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.     

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.     

Nucleic acid homologies of selected bacteria, L forms, Mycoplasma species

Rogul, M. (Walter Reed Army Institute of Research, Washington, D.C.), Z. A. McGee, R. G. Wittler, and Stanley Falkow. Nucleic acid homologies of selected bacteria, L forms, and Mycoplasma species. J. Bacteriol. 90:1200-1204. 1965.-The molar per cent of guanine plus cytosine (G + C) in the deoxyribonucleic acids (DNA) of Proteus mirabilis, strain 9, and its stable L form was determined by thermal denaturation and found to be approximately 39.5% G + C. The DNA homologies of this bacterium and its L form were estimated by the agar-column technique and were equivalent in their abilities to anneal and form specific duplexes. The next series of comparisons were performed between two Mycoplasma species and their often suggested bacterial parent. The G + C ratios of M. gallisepticum (32.7%), M. gallinarum (28.1%), and Haemophilus gallinarum (41.9%) varied to a high degree. In the homologous system, the denatured DNA of H. gallinarum trapped in agar bound approximately 40% of its sheared, denatured, and H(3)-labeled DNA. In comparison, the nucleic acids of M. gallinarum and M. gallisepticum were incapable of binding the labeled DNA of H. gallinarum. These findings provided evidence that the two strains of Mycoplasma were not derived from H. gallinarum.     

Comparative biosynthesis of ornithine and lysine by Mycoplasma and L forms

Smith, Paul F. (University of South Dakota, Vermillion). Comparative biosynthesis of ornithine and lysine by Mycoplasma and L forms. J. Bacteriol. 92:164-169. 1966.-Seven species of Mycoplasma, two L forms not requiring salt and their parent bacteria, and two yeasts were examined for enzymes involved in the biosynthesis of ornithine and lysine. All organisms tested, except two species of Mycoplasma and the yeasts, decarboxylated meso-alpha, epsilon-diaminopimelic acid. None of the Mycoplasma species or L forms was capable either of reducing alpha-aminoadipic acid to its semialdehyde or of incorporating alpha-aminoadipic acid-6-C(14) into lysine. All organisms, except the yeasts and Mycoplasma sp. caprine strain 14, acetylated glutamic acid, and all organisms possessed N(alpha)-acetyl-l-ornithine:2-oxo-glutarate aminotransferase activity. N(alpha)-acetylornithase activity was negligible in all organisms except Proteus and its L form. No transacetylation between acetylglutamic acid and ornithine, and vice versa, was demonstrable in any of the organisms. Mycoplasma species appear to possess the bacterial pathway to lysine. Ornithine does not appear to arise from glutamic acid.     

Sedimentation counting and morphology of Mycoplasma

Clark, Harold W. (The George Washington University School of Medicine, Washington, D.C.). Sedimentation counting and morphology of Mycoplasma. J. Bacteriol. 90:1373-1386. 1965.-The sedimentation technique for counting viral particles was applied to the quantitation and morphological identification of Mycoplasma in broth cultures. Mycoplasma, apparently in their native form, firmly adhered to the surface, when sedimented on glass cover slips or onto electron microscope grids. The sedimented cover slip preparations stained with crystal violet could be readily counted in the light microscope. The cultures sedimented onto electron microscope grids were readily counted at low magnification and provided excellent preparations for morphological examination at higher magnifications. It was found that air-dried Mycoplasma particles were enlarged considerably because of excessive flattening. Fixation of sedimented Mycoplasma particles in diluted OsO(4) prior to air drying yielded a more realistic morphology, with various sizes and shapes in the stages of the growth cycle exhibited. A new technique of differentially staining Mycoplasma colonies on agar plates was developed to facilitate the quantitation of viable colony-forming units for comparison with total counts. The use of plastic or Parafilm gaskets for dry mounting was developed to facilitate the handling and examination of the stained cover slip preparations. The results of this investigation indicated that the growth cycle of some Mycoplasma species includes a stage of hexadic fission with the cleavage of minimal reproductive units (less than 100 mmu) containing a limited deoxyribonucleic acid genetic coding molecule (approximately 4 x 10(6)).     

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.     

Chemotactic activity of L-forms and mycoplasma.

L-forms of Streptococcus faecalis, Escherichia coli and Proteus mirabilis showed significantly less chemotactic activity for normal human leucocytes than did parent bacterial forms which were strongly chemotactic. Mycoplasma pneumoniae and Mycoplasma hominis did not demonstrate chemotactic activity.     

A collagen film microassay for tissue collagenase.

A quantitative microassay for the detection of bacterial and tissue collagenase is presented. Collagen in acetic acid solution forms a thin, tenacious film upon contact with a dried agar surface. The digestion of the film by collagenase is detected by subsequent staining with Coomassie blue. Undigested film is stained dark blue while areas of collagenase digestion remain clear. The technique can be employed in two ways: with collagen-coated glass coverslips as a rapid screening method or with collagen-lined capillary tubes for precise quantitation. The assay has a sensitivity comparable to those assays using radioactively labeled collagen substrates and requires only 5 to 60 min of incubation.     

Studies on the susceptibility of different culture morphotypes of Listeria monocytogenes to uptake and survival in human polymorphonuclear leukocytes

This study demonstrated that atypical virulent filaments of Listeria monocytogenes (rough variant type II and designated FR for this study), isolated from clinical specimens or generated during exposure to pulsed-plasma gas discharge in liquids, were shown to be capable of survival when engulfed by human polymorphonuclear leukocytes (PMNLs). Factors shown to significantly influence the maximal respiratory burst response in PMNLs and survival of different internalized cell or filament forms of L. monocytogenes were bacterial strain, culture form, degree of opsonization (with and without the use of 10% serum) and composition of the bacterial growth media used before uptake by PMNLs. Opsonized regular-sized L. monocytogenes cells grown on blood agar (BA) elicited the greatest respiratory burst response and survived best in PMNLs. The filamentous (FR) and multiple cell chain (MCR) rough variants were significantly less susceptible to uptake and survival in PMNLs. Supplementation of tryptone soya agar with hemin resulted in significantly reduced chemiluminescence responses in phagocytosing PMNLs compared with the maximal levels observed from prior bacterial growth on BA or brain heart infusion agar that also contained a source of iron. The MCR variants secreting decreased levels of a peptidoglycan hydrolase CwhA protein exhibited the lowest percentage survival when internalized in PMNLs compared with wild-type smooth or FR culture variants as determined by the macrophage-killing assay.     

Factors affecting growth of Staphylococcus aureus L forms on semidefined medium

Banville, Robert R. (The Catholic University of America, Washington, D.C.). Factors affecting growth of Staphylococcus aureus L forms on semidefined medium. J. Bacteriol. 87:1192-1197. 1964.-A semidefined agar medium was found suitable for production and cultivation of the L form of Staphylococcus aureus. In semidefined liquid medium, growth of the L form took place in the form of a sediment containing large masses of cells, but heavy and diffuse growth occurred in the same medium with 0.05% agar. The optimal pH for L-colony formation on solid medium was 6.5. More L colonies developed on 0.75% agar than at higher agar concentrations. L colonies developed in greater numbers on pour plates than on streak plates, and in some cases more L colonies appeared under anaerobic incubation. L-colony formation appeared to be inhibited by sodium citrate. The vitamin requirements of the L forms studied were similar to those of the classical form.     

How endo- is endo-? Surface sterilization of delicate samples: a <Emphasis Type="Italic">Bryopsis</Emphasis> (Bryopsidales,Chlorophyta) case study

In the search for endosymbiotic bacteria, elimination of ectosymbionts is a key point of attention. Commonly, the surface of the host itself or the symbiotic structures are sterilized with aggressive substances such as chlorine or mercury derivatives. Although these disinfectants are adequate to treat many species, they are not suitable for surface sterilization of delicate samples. In order to study the bacterial endosymbionts in the marine green alga Bryopsis, the host plant’s cell wall was mechanically, chemically and enzymatically cleaned. Merely a chemical and enzymatic approach proved to be highly effective. Bryopsis thalli treated with cetyltrimethylammonium bromide (CTAB) lysis buffer, proteinase K and bactericidal cleanser Umonium Master showed no bacterial growth on agar plates or bacterial fluorescence when stained with a DNA fluorochrome. Moreover, the algal cells were intact after sterilization, suggesting endophytic DNA is still present within these algae. This new surface sterilization procedure opens the way to explore endosymbiotic microbial communities of other, even difficult to handle, samples.     

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.     

Effect of reactor surface on production of bacterial cellulose and water soluble oligosaccharides by <Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis> PJK

The effect of agar plates on the bacterial cellulose (BC) production in a static culture was investigated in order to find the role of agar component as a surface modifying agent. Two types of surface modified reactors (SMRs: SMRD and SMRB) were prepared by coating the bottom of the reactors with agar dissolved in distilled water and basal medium, respectively. The SMRs were used for BC and water soluble oligosaccharides (WSOS) production. Control was done by the same procedure using reactors without agar plate. In both types of SMRs, the maximum production rate was observed after the second day of cultivation compared to third day of cultivation in the case of the control. The maximum productions of BC 5.308 and 5.472 g/L were observed at the first batch using SMRs prepared with agar dissolved in distilled water (SMRDs) and SMRs prepared with agar dissolved in a basal medium (SMRBs), respectively. Similarly, in the daily-culture and successive batch strategy experiments the maximum amount of WSOS produced in the SMRs was almost double that of the control. The highest water holding capacity value 92.21 g/g was observed for BC formed in the SMRs prepared with 3.0% of agar. FTIR and XRD analyses were carried out to study the structural features of the prepared BC.     

Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2-3μm-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.     

On the nature of P.P.L.O.

Summary In a number of strains of the three species of P.P.L.O. of human origin coccoid elements have been observed, which are Gram positive or Gram variable and of very small size (<0.5 μ). They are able to grow in pure culture on the surface of nutrient agar in very small colonies. Although they have bacterial characteristics, they could not be identified as known bacteria. Agglutination tests are in favour of a serological relationship between the coccoid elements and P.P.L.O. The suggestion that these elements might be an outside contamination is made unlikely by several observations. The supposition that P.P.L.O.s are L forms of these coccoid elements should be considered further.     

Quantitative characterization of antagonistic activity of lactobacilli

In this work the method of serial dilutions of lactobacilli in two-layer agar was used. On the agar surface bacterial or fungal cultures were applied at different time intervals. A special quantitative characteristic was introduced. L. plantarum strain 8P-A3 was shown to have the maximum antagonistic activity. In great amounts L. casei and L. reuteri are capable to suppress the growth of bacteria and fungi. All lactobacilli under study produced a pronounced bactericidal effect on Pseudomonas, had different influence on the viability of Escherichia and staphylococci and exhibited fungistatic and fungicidal action only when inoculated at high concentrations.     

中国青凤蝶变异型初步观察

通过对中国青凤蝶Graphium sarpedon(L.)标本的观察,归纳出6种中国青凤蝶的变异型,提供了各变异型的主要形态特征和彩色照片,并综合大量文献资料对中国青凤蝶的亚种分布进行了探讨。     

Improved Microscopy of Mycoplasma In Vitro

Techniques were developed for continuous microscopic observation of mycoplasmata growing in vitro in Rose chambers by using an inverted phase microscope. The methods permitted direct microscopic observation of undisturbed growth of mycoplasmata in liquid medium. Inocula of mycoplasmata were passed through 0.22-mum filters before culture to provide a suspension of discrete particles. The sequential growth of Mycoplasma pneumoniae was followed from points or single straight lines, with development of branching, a net-like confluence of filaments, large bodies occurring in the center of developing colonies, and finally coccoid forms. Other species of Mycoplasma which did not attach as readily to glass could be observed also by inverted phase microscopy. Umbonation of colonies (a "friedegg" appearance) occurred in liquid medium, indicating that this appearance was not due simply to interaction with the agar medium, but may reflect a qualitative difference in growth patterns between center and periphery. For growth on solid medium, direct observation of colonies in uncovered plates of agar medium was made by using inverted phase microscopy. This was found helpful in detecting small colonies and in observing relationships between colonies.     

Staining method of poly(3-hydroxyalkanoic acids) producing bacteria by nile blue

Summary Using an ethanol solution of nile blue, we have developed an efficient method to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar plate. When the bacterial colonies with PHA granules were stained with nile blue, the stained colonies fluoresced bright orange on the irradiation of UV light. In the fluoresce emission spectra, fluorescence intensity increased with an increase in the PHA content of bacterial cells.Alcaligenes eutrophus andA.latus colonies with poly(3-hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nm on the excitation at 490nm. On the other hand,Pseudomonas oleovorans andP.putida with medium-chain-length (mcl-) PHA copolymers of C6, C8 and C10 units exhibited an emission maximum at 570nm.     

The effects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria

The growth and morphology of submerged bacterial colonies was investigated. Five separate colonial forms were recognized depending both on species and on agar concentration. These were (i) branched, dendritic structures seen only with Bacillus cereus ; (ii) lenticular colonies for all other species at high agar concentrations; (iii) small lobed to spherical colonies for non-motile organisms at low agar concentrations; (iv) and (v) large diffuse spherical colonies which can be further subdivided into 'snowball' or 'wispy' types for motile bacteria growing at agar concentrations below about 0·65% w/v. Viable count determinations suggested that agar concentration had little effect in the early stages of growth but that motile cells at low agar concentrations achieved higher cell numbers than did those in concentrations greater than 0·65% w/v. Transmission electron microscopy indicated that bacteria in lenticular colonies were tightly packed within lens-shaped splits in the agar whilst at low agar concentrations motile cells were well separated and appeared to move through the agar matrix.