Morphology of Mycoplasma laidlawii type A. II. Effect of glucose on growth and cellular morphology

Anderson, D. L. (University of Minnesota, Minneapolis), M. E. Pollock, and L. F. Brower. Morphology of Mycoplasma laidlawii type A. II. Effect of glucose on growth and cellular morphology. J. Bacteriol. 90:1768-1777. 1965.-Cells of Mycoplasma laidlawii A grown in soy peptone-yeast extract (SP-YE) broth reached higher maximal titers than cells grown in SP-YE supplemented with 0.2% glucose, and remained viable longer than glucose-grown cells. Moreover, addition of glucose to SP-YE markedly affected the morphological developmental sequence of the organism. The diameter of glucose-grown cells at 28 to 40 hr was often two to four times that of cells grown without glucose; these large glucose-grown cells deteriorated progressively, and presumably liberated 0.1- to 0.25-mu small bodies. In contrast, pleomorphic 22- to 28-hr cells grown in SP-YE without glucose gave rise to uniform bodies 0.5 to 1.0 mu in diameter; limited numbers of 0.1- to 0.25-mu small bodies were also liberated in nonglucose cultures after 45 to 64 hr. Electron-microscopic counts of 16- to 64-hr cultures agreed well with counts of colony-forming units (CFU) when organisms larger than 0.25 mu were included in the microscopic counts. Small bodies, 0.1 to 0.25 mu in diameter, apparently did not contribute substantially to numbers of CFU. Droplet patterns of cells grown in Arginine Assay Medium and L15 Tissue Culture Medium were cleaner than patterns of cells grown in SP-YE.     

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)).     

Mycoplasmal infection of insect cell cultures

Twenty-five cell cultures of three insect orders from eight laboratories were tested for mycoplasmal infection. Acholeplasma laidlawii was detected in one culture, an incidence of 4.0%. A. laidlawii, Mycoplasma orale, M. arginini, but not M. hyorhinis, could establish infections of drosophila Dm-1 cell cultures at 25 degrees C. In prospective studies, drosophila Dm-1 cultures were intentionally infected with broth-propagated A. laidlawii and M. hyorhinis. M. hyorhinis did not grow and was eliminated from the Dm-1 cultures during consecutive passages. A. laidlawii grew without obvious cytopathic effects during six weekly passages; titers of over 10(7) CFU/ml were recorded at Passages 2 and 5 (p2 and p5). Minimal cell culture infectious doses were also determined during these studies. 0.1 milliliter cell samples were inoculated into Leighton tubes containing either fresh M1A culture medium or 3T6 indicator cells in McCoy's 5a medium. After 4 d of incubation at 25 and 37 degrees C, respectively, the cover slips were stained by DNA fluorochrome Hoechst 33258 (A. laidlawii) or by specific fluorescein-conjugated antiserum (M. hyorhinis). At p2 with both mycoplasma species, the procedure using M1A medium and incubation at 25 degrees C without 3T6 cells was inferior to indicator cells. In five of six experiments at least a two-log higher titer of mycoplasmas was needed to be detected with M1A and 25 degrees C. At p5 no difference could be found. Uridine phosphorylase assays of Dm-1 cultures infected with A. laidlawii, M. hyorhinis, M. orale, and M. arginini gave clearly positive results only with A. laidlawii. The ratio of incorporated uridine to incorporated uracil method yielded false positives with two drosophila cell lines. Suggestions for assay of mycoplasmas in invertebrate cell cultures are given.     

Characterization of Mycoplasma aerosols as to viability, particle size, and lethality of ultraviolet irradiation

Kundsin, Ruth B. (Harvard Medical School, Boston, Mass.). Characterization of Mycoplasma aerosols as to viability, particle size, and lethality of ultraviolet irradiation. J. Bacteriol. 91:942-944. 1966.-Viable aerosols of four strains of Mycoplasma: M. hominis II, M. pharyngis, M. pneumoniae, and an undetermined strain recovered from a lung at autopsy were dispersed, and the particle size was determined. The median diameter of the droplet nuclei ranged from 1.5 to 3.1 mu. M. pharyngis had a dieaway constant k of 0.008, indicating a survival potential of 6 hr from 10,000 colony-forming units at 23% relative humidity. Ultraviolet irradiation destroyed over 99% of the aerosols of all four strains concurrently with spraying. The particle size and viability of the droplet nuclei carrying Mycoplasma were consistent with the theory of airborne transmission of lower respiratory-tract infection.     

UV survival of human mycoplasmas: evidence of dark reactivation in Mycoplasma buccale.

The inactivation by ultraviolet (UV) light irradiation of mycoplasma cells of five human strains was monitored by investigating the colony-forming ability. The survival curves of five strains tested indicated that the cells of Mycoplasma buccale only are single and homogenously susceptible to UV light. The effect of the repair inhibitor, caffeine, on the colony-forming ability of UV-irradiated cells was investigated with M. buccale because of its homogenous susceptibility to UV light. The colony formation of irradiated cells was markedly depressed by post-irradiation treatment with caffeine at concentrations that had little or no effect on the colony formation of unirradiated cells. The colony-forming units (CFU) of UV-irradiated cells which were kept in broth without caffeine in the dark increased without a lag as the time in the dark increased. The colony-forming ability of the irradiated cells completely recovered after 3 hr in the dark. However, when irradiated cells were kept in the presence of caffeine, no increase in their CFU was observed. The mode of action of caffeine on UV-irradiated cells closely resembles that described for other organisms which possess dark reactivation systems for UV-induced damage in deoxyribonucleic acid (DNA). Thus, the results obtained provide evidence for the existence of a dark repair function in M. buccale.     

A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture

A detection system that utilizes a primer mixture in a nested polymerase chain reaction for detecting Mycoplasma contaminants in cell cultures is described. Primers were designed to amplify the spacer regions between the 16S and 23S ribosomal RNA genes of Mycoplasma and Acholeplasma. This detection system was able to detect 20-180 colony forming units per milliliter of sample. Eight commonly encountered Mycoplasma and Acholeplasma contaminants, which include Mycoplasma (M.) arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, and Acholeplasma laidlawii, were consistently amplified. Mycoplasma contaminants generated a single DNA band of 236-365 base pairs (bp), whereas A. laidlawii produced a characteristic two-band pattern of 426 and 219 bp amplicons. Species identification could be achieved by size determination and restriction enzyme digestion. Minor cross-reactions were noted with a few closely related gram positive bacteria and DNA from rat cell lines. A Mycoplasma Detection Kit for detecting Mycoplasma contaminants in cell cultures has been developed based on this approach.     

Behavior of Mycoplasma hominis in a Human Diploid Cell Culture System

The behavior of Mycoplasma hominis in normal human embryonic lung fibroblast (HAIN-55) cell cultures was investigated. Multiplication patterns of cell-associated mycoplasmas and of extracellular mycoplasmas in the HAIN-55 cultures depended upon the size of the inoculum. This relationship did not vary with the number of days the cells had been cultured, nor with the number of HAIN-55 cell passages. The maximum mycoplasmal growth was obtained with inoculum sizes of 10⁵ to 10⁶ colony-forming units (CFU)/ml. The recovery of mycoplasmas decreased rapidly with inoculum size beyond 10⁷ CFU/ml, and growth of the HAIN-55 cells was inhibited. Growth of the cells was also inhibited by the addition of the cytoplasmic fraction of Mycoplasma hominis.     

Enumeration of mycoplasmas after acridine orange staining.

Samples of liquid mycoplasma cultures were mixed with equal part of a 0.01% solution of acridine orange and placed on agar plates. The number of fluorescing organisms per field was counted in an epifluorescence microscope at an X 1,000 magnification. When the number of fluorescing organisms per field was related to the number of colony-forming units per milliliter during the growth cycle, highly significant correlation was found in cultures with greater than or equal to 10(6) colony-forming units per ml during the exponential growth phase. The counts were weakly correlated during the stationary phase and not correlated during the death phase. This technique provides a mean to enumerate mycoplasmas in liquid cultures.     

Evaluation of lactic acid bacterium fermentation products and food-grade chemicals to control Listeria monocytogenes in blue crab (Callinectes sapidus) meat.

Fresh blue crab (Callinectes sapidus) meat was obtained from retail markets in Florida and sampled for viable Listeria monocytogenes. The pathogen was found in crabmeat in three of four different lots tested by enrichment and at levels of 75 CFU/g in one of the same four lots by direct plating. Next, crabmeat was steam sterilized, inoculated with a three-strain mixture of L. monocytogenes (ca. 5.5 log10 CFU/g), washed with various lactic acid bacterium fermentation products (2,000 to 20,000 arbitrary units [AU]/ml of wash) or food-grade chemicals (0.25 to 4 M), and stored at 4 degrees C. Counts of the pathogen remained relatively constant in control samples during storage for 6 days, whereas in crabmeat washed with Perlac 1911 or MicroGard (10,000 to 20,000 AU), numbers initially decreased (0.5 to 1.0 log10 unit/g) but recovered to original levels within 6 days. Numbers of L. monocytogenes cells decreased 1.5 to 2.7 log10 units/g of crabmeat within 0.04 day when washed with 10,000 to 20,000 AU of Alta 2341, enterocin 1083, or Nisin per ml. Thereafter, counts increased 0.5 to 1.6 log10 units within 6 days. After washing with food-grade chemicals, modest reductions (0.4 to 0.8 log10 unit/g) were observed with sodium acetate (4 M), sodium diacetate (0.5 or 1 M), sodium lactate (1 M), or sodium nitrite (1.5 M). However, Listeria counts in crabmeat washed with 2 M sodium diacetate decreased 2.6 log10 units/g within 6 days. In addition, trisodium phosphate reduced L. monocytogenes counts from 1.7 (0.25 M) to > 4.6 (1 M) log10 units/g within 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Responses of Single Cells and Aggregates of Mycoplasmas to Ultraviolet Irradiation

Except for Mycoplasma fermentans strain PG 18, single-cell suspensions of M. arthritidis, M. fermentans (ATCC 19989), M. hominis type 1, M. orale types 1 and 2, M. pneumoniae, and M. salivarium were inactivated exponentially by ultraviolet (UV) irradiation, in contrast to broth cultures containing clusters of elementary bodies. The susceptibility of the mycoplasmas was unaffected by storage at 2-4 C and at -70 C, by sonication, and by filtration. The rate of inactivation was dependent on the intensity of the radiations but independent of the concentration of the cells. Therefore, single-cell suspensions of these mycoplasmas could be differentiated from aggregates of cells by exponential inactivation of the colony-forming units (CFU). By this criterion, the CFU of M. arthritidis in the exponential phase of growth consisted of single cells, in contrast to the other species in which the CFU contained two or more elementary bodies. Even though the cultures of M. fermentans (PG 18) were grown from single cells, they were not homogeneous in their susceptibility to UV light. Neither were cultures of M. arthritidis and M. orale type 1 grown from single cells which had survived irradiation.     

Numbers and viability of bacteria in ornithogenic soils of Antarctica

Summary Bacteria in ornithogenic soils from Ross Island, Antarctica, were counted by direct observation, and the percentages of viable organisms were assessed by incubation with ³H-glucose and by enumerating numbers of colony-forming units. The effects of incubation times and temperatures, and of storage of the samples, on the uptake of ³H-glucose were determined. Direct counts showed that large total numbers of bacteria were present in samples from occupied penguin colonies and recentlyabandoned sites. The percentages of bacteria metabolizing ³H-glucose increased when incubation was extended from 2 h to 8 h at field (average 4–5°C) or laboratory (average 18.5°C) temperatures to a maximum of 22%; storage of the samples for 31 days had no significant effect. The numbers of colony-forming units (CFU) were less than 0.058% of the direct counts. There were 77 times as many CFU in samples from the abandoned site compared to the inhabited colony. About 10% of the CFU were cocci compared with about 48% visible by direct microscopy. The glucose utilization data indicated that far more of the bacteria were viable than were cultured.     

Enumeration of Viable Bacteria in the Marine Pelagic Environment

The low percentage of living bacteria commonly obtained when comparing viable counts with total direct counts in seawater could be due more to inappropriate techniques for appreciating the growth ability of living cells than to unadapted culture conditions. The most-probable-number counts in filtered seawater cultures and the microscopic counts of 4(prm1),6-diamidino-2-phenylindole (DAPI)-stained aggregate-forming units grown on black polycarbonate filters appeared significantly correlated to the direct counts. Both these techniques show that in the superficial and intermediate water masses, the living cells may constitute an important (frequently higher than 20%) but highly variable part of the total populations. These viable counts appear more realistic than the conventional CFU counts, which provide only 0.001 to 0.2% of the total counts.     

Enumeration of mycoplasmas after acridine orange staining

Samples of liquid mycoplasma cultures were mixed with equal part of a 0.01% solution of acridine orange and placed on agar plates. The number of fluorescing organisms per field was counted in an epifluorescence microscope at an X 1,000 magnification. When the number of fluorescing organisms per field was related to the number of colony-forming units per milliliter during the growth cycle, highly significant correlation was found in cultures with greater than or equal to 10(6) colony-forming units per ml during the exponential growth phase. The counts were weakly correlated during the stationary phase and not correlated during the death phase. This technique provides a mean to enumerate mycoplasmas in liquid cultures.     

The use of spiral plating and microscopic colony counting for the rapid quantitation of Mycobacterium paratuberculosis

AIMS: To evaluate a spiral plating and microscopic colony counting technique to hasten the quantitation of Mycobacterium paratuberculosis. METHODS AND RESULTS: Broth and milk cultures of M. paratuberculosis were spirally plated onto Middlebrook agar plates and microscopically counted at 8 and 14 days of incubation. The same plates were recounted at 27-28 days of incubation when grossly visible colonies were present. The results were statistically compared with no difference in CFU ml-1 derived from the shorter vs longer incubation times. Other mycobacteria isolates were also plated and microscopically examined and found to be easily distinguishable from M. paratuberculosis. CONCLUSIONS: Microscopic quantitation of spirally plated M. paratuberculosis cultures can be achieved within 8-14 days of plate incubation and compare favourably to counts derived after prolonged incubations. SIGNIFICANCE AND IMPACT OF THE STUDY: The technique could greatly hasten the quantitation of viable M. paratuberculosis.     

Scanning Electron Microscopy of the Influence of Fixation Vehicle Osmolality on Cell Morphology of Spiroplasma, Acholeplasma, and Mycoplasma spp. grown on Agar

Young Spiroplasma citri, corn stunt spiroplasma, and honey bee spiroplasma colonies fixed in 5% glutaraldehyde in M 199 cell culture medium with 0.25 M sucrose showed elongated mycelium-like cells which were sometimes branched or helical. In older colonies beaded chains and rounded bodies were formed. Fixation in 6 % glutaraldehyde in distilled water resulted in amorphous masses in which rounded bodies were present. The spiroplasma cells did not remain osmotically active after glutaraldehyde fixation. Acholeplasma laidlawii and Mycoplasma hyorhinis colonies fixed in glutaraldehyde with or without M 199 medium with 0.25 M sucrose showed little difference in cell morphology.     

Heat shock response in mycoplasmas, genome-limited organisms.

We have measured the effect of heat shock on three mycoplasmas (Acholeplasma laidlawii K2 and JA1 and Mycoplasma capricolum Kid) and demonstrated the induction of mycoplasma heat shock proteins under these conditions. Increased synthesis of at least 5 heat shock proteins in A. laidlawii K2, 11 heat shock proteins in A. laidlawii JA1, and 7 heat shock proteins in M. capricolum was observed by electrophoretic analysis of proteins from heat-shocked cells in sodium dodecyl sulfate-polyacrylamide gels. In all three strains, major heat shock proteins (66 to 68 and 26 to 29 kilodaltons [kDa]) were found. The 66- to 68-kDa protein cross-reacted with antibody to Escherichia coli DnaK protein, suggesting that this heat shock protein has been conserved in spite of major reductions in genetic complexity during mycoplasma evolution. A. laidlawii also contained a 60-kDa protein that cross-reacted with eubacterial GroEL protein and a 40-kDa protein that cross-reacted with E. coli RecA protein. Unlike with coliphages, the mycoplasma virus L2 progeny yield was not increased when virus was plated on heat-shocked A. laidlawii host cells. However, UV-irradiated L2 virus could be host cell reactivated by both A. laidlawii SOS repair and heat shock systems.     

Effect of pH on Growth and Survival of Three Avian and One Saprophytic Mycoplasma Species

The ability of Mycoplasma meleagridis, M. laidlawii, an unnamed, nonpathogenic avian species, and three isolates of M. gallisepticum to grow and survive in liquid media of various pH values was investigated. All species grew over a pH range that was generally about two units. The most significant finding was that cultures initiated in media within a pH range of approximately 8.5 to 8.9 remained viable for 45 to 60 days at 37 C, whereas cultures initiated at lower values survived for shorter periods.     

Characteristics of a New Sterol-nonrequiring Mycoplasma

Two Mycoplasma strains recovered from tissue culture environments were found to grow in complex media devoid of serum or serum fractions containing cholesterol and in a cholesterol-free synthetic medium. Neither strain was capable of synthesizing pigmented carotenoids, although these compounds are present in, and characteristic of, other sterol-nonrequiring mycoplasmas. Serological tests and an analysis of their cell protein patterns obtained by gel electrophoresis indicated that the isolates were similar to each other but distinct from other sterol-nonrequiring serotypes, Mycoplasma laidlawii and M. granularum, as well as from sterol-requiring species. The existence of Mycoplasma other than M. laidlawii and M. granularum without sterol requirements suggested the need for some taxonomic changes in this group of organisms.     

pH-controlled continuous cultivation of mycoplasmas.

The continuous cultivation of mycoplasmas in a pH-controlled metabolistat was investigated with the fermentative strain Mycoplasma mobile 163K and the nonfermentative strain Mycoplasma arthritidis ISR1. The addition of medium and the removal of culture suspension were regulated by acid production from glucose by M. mobile 163K and by ammonium production from arginine by M. arthritidis ISR1, respectively. For both strains the optimal pH for continuous growth was 7.0. The steady state could be maintained for at least 21 days. With CFU of 8.4 X 10(9) ml-1 (M. mobile 163K) and 3.2 X 10(9) ml-1 (M. arthritidis ISR1), the cell concentrations were slightly higher than those obtained in batch cultures. The dependence on the adjusted pH values was measured for several parameters, such as flow rate, CFU, glucose fermentation or production of ammonia, and gliding velocity. Since the long lag phases of batch cultures can be avoided, pH-controlled continuous cultures provide an appropriate system for the production of mycoplasma cells.     

Growth and survival of Mycoplasma neurolyticum in liquid media

Hottle, G. A. (Naval Biological Laboratory, University of California, Berkeley), and D. N. Wright. Growth and survival of Mycoplasma neurolyticum in liquid media. J. Bacteriol. 91:1834-1839. 1966.-Maximal growth of Mycoplasma neurolyticum (between 10(8) and 10(9) colony-forming units per ml) was obtained after 3 days of incubation at 36 C in broth media containing 10% agamma horse serum. When whole horse serum was used in the medium, a complement-mediated inhibition was observed. This inhibition could only be detected when growth was followed by daily plate counts. Maximal growth was delayed for about 24 hr by the horse serum, and the inhibition was spontaneously reversed at the temperature of incubation. Penicillin G was also found to have a temporary inhibitory effect. This was detected with as little as 40 units per ml. Maximal growth was delayed until the 6th day of incubation, when 200 units per ml was present, and until the 16th day, when 1,000 units per ml was present. The survival of M. neurolyticum at undetectable levels in cultures during the incubation period presented an "eclipse" phenomenon which has not been explained. The recrudescence of growth in such cultures late in the incubation period illustrates the events which may occur when mycoplasmas are isolated from clinical material by prolonged incubation in the presence of inhibitors. Survival data showed that M. neurolyticum had greatest stability at pH 8.0, with reduced viability at pH 9.0, 7.0, 10.0, and 6.0, in that order The data on growth and stability suggest a close relationship between the species. of Mycoplasma studied and bacteria.