FEMS Microbiology Letters 142 (1996) 173-177

A technique for the effective enrichment and isolation of Bacillus thuringiensis

Clare Johnson, Alistair H. Bishop *

School of Chemical 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 ap-

proach, even if successful, is the approval of regula- tory 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: A.H.Bishop@gre.ac.uk

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 nov-

el insecticidal activity has a much higher success rate compared to screening chemical compounds

with similar properties [9]. The isolation of B. thu-

ringiensis from environmental samples can, however,

be a very laborious and time-consuming procedure, requiring the microscopic examination of many col- onies presumed to be B. thuringiensis based upon colony morphology. Methods which increase the proportion of colonies which really are B. thuringien-

sis amongst those with a similar appearance save

valuable time and allow a more efficient screening

037%1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00261-3

174 C. Johnson, A.H. Bishop1 FEMS Microbiology Lepers 142 (1996) 173-177

of environmental samples for novel strains. We re-

port here a simple technique which, when compared

to published methods for the selection of B. thurin-

giensis, results in a higher proportion of such colo-

nies 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 vary-

ing 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 sam- ple 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 3OOOE cen-

trifuge. 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) contain- ing CCY salts and supplemented with 20 IU ml-’ of penicillin G. The cultures were incubated at 30°C until sporulation was complete. Individual beige col- onies with a matt texture were examined micro- scopically for the characteristic spores and crystals of B. thuringiensis [l 11.

The efficiency of the above method was compared

with the previously published methods for the isola-

tion 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 ho-

mogenising soil samples in sterile distilled water fol-

lowed by plating out onto nutrient agar supplement- ed 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 pro-

cedure were selected at random and examined micro-

scopically for the characteristic presence of a spore

with a crystalline body within the spore mother cell wall [ll]. Such colonies were classified as B. thurin-

giensis 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 tenebrio-

nis was obtained from DSM, Germany.

2.3.2. Growth of bacteria for bioassay

Isolates of B. thuringiensis were grown in the liq- uid growth medium at 30°C and 200 rpm until spor- ulation 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 con- centration with respect to the initial culture. These preparations were referred to as ‘50-fold concen- trates’.

C Johnson, A.H. Bishop1 FEMS Microbiology Letters I42 (1996) 173-l 77 175

2.3.3. Bioassay procedure

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 pos-

itive 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. Results

3.1. Comparison of isolation methods

It is apparent from these results that the rate of isolation of B. thuringiensis varies considerably be-

tween the procedures assessed (Table 1 and Fig. 1).

For both soil types the penicillin cycling method de- scribed in this paper was found to be the most effi-

cient. Statistical analysis using analysis of variance at

the 0.05 significance level shows the difference be-

tween procedures to be highly significant

(P< 0.001) for each soil type. The interaction be-

tween soil type and isolation procedure was also sig- nificant (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 meth-

ods. In accordance with this method the pH of the

Table 1 Rate of isolation of B. thuringiensis with each procedure assessed

Procedure Mean number of B.t. colonies from

60 presumptive colonies

Soil A Soil B

Meadows et al. [12] 6.6 (2.1) 4.7 (3.8)

Donovan et al. [S] 5.0 (1.7) 9.5 (5.6)

Carrozzi et al. [13] 5.0 (1.0) 14.0 (3.6)

Ohba and Aizawa [14] 2.7 (4.6) 6.0 (3.6)

Homogenate 1.0 (0.0) 1.0 (1.0)

Penicillin 19.7 (7.4) 34.7 (5.5)

Figures in parentheses are standard deviations of three replicates.

80

50

s i 40

iii 3 2

t 30

‘C 0

3 20 2

IO

0

T--

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 hu-

mus-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 colo-

nies isolated is the percentage of colonies which were identified

by microscopic examination as B. thuringiensis out of 60 ran-

domly 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 compara-

tively poor performance of this method for soil A. When compared to the next most efficient isola-

tion 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 isola-

tion 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 signifi- cantly more B. thuringiensis colonies being recovered (Fig. 2). The rate of increase, however, declines after

176 C, Johnson, A. H. Bishop I FEMS Microbiology Letters 142 (1996) 173-I 77

1 cycle 2 cycle 3

Fig. 2. Effect of increasing numbers of penicillin cycles on the ef-

ficiency of isolating Bacillus thuringiensis. 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 inten-

sive livestock unit. The percentage of B. thuringiensis colonies

isolated is the percentage of colonies which were identified by mi-

croscopic examination as B. thuringiensis out of 60 randomly as-

sessed 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 con-

sequence 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

isolation runs, again suggesting that the method does

not benefit one strain or sub-group of organisms

over the others.

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. thu-

rirzgiensis is debatable [14,16,17]. The ability to grow in the presence of p-lactam antibiotics would, how-

ever. presumably be of value to an organism which

replicated in soils where a variety of organisms pro- ducing penicillin would naturally be found.

The effectiveness of this method is clearly demon-

strated when compared to other published isolation

methods. A significant improvement in the rate of

isolation was obtained when using the penicillin cy-

cling 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 ob-

vious 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 colo-

nies. When comparing the rate of isolation following

each cycle of the procedure, the benefits of increased

selectivity following two or more cycles must be bal- anced against the increased delay in obtaining the isolates. In our experience a second cycle is worth-

while in terms of the improved isolation rate but a

Table 2

Bioactivity of isolated strains of B. thuringiensis against larvae of M. dumrstira

Activity against Isolation run I Isolation run 2 Isolation run 3 Isolation run 4

M. domestica (90 isolates) (90 isolates) (90 isolates) (90 isolates)

High” 3 (3%) 4 (4%) 0 22 (24%)

Medium” 6 (6%) 0 0 17 (18%)

Negligible’ 81 (90%) 86 (95%) 90 (1OO’X) 51 (56%)

*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.

Isolation run 5

(90 isolates)

0

11 (12%)

79 (87%)

C. Johnson, A.H. BishoplFEMS Microbiology Letters 142 (1996) 173-I 77 111

third cycle was not sufficiently more effective to jus-

tify the increase in time taken. Increasing the number of cycles, furthermore, may start to select preferen-

tially 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 ex-

pressed 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 se-

lective 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 by the Post-harvest Fisheries Research Programme of the Overseas De-

velopment Administration.

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