 6PF, UK Received 19 April 1996; revised 21 June 1996; accepted 28 June 1996
Abstract An isolation method is described which results in the enrichment of Bacillus thuringiensis compared to other members of the genus BaciZIus. This method was compared to previously published methods claimed to have the same effect. We show that our method produced significantly more colonies of B. thuringiensis out of those with a ‘presumptive-positive’ morphology than the other methods tested. The diversity of the strains isolated by our method was investigated. The variability of the strains isolated suggests that the method does not preferentially select one strain or sub-group of B. thuringiensis over others. Keywords: Selective isolation; Bacillus thuringiensis; Penicillin
1. Intruduction Bacillus
thuringiensis
is without
doubt the most successful agent of microbial control discovered to date. It has enjoyed wide-spread use over many years [l]. One of the drawbacks to its greater usage is a narrowness in the spectrum of invertebrate species which many isolates can kill. Much work has been carried out at a molecular level to try to remedy this situation [2-4]. A problem associated with this approach, even if successful, is the approval of regulatory bodies for the release of genetically engineered organisms [5,6]. An alternative and longer-established approach is to isolate naturally existing strains of B. thuringien-
* Corresponding author. Tel.: +44 (181) 331 8427; Fax: +44 (181) 331 8305; E-mail: [email protected]
sis. This has, over the years, proved very successful with, for example, the discovery of strains toxic to the larvae of some members of the genera Diptera [7] and Coleoptera [8]. Such discoveries have recently resulted in a surprisingly diverse and comprehensive list of invertebrate species which are susceptible to newly isolated strains [l]. The search for strains of B. thuringiensis with novel insecticidal activity has a much higher success rate compared to screening chemical compounds with similar properties [9]. The isolation of B. thuringiensis from environmental samples can, however, be a very laborious and time-consuming procedure, requiring the microscopic examination of many colonies presumed to be B. thuringiensis based upon colony morphology. Methods which increase the proportion of colonies which really are B. thuringiensis amongst
valuable
time
those and
with
a similar
allow
a more
appearance efficient
037%1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00261-3
save
screening
174
C. Johnson, A.H. Bishop1 FEMS
of environmental samples for novel strains. We report here a simple technique which, when compared to published methods for the selection of B. thuringiensis, results in a higher proportion of such colonies out of the presumptive positives.
2. Materials and methods 2.1. Origin of soil samples Soil samples were collected from two independent locations to provide contrasting soil types and varying microbial populations. Soil A was a humus-rich soil of pH 3, collected at a reclaimed landfill site in Northamptonshire, UK. Soil B was a sandy soil of pH 7, collected from land adjacent to an intensive livestock unit in Warwickshire, UK. Both locations had large resident populations of blowflies and houseflies. 2.2. Isolation procedures About 0.25 g (dry weight) of environmental sample was placed in test tubes containing 2 ml of liquid growth medium: this was nutrient broth (Oxoid) supplemented with CCY salts [IO], added at 1 m 1-l to aid sporulation and 20 IU ml-i of penicillin G (Sigma). The tubes were heat-shocked at 70°C for 10 min in a water-bath. The contents of each tube were poured into 250 ml flasks containing 50 ml of liquid growth medium. The flasks were incubated at 30°C at 200 rpm until sporulation was complete. The particulate matter was recovered by centrifugation for 1 h at 3600 rpm in an MSE Mistral 3OOOEcentrifuge. The pellets were resuspended in 2 ml aliquots of liquid growth medium and put through a second cycle of the procedure above. The pelleted matter from the second centrifugation step was resuspended in 2 ml of broth. Serial dilutions of these suspensions were plated out onto nutrient agar (Oxoid) containing CCY salts and supplemented with 20 IU ml-’ of penicillin G. The cultures were incubated at 30°C until sporulation was complete. Individual beige colonies with a matt texture were examined microscopically for the characteristic spores and crystals of B. thuringiensis [l 11. The efficiency of the above method was compared
Microbiology Lepers 142 (1996) 173-177
with the previously published methods for the isolation of B. thuringiensis described by Donovan et al. [8], Meadows et al. [12], Carrozzi et al. [13] and Ohba and Aizawa [14]. A further comparison for all of these methods was provided by simply homogenising soil samples in sterile distilled water followed by plating out onto nutrient agar supplemented as before with CCY salts. To avoid bias plates were identified by random numbers allocated by a person not involved in the study. After incubation at 30°C for 48 h, or until sporulation was complete, the resultant colonies were examined for colour and morphology. Those which were beige with a matt appearance were considered to be presumptive B. thuringiensis colonies. Sixty of these from each procedure were selected at random and examined microscopically for the characteristic presence of a spore with a crystalline body within the spore mother cell wall [ll]. Such colonies were classified as B. thuringiensis strains, i.e. positives; those where only spores were visible were regarded as non-B. thuringiensis strains, i.e. negatives. This investigation was repeated twice. 2.3. Assessment of strain variance To assess the diversity of the B. thuringiensis strains isolated using the penicillin cycling method, a total of 450 isolates from five separate isolation procedures from soil B were assayed for activity against Musca domestica. 2.3. I. Bacterial strains B. thuringiensis strain 4412 was obtained from Dr. Peter Luthy and B. thuringiensis subspecies tenebrionis was obtained from DSM, Germany.
2.3.2. Growth of bacteria for bioassay Isolates of B. thuringiensis were grown in the liquid growth medium at 30°C and 200 rpm until sporulation was complete. Cultures were centrifuged for 1 h at 4000 rpm in a MSE Mistral 3000E centrifuge and the pellets washed twice with deionised water. They were finally resuspended in deionised water to give a final volume which represented a 50-fold concentration with respect to the initial culture. These preparations were referred to as ‘50-fold concentrates’.
C Johnson, A.H. Bishop1FEMS Microbiology Letters I42 (1996) 173-l 77
2.3.3.
Bioassay procedure
80
The activity of the 50-fold concentrates of spores and crystals against neonate larvae of M. domestica was determined as described by Levinson et al. [15] with the following changes: B. thuringiensis subsp. tenebrionis was used as a negative control, at a dose equal to the highest dose used in the test range. B. thuringiensis strain 4412 was used as a positive control, at a dose of 40 ~1 ml-‘. All doses were replicated 5 times using 20 neonate larvae per sample and assays were repeated at least twice.
3.1.
T--
50
s i iii 3 2 t ‘C 0 3 2
3. Results
175
40
30
20
IO
Comparison of isolation methods 0
It is apparent from these results that the rate of isolation of B. thuringiensis varies considerably between the procedures assessed (Table 1 and Fig. 1). For both soil types the penicillin cycling method described in this paper was found to be the most efficient. Statistical analysis using analysis of variance at the 0.05 significance level shows the difference between procedures to be highly significant (P< 0.001) for each soil type. The interaction between soil type and isolation procedure was also significant (P = 0.003) indicating that some procedures may be more suited to a particular soil type than are others. Using the method of Carrozzi et al. [13] the isolation rate from soil B was noticeably more efficient than with the other, previously published methods, whilst with soil A isolation rates were only equivalent to those of the other published methods. In accordance with this method the pH of the Table 1 Rate of isolation
of B. thuringiensis with each procedure
Procedure
Meadows et al. [12] Donovan et al. [S] Carrozzi et al. [13] Ohba and Aizawa [14] Homogenate Penicillin Figures in parentheses
Mean number 60 presumptive
of B.t. colonies colonies
Soil A
Soil B
6.6 5.0 5.0 2.7 1.0 19.7
4.7 9.5 14.0 6.0 1.0 34.7
(2.1) (1.7) (1.0) (4.6) (0.0) (7.4)
are standard
assessed
deviations
from
(3.8) (5.6) (3.6) (3.6) (1.0) (5.5)
of three replicates.
1
2
3
4
5
Isolation Method
I 6
Fig. 1. Comparison of the rate of isolation of B. thuringiensis colonies using six isolation methods: 1 Meadows et al. [12]; 2 Donovan et al. [S]; 3 Carozzi et al. [13]; 4 Ohba and Aizawa [14]; 5 Homogenisation; 6 Penicillin cycling. 0, Soil A: a humus-rich soil of pH 3 collected from a reclaimed landfill site. n, Soil B: a sandy soil of pH 7 collected from land adjacent to an intensive livestock unit. The percentage of E. thuringiensis colonies isolated is the percentage of colonies which were identified by microscopic examination as B. thuringiensis out of 60 randomly assessed presumptive colonies.
incubation medium was readjusted to pH 6.8 after autoclaving. After incubation with soil A however, it was found that the pH had dropped to that of the soil. As the method appears to be reliant upon an optimum pH of 6.8 this may explain the comparatively poor performance of this method for soil A. When compared to the next most efficient isolation method for each soil (i.e. for soil A the method of Meadows et al. [12]; for soil B the method of Carozzi et al. [13]) the increase in the rate of isolation for our method was still significant at the 0.05 significance level (P = 0.04, P = 0.005). 3.2. Investigation into the optimisation of the penicillin cycling method
Repeat cycles of the procedure do result in significantly more B. thuringiensis colonies being recovered (Fig. 2). The rate of increase, however, declines after
C, Johnson,
176
A. H. Bishop I FEMS
Microbiology
Letters
142 (1996)
173-I 77
isolation runs, again suggesting that the method does not benefit one strain or sub-group of organisms over the others.
1 cycle 3
cycle 2
Fig. 2. Effect of increasing numbers of penicillin cycles on the efficiency of isolating Bacillus thuringiensis. 0. Soil A: a humusrich soil of pH 3 collected from a reclaimed landfill site. n , Soil B: a sandy soil of pH 7 collected from land adjacent to an intensive livestock unit. The percentage of B. thuringiensis colonies isolated is the percentage of colonies which were identified by microscopic examination as B. thuringiensis out of 60 randomly assessed presumptive colonies.
the second cycle, although it is still significant. It is likely that any increase in isolation rate is offset by the increase in work and delay in isolation as a consequence of undertaking a third penicillin cycle. 3.3. Diversity of strains of B. thuringiensis isolated Of the 450 isolates screened for activity against M. domestica larvae, 56 strains (12%) exhibited activity (Table 2). The dose required to produce an LDss effect varied between the isolates, thus verifying the diversity in the strains isolated. The percentage of strains showing activity at each level varied between
Table 2 Bioactivity
of isolated strains of B. thuringiensis
Activity against M. domestica
Isolation run (90 isolates)
High” Medium” Negligible’
3 (3%) 6 (6%) 81 (90%)
I
4. Discussion The isolation method reported here relies upon the apparent intrinsic resistance of B. thuringiensis to the antibiotic penicillin. Whilst it is not known how wide-spread this characteristic is within the species, all strains so far tested have proved resistant (A.H. Bishop, unpublished results). The ecology of B. thurirzgiensis is debatable [14,16,17]. The ability to grow in the presence of p-lactam antibiotics would, however. presumably be of value to an organism which replicated in soils where a variety of organisms producing penicillin would naturally be found. The effectiveness of this method is clearly demonstrated when compared to other published isolation methods. A significant improvement in the rate of isolation was obtained when using the penicillin cycling method, regardless of soil type. In contrast, the method of Carozzi et al. [13] was highly dependent on soil type, favouring soil B over soil A. An obvious drawback with the penicillin cycling method is the length of time involved between commencing the isolation and obtaining any isolates. In our opinion, this limitation is far outweighed by the reduction of input by skilled personnel required for the laborious task of microscopic examination of potential colonies. When comparing the rate of isolation following each cycle of the procedure, the benefits of increased selectivity following two or more cycles must be balanced against the increased delay in obtaining the isolates. In our experience a second cycle is worthwhile in terms of the improved isolation rate but a
against larvae of M. dumrstira Isolation run 2 (90 isolates)
Isolation run 3 (90 isolates)
Isolation run 4 (90 isolates)
Isolation run 5 (90 isolates)
4 (4%) 0 86 (95%)
0 0 90 (1OO’X)
22 (24%) 17 (18%) 51 (56%)
0 11 (12%) 79 (87%)
*LD,oclS ~1 of 50-fold concentrate spores and crystals per ml of larval diet. bLD~” > 15 ~1 of SO-fold concentrate spores and crystals per ml of larval diet. ‘No toxicity observed up to a dose of 120 ~1 of 50-fold concentrate spores and crystals per ml of larval diet.
C. Johnson, A.H. BishoplFEMS
Microbiology Letters 142 (1996) 173-I 77
third cycle was not sufficiently more effective to justify the increase in time taken. Increasing the number of cycles, furthermore, may start to select preferentially for some strains of B. thuringiensis over others, thus reducing the diversity of the strains eventually isolated. Any method for the isolation of micro-organisms will, without doubt, favour one group of organisms over another. The method reported here involves two phases of growth in a liquid medium, in the presence of penicillin, and, as such, it is possible that the culture could become dominated by one, highly suited strain. This would result in many or all of the colonies on the solid medium being of the same strain of B. thuringiensis. Results obtained from the bioassays show this not to be the case. Isolates obtained from an individual isolation run revealed large variations in the level of activity expressed against M. domestica. When comparing the activity range of isolates between isolation runs the number of isolates obtained within each activity range was variable. If the procedure were highly selective for a limited number of strains it would be likely that these strains would be repeatedly selected in subsequent isolation runs. Since this was not found the strain diversity of the final culture fluid would not appear to be noticeably affected by the procedure.
Acknowledgments This work was supported Fisheries Research Programme velopment Administration.
by the Post-harvest of the Overseas De-
References [I] Feitelson, J.S., Payne, J. and Kim, L. (1992) Bacillus thuringiensis: insects and beyond. Biotechnology 10, 271-275. [2] Bosch, D., Schipper, B., van der Kleij, H., de Maagd, R.A. and Stiekema, W.J. (1994) Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Biotechnology 12, 915-918. [3] Crickmore, N., Nicholls, C., Earp, D.J., Hodgman, T.C. and Ellar, D.J. (1990) The construction of strains of Bacillus fhu-
111
ringiensis strains expressing novel entomocidal Gendotoxin combinations. B&hem. J. 270, 133-I 36. [4] Porter, A.G., Davidson, E.W. and Liu, J.L. (1993) Mosquitotidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes. Microbial. Rev. 57, 838-861. [5] Pickup, R.W., Morgan, J.A.W., Winstanley, C. and Saunders, B. (1991) Implications for the release of genetically engineered organisms. J. Appl. Bacterial. 70, 19S3OS. [6] Faust, R.M. and Jayaraman, K. (1990) Current trends in the evaluation of the impact of deliberate release of microorganisms in the environment: a case study with a bioinsecticidal bacterium. In: Introduction of Genetically Modified Organisms into the Environment (Mooney, P.F. and Bernadi, G., Eds.), pp. 167-189. J. Wiley and Sons, Chichester. [7] Goldberg, L.H. and Margalit, J. (1977) A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Vranoraenia unguiculata, Culex univittatus, Aedes aegypti and C&x pipiens. Mosquito News. 37, 335358. 181Donovan, W.P., Gonzalez, J.M. Jr., Gilbert, M.P. and Dankocsik, C. (1988) Isolation and characterisation of EG2158, a new strain of BaciZlusthuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene. Mol. Gen. Genet. 58, 13441350. [91 Lambert, B. and Peferoen, M. (1992) Insecticidal promise of Bacillus thuringiensis. Facts and mysteries about a successful biopesticide. Bioscience 42, 112-122. [lOI Stewart, G.S.A.B., Johnstone, K., Hagelberg, E. and Ellar, D.J. (1981) Commitment of bacterial spores to germinate. Biochem. J. 198, 101-106. 1111 Bulla, L.A. Jr., Bechtel, D.B., Kramer, K.J., Shethna, Y.I., Aronson, A.I. and Fitz-James, P.C. (1980) Ultrastructure, physiology and biochemistry of Bacillus thuringiensis. Crit. Rev. Microbial. 8, 147-204. [W Meadows, M.P., Ellis, D.J., Butt, J., Jarrett, P. and Burges, H.D. (1992) Distribution, frequency and diversity of Bacillus thuringiensis in an animal feed mill. Appl. Environ. Microbial. 55, 2437-2442. u31 Carozzi, N.B., Kramer, V.C., Warren, G.W., Evola, S. and Koziel, M.G. (1991) Prediction of insecticidal activity of Bacillicsthuringiensis strains by polymerase chain reaction product profiles. Appl. Environ. Microbial. 57, 3057-3062. of Bacillus [I41 Ohba, M. and Aizawa, K. (1986) Distribution thuringiensis in soils of Japan. J. Invert. Pathol. 47, 277-282. 1151 Levinson, B.L., Kasyan, K.J., Chiu, S.S., Currier, T.C. and Gonzalez, J.M. (1990) Identification of B-exotoxin production, plasmids encoding g-exotoxin and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography. J. Bacterial. 172, 3172-3179. 1161 Martin, P.A.W. and Travers, R.S. (1989) Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl. Environ. Microbial. 55, 2437-2442. [I71 Smith, R.A. and Couche, G.A. (1991) The phylloplane as a source of Bacillus thuringiensis variants. Appl. Environ. Microbiol. 57, 311-315.
A technique for the effective enrichment and isolation of Bacillus thuringiensis Clare Johnson, Alistair H. Bishop * School
ofChemical and Life Sciences. University of Greenwich, Wellingron Street, London SE18 6PF, UK Received 19 April 1996; revised 21 June 1996; accepted 28 June 1996
Abstract An isolation method is described which results in the enrichment of Bacillus thuringiensis compared to other members of the genus BaciZIus. This method was compared to previously published methods claimed to have the same effect. We show that our method produced significantly more colonies of B. thuringiensis out of those with a ‘presumptive-positive’ morphology than the other methods tested. The diversity of the strains isolated by our method was investigated. The variability of the strains isolated suggests that the method does not preferentially select one strain or sub-group of B. thuringiensis over others. Keywords: Selective isolation; Bacillus thuringiensis; Penicillin
1. Intruduction Bacillus
thuringiensis
is without
doubt the most successful agent of microbial control discovered to date. It has enjoyed wide-spread use over many years [l]. One of the drawbacks to its greater usage is a narrowness in the spectrum of invertebrate species which many isolates can kill. Much work has been carried out at a molecular level to try to remedy this situation [2-4]. A problem associated with this approach, even if successful, is the approval of regulatory bodies for the release of genetically engineered organisms [5,6]. An alternative and longer-established approach is to isolate naturally existing strains of B. thuringien-
* Corresponding author. Tel.: +44 (181) 331 8427; Fax: +44 (181) 331 8305; E-mail: [email protected]
sis. This has, over the years, proved very successful with, for example, the discovery of strains toxic to the larvae of some members of the genera Diptera [7] and Coleoptera [8]. Such discoveries have recently resulted in a surprisingly diverse and comprehensive list of invertebrate species which are susceptible to newly isolated strains [l]. The search for strains of B. thuringiensis with novel insecticidal activity has a much higher success rate compared to screening chemical compounds with similar properties [9]. The isolation of B. thuringiensis from environmental samples can, however, be a very laborious and time-consuming procedure, requiring the microscopic examination of many colonies presumed to be B. thuringiensis based upon colony morphology. Methods which increase the proportion of colonies which really are B. thuringiensis amongst
valuable
time
those and
with
a similar
allow
a more
appearance efficient
037%1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00261-3
save
screening
174
C. Johnson, A.H. Bishop1 FEMS
of environmental samples for novel strains. We report here a simple technique which, when compared to published methods for the selection of B. thuringiensis, results in a higher proportion of such colonies out of the presumptive positives.
2. Materials and methods 2.1. Origin of soil samples Soil samples were collected from two independent locations to provide contrasting soil types and varying microbial populations. Soil A was a humus-rich soil of pH 3, collected at a reclaimed landfill site in Northamptonshire, UK. Soil B was a sandy soil of pH 7, collected from land adjacent to an intensive livestock unit in Warwickshire, UK. Both locations had large resident populations of blowflies and houseflies. 2.2. Isolation procedures About 0.25 g (dry weight) of environmental sample was placed in test tubes containing 2 ml of liquid growth medium: this was nutrient broth (Oxoid) supplemented with CCY salts [IO], added at 1 m 1-l to aid sporulation and 20 IU ml-i of penicillin G (Sigma). The tubes were heat-shocked at 70°C for 10 min in a water-bath. The contents of each tube were poured into 250 ml flasks containing 50 ml of liquid growth medium. The flasks were incubated at 30°C at 200 rpm until sporulation was complete. The particulate matter was recovered by centrifugation for 1 h at 3600 rpm in an MSE Mistral 3OOOEcentrifuge. The pellets were resuspended in 2 ml aliquots of liquid growth medium and put through a second cycle of the procedure above. The pelleted matter from the second centrifugation step was resuspended in 2 ml of broth. Serial dilutions of these suspensions were plated out onto nutrient agar (Oxoid) containing CCY salts and supplemented with 20 IU ml-’ of penicillin G. The cultures were incubated at 30°C until sporulation was complete. Individual beige colonies with a matt texture were examined microscopically for the characteristic spores and crystals of B. thuringiensis [l 11. The efficiency of the above method was compared
Microbiology Lepers 142 (1996) 173-177
with the previously published methods for the isolation of B. thuringiensis described by Donovan et al. [8], Meadows et al. [12], Carrozzi et al. [13] and Ohba and Aizawa [14]. A further comparison for all of these methods was provided by simply homogenising soil samples in sterile distilled water followed by plating out onto nutrient agar supplemented as before with CCY salts. To avoid bias plates were identified by random numbers allocated by a person not involved in the study. After incubation at 30°C for 48 h, or until sporulation was complete, the resultant colonies were examined for colour and morphology. Those which were beige with a matt appearance were considered to be presumptive B. thuringiensis colonies. Sixty of these from each procedure were selected at random and examined microscopically for the characteristic presence of a spore with a crystalline body within the spore mother cell wall [ll]. Such colonies were classified as B. thuringiensis strains, i.e. positives; those where only spores were visible were regarded as non-B. thuringiensis strains, i.e. negatives. This investigation was repeated twice. 2.3. Assessment of strain variance To assess the diversity of the B. thuringiensis strains isolated using the penicillin cycling method, a total of 450 isolates from five separate isolation procedures from soil B were assayed for activity against Musca domestica. 2.3. I. Bacterial strains B. thuringiensis strain 4412 was obtained from Dr. Peter Luthy and B. thuringiensis subspecies tenebrionis was obtained from DSM, Germany.
2.3.2. Growth of bacteria for bioassay Isolates of B. thuringiensis were grown in the liquid growth medium at 30°C and 200 rpm until sporulation was complete. Cultures were centrifuged for 1 h at 4000 rpm in a MSE Mistral 3000E centrifuge and the pellets washed twice with deionised water. They were finally resuspended in deionised water to give a final volume which represented a 50-fold concentration with respect to the initial culture. These preparations were referred to as ‘50-fold concentrates’.
C Johnson, A.H. Bishop1FEMS Microbiology Letters I42 (1996) 173-l 77
2.3.3.
Bioassay procedure
80
The activity of the 50-fold concentrates of spores and crystals against neonate larvae of M. domestica was determined as described by Levinson et al. [15] with the following changes: B. thuringiensis subsp. tenebrionis was used as a negative control, at a dose equal to the highest dose used in the test range. B. thuringiensis strain 4412 was used as a positive control, at a dose of 40 ~1 ml-‘. All doses were replicated 5 times using 20 neonate larvae per sample and assays were repeated at least twice.
3.1.
T--
50
s i iii 3 2 t ‘C 0 3 2
3. Results
175
40
30
20
IO
Comparison of isolation methods 0
It is apparent from these results that the rate of isolation of B. thuringiensis varies considerably between the procedures assessed (Table 1 and Fig. 1). For both soil types the penicillin cycling method described in this paper was found to be the most efficient. Statistical analysis using analysis of variance at the 0.05 significance level shows the difference between procedures to be highly significant (P< 0.001) for each soil type. The interaction between soil type and isolation procedure was also significant (P = 0.003) indicating that some procedures may be more suited to a particular soil type than are others. Using the method of Carrozzi et al. [13] the isolation rate from soil B was noticeably more efficient than with the other, previously published methods, whilst with soil A isolation rates were only equivalent to those of the other published methods. In accordance with this method the pH of the Table 1 Rate of isolation
of B. thuringiensis with each procedure
Procedure
Meadows et al. [12] Donovan et al. [S] Carrozzi et al. [13] Ohba and Aizawa [14] Homogenate Penicillin Figures in parentheses
Mean number 60 presumptive
of B.t. colonies colonies
Soil A
Soil B
6.6 5.0 5.0 2.7 1.0 19.7
4.7 9.5 14.0 6.0 1.0 34.7
(2.1) (1.7) (1.0) (4.6) (0.0) (7.4)
are standard
assessed
deviations
from
(3.8) (5.6) (3.6) (3.6) (1.0) (5.5)
of three replicates.
1
2
3
4
5
Isolation Method
I 6
Fig. 1. Comparison of the rate of isolation of B. thuringiensis colonies using six isolation methods: 1 Meadows et al. [12]; 2 Donovan et al. [S]; 3 Carozzi et al. [13]; 4 Ohba and Aizawa [14]; 5 Homogenisation; 6 Penicillin cycling. 0, Soil A: a humus-rich soil of pH 3 collected from a reclaimed landfill site. n, Soil B: a sandy soil of pH 7 collected from land adjacent to an intensive livestock unit. The percentage of E. thuringiensis colonies isolated is the percentage of colonies which were identified by microscopic examination as B. thuringiensis out of 60 randomly assessed presumptive colonies.
incubation medium was readjusted to pH 6.8 after autoclaving. After incubation with soil A however, it was found that the pH had dropped to that of the soil. As the method appears to be reliant upon an optimum pH of 6.8 this may explain the comparatively poor performance of this method for soil A. When compared to the next most efficient isolation method for each soil (i.e. for soil A the method of Meadows et al. [12]; for soil B the method of Carozzi et al. [13]) the increase in the rate of isolation for our method was still significant at the 0.05 significance level (P = 0.04, P = 0.005). 3.2. Investigation into the optimisation of the penicillin cycling method
Repeat cycles of the procedure do result in significantly more B. thuringiensis colonies being recovered (Fig. 2). The rate of increase, however, declines after
C, Johnson,
176
A. H. Bishop I FEMS
Microbiology
Letters
142 (1996)
173-I 77
isolation runs, again suggesting that the method does not benefit one strain or sub-group of organisms over the others.
1 cycle 3
cycle 2
Fig. 2. Effect of increasing numbers of penicillin cycles on the efficiency of isolating Bacillus thuringiensis. 0. Soil A: a humusrich soil of pH 3 collected from a reclaimed landfill site. n , Soil B: a sandy soil of pH 7 collected from land adjacent to an intensive livestock unit. The percentage of B. thuringiensis colonies isolated is the percentage of colonies which were identified by microscopic examination as B. thuringiensis out of 60 randomly assessed presumptive colonies.
the second cycle, although it is still significant. It is likely that any increase in isolation rate is offset by the increase in work and delay in isolation as a consequence of undertaking a third penicillin cycle. 3.3. Diversity of strains of B. thuringiensis isolated Of the 450 isolates screened for activity against M. domestica larvae, 56 strains (12%) exhibited activity (Table 2). The dose required to produce an LDss effect varied between the isolates, thus verifying the diversity in the strains isolated. The percentage of strains showing activity at each level varied between
Table 2 Bioactivity
of isolated strains of B. thuringiensis
Activity against M. domestica
Isolation run (90 isolates)
High” Medium” Negligible’
3 (3%) 6 (6%) 81 (90%)
I
4. Discussion The isolation method reported here relies upon the apparent intrinsic resistance of B. thuringiensis to the antibiotic penicillin. Whilst it is not known how wide-spread this characteristic is within the species, all strains so far tested have proved resistant (A.H. Bishop, unpublished results). The ecology of B. thurirzgiensis is debatable [14,16,17]. The ability to grow in the presence of p-lactam antibiotics would, however. presumably be of value to an organism which replicated in soils where a variety of organisms producing penicillin would naturally be found. The effectiveness of this method is clearly demonstrated when compared to other published isolation methods. A significant improvement in the rate of isolation was obtained when using the penicillin cycling method, regardless of soil type. In contrast, the method of Carozzi et al. [13] was highly dependent on soil type, favouring soil B over soil A. An obvious drawback with the penicillin cycling method is the length of time involved between commencing the isolation and obtaining any isolates. In our opinion, this limitation is far outweighed by the reduction of input by skilled personnel required for the laborious task of microscopic examination of potential colonies. When comparing the rate of isolation following each cycle of the procedure, the benefits of increased selectivity following two or more cycles must be balanced against the increased delay in obtaining the isolates. In our experience a second cycle is worthwhile in terms of the improved isolation rate but a
against larvae of M. dumrstira Isolation run 2 (90 isolates)
Isolation run 3 (90 isolates)
Isolation run 4 (90 isolates)
Isolation run 5 (90 isolates)
4 (4%) 0 86 (95%)
0 0 90 (1OO’X)
22 (24%) 17 (18%) 51 (56%)
0 11 (12%) 79 (87%)
*LD,oclS ~1 of 50-fold concentrate spores and crystals per ml of larval diet. bLD~” > 15 ~1 of SO-fold concentrate spores and crystals per ml of larval diet. ‘No toxicity observed up to a dose of 120 ~1 of 50-fold concentrate spores and crystals per ml of larval diet.
C. Johnson, A.H. BishoplFEMS
Microbiology Letters 142 (1996) 173-I 77
third cycle was not sufficiently more effective to justify the increase in time taken. Increasing the number of cycles, furthermore, may start to select preferentially for some strains of B. thuringiensis over others, thus reducing the diversity of the strains eventually isolated. Any method for the isolation of micro-organisms will, without doubt, favour one group of organisms over another. The method reported here involves two phases of growth in a liquid medium, in the presence of penicillin, and, as such, it is possible that the culture could become dominated by one, highly suited strain. This would result in many or all of the colonies on the solid medium being of the same strain of B. thuringiensis. Results obtained from the bioassays show this not to be the case. Isolates obtained from an individual isolation run revealed large variations in the level of activity expressed against M. domestica. When comparing the activity range of isolates between isolation runs the number of isolates obtained within each activity range was variable. If the procedure were highly selective for a limited number of strains it would be likely that these strains would be repeatedly selected in subsequent isolation runs. Since this was not found the strain diversity of the final culture fluid would not appear to be noticeably affected by the procedure.
Acknowledgments This work was supported Fisheries Research Programme velopment Administration.
by the Post-harvest of the Overseas De-
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