J Clin Pathol: first published as 10.1136/jcp.17.5.504 on 1 September 1964. Downloaded from J. clin. Path. (1964), 17, 504 The evaluation and modification of a technique for comparing the efficiency of antiseptics against subcutaneously deposited bacteria in mice LEONARD JEFFRIES AND S. A. PRICE Walton Oaks Experimental Station, Dorking Road, Tadworth, Surrey SYNOPSIS An attempt has been made to study the action of antiseptics on artificial streptococcal 'wounds' in mice by a published technique (Martin, 1959). The results were not in agreement with those of Martin and the possible reasons for this are discussed. Suggestions are made for modi- fying the original method to increase its scope and precision. The production and treatment of Ps. pyocyanea and K!. aerogenes 'wounds' in mice is described and discussed. Martin (1959) described a method for studying the EXPERIMENTAL MATERIALS action of antiseptics on subcutaneously deposited streptococci in mice. In the basic technique mice ANAESTHETICS Two were tested. were anaesthetized with Avertin and a dose of Tribromethanol solution Avertin (Bayer Products Ltd., Kingston-on-Thames) was diluted 1 in 40 in 10% ethylcopyright. Strep. pyogenes, sufficient to cause death in untreated alcohol to give a 2-5 % solution immediately before use, mice, was injected subcutaneously; the needle was and tested with congo red to ensure that it had not left in position and the antiseptic was injected decomposed. through it into the same site 20 minutes later. Pentobarbitone sodium, B.P. solution Nembutal, Efficiency ofthe antiseptic wasjudged by comparison veterinary (Abbott Laboratories Ltd. Queensborough, of mortality rates and mean survival times of treated Kent), was diluted 1 in 10 in isotonic saline, to give a mice which died with that of untreated controls. 0-6% solution, which was freshly prepared before the In some experiments attempts were made to simu- start of each experiment. http://jcp.bmj.com/ late natural wound conditions by suspending both MICE A closed colony of randomly-mated albino mice the streptococcus and antiseptic separately in horse has been maintained for four years from stock supplied blood. When antiseptics were dissolved in horse by Mr. F. Sullivan, Pharmacology Department, Guy's blood at the concentrations recommended by their Hospital Medical School. Mice within the weight range respective manufacturers for the treatment ofwounds of 17 to 20 g. were used. chlorhexidine was stated by Martin to be markedly effective when compared with four quaternary ANTISEPTICS Solutions or suspensions were prepared in ammonium compounds, the effects of which were 90% defibrinated horse blood or water by adding 1 on September 28, 2021 by guest. Protected to be Of 14 'conventional volume of aqueous antiseptic solution at 10 times the judged slight. antiseptics', required strength to 9 volumes of blood or water. Of presumably dissolved or suspended in water, eight these, 0-2 ml. was administered subcutaneously in all were without effect, two were slightly effective, experiments. The following antiseptics were tested: three were doubtful, and only one (acriflavine 0 1 %) Chlorhexidine, phenoctide, domiphen bromide, benz- was clearly active. alkonium chloride, dequalinium chloride, and compound When we attempted to screen compounds 1162 (synthesized by, and under experimental investi- synthesized in these laboratories by this method it gation in, these laboratories). became clear that there were complicating factors leading to death of mice such as anaesthetic and INFECTING ORGANISMS Cultures were diluted in either toxicity. In our investigations we used a nutrient broth or 90% defibrinated horse blood, according antiseptic to the experiment, and injected subcutaneously in a different strain of mouse and Streptococcus haemo- volume of 0-05 ml. lyticus of a different Lancefield group from those Streptococcus haemolyticus, group C(Pion strain, C.N.4. used by Martin. Wellcome Collection of Bacteria). This strain was main- Received for publication 10 January 1964. tained by regular passage through mice at weekly intervals. 504 J Clin Pathol: first published as 10.1136/jcp.17.5.504 on 1 September 1964. Downloaded from Evaluation and modification technique for comparing antiseptics against subcutaneous bacteria in mice 505 Heart blood from a moribund infected mouse was with Strep. haemolyticus and treated with 0-1 % inoculated into brain-heart infusion broth (Difco) chlorhexidine, 0-1% compound 1162, or 0-5% containing 10% defibrinated horse blood and incubated phenoctide the mortality rates were high in all overnight. On the following day 1 ml. was added to 10 ml. groups whether or not 90% horse blood was used brain-heart infusion broth containing 10% normal horse for the and serum, and incubated for six hours. For use the culture suspending antiseptics bacteria. All mice was diluted 1 in 1,000; this produced a fatal septicaemia in the saline-treated infection control groups died in mice in 2 to 2 5 days. and so also did those treated with 0-5 % phenoctide. Klebsiella aerogenes (N.C.T.C. 5055) This organism The phenoctide-treated mice in fact died earlier than was deposited in the National Collection of Type the saline-treated controls, but whereas Strep. Cultures in 1937 as Klebsiella pneumoniae. In accordance haemolyticus was recovered from the heart blood of with the classification proposed by Cowan, Steel, Shaw, all the control mice it could not be recovered from and Duguid (1960) it is now regarded as a strain of most of the mice that had been treated with Klebsiella aerogenes (Steel, 1963). An overnight brain- phenoctide. It was apparent therefore that the heart infusion broth culture diluted 1 in 1,000 regularly been caused a septicaemia in mice and was fatal in an average deaths may have due to the toxicity of this time of 3-5 days. The virulence of this strain remained antiseptic which nevertheless killed the streptococcus constant over seven months without 'mouse passage'. at the injection site. Subsequent experiments with Pseudomonaspyocyanea Two laboratory stock strains, unanaesthetized mice have confirmed that 0 5 % one of which, W.O. 83, was originally isolated from a phenoctide is indeed toxic and that anaesthetized human wound infection, and W.O. 126 isolated from mice are killed by even lower concentrations. a case of bovine mastitis, were chosen. Scrutiny of Martin's results reveals that he too A six-hour brain-heart infusion broth culture of either found the survival times of infected mice treated strain diluted 1 in 10 in horse blood produced a severe with 0-5 % phenoctide to be shorter than those of abscess at the injection site within six days in the majority of mice and a few mice died from septicaemia before the infected controls. He assumed that deaths were due sixth day. The injection of fewer organisms resulted in to infection and concluded that the activity of lesions in only a small proportion of mice. Two other phenoctide, at the recommended level of 0 5 %, was strains of Ps. pyocyanea were found to be unsuitable as very slight. We consider that the antiseptic is toxic their virulence for mice, by the subcutaneous route, was at that level and that it is necessary to test at lower copyright. too low. levels in order to demonstrate its antibacterial activity uncomplicated by toxicity. THE TECHNIQUE OF SUBCUTANEOUS We found that Avertin solution, diluted as INJECTION AND TREATMENT WITH ANTISEPTICS recommended by Martin and injected intra- During the periods of induction of anaesthesia and peritoneally in a dose of 0-2 ml., was toxic to recovery mice were placed on several layers of cellulose approximately 30% of our mice within the weight wadding in Harwell-pattern cages; shallow trays, range 17-22 g. Ten per cent of mice died within six similarly lined, were used in the actual experiments. hours of injection and a 20 % http://jcp.bmj.com/ Groups of mice were anaesthetized by the intraperitoneal further regained injection of either Avertin of Nembutal. When all mice consciousness but subsequently died during the in a group were completely relaxed each animal was following five days. placed on its right side and rested against the inner edge From Martin's original description and discussion of the tray. this ingenious technique appeared to represent a Each mouse was injected subcutaneously in the region potential advance on previously described methods. of the left shoulder with 0-05 ml. of bacterial suspension We found, however, that toxicity, both of anaesthetic through a gauge 25 hypodermic needle attached to a and antiseptic, may be serious complicating factors on September 28, 2021 by guest. Protected tuberculin syringe. With care to avoid altering the that may readily vitiate results. position of its point in the subcutaneous tissues the needle was detached from the syringe and the hub was rested on the body of the mouse. Twenty minutes later MODIFICATIONS TO THE BASIC TECHNIQUE 0-2 ml. of antiseptic solution was injected through the same needle, which was then removed. In the earlier CHOICE OF ANAESTHETIC In his description of experiments one group of control mice was used; these Avertin anaesthesia Martin stated: 'This anaesthetic were infected and subsequently treated with 90% horse was found superior to pentobarbitone for this blood or saline. Later experiments included an additional purpose, because deaths from the anaesthetic did group of mice which were also injected with 90% horse not occur'. blood as a control of anaesthetic toxicity. Some strains of mice are known to be more EXPERIMENTS AND RESULTS sensitive to Avertin than to Nembutal (pento- barbitone) and vice versa and, for this reason, EXPERIENCES WITH THE TECHNIQUE AS DESCRIBED BY different workers favour one or other anaesthetic MARTIN In Avertin-anaesthetized mice infected (Brown, 1963). It is possible that some of the deaths J Clin Pathol: first published as 10.1136/jcp.17.5.504 on 1 September 1964.