Effect of the composition of reversion medium on change of Staphylococcus aureus lysostaphin protoplasts to coccal forms and L-forms

The experimental conditions under which protoplasts of Staphylococcus aureus strain MS353 (pCp) are converted to the coccal or L-form were investigated. Protoplasts prepared by treating coccal MS353 (pCp) strain with Lysostaphin formed various types of colonies (coccal form, L-form and mixed types) in about 50% yield when they were plated on reversion (R) medium consisting of 2% brain heart infusion, 0.5M sodium succinate, 0.01% bovine serum albumin, 20 mM MgCl2 and 0.6% agar. The L-form type colonies with a typical fried-egg appearance that developed on the R medium at an early stage gradually reverted to the coccal form through a mixed type stage in which a high density area first appeared in the periphery of the colony and then spread throughout the colony. The use of modified R medium without MgCl2 or R medium in which 0.5M sodium succinate as an osmotic stabilizer was replaced by 7.5% NaCl resulted in marked delay in the appearance of reverted cells. R medium without bovine serum albumin yielded atypical L-form type colonies, which contained masses of coccal cells with very irregular margins. On the other hand, R medium without MgCl2 but with penicillin G supported development of L-form type colonies at high rate (13-15%) from the inoculated protoplasts.     

Improved method for detection of Vibrio parahaemolyticus in seafood.

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.     

Listeria monocytogenes L Forms I. Induction, Maintenance, and Biological Characteristics

L forms were induced from 15 of 16 strains of Listeria monocytogenes on penicillin gradient plates incubated under aerobic conditions. The culture medium for maintenance of these L forms must contain an electrolyte in a concentration of 1% or sucrose in a concentration of 10%. The electrolytes NaCl, KCl, or MgSO(4) were used in both induction and maintenance media. Induction of L forms occurred more rapidly on media containing KCl. Listeria L forms had the same fermentation reactions as the parent bacterium. The L-form growth in liquid medium was slow, not extensive, and appeared as clumps on the bottom of culture tubes. The morphology of Listeria L forms was similar to that reported for other bacterial L forms. The L forms derived from strain 10403, serotype 1, were stable after two or more passages on penicillin media. They did not revert to the bacterial form after 40 subcultures on penicillin-free media. Some L-form colonies derived from strain 10403 did revert to the bacterial form when transferred directly from induction plates to penicillin-free media. Studies of the growth characteristics for L forms derived from strain 10403 gave the following results: an optimal temperature of 30 C, high electrolyte or sucrose concentration necessary for induction and maintenance, and no requirement for serum.     

Salt-Induced L-Forms of Staphylococcus aureus

When growing cultures of a salt-sensitive strain of Staphylococcus aureus were inoculated on nutrient agar containing 0.8 m NaCl and 0.5% bovine serum albumin, typical colonies of L-form developed extensively after 2 days of incubation at 30 C. Incubation of growing cultures with lipoteichoic acid, sodium polyanethole sulfonate and subtilisin resulted in inhibition of L-form induction.     

Serum inhibits penicillin-induced L-form growth in Staphylococcus aureus: a note of caution on the use of serum in cultivation of bacterial L-forms.

Heat-inactivated horse serum inhibited penicillin-induced L-form colony formation in Staphylococcus aureus when included in an osmotically stabilized culture medium. Most, perhaps all, L-form colonies that appeared with low frequencies on the serum-supplemented medium were of the penicillin-independent, stable type. This relationship must be taken into account when use of serum is considered for L-form cultivation.     

Cell fusion between L-forms and protoplasts of Staphylococcus aureus

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.     

Effect of medium composition on the susceptibility of oral streptococci to mercuric chloride

Although there is considerable interest in identifying mercury-resistant bacteria, no standardized assay exists for this purpose. In this study, the effect of the composition of the medium on the susceptibility of oral streptococci to HgCl₂ was investigated. The minimum inhibitory concentration (MIC) of HgCl₂ for 52 streptococcal strains and the reproducibility of MIC values for Hg-sensitive and Hg-resistant strains was determined with 11 different media. Addition of blood increased the MIC values, and some media (tryptone soya agar, with or without blood) could not discriminate between Hg-sensitive and Hg-resistant strains. The proportion of streptococci that appeared to be resistant to Hg was very high (>70%) on some media (mitis-salivarius, tryptone soya, Columbia), but not on others (Mueller-Hinton, Brain Heart Infusion, Isosensitest). The MICs of the control strains varied considerably on different testing occasions for tryptone soya agar (with and without blood), Isosensitest agar, and Columbia agar (with blood). Mueller-Hinton (without blood) appeared to be the most suitable medium for isolating Hg-resistant oral streptococci. Received: 21 December 2001 / Accepted: 17 January 2002     

Improved Method for Detection of Vibrio parahaemolyticus in Seafood

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.     

Studies in the differentiation between Microsporum ferrugineum Ota and Trichophyton soudanense Joyeux

A study, conducted with 20 isolates of Microsporum ferrugineum and 12 isolates of Trichophyton soudanense, revealed that some of the discrepancies in the literature regarding their characteristics and differentiation were due to methodology, strain variation and the use of an insufficient number of isolates. We found all isolates of T. soudanense to be urease negative and gelatinase positive (usually by the first week); to produce brown to black colonies on Lowenstein-Jensen medium; to rapidly decompose casein and more slowly tyrosine; to grow well or better at 37°C as compared to room temperature; to produce reflexive branching on cornmeal Tween agar and abundant microconidia on casero medium and to exhibit no sexual reaction with either mating type of Arthroderma simii. All but one isolate demonstrated restricted growth on lactose agar and only three isolates perforated hair.In contrast, we found 18 of 20 isolates of M. ferrugineum to be urease positive in urea broth (most isolates were negative on urea agar); all produced light-colored colonies on Lowenstein-Jensen medium; spreading colonies on lactose agar and failed to perforate hair in vitro or to produce reflexive branching. Most isolates manifested poorer to no growth at 37°C compared to room temperature and all but one failed to decompose casein and tyrosine. A few strains produced macroconidia and/ or microconidia on casero medium and some reacted sexually with A. simii (a) or (–) mating type. Gelatin hydrolysis was variable.We suggest the following key tests to differentiate M. ferrugineum from T. soudanense: urease activity in urea broth; colony color on Lowenstein-Jensen medium; growth on lactose agar; growth at 37° C compared to room temperature; presence of reflexive branching on cornmeal Tween agar.     

Regeneration of protoplasts of Bacillus subtilis 168 and closely related strains

Regeneration of protoplasts to bacilli was attempted in several strains of Bacillus closely related to Bacillus subtilis 168. On DM3 and similar media using succinate as osmotic support, only B. subtilis 168 and Bacillus natto ATCC 15245 were able to regenerate. Media containing mannitol as osmotic support, and agar as gelling agent gave rise to L-form colonies with Bacillus licheniformis NCTC 6346. Many of the L-form colonies were able to regenerate to the bacillary form when plated on the mannitol medium solidified with gelatin. All of the Bacillus species tested were able to regenerate on the latter medium at rates sufficient to allow protoplast transformation and fusion experiments.     

Experimental Salmonellosis

Mice were infected with smooth or rough strains of Salmonella enteritidis and Salmonella typhimurium and viable bacterial cells found in the liver of the inoculated animals were enumerated by plating homogenates of tissues on nutrient agar plates containing 0.35 M sucrose. Some rough strains of these Salmonella were recovered in the bacteria seen on these plates and appeared able to form colonies only on the sucrose-containing medium but not on an identical medium without added sucrose. This population did not appear in the liver of animals until at least 24 hr after infection. The number of bacteria capable of forming colonies only on the hypertonic medium was found to vary with the time after infection and the strain of bacteria used for infection. From the results of morphological examination of cells of the colonies developing on the hypertonic plates, these bacterial forms were thought to result from unstable L forms in the infected tissues. Possible processes of the formation of these L forms in vivo and their significance in induction of anti-infectious immunity are discussed.     

细菌L型的厌氧诱导和培养

厌氧条件下以羧卡青霉素诱导金黄色葡萄球菌、大肠杆菌和蜡样芽胞杆菌形成Ｌ型,观察细菌Ｌ型在厌氧条件下的形成、形态、生长及时渗透压的敏感性等特性。结果表明：蜡样芽胞杆菌在厌氧条件下不能形成Ｌ型或其Ｌ型在厌氧条件下亦不能返祖。金黄色葡萄球菌和大肠杆菌在厌氧条件下虽能诱生Ｌ型,但形成丝状体的构成Ｌ型菌落难以传代培养,厌氧培养未见Ｌ型圆球体和典型Ｌ型油煎蛋样菌落。金黄色葡萄球菌Ｌ型在含１％～１０％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体;大肠杆菌和蜡样芽胞杆菌的Ｌ型在含２％～６％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体。涂片染色或返祖试验证实细菌Ｌ型在含０．５％ＮａＣｌ的Ｌ型培养基或常规细菌学培养基上亦可生存。非菌落性Ｌ型巨形体和丝形体是细菌Ｌ型在琼脂培养基上广泛的存在形式。     

Morphology of Ureaplasma urealyticum (T-mycoplasma) organisms and colonies.

The morphology of Ureaplasm urealyticum in broth cultures was studied by phase-contrast microscopy. Most organisms appeared singly or in pairs. Long filaments and long chains of cocci, common in classical mycoplasma cultures, were not observed. On solid medium, U. urealyticum produced "fried-egg" colonies which developed according to the scheme suggested by Razin and Oliver (J. Gen. Microbiol., 1961) for the morphogenesis of the classical mycoplasma colonies. The formation of the peripheral zone of the colonies followed that of the central zone only when growth conditions were adequate, Hence, the appearance of peripheral zones, and consequently the larger colony size, can be taken as an indicator of improved growth conditions. Incubation in an atmosphere of 100% CO2 resulted in significantly larger colonies than in an atmosphere of N2, O2, or air. CO2 acts as a buffer, keeping the pH at the optimal range for Ureaplasma growth (pH 6.0 to 6.5) in the presence of the ammonia produced from the urea hydrolyzed by the organisms. The addition to the medium of 0.01 M urea together with 0.01 M putrescine enabled better growth than with urea alone. Small amounts of phosphate improved growth in an atmosphere of CO2, apparently fulfilling a nutritional role. Under nitrogen, higher phosphate concentrations were required for good growth, apparently serving as a buffer as well as a nutrient. Sodium chloride and sucrose which had been added to increase the tonicity of the medium inhibited growth above 0.1 M. An increase in the agar concentration above 2% resulted in decreased colony size. Likewise, prolonged drying of the agar plates caused a marked decrease in colony size, mostly affecting the peripheral zone. The addition of both urea and putrescine to the growth medium and incubation in a humidified CO2 atmosphere are recommended for improved growth and formation of fried-egg colonies of U. ureaplyticum on agar. It must be emphasized that these experiments were carried out with a laboratory-adapted strain.     

Note: Comparison of media for the isolation of lactose-positive Salmonella

We studied the capacity of 10 selective media (Rambach agar, RB; salmonella-shigella agar, SS; SM-ID medium, SM; Hektoen enteric agar, HE; modified semisolid Rappaport-Vassiliadis agar, MSRV; bismuth sulphite agar, BS; MacConkey agar, MC; brilliant green agar, BG; novobiocin-brilliant green-glucose agar, NBG; and novobiocin-brilliant green-glycerol-lactose agar, NBGL), and the C₈-esterase test (MUCAP test, Biolife, Italy) to detect the growth of 14 strains of lactose-positive Salmonella (12 Salm. virchow and two Salm. montevideo ) and 16 Salm. arizonae. Suspensions of pure strain were plated on the aforementioned media and on Mueller-Hinton, used as a control, with inocula of 3 x 10² cfu ml^-1. The performance of BS was excellent, determining the 30 strains as typical Salmonella colonies (H₂S+). On NBG, 27 strains were detected. On MSRV, only some strains grew and only one produced swarming. On the other media, the two Salm. montevideo and the 12 Salm. virchow strains produced coliform colonies. Some of these latter were inhibited on BG and NBGL. The 16 Salm. arizonae strains produced typical colonies on all the media, except on RB, SM and MSRV. On NBGL, two strains did not produce H₂S. The C₈-esterase test was only successful with Salm. montevideo and Salm. virchow on NBG and RB (with a few exceptions on the latter). However, with Salm. arizonae the test was positive on SS, MC, HE, BG and NBG. In summary, BS was the best medium of those used (all the 30 strains were isolated), followed by NBG (27 isolates).     

Development of Streptococcal L-Form Colonies

The development and architecture of L-form agar colonies produced from protoplasts and L-phase bodies were studied by both light and scanning electron microscopy. Agar blocks containing L-phase microcolonies of group A Streptococcus strains ADA and GL8 and group D Streptococcus strain F24 as well as longitudinal sections of mature colonies were used as samples. Initially, granules of about 0.5 mum in diameter were produced by multiple condensation and fragmentation of protoplasts and large bodies. Surface growth by granules ensued and infiltration into agar occurred only after 10 to 11 hr of incubation at 37 C. Club-shaped granules were noted and division seemed to take place by simple fission. The configuration of large bodies and granules in mature colonies suggested budding as another means of replication. Acellular spaces inside the colonies appeared to have been formed by lysis of large bodies or by the envelopment of space by the extending growth of minute granules. Whereas no significant strain variation was noted in colonies of less than 24 hr of incubation, fully mature colonies were differentiated on uniform media.     

Growth of Helicobacter pylori in various liquid and plating media

The objectives of this research were to compare commonly used liquid and plating media to elucidate whether one medium provided superior growth of Helicobacter pylori in vitro. The liquid media compared were Mueller-Hinton broth, brain heart infusion broth and H. pylori special peptone broth, formulated in this laboratory. No significant differences in growth rates were noted and shaking during the incubation of broths was not essential for good growth. The plating media compared included Columbia agar, Mueller-Hinton agar, modified Glupczynski's Brussels campylobacter charcoal agar, Johnson-Murano agar and H. pylori special peptone agar (HPSPA). None of the non-specific plating media that have been used historically to culture H. pylori exhibited any particular advantage. However, HPSPA provided an obvious advantage in colony size. Helicobacter pylori special peptone agar enhances the cultivation of H. pylori and could improve the recovery of the bacterium from clinical samples in vitro.     

Calcium and magnesium in Mueller-Hinton agar and their influence on disk diffusion susceptibility results

Concentrations of calcium and magnesium were determined for 39 lots of media, including broth and agar media used for susceptibility tests and plain agar. In addition, the effect that media with and without physiological levels of these divalent cations would have on the disk diffusion susceptibility of 21 strains ofPseudomonas aeruginosa to four antimicrobics was also ascertained. Mueller-Hinton agar showed a wide variation in calcium and magnesium content. Mueller-Hinton broth contained lower concentrations of calcium and magnesium, and there was little lot-to-lot variation. Lots of Mueller-Hinton agar with higher concentrations of calcium and magnesium yielded smaller zone diamaters than those with lower concentrations. Even at equal cation concentration, zones of inhibition varied from lot to lot. Since the addition of calcium and magnesium to Mueller-Hinton agar to obtain a predetermined level did not result in equivalent zone diameters, performance testing of susceptibility media is recommended.     

Inhibitory Action of Various Plating Media on the Growth of Certain Salmonella Serotypes

The growth of 68 strains of Salmonella typhi , 697 other Salmonella strains and 213 strains of other Gram negative intestinal bacteria on 8 plating media was assessed semi-quantitatively. These media were found to be differentially inhibitory to different Salmonella serotypes. The combined use of two plating media, brilliant green MacConkey agar and deoxycholate citrate agar, allowed potentially the recovery of the maximum number of Salmonella strains. If only one medium was used, brilliant green MacConkey agar would appear to be the best plating medium for the isolation of non-typhoid salmonellas in general and S. choleraesuis in particular. Xylose lysine deoxycholate agar, on which a certain proportion of salmonellas failed to yield typical, recognizable colonies, was found not to be a good plating medium for their isolation.     

A stable L-form of the fish pathogen Aeromonas salmonicida

A stable L-form of Aeromonas salmonicida , which resulted from induction with benzylpenicillin, grew on brain heart infusion agar at 0–5°C. The L-form was stored successfully for 10 months in phosphate-buffered saline supplemented with 150 g l^-1 glycerol at -70°C. Reversion of the L-form to parental-type walled cells was achieved by transfer to brain heart infusion broth with incubation at 25°C.     

Induction of L-Phase Variant from Protoplast of Staphylococcus aureus

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature. Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCl) formed typical L-form colonies. Horse serum (0.25%) and Mg²⁺ (109 mm) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan 1, Mg²⁺ was not required. The active factor(s) in horse serum was heat-resistant and protein in nature.