Induction of stable L-forms of Staphylococcus aureus and Streptococcus faecalis

Stable L-forms were induced from Staphylococcus aureus and Streptococcus faecalis. These formed typical foamy L-colonies and showed large and small round bodies. They grew continuously on routine antibiotic-free nutrient broth and blood agar media for 12 passages without reversion to their parental forms. At different concentrations of penicillin various morphological forms were observed. Effect of sucrose, normal horse serum and penicillin on their adaptation and stabilization is discussed.     

Morphology and Reproductive Processes of the L Forms of Bacteria II. Comparative Study of L Forms and Mycoplasma with the Electron Microscope

Representative electron micrographs, from the study of eight strains of L forms and one strain of Mycoplasma, are presented. A- and B-type L forms were derived from two strains of Proteus, two other L forms were derived from a diphtheroid and from a staphylococcus strain, and two strains (designated as LX) were isolated from L forms derived from a group A beta-hemolytic streptococcus and from a staphylococcus. The Mycoplasma strain was isolated from goats. Sections were made of young colonies grown within agar and from parts of surface colonies embedded in the agar. B-type L colonies of Proteus were produced by inoculation of bacteria into media containing penicillin. The large bodies developing from the bacteria and the organisms in B-type L colonies of Proteus, like the parent bacteria, had a cell wall consisting of a plasma membrane and an outer cell wall. The loss of rigidity in the cell wall indicated an alteration in its structure. The A-type L cultures of Proteus consisted of irregular branching masses extending in several directions, of small dense organisms corresponding to the elementary corpuscles present in cultures of Mycoplasma, and of intermediary forms. In contrast to the B-type, all organisms in the A-type colonies were surrounded by a single unit membrane corresponding to the plasma membrane of bacteria. The structures inside the cell membrane, both in the A- and B-type, seemed to correspond to the structure of the parent bacteria, which contained ribosomes and threads of DNA. The elementary corpuscles formed chains and filaments, and, apparently, these corpuscles took part in the multiplication by gradual enlargement. The organisms seen in the cultures of all L forms and Mycoplasma studied, except in the B-type L forms of Proteus, corresponded in size, shape, and structure, as well as in the development of elementary corpuscles, to the organisms in the A-type L form of Proteus. In contrast to the spherical organisms usually seen in broth cultures, the organisms in young cultures of Mycoplasma, which were grown within the agar, were similar in morphology, as well as in the discernible structure of the organisms, to L forms. Significant morphological and structural differences were not apparent between the L forms and Mycoplasma (in cultures grown within agar media) under the conditions of this investigation.     

Permanent alterations of the L-forms of proteus and salmonella under various conditions

L-forms obtained from three strains of Proteus and from one strain of Salmonella have been kept for 15 to 20 years by weekly or monthly transfers on agar plates containing penicillin. The morphology and growth requirements of these strains have changed. They now grow abundantly on the surface of agar and in broth. The cultures consist of large bodies, small granules, and transitional forms. These organisms are more resistant to distortion and stain more deeply than organisms of the usual L-forms. In broth and to a lesser extent on agar, branching filaments develop, on the ends of which both the large, round organisms and small organisms are produced. The filaments are a transitional stage in the development of the cultures. Usual bacillary forms were not present in the culture and did not appear in successive transfers in the absence of penicillin. Bacilli reappeared on exposure of the L cultures to the influence of a spore-bearing bacillus. A similar transformation of L-forms has also been observed developing within a short time in recently isolated A and B type L cultures of one Proteus strain during the process of reversion to the bacterial form. The altered cultures are fixed in a stage of transition between the B type L-form and the regular bacteria.     

Colonial Morphology of Treponemes Observed by Electron Microscopy

The colonial morphology of three strains of cultivable, nonpathogenic treponemes including a human oral treponeme was examined by light and electron microscopy. Treponema phagedenis strains Kazan and Reiter produced large white colonies on the surface of solid media composed of sterility test broth, 0.9 to 3.1% agar, rifampin, and 12.5% rabbit or horse serum. A human oral treponeme, strain G7201, grew as diffused white zones on 0.9 to 3.1% agar plates. Under the cultural conditions employed agar concentrations slightly affected the time of appearance of colonies of the three strains of treponemes. When the colonies of these three strains were viewed by scanning electron microscopy, differences in their colonial morphology were observed. The 11-day-old colonies of human oral strain G7201 were very small, 5 to 15 μm in diameter, and had a slight irregular border. Kazan treponemes developed circular, entire and low convex colonies. Scanning and transmission electron microscopy revealed that the colonies of Reiter treponemes contained spherical forms almost up to 5 μm in diameter, each consisting of an outer membrane and a treponemal main body. They were very similar to the spherical bodies produced by strain G7201 in sucrose-containing broth.     

CULTURE OF HUMAN GENITAL "T-STRAIN" PLEUROPNEUMONIA-LIKE ORGANISMS.

Ford, Denys K. (University of British Columbia, Vancouver, Canada). Culture of human genital "T-strain" pleuropneumonia-like organisms. J. Bacteriol. 84:1028-1034. 1962.-The conditions under which "T-strain" pleuropneumonia-like organisms, as described by Shepard, are best cultured were investigated. The organisms were found to grow on several types of nutrient agar and broth, of which PPLO medium supplemented with yeast extract and horse serum was the simplest. Subculture was possible through broth cultures, provided the broths were not incubated longer than 16 hr. The organisms on agar required either Fortner's anaerobic atmosphere or 10% CO(2), but broth cultures grew aerobically. "T-strains" grew over a pH range of 6.8 to 7.8, and a temperature range of 30 to 36 C. They were viable after storage for 16 days at 4 C and for 90 days at -20 C, and they resisted lyophilization. They were sensitive to 1.5 mug per ml of tetracycline and streptomycin, but were resistant to ampicillin and penicillin. Quantitative studies showed maximal concentration in broth of 10(6) to 10(7) organisms per ml, and logarithmic multiplication for the first 12 hr of broth culture, with a subsequent rapid decline in number. Colonial morphology was maintained after numerous subcultures.     

Induction and Propagation of a Bacillus subtilis L Form in Natural and Synthetic Media

The L form of Bacillus subtilis NRRL B-3275 was induced in a 7% NaCl broth medium and subsequently propagated in natural and synthetic media. The L form grew readily in tryptone broth supplemented with glucose, NaCl, and phosphate buffer, and in a synthetic medium containing only glucose and biotin, in addition to the required salts. Successive transfers from the bacillus inoculum and subsequent large bodies in the tryptone broth with 7% NaCl resulted in gradual selection or transition from the bacillary form to a stable L form without the addition of an antibiotic. The number of viable granules attained in the broth culture exceeded 9 x 10(7) per ml, and numerous large bodies were always present in rapidly growing cultures.     

Multiplication of different Legionella species in Mono Mac 6 cells and in Acanthamoeba castellanii.

Survival and distribution of legionellae in the environment are assumed to be associated with their multiplication in amoebae, whereas the ability to multiply in macrophages is usually regarded to correspond to pathogenicity. Since most investigations focused on Legionella pneumophila serogroup 1, we examined the intracellular multiplication of different Legionella species in Mono Mac 6 cells, which express phenotypic and functional features of mature monocytes, and in Acanthamoeba castellanii, an environmental host of Legionella spp. According to the bacterial doubling time in Mono Mac 6 cells and in A. castellanii, seven clusters of legionellae could be defined which could be split further with regard to finer differences. L. longbeachae serogroup 1, L. jordanis, and L. anisa were not able to multiply in either A. castellanii or Mono Mac 6 cells and are members of the first cluster. L. dumoffi did not multiply in Mono Mac 6 cells but showed a delayed multiplication in A. castellanii 72 h after infection and is the only member of the second cluster. L. steigerwaltii, L. gormanii, L. pneumophila serogroup 6 ATCC 33215, L. bozemanii, and L. micdadei showed a stable bacterial count in Mono Mac 6 cells after infection but a decreasing count in amoebae. They can be regarded as members of the third cluster. As the only member of the fourth cluster, L. oakridgensis was able to multiply slight in Mono Mac 6 cells but was killed within amoebae. A strain of L. pneumophila serogroup 1 Philadelphia obtained after 30 passages on SMH agar and a strain of L. pneumophila serogroup 1 Philadelphia obtained after intraperitoneal growth in guinea pigs are members of the fifth cluster, which showed multiplication in Mono Mac 6 cells but a decrease of bacterial counts in A. castellanii. The sixth cluster is characterized by intracellular multiplication in both host cell systems and consists of several strains of L. pneumophila serogroup 1 Philadelphia, a strain of L. pneumophila serogroup 2, and a fresh clinical isolate of L. pneumophila serogroup 6. Members of the seventh cluster are a strain of agar-adapted L. pneumophila serogroup 1 Bellingham and a strain of L. pneumophila serogroup 1 Bellingham which was passaged fewer than three times on BCYE alpha agar after inoculation and intraperitoneal growth in guinea pigs. In comparison to members of the sixth cluster, both strains showed a slightly enhanced multiplication in Mono Mac 6 cells but a reduced multiplication in amoebae. From our investigations, we could demonstrate a correlation between prevalence of a given Legionella species and their intracellular multiplication in Mono Mac 6 cells. Multiplication of members of the genus Legionella in A. castellanii seems to be dependent on mechanisms different from those in monocytes.     

Intracellular Production of Brucella L Forms I. Recovery of L Forms from Tissue Culture Cells Infected with Brucella abortus

Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. I. Recovery of L forms from tissue culture cells infected with Brucella abortus. J. Bacteriol. 91:285-296. 1966.-Infectivity of virulent Brucella abortus strain 3183 was less for hamster macrophages after a 2-hr adsorption period than for an attenuated strain (S19) and its tissue culture variant (30). Both strains S19 and 30 were very toxic for the cells, but 3183 was not toxic. Two types of L forms were recovered from a large percentage of hamster kidney cell cultures when disintegration of infected cells was accelerated by tissue culture medium of high pH. One type grew in finely granular microcolonies, was isolated from cells infected for short periods of time, and often reverted to the bacterial form. The other type occurred in small irregularly shaped forms which later developed into round bodies. Both stained specifically with fluorescein-conjugated B. abortus antiserum. Semisolid media containing 0.7% agar provided optimal subsurface L-form growth. L forms also grew well in Thioglycollate Medium but grew poorly in other liquid media. Surface L-form growth was supported by several agar media, but CO(2) was required for optimal growth. Monolayers infected with strain 3183 and examined immediately after adsorption contained occasional small, round bodies. Bizarre forms increased in number with time and, after 24 to 72 hr, large pink-staining inclusions were often present which persisted for several days. Also appearing at about the same time were smaller, dark-staining forms which were first seen in clusters but later dispersed and finally occurred in chainlike configurations. Direct fluorescent-antibody stains of infected cells established that the intracellular forms were related to the infecting strain of B. abortus.     

Mycoplasma pneumoniae and Mycoplasma salivarium: Electron Microscopy of Colony Growth in Agar

Colonies of Mycoplasma pneumoniae and Mycoplasma salivarium grown in PPLO agar were examined by light and electron microscopy. The main objective of the investigation was to attempt in situ fixation and minimize tonic changes in the organisms. Microscopy revealed that both organisms grew both in and upon the agar. The agar and surface growths of M. pneumoniae exhibited similar profiles, whereas those of M. salivarium differed strikingly. Both organisms are highly pleomorphic, but their matrix was denser and appeared more intact than in previously reported profiles. Cells which resemble the commonly reported mycoplasma were occasionally observed. The significance of these discrepant profiles remains unanswered. It is suggested that they may represent aged or osmotically damaged cells.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

Ultrastructure of Mycoplasma hominis.

Anderson, Douglas R. (National Cancer Institute, Bethesda, Md.), and Michael F. Barile. Ultrastructure of Mycoplasma hominis. J. Bacteriol. 90:180-192. 1965.-Both thin-sectioning and negative staining were used in an electron microscopic study of the morphology of pleuropneumonia-like organism (PPLO) strain HEp-2 (Mycoplasma hominis, type I) grown in an artificial liquid medium. The morphology is quite variable and seems to depend, in part, on the age of the culture. The smallest form observed ("elementary body") is 80 to 100 mmu in diameter. The internal components of the larger PPLO cells (0.5 to 1 mu) are variable-some have ribosomelike granules and nuclear areas of netlike strands, and others have only irregular dense areas in a pale groundplasm. Some of the forms have dense cytoplasmic bodies which look much like elementary bodies. Others have vacuoles which may contain structures which look like smaller organisms. Especially in older cultures, very large (10 mu) vacuolated organisms are seen, probably corresponding to the "large bodies" described by light microscopists. Filamented forms are also seen. These observations suggest several possible modes of reproduction, each perhaps operating under different cultural conditions or at different ages of the culture.     

Evidence for a Viral Macronuclear Endosymbiont in Paramecium caudatum

ABSTRACT. Some strains of P. caudatum contain macronuclear inclusion bodies that are morphologically distinct from bacteria. They vary in number as well as in size in each macronucleus. The inclusion bodies are basically divided into peripheral and inner areas. The peripheral area consists of fibrillar proteins of 22–24 nm in thickness, which are specifically stained with fast green in 45% acetic acid. On the other hand, chromatin-like granules are within the inner area of large inclusion bodies. The granules within the inner area changed their distribution depending upon the physiological state of their host cells. Transplantation experiments and crossbreeding analyses revealed that genetic factors responsible for the multiplication of the inclusion bodies can 'infect' other macronuclei (or cells) via the cytoplasm. These results suggest that the inclusion bodies are a non-bacterial macronuclear endosymbiont, possibly produced by a virus or a virus-like element.     

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.     

Analysis of colonies developing in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum

An analysis has been made of cell colonies developing in agar cultures from mouse bone marrow cells following stimulation either by neonatal kidney cell feeder layers or AKR lymphoid leukemia serum. Colonies arose by cell proliferation and were mixtures of granulocytic and mononuclear cells. Colonies stimulated by kidney feeder layers reached a mean size of 2000 cells by day 10 of incubation and remained predominantly granulocytic in nature. When bovine serum was substituted for fetal calf serum, cell colonies grew to a smaller size and lost their granulocytic nature, finally becoming almost pure populations of mononuclear cells. Colonies stimulated by AKR leukemic serum reached a mean size of 350 cells by day 10 of incubation. Although these colonies initially were granulocytic in nature, they finally became almost pure populations of mononuclear cells. The colony mononuclear cells actively phagocytosed carbon, and contained metachromatic granules probably derived from ingestion of agar. The mononuclear cells in these colonies may not have been members of the original colony, but may have been incorporated in the colony as it expanded in size, subsequently proliferating in the favourable environment of the colony.     

Ultrastructure of Brucella abortus L-forms induced by penicillin in a liquid and in a semisolid medium

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.     

Selection of Media for the Isolation of Common Bacterial L-Phase Organisms from a Clinical Specimen

The L-phase of 13 bacteria commonly associated with disease were induced by penicillin and inoculated into various solid and broth media; their growth was recorded for a period of 14 days. Plates containing highly purified agar and sucrose as the stabilizing agent and those incubated under aerobic conditions gave the best results. Magnesium seems to be necessary for growth in broth media on primary isolation, although it may not be necessary on multiple transfers after a more stable state has been reached. Growth in broth media is much more difficult to achieve. Reversion is aided by using a higher concentration of agar in plates, by decreasing the sucrose concentration, and by omitting the antibiotics and horse serum. A procedure has been outlined for the routine culture and identification of L-phase organisms from a clinical specimen.     

Emulsification of Hydrocarbon Fermentation Broth by Surfactants

In order to perform hydrocarbon fermentation satisfactorily, it was necessary to get the sample representing the real feature of the broth. However, Corynebacterium hydrocarboclastus S10B1, which could accumulate a good deal of l-glutamic acid, formed a coagulated mass of cells like pellet as they grew up, making the broth heterogeneous, To obtain homogeneous broth, addition of some kinds of surfactants was examined. It was made clear that addition of higher alcohol ethers of polyoxyethylene having H.L.B. value of about 15 was effective and emulsified broth could be obtained. Thus time course studies could be performed in hydrocarbon fermentation.     

Atkinsiella awabi sp. nov. isolated from stocked abalone,Haliotis sieboldii

A fungal disease in the abalone,Haliotis sieboldii, stocked in Yamaguchi Prefecture, Japan, showed external signs of infection of tubercle-like swelling on the mantle and melanized lesions on the peduncle. The fungus responsible was isolated by inoculating materials taken from the lesions onto PYGS agar with streptomycin sulphate and ampicillin, and incubation at 20°C. For morphological observation and spore formation study, the fungus was transferred respectively into PYGS broth and sterilized artificial seawater and incubated at 20°C. Resulting, hyphae were stout, irregular, branched, 16–140µm diam, sporadically consisting of dense cytoplasmic swollen hyphae. Sporangia were formed through the formation of septa and lateral or terminal discharge tubes which were wavy or coiled. Zoospores were pyriform, biflagellate and diplanetic. The encysted spore generally developed a hairlike filament with globular enlarged tip in PYGS broth. Direct germination without filament formation also occurred occasionally. This fungus was identified as belonging to the genusAtkinsiella, and was designatedAtkinsiella awabi sp. nov. The fungus was exclusively a marine fungus and grew best in shrimp extract medium at 20°C. Five chemicals were tested for their effects against fungal zoospores.     

A NOTE ON THE USE OF ROGOSA AND MODIFIED ROGOSA MEDIA FOR THE ENUMERATION AND CULTURE OF LACTOBACILLI

SUMMARY: The modified Rogosa media (acetate broth and acetate agar) developed by Mabbitt & Zielinska (1956) have been compared with tomato juice media for the growth of representative Lactobacillus species.
Most of the species tested grew markedly better in acetate broth than in tomato dextrose broth, but some cultures did not grow well in the acetate broth although they grew normally in tomato dextrose broth. The acetate agar was satisfactory for counting those lactobacilli cultured at 30° but was inferior to tomato dextrose agar for counting those grown at 37°.     

Comparison of seven plating media for enumeration of Listeria spp.

The suitability of seven media for the enumeration of Listeria spp. was evaluated at 30 degrees C for 48 h. The media tested were (i) the original McBride Listeria agar formulation (with glycine); (ii) modified McBride agar containing glycine anhydride; (iii) LiCl-phenylethanol-moxalactam (LPM) agar; (iv) acriflavine-ceftazidime agar; (v) Rodriguez isolation agar (RISA); (vi) modified Vogel-Johnson (MVJ) agar; (vii) cyclohexanedione-nalidixic acid-phenylethanol agar; and tryptose agar as control. A total of 66 organisms were used including 11 Listeria monocytogenes strains and 5 other Listeria spp. For L. monocytogenes strains only, all media performed highly similarly. Of the other Listeria spp., only two grew on MVJ agar and three each grew on LPM and RISA. Only LPM agar inhibited the 50 non-listeriae, including five yeasts, while MVJ agar inhibited all but one yeast. The McBride Listeria agar formulation that contained glycine anhydride was less selective than the original. When pure cultures of 10 bacteria (including one L. monocytogenes strain) were combined and plated on four media, L. monocytogenes colonies were easiest to enumerate on MVJ agar, followed by LPM and RISA. These media ranked in the same order when plated with homogenates of various foods to which was added L. monocytogenes Scott A, but LPM agar was the best overall since Scott A was inhibited by MVJ. Upon microscopic examination of listerial colonies from the plating media, atypical cell morphology was noted with cells being about twofold in size on LPM, MVJ, and acriflavine-ceftazidime agars. Overall, LPM agar was the most suitable of the media tested even though it was inhibitory to Listeria grayi and Listeria murrayi.