The FH strain of Mycoplasma pneumoniae was studied in experimentally infected cultures of lung diploid fibroblasts. The multiplicity of infection was found to have a profound influence on the distribution of the cell-associated and extra-cellular mycoplasmas. At a high multiplicity there was very little difference between the ratio of cell-associated and extracellular mycoplasmas. In contrast, with a low multiplicity the cell-associated mycoplasmas predominated and with further reductions of the inoculum only the cell-associated mycoplasmas could be detected. The significance of these findings is discussed. It is suggested that caution has to be exercised in the determination of the potency of antibiotics against mycoplasma by methods employing acellular media alone, and it is desirable to utilize also appropriate cell culture models. A possible approach to designing such models is provided by this study.

We express our appreciation to the Tissue Culture Laboratory, Pfizer Limited, Sandwich, for kindly supplying us with cell cultures. We furthermore thank the Board of Directors, Pfizer Limited, for permission to publish this study. The helpful discussions with our colleagues Drs D. Jackson and G. M. Williamson in preparing the manuscript are acknowledged with thanks.

Two cyclones for sampling dilute aerosols in the field are described. Their advantages are their robustness and reliability, the high degree of concentration achieved, and the ease with which they will provide a long uninterrupted sequence of samples.

Methods of correcting estimates of cloud concentration for transfer lag in the cyclone are derived.

The cyclones were constructed by J. Nott. We are indebted to G. J. Harper and E. J. Morris for allowing us to make use of their observations, and to Dr J. S. Paterson (Superintendent, Allington Farm) for providing the realistic aerosol at our convenience.

One hundred and twenty-nine samples of fresh cream collected in Worcestershire were examined bacteriologically. Sixty (46·5 %) creams had counts of over 100,000 bacteria/ml. The bacteria present were of many varieties, the commonest being Bacillus spp. (aerobic spore formers), Gram-negative bacilli, staphylococci and micrococci. Since most of the creams had been either pasteurized as cream or manufactured from pasteurized milk it was thought that the many bacteria were present because of contamination after pasteurization due to three main causes; unsatisfactory or unhygienic premises, unsuitable equipment, manual handling during the filling and capping process.

The methylene blue test results related well with bacterial counts but there were seven (5·4 %) anomalous results. Although the methylene blue reduction test therefore could serve as a simple and reasonable guide to the hygienic quality of fresh cream, 5·4 % of anomalous results would perhaps make it unsuitable as a statutory test.

We thank Mr R. Colenso, M.A.P.H.E., M.A.P.H.I., Chief Public Health Inspector, and Mr H. Beckett, Milk Sampling Officer of the Worcestershire County Council, for arranging the supply of samples.

A serological survey for brucella antibodies has been carried out in Northern Ireland in an attempt to assess the effects of the brucellosis eradication programme in cattle which has been in progress since 1959. One thousand eight hundred and ninety four sera from blood donors, from farmer's lung tests, and from various groups occupationally exposed to cattle were examined by the serum agglutination test, the anti-human globulin (A.H.G.) (Coombs) test and the complement-fixation test.

The incidence of brucellosis in Northern Ireland is now less than it was 20 years ago, but is similar to that reported in a recent survey in England. It now appears that the principal method of transmission of brucellosis in the province is by occupational exposure rather than by drinking infected milk. Though it may be too early to make a serological assessment of the effects on human infections of brucellosis eradication in cattle, this survey provides a basis for future evaluation.

We thank Prof. O. L. Wade, Prof. P. C. Elmes and Dr W. R. Kerr for their encouragement and helpful advice; the Medical Officers of Health in Antrim, Armagh, Down and Fermanagh, Londonderry, and Tyrone, and in Belfast and Londonderry County Borough for help in obtaining blood samples; Dr M. C. Huth of the Northern Ireland Blood Transfusion Service, for sera; Dr D. W. R. Mackenzie and Miss H. E. Cairns for sera from the Mycology Diagnostic Laboratory; Dr J. H. Connolly for sera from the Virus Reference Laboratory, and for helpful advice; the farm workers and veterinary surgeons for volunteering blood samples; the technical staff of the Veterinary Research Laboratories for carrying out the tests.

The value of clean zones and of transfer areas in operating suites was assessed by comparisons of the amounts of contamination on floors, trolleys and footwear in suites with and without a clean zone and a transfer area; counts of Clostridium welchii were used as an index of bacterial contamination introduced into the aseptic zone from outside.

The mean counts of Cl. welchii on contact plates from the wheels of trolleys used to convey patients from wards to the operating suite (67·9 ± 7·68 per plate) were significantly higher than those from theatre trolleys (i.e. those used only inside a theatre suite provided with a transfer area) (3·13 ± 0·47 per plate); mean counts of total bacteria were only slightly lower on the wheels of theatre trolleys than on those of hospital trolleys. Other surfaces of hospital trolleys showed counts similar to those found on theatre trolleys.

Contact plates from floors showed significantly lower counts of Cl. welchii in the aseptic zone and the clean zone than in the hospital corridor, the protective zone and (when present) the transfer area.

The mean counts per 100 cm2 of Cl. welchii were approximately the same on the floor of a theatre with a clean zone and a transfer area (0·83) as in one with a clean zone but no transfer area (0·5). Counts of total bacteria were higher in the latter. A suite with no clean zone or transfer area showed a higher mean count of Cl. welchii on contact plates from the aseptic zone (operating theatre) (20·5 ± 12·33 per 100 cm2). These higher levels of contamination were due to sporadic high counts of Cl. welchii found near the door of the theatre with no clean zone; in another theatre with no clean zone the level of Cl. welchii on the floor was not higher than that in the theatres with clean zones.

Theatres with plenum ventilation had lower mean counts of airborne Cl. welchii than those ventilated by windows: there was no significant difference in the levels of Cl. welchii on the floors of theatres with the two forms of ventilation.

On sampling with contact plates, theatre footwear yielded fewer total organisms, Staphylococcus aureus and Cl. welchii than outdoor shoes removed before entering the clean zone.

The hygienic value of transfer areas and clean zones is discussed. Bacteriological support could not be obtained for the former, but the latter appeared to contribute something to the cleanliness of the theatre by preventing heavy sporadic contamination.

We wish to thank Mr M. Wilkins for valuable assistance, the staff of the operating theatres for their co-operation and Alne Engineering Limited, 57 High Street, Henley-in-Arden, Solihull, for supplying disposable contact plates.

A survey of 13 linen chutes in eight hospitals was carried out to assess the amount of bacterial contamination on the inner walls of these chutes. It was shown that the average bacterial count in these chutes was low by general hospital standards (19·7 bacterial colonies per Rodac plate). This concentration was eight times less than the average concentration found on the floor surface at each linen disposal and collection point (153 bacterial colonies per Rodac plate).

Three chutes sampled during the survey were periodically cleaned but appeared to derive only very limited benefits from the cleaning method they used.

Tests carried out on three linen chutes showed that the bacterial surface contamination of a sterilized chute could normally reach a maximum concentration in a period of between 4 and 24 hr.

It is concluded that so long as good engineering practices are used in the construction of linen chutes the bacterial contamination on their inner surface should not contribute to the problem of hospital cross infection. For this reason, and because of the impracticability of sterilizing linen chutes at very short intervals of time, it is considered that cleaning of chutes would serve no practical purpose.

Thanks must be recorded to the authorities of all those hospitals who gave their permission for us to study their linen disposal chutes and afforded us every assistance.

We should also like to thank Mr W. Carson, Leader of the Building Services Research Unit, for his help and encouragement.

The work reported was a part of an investigation into the design of linen disposal chutes sponsored by the Western Regional Hospital Board, Scotland.

A modification of the Henderson apparatus is described which is mobile and which can produce bacterial or viral aerosol in air whose relative humidity can be chosen in the range 20–95 %. The apparatus forms a basic utility for microthread, rotating drum or other aerosol studies.

Much help and advice has been given by Messrs. J. F. H. Peel and D. J. Garriock of the Engineering Section M.R.E. throughout the development of this apparatus. The proportioning valve is a modification of one originally used for a similar purpose by Mr K. R. May. Thanks are due to Messrs. G. J. Harper and A. M. Hood for criticism of the script.

After a single subcutaneous injection into baboons the MRC-4 strain of trachoma/inclusion conjunctivitis (TRIC) agent underwent limited multiplication at the injection site, but was then eliminated rapidly from the skin and regional lymph nodes. Forty-eight hours after a single intravenous injection, but not thereafter, it appeared in the peripheral lymph nodes and spleen. The single parenteral injections failed to immunize baboons against conjunctival challenge with the homologous strain. These findings contrasted with those previously reported for the more virulent mutant, MRC-4 f, which multiplied readily in the skin, lymph nodes and spleen, persisted in these tissues up to 3 weeks after injection, and conferred good immunity to conjunctival challenge with MRC-4. The difference in behaviour of MRC-4 and MRC-4 f might be accounted for, at least in part, by the use of a smaller inoculum of live MRC-4; but similar findings in guinea-pigs, reported elsewhere, suggest that the differences observed are real. In conjunction with previous work, the present study suggests that the immunogenicity of TRIC agent is closely related to the mass of antigen that can be administered to or propagated within the recipient.

We are grateful to Mr D. Venters for his able technical assistance.

Two new media, strontium chloride and strontium selenite broths, are described for the enrichment culture of Salmonella from human and animal material.

In comparative trials, strontium chloride was found to be comparable to Rappaport medium for the recovery of a wide range of Salmonella serotypes from human faeces and sewage, and from pig faeces and glands. Both were superior to selenite F medium.

Strontium selenite was found to be superior to selenite F in the recovery of S. typhi from human faeces and sewage.

We are greatly indebted to Mr M. H. Lello for preparing the samples of strontium selenite used in the investigation and for allowing us to publish the notes on his method of preparing the substance.

Our thanks are due to Pro. C. T. Huang of Hong Kong for sending samples of faeces from typhoid cases under his care; and to Dr Joan Taylor for her continuing help in confirming or identifying the serotypes of salmonella isolated.

We thank Dr W. S. Davidson, Commissioner of Public Health, Western Australia, for permission to publish.

Hysterectomy-produced, colostrum-deprived pigs were injected twice with formalinized antigen prepared from the J strain of Mycoplasma suipneumoniae; the first injection was with Freund's adjuvant and the second injection without adjuvant. The immunity of these animals was tested by inoculating them intranasally with different doses of lung suspension prepared from cases of enzootic pneumonia. Two of the pigs were not killed shortly after infection, but were kept and challenged with enzootic pneumonia in order to compare the serology of experimentally-injected animals with the serology of the immune state following the experimentally-induced disease.

In a second main experiment, a pregnant sow was injected twice with nonformalinized antigen without adjuvant, and her litter was subsequently exposed to the disease at 7 days of age after suckling naturally from birth.

There was no evidence to suggest that the injections had protected the pigs in the first experiment against a high dose of infection, but they may have given some protection against low doses. The piglets suckled by the injected sow were not protected against two different doses of infection.

Serum samples taken at different stages were examined by the metabolic inhibition (MI) test, the indirect-haemagglutination (IHA) test, the complement fixation (CF) test and the gel-diffusion precipitin test, using M. suipneumoniae as antigen.

Serum samples taken before injection in the first experiment were all negative in the MI test and they became positive after the injections of antigen. However, 1 the highest MI titres obtained were not associated with obvious immunity; nor was the development of true immunity after experimental infection associated with a change in MI titre.

In the first experiment, substantial IHA titres (over 20,000) were recorded by 14 days after the second injection of antigen. Again, there was no correlation between the IHA titres and the area of pneumonia following experimental infection. In the sow experiment, IHA titres developed after the first injection and increased after the second; a high IHA titre occurred in the colostrum and titres of 320 or more were present in the piglets 7 days after birth.

The CF titres appeared earlier than the IHA titres but did not increase so markedly thereafter. Once more, there was no correlation between the titre before infection and the area of pneumonia afterwards.

In the gel-diffusion test, precipitins were demonstrated in all the post-injection serum samples tested, most of the samples being positive after the first injection. Precipitins were also demonstrated in the colostrum of the injected sow and in her uninjected litter at 7 days of age.

From these experiments it was concluded that, as judged by the development of pneumonic lesions and in marked contrast to the known immunizing effect of the disease itself, injections of antigen given in this manner had little or no protective effect against the dose levels of infection used. Nevertheless, the titres obtained in the MI, IHA and CF tests were comparable with those obtained earlier in pigs that were strongly immune, which provides further evidence for the suggestion that these tests do not measure protective immunity.

Miss Elaine Repworth provided technical assistance. Most of the expenses of this work, including the salary of two of the authors (R. G. H. and R. L. W.) were met by a grant from the Agricultural Research Council.

One hundred and eleven consecutive deaths in seven mental hospitals during the months of February to October 1967 were investigated histologically and bacteriologically.

Bacteria were present in the lung tissue significantly more frequently when inflammation was present than when it was absent and the differences were significant for both Escherichia coli and Staphylococcus aureus; E. coli was isolated from 22(34%) of the 65 patients with inflamed lungs compared with two (4%) of 46 control patients and the corresponding figures for Staph. aureus were 20(31%) of 65 patients compared with seven (15%) of 46 control patients.

Some strains of E. coli were serotyped using antisera against 0 antigens 1, 4, 6, 18ab, 18ac, 25, 50, 65, 69 and 75 and the strains most commonly found in the inflamed lungs, spleens and other inflammatory lesions of 25 patients were 06, 018 and 075. These strains were isolated more frequently from these sites than from the faeces of a group of 19 patients with no such lesions.

In this series E. coli was the commonest organism to be associated with terminal bronchopneumonia and the possible reasons for this are discussed.

Influenza vaccines prepared with a mineral oil adjuvant induce a substantial and durable antibody response. However, vaccines containing mineral oil in simple emulsion (water-in-oil emulsion) sometimes produce persistent local reactions (Medical Research Council, 1964). The development of a redispersed ‘multiple’ emulsion in which antigen is incorporated as an oil-in-water emulsion starting from an original water-in-oil emulsion was described by Herbert (1965). Such multiple emulsion vaccine has a lesser viscosity than simple emulsions and might therefore produce a substantial antibody response with fewer reactions. In this investigation a comparison has been made of the antibody response and short-term vaccination reactions after aqueous influenza vaccine, influenza vaccine containing a mineral oil in simple emulsion, and influenza vaccine containing mineral oil in multiple emulsion.

Phagocytosis prevents the multiplication of Streptococcus suis in blood samples from conventionally reared adult pigs. It is mediated by antibody, probably IgM, present in the serum of adults and specifically directed against the capsular polysaccharide of S. suis. The blood of young pigs reared in a ‘pathogen-free’ environment allows multiplication of encapsulated S. suis. Of 160 piglets conventionally reared, the blood of 92(58%) did not permit multiplication of S. suis. Blood from the remaining 68(42%) allowed multiplication but became bacteriostatic when the animals reached the age of 6 to 8 weeks. Serum from adult pigs confers bacterio static activity on blood from susceptible piglets in vitro and passively protects such piglets against experimental S. suis infection in vivo.

We thank Dr T. J. L. Alexander, formerly of Ontario Veterinary College, and Mr P. C. Trexler of the Royal Veterinary College, London, for providing us with serum from gnotobiotic pigs.

The work described in this report was carried out by one of us (K. K. A.) during the tenure of a Commonwealth Scholarship and was incorporated in a dissertation submitted for the Ph.D. degree of the University of Cambridge.

The infection of a child with Salmonella paratyphi B phage type Battersea caught from his pet terrapins is recorded. In a small subsequent survey a relatively large proportion of these animals was found to be excreting salmonellas. The danger of these seemingly harmless pets is emphasized.

It is a pleasure to acknowledge the help and encouragement given by Dr E. H. Gillespie at all stages in the production of this manuscript. We also thank Dr Joan Taylor and Dr J. Wallace for final identification of some of the organisms isolated and Dr E. S. Anderson for phage typing the Salmonella paratyphi B strains.

In a herd affected with bovine mastitis, dwarf colony variants of Staphylococcus aureus, auxotrophic for Ca-pantothenate, were isolated from the milk of four cows with mastitis. From one of these cows, a dwarf variant with an unknown metabolic disorder was isolated during the same period. Infections with auxo trophic staphylococci were always chronic and proved to be refractory to anti biotic treatment, though the causative micro-organism was highly susceptible to the drugs used as judged by in vitro tests.

The authors acknowledge with appreciation the skilful help of Miss Nili Abramova.

In a residential estate of 4000 persons, containing neither industry nor retail butchers shops, salmonellas were regularly found in the sewerage system. They were frequently found in the sewage of a portion of the estate housing 1000 persons. The range of serotypes found was wide and some types suggested an exotic origin. No overt salmonella infection in the estate was reported during the period of survey, although local general practitioners had been previously alerted. Overt infection due to serotypes found in the survey were, however, reported in other areas of Glamorgan. Multiple sampling points in the sewerage system and a serological technique for examining samples contaminated with multiple salmonella serotypes were essential for the technical success of the survey.

We should like to thank Prof. Scott Thomson for his advice in the preparation of this paper; Dr E. S. Anderson of the Central Enteric Reference Laboratory and Bureau, Colindale, for phage-typing the strains of S. typhimurium and S. paratyphi B; and Dr G. J. G. King of the Public Health Laboratory, Bournemouth, for identifying the serotypes isolated. We should also like to thank Mr T. R. Liddington and Mr J. H. Price for their technical assistance.

In a survey undertaken in an intensive care unit, coliform bacilli were found to be responsible for most infections, Pseudomonas aeruginosa and Staphylococcus aureus being isolated much less frequently. Tracheostomy or endotracheal intubation predisposed to infection, but in our experience intermittent positive pressure ventilation did not significantly affect its incidence. Little cross-infection has occurred, and it has never been possible to incriminate the ventilators in its transmission.

We gratefully acknowledge the assistance we have received from colleagues in the Intensive Therapy Unit and the Department of Bacteriology during the course of this investigation.

The theory of assigning an upper limit to the estimate of the degree of contamination of a medium is briefly explained. Tables to save computational labour are presented and their use elucidated by examples.

The antigens present in the preparation of cholera toxin consisting of the culture filtrate of 569 B strain of V. cholerae have been analysed by gel-diffusion, intra-gel absorption, and immuno-electrophoresis. The antiserum raised against the toxin was tested using the following antigens: whole cell lysates of V. cholerae 569 B and an El Tor (water) strain W 6 (proposed as a vaccine strain), suspensions of the same strains of vibrios with and without heat-denaturation, suspension of an El Tor (case) strain (GS-9) and a preparation of vibrio polysaocharide. The antitoxic serum gave four precipitin bands against the toxin preparation and nine bands against the whole cell lysate of V. cholerae. Two bands could be identified as being due to the heat-labile specific antigens present in the cholera toxin. These two bands were abolished on absorption of the antitoxic serum with a live suspension of W 6 but not on absorption with heat-denatured W 6. It has, therefore, been concluded that the proposed vaccine strain W 6 contains the specific antigens of cholera toxin. The significance of these results has been discussed in relation to the residual virulence of the proposed vaccine strain and antitoxic immunity produced in the intestinal tract of the adult rabbit following exposure to the vaccine strain.

We gratefully acknowledge the gift of purified vibrio polysaccharide from Dr C. V. N. Rao of the Indian Association for the Cultivation of Science, Jadavpur, Calcutta-32. The technical assistance of Mr B. Sanyamat and Mr I. Guhathakurta is also thankfully acknowledged.

The identification of 18 strains of haemadsorbing viruses as parainfluenza type 4 viruses subtypes A and B is reported, as is a serological survey to show the distribution of antibody to these viruses in various age groups. The results suggest that although isolation of the virus is infrequent, infection in early childhood is common and the viruses may be important respiratory pathogens.

I wish to thank Dr G. B. Bruce White for kindly providing sera and also Dr B. E. Andrews, Dr Suzanne K. R. Clarke, Dr F. O. MacCallum, Dr E. M. Mackay Scollay, Dr Hélène J. Mair and Dr J. O'H. Tobin for sending the strains.