Rapid serotyping of campylobacters based on heat-stable antigens, using a combined passive haemagglutination/co-agglutination technique

Tryptic soy broth/agar, brain heart infusion agar and Columbia broth/agar, all widely used in the bacteriological laboratory, were radiation-sterilized at a dose of 10–15 kGy. The media were tested for the growth of 12 different micro-organisms, including fastidious pathogens such as Streptococcus pyogenes, Strep, pneumonias, Haemophilus influenzas and Neisseria meningitidis. Solid and fluid media supplemented with catalase before irradiation performed well in comparison with heat-sterilized media.     

Nitrate-Sensitive ATPase Activity and Proton Pumping in Guard Cell Protoplasts of Commelina

ATPase activity was measured in crude homogenates of guard cellprotoplasts of Commelina communis L. using a linked enzyme assay.A low level of azide-sensitive ATPase activity was detectedwith a pH optimum of 6.8. This activity was stimulated by 0.01%(v/v) Triton X-100, and the pH optimum shifted to pH 7.4. Nitrate-sensitiveATPase activity was measured in the presence of azide and showeda pH optimum around pH 8.0. Proton pumping activity in a mixedpopulation of vesicles from GCP was monitored using fluorescencequenching of quinacrine. Mg-ATP dependent proton pumping wasobserved at pH 8.0, but not at pH 6.6. The activity at pH 8.0was inhibited by nitrate and DCCD but not vanadate. These dataindicate that activity of the tonoplast proton pump was beingmeasured. There was, however, no evidence for a tonoplast cation(K⁺)/proton antiporter under these assay conditions as potassiumdid not reduce the initial rate of pH gradient formation orincrease the rate of collapse of a pre-formed gradient afterinhibition of the pump. Key words: Tonoplast ATPase, proton pump, guard cell protoplasts, Commelina     

Vanadate Sensitive ATPase and Phosphatase Activity in Guard Cell Protoplasts of Commelina

Flicker, M. D. and Willmer, C. M. 1986. Vanadate sensitive ATPaseand phosphatase activity in guard cell protoplasts of Commelina.—J.exp. Bot. 38: 642–648. Phosphatase activity was measured in extracts of guard cellprotoplasts of Commelina communis L. using the artificial substratep-nitrophenylphosphate. A pH optimum of 5.8 to 6.3 was determined.Ammonium molybdate (Ol mol m^–3) and sodium vanadate (1–0mol m^–3) gave almost complete inhibition of phosphataseactivity at pH 60. ATPase assays were, therefore, conductedin the presence of 0–2 mol m ^–3 molybdate and vanadatewas used as a specific inhibitor of plasmamembrane ATPase activity.Vanadate sensitive ATPase activity showed a pH optimum of 6.6and activity was stimulated by KC1. These properties are characteristicof plasmamembrane proton pumping ATPases in other systems andsuggest that proton extrusion in guard cells could be mediatedby a similar enzyme. The maximum ATPase activity is sufficientto account for all the proton flux observed during the stomatalopening response. Key words: ATPase, Commelina, guard cell protoplasts, phosphatase, vanadate     

A two-year study of the distribution of ''thermophilic'' campylobacters in human, environmental and food samples from the Reading area with particular reference to toxin production and heat-stable serotype

The incidence of 'thermophilic' campylobacters in foods and environmental samples has been studied over a two-year period. Of 781 environmental samples, 529 (67%) were found to contain campylobacters, and campylobacters were isolated from 835 (39%) of 2116 food samples. Sewage was almost always contaminated with campylobacters (96·6% of samples) and of the food samples both poultry (55·5%) and offal (47·0%) were commonly contaminated. Determination of the heat-stable serotypes of all strains isolated from these sources and of 921 strains isolated from human faeces showed that there was a wide distribution of serotypes in most types of sample. Serotype Pen 2 was the commonest type found in human faeces (18·9%) and it was also commonest in offal (21·3%), beef (40·0%), sewage (17·7%) and was the third commonest type in poultry. A comparison of culture media and conditions for optimal production of both cytotoxic and cytotonic enterotoxins showed that Brucella Broth incubated under microaerobic conditions for 24 h at 42°C was suitable for both toxins. Detection of cytotoxic activity was most sensitive using HeLa cells. The sensitivities of two ELISA systems and a Chinese Hamster Ovary tissue culture assay for detection of cytotonic enterotoxin were comparable. Not all strains isolated from cases of enteritis in human beings produced toxin; 23·1% produced cytotonic enterotoxin and 17·5% produced cytotoxin. There was no correlation between serotype and toxin production. The wide distribution of campylobacters, indistinguishable from those isolated from cases of enteritis in human beings, leads us to conclude that simplistic statements suggesting that one particular type of food is primarily responsible for cases of human disease should not be made.     

Serotyping of mesophilic Aeromonas spp. on the basis of lipopolysaccharide antigens

A serotyping system has been developed for Aeromonas hydrophila, A. sobria and A. caviae based on lipopolysaccharide (LPS) antigens. Antigens are detected by slide agglutination of boiled cells and the serotype is confirmed by tube agglutination. The antigens involved in the serotyping reactions were shown to be capable of sensitizing chicken red blood cells and were extractable by ethyl-enediaminetetraacetate. Furthermore, the reactions could be prevented by absorb-ing antisera with purified LPS. Using 16 antisera, 63 of 137 (46%) strains isolated from human faeces could be serotyped.     

A note on the effect of different storage procedures on the ability of Preston medium to recover campylobacters

The effect of different storage procedures on the ability of Preston medium to recover campylobacters was investigated. Freshly poured media was shown to recover more campylobacters than media stored under aerobic or anaerobic conditions at room temperature or at 4dEC. The growth of Campylobacter laridis was greatly reduced by storage of media and although most strains of C. jejuni and C. coli were not markedly affected, the growth of one strain of C. jejuni was considerably reduced. It is recommended that freshly prepared media be used whenever possible, but if storage is necessary, then plates should be held at 4dEC, preferably under anaerobic conditions. These precautions may not be necessary for workers interested solely in C. jejuni or C. coli , but are essential for the optimum isolation of C. laridis.     

A note on the isolation of salmonellas from environmental samples using three formulations of Rappaport's broth

The efficiency of isolation of salmonellas from 412 seagull intestines and 247 polluted water samples was compared using three formulations of Rappaport's medium (RV, RV-soya and R25). A modification of RV medium (RV-soya) was shown to be the most efficient of the three media and it was shown that the duration of incubation of this medium could be restricted to 24 h. Inoculation ratios were compared for RV and RV-soya and 1:100 was shown to be significantly better than 1:20. It is concluded that RV-soya is at least as efficient as the standard RV medium and its use can therefore be recommended.     

Stomatal Responses Measured Using a Viscous Flow (Liquid) Porometer

The design of a liquid flow porometer for the measurement ofstomatal responses in epidermal peels is described. The systemis computerized and allows automatic measurement of about 1000stomata (of Commelina communis L.) in three parallel experiments.Simultaneous microscopic observations of the stomata is alsopossible. Conditions are described for rapid measurement ofstomatal responses with greater resolution than that achievedby conventional microscopic measurement techniques. To determinewhich feature(s) of the stomatal pore influenced the flow ratethe most precise estimates of pore diameter and area were madeusing a cryo-fixation technique, scanning electron microscopyand image analysis. Flow rate showed a near linear dependenceon pore area. The LFP was evaluated for measuring stomatal responsesby comparison with conventional Petri-dish experiments. Responsesto KCl, CO₂, fusicoccin, and ABA were similar using the twomethods, though there was little or no response to light inthe LFP. Pore widths were also lower in the LFP under similarexperimental conditions. The probable causes of these phenomenaare discussed. Key words: Liquid flow porometer, stomatal responses, Commelina