Some Cultural Reactions Of Clostridium Tetani

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PLATES XXXII-XXXVIN earlier papers (Williams and Willis, 1970;Willis and Williams, 1970;Williams, 1971) we reported that the surface swarming of Clostridiurn tetani in plate cultures is prevented by incorporation into the medium of commercial tetanus antitoxic serum, and that this technique provides a superior alternative to firm agar for obtaining surface viable counts of CZ. tetani. Because all strains of CZ. tetani are known to share a comrnon 0 antigen, it seemed likely that this inhibitory effect on swarming was due to the presence in the antiserum of agglutinating 0 antibody to the vegetative cell.Strains of CZ. septicurn are divisible into two groups on the basis of specific components of the 0 antigen, neither of which is shared with CZ. chauvoei (Moussa, 1959). Batty and Walker (1963) applied this difference in the 0 antigenic components of the two organisms to the preparation of fluorescent labelled antibodies which may be used as specific stains to differentiate between CI. chauvoei and CZ. septicurn in smears and in tissue sections.In the present study we report upon the use of CZ. septicurn 0 antiserum for the prevention of swarming growth of CZ. septicurn in plate cultures.MATERIALS AND METHODS Organisms. Twenty-five strains of Cl. septicurn were used. These were kindly provided by Dr S. P. Lapage from the National Collection of Type Cultures (NCTC), and Dr P. D. Walker from the Wellcome Research Laboratory Culture Collection (CN). The strains were respectively nos. NCTC281,282,283,284,286,501,504,547,549,550,551; and nos. CN424, 2779, 3232, 3608, 3610, 3847, 3848, 3941, 3942, 3957, 4212, 5115, 5421, 5545. In addition, two strains of CI. chauvoei (NCTC8070 and 8590) and stock strains of C Z . welchii type A, CZ. biferrnentans, CI. oedematiens type A and CI. histolyticum were used.Preparation of 0 antigen. This was as described by Batty and Walker. Preparation of antiserum. The method used was that described by Batty and Walker.Antiserum was prepared against a mixture of four strains of CI. septicum, each of the two serological groups of Moussa being represented by two strains. The strains used were nos. NCTC501, 551, 281 and 282. Courses of immunisation repeated over a period of months yielded sera with agglutinating titres of the order of 2560.Media. Fresh horse blood agar was used as the plating medium, and was prepared in the usual way from an ox heart digest broth base (Cruickshank, 1965); layer plates containing 5 per cent. of defibrinated horse blood (Wellcome) were used. The ordinary agar medium contained 1.5 per cent. of New Zealand agar; firm agar plates contained 4 per cent. of New Zealand agar. For the plate culture of CZ. chauvoei the medium was enriched with

Section: Plates Xxxii-xxxvmentioning

confidence: 99%

Prevention Of Swarming Of Clostridium Septicum

Willis¹,

Williams²

1972

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“…Thus flagellotropic-phage preparations, spread on the surfaces of media, inhibit the swarming of Proteus spp. (Roux and Applebaum, 1972), commercial tetanus antitoxin inhibits the swarming of C. tetani (Willis and Williams, 1970;Williams, 1971) and an antiserum to selected strains inhibits the swarming of C. septicurn (Willis and Williams, 1972). Williams (1971) and Willis and Williams (1 972) found that antiserum-treated media were marginally better than FA for producing discrete colonies of C. tetani and C. septicurn.…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

“…Some medical bacteriologists have favoured FA to prevent the swarming of Proteus spp. (Stokes, 1960;Crowley, Bradley and Darrell, 1969), but more recently its value has been questioned because it sometimes failed to prevent swarming (Stokes, 1975) and reduced the growth of certain clostridia (Williams, 1971 ;Willis and Williams, 1972).The ideal anti-swarming medium should be suitable for all swarming bacteria, non-inhibitory to the growth of any bacterium to be isolated from mixtures that include swarmers, and easy to prepare. No medium satisfies all of these criteria.…”

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confidence: 99%

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Effect of firm agar on the swarming of Proteus and Clostridium species and on the colonies of clinically important bacteria (Plates IX-XI)

Hayward

Incledon

Spragg

1978

PLATES IX-XI THE use of firm agar (FA) to inhibit the swarming of Proteus and Clostridium spp. was first reported by Hayward and Miles (1943). A description of its preparation and use followed (Hayward, 1945(Hayward, , 1947. Some medical bacteriologists have favoured FA to prevent the swarming of Proteus spp. (Stokes, 1960;Crowley, Bradley and Darrell, 1969), but more recently its value has been questioned because it sometimes failed to prevent swarming (Stokes, 1975) and reduced the growth of certain clostridia (Williams, 1971 ;Willis and Williams, 1972).The ideal anti-swarming medium should be suitable for all swarming bacteria, non-inhibitory to the growth of any bacterium to be isolated from mixtures that include swarmers, and easy to prepare. No medium satisfies all of these criteria. The chief disadvantages of FA are the experience and special attention required for its preparation, and the unfamilar appearance it gives to colonies of common bacteria (Crowley et al., 1969;Smith, 1972).The present work was undertaken to provide a detailed description of the preparation of FA media, an assessment of the effectiveness of FA in preventing the swarming of proteus colonies, information on the effect of FA on surface viable counts of clinically important bacteria, and an account of the colonial morphology on FA of a variety of bacteria likely to be isolated in clinical laboratories. MATERIALS AND METHODSOrganisms Proteus mirabilis and Proteus vulgaris cultures (167 and 14 respectively), all of which swarmed on " routine " nutrient agar (see below), were used to test the inhibition of swarming by FA. Of the P. mirabilis cultures, 164 were freshly isolated; they came from faeces (87), urine (56), respiratory tract (7), wounds (6), genital tract (4), ear (l), eye (l), water (1) and sewage (1). The remaining three were from culture collections, namely, the National Collection of Type Cultures, London (strain NCTC9559), the Monash University Culture Collectioii and the Queensland University Culture Collection. Of the P. vuZgaris cultures, nine were freshly isolated; they came from faeces (6), urine (2) and the nose (1). The remaining five were from culture collections, namely, the National Collection of Type Cultures, London (strain NCTC4636), the Monash University Culture Collection, the The organisms used for experiments on viable counts and colonial morphology were from culture collections, except for the culture of Neisseria gonorrhoeae which was freshly isolated.The identities of all cultures were confirmed by the methods of Cowan and Steel (1974). MediaThe term " routine " is used to describe media containing 1.2% agar. The preparation of FA media required attention to detail. Care was needed to ensure that the period of steaming was sufficient to dissolve the agar completely. Boiling-over during autoclaving was prevented by the use of flasks that were large in proportion to the amount of medium, e.g., a 500-ml flask was used for 100 ml of medium and a 750-ml flask for 200 ml of medium; it was also necessary to...

“…septicum strains, the spreading surface growth rendered accurate colony counting difficult. This was controlled by a procedure based on that of Willis and Williams (1970): well-dried plates were treated with a selected Cl. septicurn antitoxin before they were seeded (see Materials and methods).…”

Section: Nilmentioning

confidence: 99%

The Recovery Of Clinically Important Anaerobes On Solid Media

Watt

1972

IT was shown by Collee, Rutter and Watt (1971) that the difficulties encountered in the surface culture of Clostridium oedematiens (CZ. novyi) types B and D can be overcome by the use of modified Moore's medium (Moore, 1968) or by sprinkling sterile iron filings on the surface of a seeded blood agar plate. This approach made it possible to study quantitatively whether the growth was derived from spores or from vegetative cells. It was found for type D of this exacting organism that vegetative cells can account for an appreciable part of the growth obtained when a broth culture is subcultured on solid medium. This finding may be of practical importance also in the case of other anaerobes. It raises the question whether the traditional methods give adequate recovery of anaerobic organisms for clinical purposes or whether more elaborate procedures such as those of Hungate (1950) The following organisms were subcultured in nutrient broth after isolation from clinical material and identification : two strains of Staphylococcus aureus; one strain of Streptococcus pyogenes (group A), one strain of Bacillus anthracoides; one strain of Neisseria gonorrhoeae ; one strain of Veillonella sp.; and two strains of Haernophilus influenzae.Media. Cooked-meat broth was prepared as described by Cruickshank (1968, p. 757), but the infusion broth component was made from Nutrient Broth no. 2 (Oxoid). Oxoid Nutrient Broth was also used for the subculture of some of the test organisms. "Presteamed nutrient broth " was used as a diluent in the quantitative studies; it was heated by steaming at 100°C for 30 min. to remove dissolved air, then cooled rapidly to 37°C.In the initial studies, Watt's modification of Moore's medium (cysteine-dithiothreitol human blood agar) was used (Collee et ~1 . ) . In later studies, a further modification (cysteinedithiothreitol equine blood agar was prepared by substituting 10 per cent. horse blood for 33 per cent. human blood.Blood agar plates were prepared with Oxoid Blood Agar Base no. 2 enriched with equine blood (10 per cent.) or human blood (33 per cent.).Blood. Outdated citrated human blood was obtained from the Blood Transfusion Department, Edinburgh Royal Infirmary. Defibrinated equine blood was supplied by Wellcome Laboratories.