The environmental and microbiological factors that can influence heavy metal toxicity are discussed with a view to understanding the mechanisms of microbial metal tolerance. It is apparent that metal toxicity can be heavily influenced by environmental conditions. Binding of metals to organic materials, precipitation, complexation, and ionic interactions are all important phenomena that must be considered carefully in laboratory and field studies. It is also obvious that microbes possess a range of tolerance mechanisms, most featuring some kind of detoxification. Many of these detoxification mechanisms occur widely in the microbial world and are not only specific to microbes growing in metal-contaminated environments.

Cefuroxime is a new broad-spectrum cephalosporin antibiotic with increased stability to f8-lactamases. This stability, although no absolute in all cases, has the effect of widening the antibacterial spectrum of the compound so that many organisms resistant to the established cephalosporins are susceptible to cefuroxime. It is active against gram-positive organisms, including penicillinaseproducing staphylococci, but it is less active against methicillin-resistant strains. In addition to its high activity against non-,8-lactamase-producing gram-negative bacteria, cefuroxime effectively inhibits the growth of many 1B-lactamase-producing strains, including Enterobacter, Klebsiella, and indole-positive Proteus spp. It is highly active against Neisseria gonorrhoeae, Neisseria meningitidis, and also Haemophilus influenzae, including ampicillin-resistant strains. Cefuroxime is rapidly bactericidal and induces the formation and subsequent lysis of filamentous forms over a small concentration range.

A mechanism of mammalian teratogenesis involving inhibition of embryotrophic nutrition is suggested and exemplified by the action of trypan blue on pregnant rats. There is evidence for the localization of trypan blue in heterolysosomes of the epithelium of the visceral yolk sac, and our experiments indicate that the dye is an inhibitor of a selection of hydrolytic enzymes present in lysosomal fractions from homogenates of rat visceral yolk sac. It seems likely that trypan blue inhibits the intracellular digestion of embryotroph by the visceral yolk-sac epithelium; the conceptus may therefore be deprived of essential nutrients at critical stages of development.

SYNOPSIS. Some aspects of the physiology of encystment of the soil amoeba Hartmannella castellanii in a replacement encystment medium consisting of 5 × 10‐2 M MgCl2 have been investigated. It is suggested that measurement of the cellulose produced during encystment in the synthesis of the cyst wall is a more reliable measure of the process than other methods tried. The degree of encystment was dependent on the physiologic state of the amoebae and the composition of the growth medium, but the initial pH of the encystment medium (C. 4.0‐8.5) had little effect on the process. The requirement for Mg during encystment was probably not due to its deficiency during growth. Encystment was inhibited to varying extents by inhibitors of protein synthesis, tetracycline and chloramphenicol and also by arsenate, arsenite and iodoacetate; sodium fluoride, malonate and 2, 4‐dinitrophenol were without marked effect. Addition of glucose and α‐ketoglutarate to the replacement medium led to improvement in the encystment response. The presence of glutamate and histidine during encystment led to cell death. Other carbon and nitrogen sources had no effect. During encystment there was an increase in the metabolic activity of the amoebae, as measured by their oxygen consumption. This was accompanied by a decrease of about 40% in cellular dry weight and protein content. Of the other chemical components, there were marked initial increases in the levels of total carbohydrates and pentose which were followed by their depletion during cellulose synthesis. Encystment was completed after about 64 hr when the synthesis of cellulose was complete and the oxygen uptake of the amoebae fell to an immeasurable level.

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1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).

The structure and behaviour of cysts of a single strain of Acanthamoeba castellanii produced in different cultural conditions were investigated. There appeared to be a gradation from the type of cysts with a wrinkled cyst wall, which were produced in monoxenic culture and in replacement medium, to the smooth, refractile cells, lacking a distinct wall, which were produced in a growth medium supplemented with MgCl,. All the cyst types exhibited depressed metabolic activity and only differed in the degree to which they were resistant to heating and low temperatures. The results are discussed in relation to the use of cyst structure as a criterion for classifying the various species of Acanthamoeba.The cysts produced by amoebae at various stages of their life-cycles, particularly under conditions of starvation, are features of some taxonomic importance. This is especially true for the small soil and freshwater amoebae of the genera Acanthamoeba, Hartmannella and Mayorella. Despite reports that there can be 'considerable' variation in the form of the mature cysts, relatively small differences in their structure are still important criteria for the identification of some species of Acanthamoeba (Page, 1967(Page, , 1976.In this study a comparison has been made of the cysts produced by a strain of A. castellanii under different conditions of axenic and monoxenic culture, and the results are discussed in relation to the use of cyst structure as a taxonomic criterion. M E T H O D SThe organism and its culture. The Neff isolate of Acanthamoeba castellanii was obtained from Mrs K. M. G.Adam, Zoology Department, University of Edinburgh, and was grown axenically in 4 % (w/v) Mycological Peptone (Oxoid). Cultures (50 ml) were incubated (30 "C) in 250 ml conical flasks on a shaking water-bath and subcultures were made with 5 ml from a 2 d culture. (iii) by adding MgClz (final concentration 50 mM) to PGY growth medium [consisting of (g 1-l): Difco Proteose Peptone, 7-5; Difco yeast extract, 7.5; glucose, 15-01; and (iv) following growth in monoxenic culture. In the monoxenic cultures the amoebae were grown with Klebsiella aerogenes on a solidified medium containing (g 1-l): Oxoid Bacteriological Peptone, 1.0; Oxoid yeast extract, 0.1 ; glucose, 1.0; Oxoid agar no. 2, 20.0. An overnight culture of the bacteria was grown on the above medium (without the agar) and 0.1 ml of this was mixed with 0-05 ml of a 2 d amoeba culture which had been grown axenically as described above. The mixed suspension was spread over the surface of the solidified medium and incubated for 5 to 6 d. The cysts were then collected by flooding the plates with sterile distilled water and scraping the surface of the agar with a glass spreader. All cultures were incubated at 30 "C.Electron microscopy. Cysts were fixed for 3 h in glutaraldehyde (3 %, w/v, in 100 mM-phosphate buffer, pH 6.8) and postfixed for 1.5 h in osmium tetroxide (2 %, w/v, in 100 mM-phosphate buffer, pH 6.8).

SUMMARYComplete encystment of Acanthamoeba occurred in peptone-yeast extractglucose medium supplemented with 50 mwmagnesium chloride. There was an increase in the RNA, protein and DNA contents of the cells which coincided with an increase in the proportion of binucleate amoebae and commitment to encystation, suggesting that encystation could be a consequence of the inhibition of cell division when nutrient concentrations were limiting growth.