Mycoplasma Medium Research Articles

Spiroplasma monobiae sp. nov. from the Vespid Wasp Monobia quadridens (Hymenoptera: Vespidae)

Spiroplasma strain MQ-1T (T = type strain) from the hemolymph of the vespid wasp Monobia quadridens differed serologically from other spiroplasma species, groups, and subgroups. Cells of strain MQ-1T were helical and motile and possessed a single cytoplasmic membrane, with no evidence of a cell wall. The organism grew in conventional mycoplasma medium, in serum fraction, SM-1, MID, and SP-4 liquid media, and on SP-4 solid medium in either aerobic or anaerobic environments. The optimum temperature for growth was 32°C, but multiplication occurred over a wide temperature range (10 to 37°C). The doubling time at 32°C in MID medium was 1.9 h. Strain MQ-1T catabolized glucose but hydrolyzed neither arginine nor urea. Previous work showed that strain MQ-1T has a unique methylase, previously known only in eucaryotes. Also, strain MQ-1T induces production of tumor necrosis factor in bone marrow macrophages. The guanine-plus-cytosine content of the DNA was 28 ± 1 moI%. The genome size of strain MQ-1T was 940 kb (627 MDa); a similar strain, MQ-8, had a genome size of 985 kb (657 MDa). Strain MQ-1T and its allies have the smallest genomes known in the genus Spiroplasma. Strain MQ-1 (= ATCC 33825) is designated the type strain of a new species, Spiroplasma monobiae.

Production of mycoplasma-specific antisera in rabbits immunologically tolerized at birth to mycoplasma medium constituents

Neonatal rabbits were injected intraperitoneally (i.p.) within 12 h of birth followed by similar injections every day for 10 consecutive days and then every second day for a further 8 weeks, with mycoplasma broth medium (tolerogen), to induce immune tolerance. The rabbits were then immunized with the porcine mycoplasmas, M. hyopneumoniae or M. hyorhinis at 9 weeks of age. Immune sera obtained from these rabbits and from normal control rabbits were tested for antibodies against broth mycoplasma antigens and for antibodies to medium components by double immunodiffusion in agarose and by enzyme-linked immunosorbent assay (ELISA). Antisera obtained from the tolerized rabbits contained no antibodies to medium components as evidenced by lack of reactivity in both assays. In immunofluorescence tests the antisera obtained from tolerized rabbits permitted specific staining of colonies of the homologous mycoplasma grown on mycoplasma agarose medium. In contrast the antisera obtained from normal rabbits produced strong reactions in all of the tests and non-specific background fluorescence due to reactions with components of the culture medium.

Comparison of two methods for detection of Mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in The Netherlands

A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied.

Some observations on the indirect Elisa for antibodies toMycoplasma iowaeserovar i in sera from turkeys considered to be free from Mycoplasma infections

A number of sera from turkeys considered to be uninfected with Mycoplasma iowae were examined by indirect ELISA using whole cell Ai. iowae serovar I antigen. In an attempt to reduce the number of 'false positive' sera, assays were made (a) using plates blocked with foetal calf serum or skim milk before, or, bovine serum albumin after, coating with antigen, (b) using the absorbance difference method in which each serum was tested against mycoplasma antigen and medium antigen and (c) with unblocked plates but with serum heated to 56 C for 30 min. The upper limit of absorbance for the negative sera was determined using formulae already used by others including, the mean plus twice the standard deviation, the mean plus three times the standard deviation, (M + 3SD), twice the mean, 115% of the mean and four times the standard deviation. Some reduction in the number of 'false positive' sera occurred after heating sera or using the formula of M + 3SD. This formula might, however, reduce the sensitivity of the test.

Acholeplasmas and similar nonsterol-requiring mollicutes from insects: Missing link in microbial ecology

Wall-less prokaryotes from guts of five insect species (a corn root maggot [Plecia sp.: Diptera: Bibionidae, strain PS-1], a syrphid fly [Diptera: Syrphidae, strain YJS], two tabanid flies [Tabanus catenatus, strain TAC, andChrysops discalis, strain DF-2], and a vespid wasp [Monobia quadridens, strain MQ-3]) were characterized. The strains grew at 23°–32°C in conventional mycoplasma media containing 10% (vol/vol) serum, or in serum-free mycoplasma medium with or without fatty-acid-Tween-80 supplements. No helical forms were noted in dark-field microscopy, and electron micrographs of thin sections of the strains showed a single membrane. Two strains (DF and MQ-3) cross reacted with antiserum againstAcholeplasma florum. Although the other three strains did not react with antisera to establishedAcholeplasma orMycoplasma species, they were otherwise characteristic of acholeplasmas. This study, the first to demonstrate acholeplasmas from Arthropoda, in conjunction with previous isolations from plant surfaces, suggests that insects may constitute an important reservoir for acholeplasmas.

Serological cross-reactions between Mycoplasma genitalium and Mycoplasma pneumoniae.

The recently discovered mycoplasma species Mycoplasma genitalium was isolated from urethral specimens from men with nongonococcal urethritis (Tully et al., Lancet i:1288-1291, 1981). In a previous report (K. Lind, Lancet ii:1158-1159, 1982), prominent serological cross-reactions were demonstrated between this mycoplasma and M. pneumoniae. In the present study, the two mycoplasma species were compared more extensively. In classical mycoplasma medium without thallium acetate, M. genitalium grew more slowly than M. pneumoniae did but finally formed similar amounts of acetic acid and lactic acid from glucose. Although their colonies on solid medium were indistinguishable, transmission electron microscopy showed that the flask-formed cells of M. genitalium (especially their necks) were shorter than those of M. pneumoniae. The two species were distinct since DNA hybridization showed only 1.8% homology in base sequences, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed significantly different profiles of the two strains. However, considerable similarities were found in their antigenic reactions in various serological tests. The presence of common or closely related antigens was demonstrated in the two species with rabbit immune sera in complement fixation test with chloroform-methanol-extracted antigens by an indirect immunofluorescence test on microcolonies, and by metabolism inhibition and growth inhibition tests. Cross-reactions were also demonstrated by crossed immunoelectrophoresis. The role of M. genitalium as a human pathogen in the genital tract has not been assessed. If serological tests are to be used in this assessment, caution must be exercised due to the extensive cross-reactions demonstrated. Some of the species-specific antigens which we have demonstrated would be appropriate for use in such tests and would help to circumvent problems caused by cross-reactions.

Tenazität von Mycoplasma bovis

The survival of Mycoplasma bovis was examined on or in different sterilized carriers which normally may have contact to cows (sponge, cotton, wire, glass, wood, straw, tap water, milk). At 4°C M. bovis survived on sponges 57 days, in milk 54 and in Mycoplasma medium for 50 days. At 20°C survival time was 1 to 2 weeks, at 37°C 1 week.

Liposomes replace serum for cultivation of fermenting mycoplasmas.

Cholesterol and albumin are limiting factors in the growth of Mycoplasma species. These nutrients are usually supplied in the culture medium by the addition of serum. The growth of M. pneumoniae in a serum-free medium containing an ethanolic cholesterol suspension and albumin was about one-half the level attained in serum-containing medium. M. gallisepticum and M. fermentans were not cultivable in the cholesterol suspension medium even after supplements were included. In another culture medium containing phosphatidylcholine-cholesterol liposomes and albumin as serum replacements, the growth of M. pneumoniae was approximately equal to that in serum-containing medium, and the growth of M. gallisepticum and M. fermentans was significantly greater than that in medium containing serum. M. fermentans produced even higher yields in liposome medium supplemented with arginine. These fermenting mycoplasmas readily adapted to the liposome medium and by the fifth or sixth serial passage produced thick confluent growth on the lower surface of culture bottles. To obtain maximum growth, we serially transferred the mycoplasmas at least 10 times in serum-free medium before quantitations of growth were made. This is the first report of a serum-free mycoplasma medium of high growth-promoting ability.

Mycoplasma muris, a New Species from Laboratory Mice

Three mycoplasma strains were isolated from the vaginal tracts of strain RIII mice. These isolates had similar biochemical and serological properties and grew on SP-4 medium but not on conventional mycoplasma medium containing horse serum and yeast extract. Anaerobic incubation was required. These organisms hydrolyzed arginine but not urea, produced a film and spot reaction, and were susceptible to 1.5% digitonin. Cholesterol was required for growth, and the organisms adsorbed to guinea pig erythrocytes and exhibited uridine phosphorylase activity. The organisms were serologically distinct from 82 Mycoplasma species and unclassified serotypes. On the basis of these findings and other morphological and biological properties of the organisms, we propose that mycoplasma strains with these characteristics belong to a new species, Mycoplasma muris. Strain RIII-4 (= ATCC 33757) is the type strain.

Covert infection of a mouse mammary tumor cell line with Mycoplasma hyorhinis: cosedimentation with mouse mammary tumor virus in sucrose density gradients.

Supernatant fluids from cultures of a mouse mammary tumor (MT) cell line were found to produce a specific cell detachment effect when inoculated into HeLa cells. The cell detachment factor (CDG) responsible for this effect was examined. Repeated attempts to cultivate this CDF in bacteriologic, fungal, and mycoplasma media were unsuccessful. However, using the DNA fluorochrome staining technique and specific immunofluorescent staining procedures, the CDF was identified positively as a noncultivable strain of Mycoplasma hyorhinis. It was also noted that this CDF could be labeled with [3H]uridine in MT cell cultures, concentrated, and banded at a density of 1.18 g/cm3 when centrifuged to equilibrium in a 20 to 60% sucrose gradient. Using a multiple antibiotic treatment regimen, the MT cells were "cured" of the M. hyorhinis contaminant. Re-infection of these cells with an exogenous strain of M. hyorhinis resulted in the same cell detachment effect, and this strain when labeled with [3H]uridine also sedimented at a density of 1.18 g/cm3. The salient feature of these studies is that M. hyorhinis sediments at the same density of mouse mammary tumor virus (MMTV) in sucrose density gradients. This was demonstrated by sucrose density gradient analyses of a purified sample of MMTV, assaying for reverse transcriptase activity, and a [3H]uridine labeled sample of the M. hyorhinis present in the MT cell cultures.

Isolation of Mycoplasma hyorhinis and Mycoplasma fermentans from cell cultures

A mycoplasma medium with boiled blood and sucrose (Herderschee medium), a commercially available (Difco) PPLO medium and an enriched trypticase soy broth medium were tested for their suitability for isolation and subcultivation of a fastidious strain of Mycoplasma hyorhinis (DBS 1050) and a M. fermentans strain (IM 1) isolated on Herderschee medium. The mycoplasma strains were maintained in VERO-cell cultures, for which M. fermentans proved to be very pathogenic. It was therefore, stored at −70°C after two passages in the cell culture. M. hyorhinis was inoculated in various VERO-cell passages and subsequently propagated in the cells. On Herderschee and Difco media M. hyorhinis strain DBS 1050 could always be isolated from the cell cultures one to four days after trypsinization of the cells. Recovery succeeded better from frozen (−70°C) cell suspensions than from supernatant cell culture medium, and also better after direct inoculation on agar medium than after inoculation in broth. Primary isolations on agar medium could nearly always be subcultivated. The concentration of organisms detected on agar medium at primary isolation and the facility of subsequent subcultivation in mycoplasma medium seemed to depend partly on the number of mycoplasma passages in the cell culture, and partly on the subline of the cell culture used for inoculation. M. fermentans strain IM 1 could be isolated on Herderschee medium and on Difco medium with sucrose from supernatant cell culture medium as well as from frozen cell suspensions and equally well from one to seven days after trypsinization of the cell culture, not just after trypsinization. Both mycoplasma strains were only detected after anaerobic incubation (5% CO2 in N2). They could be cultivated as well when 2·5% instead of 10% (w/v) yeast extract was incorporated in the media. The yeast extract could be boiled and heated up to 120°C without impairing its growth promoting quality. Therefore, nicotinamide adenine dinucleotide is dispensable for these two strains. Neither mycoplasma strain could be cultivated on enriched trypticase soy broth medium. The implication of these observations when testing cell cultures and live virus vaccines for contamination with mycoplasma are discussed.

Isolation ofAcholeplasma spp. from coconut palms affected by lethal yellowing disease in Jamaica

A total of 35 isolates ofAcholeplasma spp. were recovered from phloem sap and rotting tissues of lethal yellowing-diseased coconut palms. The highest isolation rate, 33% of samples from two palms, was obtained in a conventional mycoplasma medium supplemented with Tween 80; no isolates were recovered from healthy palm tissues or from uninoculated media constituents. Metabolic and serological tests on uncloned isolates showed that about two-thirds were strains ofA. axanthum and the remainder were related toA. oculi. These results strongly suggest that acholeplasmas occur in or on plant tissues either as pathogens, epiphytes, or saprophytes.

Enhanced Isolation of Mycoplasma pneumoniae from Throat Washings with a Newly Modified Culture Medium

Two hundred throat washings, previously screened and presumed negative for Mycoplasma pneumoniae in conventional mycoplasma culture media, were retested for the organism in a modified medium (PS-4) initially developed for cultivation of a tick-derived Mycoplasma (spiroplasma). The organism was rapidly identified with an agar plate immunofluorescence procedure. M. pneumoniae was isolated from 69 (34.5%) of the 200 "negative" specimens cultured on a diphasic SP-4 medium, in contrast to 10 isolations (5%) made on conventional diphasic mycoplasma medium. This enhanced recovery of M. pneumoniae represented a combination of a superior culture medium and a more efficient identification technique. The findings suggest that these procedures might be effectively applied to the recovery of M. pneumoniae from all likely host and that improved recovery of the organism may aid in the interpretation of a number of puzzling questions about the epidemiology of M. pneumoniae infections.

Cultivation, serology, ultrastructure, and virus-like particles of spiroplasma 277F

Spiroplasma 277F, a helical, motile mycoplasma from rabbit ticks in Montana, was cloned and cultivated in liquid and solidified spiroplasma or mycoplasma media. Serum was required, glucose was fermented, and digitonin inhibited growth. Colonies of spiroplasma 277F possessed granular centers and were surrounded by smaller, subsurface “satellite” colonies. Cloned agent 277F was antigenically distinct from the suckling mouse cataract agent, the corn stunt organism, andSpiroplasma citri by growth inhibition and deformation tests, but exhibited weak cross-reactivity withS. citri in the precipitin ring tests. Although current passage levels did not cause cataracts in neonatal rats, kill embryonated hen's eggs, or cause bovine mastitis, definitive tests must await the availability of fresh isolates. Ultrastructurally, 277F closely resembledS. citri but displayed a system of 3-nm threadlike filaments in the exterior layer of its membranous covering. Two phagelike entities, similar to viruses associated withS. citri, were present.

Studies on the Effect of Butylated Hydroxytoluene on Mycoplasma synoviae

Butylated hydroxytoluene (BHT) is one of the antioxidants added to human and animal feed products in concentrations ranging from 50 to 200 ppm to delay degradation of the labile lipid components. BHT is known to be an inactivator of mammalian and bacterial viruses that contain lipid.Results of in vitro studies showed that 10 ppm of BHT in Mycoplasma medium prevented growth of each of the six Mycoplasma synoviae (Ms) isolates. BHT added at 100, 200, and 400 ppm in the feed did not have any significant in vivo effect on the Ms serological responses or show any chemoprophylactic effect on birds infected with Ms.

Suckling mouse cataract agent is a helical wall-free prokaryote (spiroplasma) pathogenic for vertebrates.

THE suckling mouse cataract agent (SMCA) is a filterable organism originally isolated from a pooled extract of rabbit ticks (Haemaphysalis leporis-palustris) collected near Atlanta, Georgia in April 1961 (ref. 1). The agent grows to high titre in the eyes and brains when inoculated intracerebrally into newborn mice in which it induces cataract, uveitis and chronic brain infection1–3. SMCA has also been grown to high titre in embryonated hen's eggs, in which it produces a lethal infection in 4–9 d (ref. 1). The agent has not been grown in tissue culture or in artificial media, but growth in a rabbit lens organ culture has been reported4. Stained SMCA-infected tissues failed to show the presence of rickettsiae and, as a result of such negative inferences and other characteristics of the agent3–5, it was considered to be a candidate slow virus6. Within the past few years however, ultrastructural and biological studies showed that a wall-free prokaryote was consistently associated with the acute and chronic disease in mice and with lethality in chick embryos7,8. Although the agent resembled mycoplasmas morphologically, it differed from them in its apparent non-cultivability on conventional mycoplasma medium. We now report that the organism associated with the suckling mouse cataract syndrome is a prokaryote similar to the spiroplasmas—a group of mycoplasmas known previously only from plants and insects.

Suitability of peracetic acid for sterilization of media for mycoplasma cultures.

The utility of peracetic acid for sterilization of serum and yeast extract additions to mycoplasma medium was studied by culturing six Mycoplasma species. Culture media containing additions that had been sterilized with peracetic acid proved to be as good as filtered components. The use of 0.05 to 0.1% peracetic acid is recommended to sterilize the serum and yeast extract additions since savings in time and equipment can be accomplished.

The interaction between Mycoplasma hominis and poliovirus in cell culture.

SUMMARY Eagle’s culture medium containing 2% heat-inactivated calf serum is inadequate for the growth of Mycoplasma hominis. However, when the medium was conditioned by remaining in contact with the monolayers for several days, or when it was supplemented with 10% mycoplasma medium, it supported the growth of this organism. The presence of Mycoplasma hominis in MK and MS cell cultures had no effect on the replication of poliovirus type II, as judged by the titration of the infectivity of the virus produced. However, the presence of poliovirus in primary cultures of monkey kidney cells enhanced the growth of mycoplasma in such a way that the delay in the logarithmic phase of growth of this organism was overcome.

Isolation of mycoplasmas from lungs by a perfusion technique.

Homogenization ofMycoplasma pulmonis-infected mouse lung tissue in order to isolate the organisms resulted in their death. Since mycoplasmas are situated almost entirely on the surface of bronchial epithelial cells it was feasible to devise a lung perfusion technique to wash out the organisms. At autopsy the trachea was exposed, a cannula tied securely in it, and mycoplasma medium (1–2 ml) injected into the lungs and then withdrawn. The number ofM. pulmonis organisms recovered from a washing by this procedure was found to be about the same in the first 3 washings, after which there was at least a 10-fold decrease. Of 29 mice infected withM. pulmonis, the organisms were isolated from the lungs of 19 by both washing and homogenization but from the remaining 10 by the perfusion method only. Similarly, in experiments in which mice were inoculated withM. gallisepticum, the organisms were isolated from the lungs of 12 mice by perfusion but from the lungs of only 2 of the same mice by homogenization.M. pneumoniae was recovered from infected hamster lungs by the perfusion method and almost as well by homogenization. Possible reasons for the differences are discussed as are other applications of the perfusion technique.

Antibiotics in mycoplasma media and the temporary storage of specimens containing mycoplasmas of the genital tract.

Antibiotics in mycoplasma media and the temporary storage of specimens containing mycoplasmas of the genital tract.