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.     

Antibiotic Control of Mycoplasma in Tissue Culture

Seven of eight strains of Mycoplasma (PPLO) were found to be sensitive to the deoxystreptamines, certain macrolides, and the tetracyclines. These antibiotics are relative noncytotoxic. Kanamycin and tetracycline were useful in eliminating PPLO (pleuropneumonia-like organisms) strain Squibb no. 1 from a HeLa cell line which was deliberately contaminated with PPLO. Repeated exposure of M. laidlawii type B cells to neomycin resulted in a 50-fold increase in resistance, and the resistant strain was also resistant to gentamicin, kanamycin, neomycin, and paromomycin. A tetracycline-resistant strain of this culture was found to be resistant to 7-chlortetracycline, 7-chlor-6-demethyltetracycline, and 5-hydroxytetracycline. One PPLO strain, Squibb no. 2, derived from a contaminated HeLa cell culture, was resistant to all antibiotics studied.     

IDENTIFICATION OF MYCOPLASMATACEAE BY THE FLUORESCENT ANTIBODY METHOD

Clark, Harold W. (The George Washington University, Washington, D.C.), Jack S. Bailey, Richard C. Fowler, and Thomas McP. Brown. Identification of Mycoplasmataceae by the fluorescent antibody method. J. Bacteriol. 85:111-118. 1963.-The conditions of the fluorescent antibody reactions were studied in relation to their application to Mycoplasmataceae or pleuropneumonia-like organisms (PPLO). Mycoplasma hominis type 1 and 2 antigens and their homologous antisera were used to determine the activity and specificity of these and other strains. Fluorescein isothiocyanate conjugated antiserum globulin preparations were used in both the direct and indirect fluorescent antibody methods. A direct tube technique was used for the detection and measurement of growth in broth cultures by the addition of conjugated antiserum. The specific fluorescent staining and recognition of hot water fixed M. hominis colonies was presented as a suitable identification standard. The antigenic activity was found to remain in the insoluble residue after exposure of M. hominis strains to sonic vibration (9 kc) for 30 min and centrifugation. Brief 2-min exposures of tissue cells to vibration (9 kc) caused the disruption of tissues, with the release of viable and "bound" nonwashable strains that reacted specifically with fluorescent antibody. It is proposed to apply both the sonic vibration and the fluorescent antibody techniques for the identification of Mycoplasmataceae in human tissues.     

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.     

Inhibition of Sindbis virus plaque formation by extracts of Escherichia coli

Vilcek, Jan (New York University School of Medicine, New York, N.Y.), and John H. Freer. Inhibition of Sindbis virus plaque formation by extracts of Escherichia coli. J. Bacteriol. 92:1716-1722. 1966.-Extracts prepared from washed cells of Escherichia coli B by sonic treatment and subsequent filtration through a 0.45-mu membrane filter significantly inhibited plaque formation with Sindbis virus in cultures or primary chick embryo cells up to a dilution of 1:20,000. The inhibitor acted on the cells rather than directly on the virus. The inhibiting substance was nondialyzable. Treatment of crude extracts with nucleases, trypsin, chymotrypsin, pepsin, or ether had no effect on the activity. Treatment with pronase destroyed the virus-inhibiting effect. Extracts prepared from two strains of E. coli B and one strain of E. coli K-12 all showed inhibitory activity against Sindbis virus. The inhibitor was present in the cytoplasmic fraction of bacteria. It was also active against Sindbis virus in human cells and showed some activity against vesicular stomatitis and vaccinia viruses in different types of cells. Interferon was not shown to be involved in the inhibition, although actinomycin D partially reversed the inhibitory activity of the extracts.     

Procurement and Maintenance of Germ-Free Swine for Microbiological Investigations

Germ-free swine were routinely procured by both hysterectomy and hysterotomy (Caesarian section). By means of light-weight portable equipment, piglets could be obtained and transported to the laboratory (without contamination) over distances in excess of 100 miles. The isolators employed in rearing were constructed of stainless steel and flexible plastic film. At weekly intervals, fecal swabs and waste from the floor of the isolator were cultured on blood-agar and in thioglycolate broth, as well as being examined microscopically for the presence of bacteria, yeast, and fungi. The presence of pleuropneumonia-like organisms (PPLO) and viruses in such material was not demonstrable, either by the use of enriched PPLO media or primary porcine-kidney cell cultures. Tissues, body fluids, and cecal contents of piglets sacrificed specifically for microbiological examination were also negative for PPLO, viruses, bacteria, yeast, and fungi. Prenatal infestations by ascarids were not observed. Nutritional problems related to rearing of germ-free piglets, such as hypoglycemia, were not encountered, and the use of an autoclaved commercial sow's milk replacer proved quite satisfactory. The temperature to which piglets were subjected during the first few days of life, however, was very important. The isolator design and application of gnotobiotic techniques to the procurement and rearing of a large germ-free animal such as the pig proved feasible and less difficult than anticipated.     

Electrical Properties of the Membranes of the Pleuropneumonia-Like Organism A 5969

The electrical properties of the pleuropneumonia-like organism A 5969 have been determined over the frequency range from 0.5 to 250 Mcps. The frequency dependence of the dielectric constant and conductivity of PPLO suspensions is completely consistent with the existence of a membrane. The PPLO has an internal conductance which in part reflects its ionic equilibrium with normal nutrient and macromolecular constituents. But it is fairly independent from variation in external ionic strength.     

Composition and enzyme activities of Spiroplasma citri membranes.

Spiroplasma citri was cultured in three different media that supplied cholesterol and fatty acids from: (i) horse serum, (ii) pleuropneumonia-like organism (PPLO) serum fraction, or (iii) bovine serum albumin-fatty acid-cholesterol. The ability of PPLO serum fraction to support growth varied by lot number. Neither PPLO serum fraction nor the bovine serum albumin medium supported growth as well as the horse serum medium. Analysis of cholesterol, lipid phosphorus, and membrane protein showed the horse serum- and PPLO-grown cells to be indistinguishable, but the bovine serum albumin-grown cells were deficient in lipid phosphorus. The three cultures did not show markedly different fatty acid compositions, but, in all cases, the cultures preferentially incorporated palmitic acid and discriminated against linoleic acid. Cultures grown for different times from logarithmic growth through a degenerative phase showed relatively constant ratios of cholesterol/protein and lipid phosphorus/protein. Fatty acid composition was also relatively constant at the different stages. Adenosine triphosphatase and p-nitrophenyl phosphatase were mainly associated with the membrane, whereas reduced nicotinamide adenine dinucleotide oxidase was either readily removed or not associated with the membrane. The reduced nicotinamide adenine dinucleotide oxidase was inactivated at temperatures above 35 degrees C.     

Isolation and characterization of filterable marine bacteria

Anderson, J. I. W. (Northeast Shellfish Sanitation Research Center, Narragansett, R.I.), and W. P. Heffernan. Isolation and characterization of filterable marine bacteria. J. Bacteriol 90:1713-1718. 1965.-By a process of double filtration of seawater, first through a membrane filter with a pore diameter of 0.45 mu and then through a membrane filter with a pore diameter of 0.22 mu, it was possible to isolate on the surface of the latter membrane a group of marine organisms not usually encountered by conventional techniques of pour plates or one-stage filtration. Many of the isolates could not be identified, but the largest single group belonged to the genus Spirillum; other isolates were placed in the genera Leucothrix, Flavobacterium, Cytophaga, and Vibrio. A group of four organisms which was not identified was characterized by the formation of large, club-shaped cells, 20 to 30 mu long. Of the 25 strains studied in detail, 22 required seawater for growth and 8 retained their filterable property after cultivation. No filterable bacteria were isolated from terrestrial samples.     

Control of Pleuropneumonia-like Organisms in Cell Culture

Mammalian cell culture systems were maintained free of mycoplasmas by using a 3-day agar plate test as a weekly routine to monitor the conditions of the cells. If contaminated cell cultures were found, they were discarded and replaced from a pleuropneumonia-like organism (PPLO)-free cell bank. PPLO-free lines were established by treatment with various antibiotics. The KB cell line was freed of mycoplasmas by treatment for 1 week with a mixture of chlortetracycline, kanamycin, and chloramphenicol. L-929 cells were cleared of contamination with either spectinomycin or tylosin, and a synovial cell line was cleared with lincomycin or tylosin. Each cell line, after eradication of the contaminant, was stored in liquid nitrogen. A number of agents were tested to determine minimal inhibitory concentration against three known and three unidentified mycoplasmas. Chlortetracycline and tetracycline were found to be highly active against all strains, whereas tylosin, spectinomycin, and lincomycin, though less active, were equally useful because of their low toxicity against cells. Kanamycin was highly active against three strains, but inactive at high levels against the KB cell contaminants. A disc plate test was used to check isolated cell contaminants for sensitivity to various agents.     

Use of Membrane Filter Technique in the Microbiological Control for the Brewing Industry

A physical method was developed involving serial filtration with membrane filters for separating yeast cells from bacteria. Such a method eliminates the need for antibiotics previously required to permit differential counting of such populations. All yeast cells filtered were successfully retained and cultivated on a 1.2-mu membrane filter by use of a synthetic medium. All bacteria filtered avoided entrapment on a 1.2-mu membrane filter and were successfully retained and cultivated on a 0.22-mu membrane filter with the same synthetic medium. Final filtrates from these serial filtrations were free from all yeast cells and bacteria when tested with Fluid Thioglycollate Medium.     

Rapid separation and quantitation of mixed microorganisms by filtration and bioluminescence

A membrane filtration/bioluminescence system was developed for the differentiation and quantitation of mixed populations of microorganisms. Samples containing microorganisms were filtered through two membrane filters of descending pore size. The microorganisms retained on the filter contain ATP that can be extracted and measured on the filter via the firefly luciferase-luciferin bioluminescence assay. Results, obtained in less than 20 min, show a good correlation (r greater than or equal to 0.95) between the light produced and the number of organisms in the sample. Using these techniques, Escherichia coli can be separated from yeast or mold and measured in samples containing both microorganisms. When lysostaphin is used to selectively lyse Staphylococci on the filter, the specific quantification of these bacteria among other microorganisms can also be accomplished. The filtration/bioluminescence technique offers the potential of being a rapid and sensitive method to differentiate and detect microorganisms, by selective sizing or lysing, in a variety of samples.     

Significance of fecal coliform-positive Klebsiella.

A total of 191 Klebsiella pneumoniae isolates of human clinical, bovine mastitis, and a wide variety of environmental sources were tested for fecal coliform (FC) response with the membrane filtration and most probable number techniques. Twenty-seven Escherichia coli cultures of human clinical and environmental origins were also tested. Eighty-five percent (49/58) of known pathogenic K. pneumoniae were FC positive, compared with 16% (19/120) of the environmental strains. E. coli results indicated 93% (13/14) of the clinical and 85% (11/13) of the environmental strains as FC positive. There was no significant difference in the incidence of FC-positive cultures between pathogenic Klebsiella and E. coli. pH measurements of K. pneumoniae and E. coli cultures growing in m-FC broth at 44.5 degrees C revealed three distinct pH ranges correlating with colony morphology. beta-Galactosidase assays of Klebsiella and E. coli cultures at 44.5 degrees C indicated all were able to hydrolyze lactose, even if they were FC negative by the membrane filtration or most probable number techniques. The FC response pattern appears stable in K. pneumoniae. Three pathogenic cultures showed no change in FC responses after 270 generations of growth in sterile pulp mill effluent. Since K. pneumoniae is carried in the gastrointestinal tract of humans and animals and 85% of the tested pathogenic strains were FC positive, the isolation of FC-positive Klebsiella organisms from the environment would indicate their fecal or clinical origin or both. The added fact that K. pneumoniae is an opportunistic pathogen of increasing importance makes the occurrence of FC-positive environmental Klebsiella, particularly in large numbers, a potential human and animal health hazard.     

A selective medium for Fusobacterium spp.

J.S. BRAZIER, D.M. CITRON AND E.J.C. GOLDSTEIN, 1991. A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 μg/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.     

Evaluation of Recovery Methods to Detect Coliforms in Water

Various recovery methods used to detect coliforms in water were evaluated by applying the membrane filter chamber technique. The membrane filter chambers, containing pure-culture suspensions of Escherichia coli or natural suspensions of raw sewage, were immersed in the stream environment. Samples were withdrawn from the chamber at regular time intervals and enumerated by several detection methods. In general, multiple-tube fermentation techniques gave better recovery than plating or membrane filtration procedures. The least efficient method of recovery resulted when using membrane filtration procedures, especially as the exposure period of the organisms to the stream environment increased. A 2-h enrichment on a rich, nonselective medium before exposure to selective media improved the recovery of fecal coliforms with membrane filtration techniques. Substantially enhanced recoveries of E. coli from pure-culture suspensions and of fecal coliforms from raw-sewage suspensions were observed when compared with recoveries obtained by direct primary exposure to selective media. Such an enrichment period appears to provide a nontoxic environment for the gradual adjustment and repair of injured cells.     

Properties of Mycoplasma hominis 4330

Mycoplasma strain 4330, one of the earliest strains of pleuropneumonia-like organisms to be isolated from man in the United States, has been found to resemble M. hominis type 1 by serological methods (the growth inhibition and latex agglutination tests). The results of earlier serological studies indicated a similarity between the Campo and 4330 strains which was not detected by use of the cultures currently available. Strain 4330 differs from strains of Mycoplasma recently isolated from man by producing acid from a variety of carbohydrates. This acquisition of biochemical properties may be the result of hundreds of transfers on artificial media during a period of more than a quarter of a century. Identification of the strain was deemed advisable, since two different cultures and a mixed culture existed under the designation "4330." The extraneous organisms were found to be closely related to M. laidlawii by their biological and serological properties.     

Polymyxin-Coagulase-Mannitol-Agar: I. A Selective Isolation Medium for Coagulase-Positive Staphylococci1

A selective, differential plating medium was developed for the isolation and identification of coagulase-positive and mannitol-fermenting staphylococci. Coagulase produced by growing Staphylococcus aureus caused an opaque zone of fibrin to form around each colony. Several strains of S. aureus produced a visible coagulase reaction by 8 hr, and all strains gave a positive reaction before 12 hr. Mannitol fermentation was usually observed between 12 and 36 hr. Rabbit serum was filtered through Sephadex G-100 to obtain plasmin- and plasminogen-free coagulase-reacting factor (CRF). False-negative reactions, caused by staphylokinase and staphylococcal Müller factor action on plasminogen, were eliminated when this CRF was used. False-positive reactions by lipolytic, coagulase-negative staphylococci were reduced, since gel filtration removed the serum lipoprotein which served as a primary source of opacity. The addition of 75 mug of polymyxin B per ml selectively retarded the growth of S. epidermidis and minimized false-positive reactions caused by citrate-utilizing gram-negative rods. The preparation, characteristics, and use of the medium are presented.     

Cholesterol uptake byPropionibacterium freudenreichii

Strains ofPropionibacterium freudenreichii were tested for growth and cholesterol uptake in brain-heart infusion (BHI) medium supplemented with pleuropneumonia-like organism (PPLO) serum fraction as the cholesterol source. Stirred cultures ofP. freudenreichii reduced the medium's cholesterol content by 50% or more after 10–14 days of incubation at 32°C. Cholesterol uptake by the propionibacteria did not require strictly anaerobic conditions or the presence of bile acids. Up to 70% of the cholesterol removed from the medium could be recovered by solvent extraction from washed cells ofP. freudenreichii.     

Filtration Method for Bacteriophage Detection

A filtration method has been developed which can be used to detect and enumerate phage in low concentrations directly from solution without the need for prior concentration. In this method, a known volume of the phage solution is mixed with a suitable host solution. Samples are filtered through membrane filters; the filter is removed and incubated, and after 24 hr the resultant plaques are counted and the titer is calculated. Escherichia coli B and the coliphage T2 were used in these studies. Host cultures less than 12 hr old produced the best results. Approximately 10(10) host organisms must be present in the sample taken for filtration. To avoid phage reproduction, all steps prior to filtration must be done in less than 45 min. The method was compared with the soft-agar technique and was shown to be less precise but able to measure phage in lower concentrations.     

An enzyme-linked immunosorbent assay-based isolation procedure for verotoxigenic Escherichia coli.

A colony enzyme-linked immunosorbent assay using the hydrophobic grid membrane filter format was developed for the isolation of verotoxigenic Escherichia coli from human and food samples. The method utilizes monoclonal antibodies directed against the verotoxins and is sensitive to all verotoxin 1- and/or 2-producing serotypes. E. coli that produced a minimum of 2 x 10(2) and 2 x 10(3) 50% cytotoxic doses per ml of verotoxins 1 and 2, respectively, were detectable. In a method comparison using human stool specimens, this procedure isolated 29% more E. coli O157 than did the standard sorbitol-MacConkey agar procedure, with no false-positive reactions. When applied to meat, 11 of 20 samples positive for verotoxin by polymyxin extraction yielded verotoxigenic E. coli of a variety of serotypes including O157:H7. Four false positives were noted. This procedure provides a sensitive means for the isolation of verotoxigenic E. coli and should facilitate recovery of those serotypes that are otherwise indistinguishable from nonpathogenic strains.