TRANSFER EXPERIMENTS Broth mating procedure

Donor and recipient strains were grown overnight at 37°C in Oxoid Nutrient Broth No. 2 supplemented with 0.2% (w/v) glucose and O.lM Tris with pH adjusted to 7.8 using 5M HCl (N2GT) which had been sterilised at

lOpsi for 10 min. An aliquot (Iml) was inoculated into 20ml fresh N2GT in 100ml Ehrlenmeyer flasks which were placed in an orbital incubator shaker at 37°C and cultures grown for 1.5 to 2h shaking at 200 rpm to give

approximately 5 x 10® colony forming units (cfu) ml~^ The procedure

described by Dunny and coworkers (1979) was used. Mating mixtures consisted of 0,05ml donor culture and 0.5ml recipient culture mixed in 4.5ml fresh N2GT broth i.e. a donor: recipient ratio of 1:10 and a total

of approximately 5 x 10 cfu ml . The actual ratio and concentration of

bacteria was checked by performing viable counts on cultures added to

mating mixtures. Serial 10-fold dilutions of culture were made in sterile

0.9% (w/v) sodium chloride and one drop from a Pasteur pipette (average volume 25yl) of dilutions 10 to 10 ^ was placed in duplicate on DST agar. After allowing the agar to dry at room temperature plates were incubated for 36h at 37 C and the cfu ml estimated.

Mating mixtures were incubated without shaking for 4h at 37°C, with cultures of donor and recipient strains alone treated in the same fashion. After incubation all cultures were mixed vigorously and the number of

viable donor and recipient bacteria estimated by viable counting as before using appropriate selective agar - selection of donors was with DST agax

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containing 25yg ml erythromycin except with strain SB69 where 20yg ml*~^ tetracycline was used, and for recipients, with DST agar containing 50yg ml"* fusidic acid or lOOyg ml rifampicin depending on the recipient strain. In fact, the number of donor cfu should be estimated for each'drug under test as the donor viable count at the end of mating will In part consist

on only one antibiotic therefore has the effect of underestimating transfer frequency of those markers where the actual frequency is less than the selected marker and vice versa when the transfer frequency is greater. Filter mating procedure

Bacterial strains were grown by the same procedure as that described for broth matings, although latterly BHI broth was used as nutrient medium. As above, the viable count of cultures was estimated before mixing 0.5ml donor and 4.5ml recipient to give a ratio of one donor to ten recipients

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and a total count of approximately 5 x 10 cfu ml . Immediately after

mixing, 1ml of the mating mixture was filtered on to a 25mm diameter 0.45ym filter (Sartorius, type SM113) which was placed on DST agar and incubated at 37°G for 18h. After incubation, the cells on the filter were re­

suspended in 1ml nutrient medium by vigorous agitation using a vortex mixer and the number of viable donor and recipient bacteria estimated on

appropriate selective solid medium. Selection for donors and recipients

was as above.

Selection of transconjugants

Transconjugants were selected on the basis of their simultaneous

resistance to both a donor antibiotic resistance trait and a recipient anti- — 1

biotic resistance trait. Concentrations of antibiotics in yg ml used

for selection were : erythromycin 25; lincomycin 100; tetracycline 20; streptomycin, kanaraycin and neomycin 1000; chloramphenicol 20; ampicillin and penicillin 10; fusidic acid 50; rifampicin 100, Transfer of haemo­ lysin activity could not be selected directly but was assessed on DST agar containing 6% (v/v) defibrinated Horse blood and fusidic acid or rifampicin.

Fusidic acid was used in preference to rifampicin in blood agar as the colour of the latter antibiotic interfered with haemolysin detection, particularly if the transfer frequency was low and the background of non-haemolytic colonies consequently high, a situation which results in almost imperceptible haemolytic zones.

After incubation of mating mixtures and thorough mixing, duplicate 0.1ml aliquots were spread using a sterile glass spreader, on well dried DST agar plates containing selective antibiotics. When necessary,

aliquots of serial lO-fold dilutions prepared for viable counts were also spread in order to achieve a suitable number of single colonies. All agar plates were allowed to dry at room temperature before incubating at

37°C for 48 to 72h. fransconjugant colonies which grew on selective

plates were counted and the number of transconjugants per ml estimated. From this count and that of the donor cells at the end of mating, the frequency of transconjugants per donor was calculated.

Determination of transconjugant phenotype

In order to determine the phenotype of transconjugant colonies, master plates were prepared on DST agar by conventional "picking and patching". To avoid bias, all single colonies on a selective plate or all single colonies of a section of a selective plate were taken for preparation of the master plate. Patches of donor and recipient strains were also included on master plates which were incubated at 37°C for 36h and repli­ cated, using sterile velvets, on to DST agar plates containing single anti­ biotics or 6% horse blood, representing non-selected donor and recipient markers. Antibiotic concentrations were the same as those used for transconjugant selection. As many as six replicas were made from each master, the last replica being a DST agar plate without antibiotic to check the efficiency of replication. Replica plates were incubated at 37°C for 36 to 48h.

activity. CIA producing strains were grown in N2GT broth at 37°C in an orbital incubator shaker at 150 rpm to late exponential phase at an

absorbance of 0.8 at 610nm. Cultures were centrifuged for 10 min in a

Sorvall SA-600 rotor at 8000 rpm, the supernatant decanted and filtered through a 25mm diameter 0.45yra filter (Millipore) and finally, the filtered supernatant was autoclaved at 15 psi for 20 min before storage at 4°C.

Response to CIA was assayed in ^U-shaped* microtiter trays (Sterilin). Autoclaved filtrate was diluted in fresh N2GT to give serial two—fold

dilutions in 50yl volumes. Strains to be tested for response to CIA

were grown in N2GT at 37 C overnight, diluted to an absorbance of 0.5 at 660nm and 50yl added to each dilution. The range of final dilutions of CIA was 1/2 to 1/256 with a well containing N2GT and responder cells alone as a control. Microtiter trays were incubated at 37°C for 90 to 120 min on a rotating table (Rotatest shaker, Luckham) before examining wells

for clumping of responder cells. The CIA titre was taken as the

reciprocal of the highest dilution in which clumping occurred.

ELIMINATION OF PLASMID DNA