Preliminary Characterization of Bacteriophages Infecting the Thermophilic Actinomycete Thermomonospora

Bacteriophages lysing strains of Thermomonospora alba and T. fusca were isolated, following specific enrichment, from vegetable composts. Four Thermomonospora phages were distinguished by plaque morphology and host range. Electron microscopy of phage particles, termperature inactivation profiles, and electrophoretic analyses of major virion proteins and genomic DNA were used in the comparison and initial characterization of these phages. The four phages studied possessed polyhedral heads and long tails; genomes were linear double-stranded DNA molecules, 35 to 45 kilobases in length, which probably contain cohesive ends. Transfection of Thermomonospora protoplasts with purified genomic DNA from one of the phages was demonstrated.     

Distinct Types of rRNA Operons Exist in the Genome of the Actinomycete Thermomonospora chromogena and Evidence for Horizontal Transfer of an Entire rRNA Operon

We describe here the presence of two distinct types of rRNA operons in the genome of a thermophilic actinomycete Thermomonospora chromogena. The genome of T. chromogena contains six rRNA operons (rrn), of which four complete and two incomplete ones were cloned and sequenced. Comparative analysis revealed that the operon rrnB exhibits high levels of sequence variations to the other five nearly identical ones throughout the entire length of the operon. The coding sequences for the 16S and 23S rRNA genes differ by approximately 6 and 10%, respectively, between the two types of operons. Normal functionality of rrnB is concluded on the basis of the nonrandom distribution of nucleotide substitutions, the presence of compensating nucleotide covariations, the preservation of secondary and tertiary rRNA structures, and the detection of correctly processed rRNAs in the cell. Comparative sequence analysis also revealed a close evolutionary relationship between rrnB operon of T. chromogena and rrnA operon of another thermophilic actinomycete Thermobispora bispora. We propose that T. chromogena acquired rrnB operon from T. bispora or a related organism via horizontal gene transfer.     

Selective Medium for the Isolation of Prototheca

A medium was devised which permitted the selective isolation of Prototheca spp. from nature.     

Polymyxin-Coagulase-Deoxyribonuclease-Agar: a Selective Isolation Medium for Staphylococcus aureus

Fibrin halos developed around coagulase-positive Staphylococcus aureus colonies, and deoxyribonuclease production was detected by using a developing solution containing 1 mg of methyl green per ml.     

Rifampin-Blood-Agar as a Selective Medium for the Isolation of Certain Anaerobic Bacteria

A selective medium which allows detection of relatively small numbers of Fusobacterium varium in fecal specimens is described. Blood-agar containing 50 mug of rifampin per ml inhibits the growth of many species of Bacteriodes and of F. fusi-forme/nucleatum but allows good growth of F. varium and most strains of F. mortiferum. Quantitative cultures of 11 fecal specimens were done on rifampin and other selective and nonselective media. F. varium was recovered in counts of 10(6) and 10(7) per gram from two specimens on rifampin only. A third specimen yielded 10(10)F. varium on several media, including rifampin. Some Eubacterium and Clostridium species also grew on rifampin, and these ordinarily were distinguished from the Fusobacterium by colony morphology. This medium is of value in fecal flora studies and should be useful with other kinds of specimens where mixtures of organisms are common.     

A Comparison of Various Selective Media, including a New Selective Medium for the Isolation of Brucellae from Milk

S ummary : A comparative trial of a new medium for brucella was made on samples of milk from individual cows and herd samples of milk. The new medium gave a higher isolation rate than any of the other 3 selective media tested. There was no evidence that the new medium inhibited any of the biotypes of Brucella abortus , especially biotype 2; it was the only medium suitable for isolating from samples of herd milk strains of biotype 2 which are sensitive to antibiotics and aniline dyes.     

A Selective Medium for Isolation and Presumptive Identification of the Bacteroides fragilis Group

A new selective medium, Bacteroides fragilis ammonium-sulfate gentamicin (BFAG) agar, for isolation and presumptive identification of the Bacteroides fragilis group is presented in this paper. This semisynthetic medium includes 0.2 g of ammonium sulfate, 0.7 g of lactose, 10 mg of gentamicin, 0.1 mg of aminobenzylpenicillin, 60 units of bacitracin, 20 mg of sodium cholate and 1 mg of sodium azide per 100 ml of medium. Stock cultures of the B. fragilis group grew well on this medium. None of the other 126 gram-positive or negative strains belonging to 40 aerobic or 45 anaerobic species tested grew on this medium. Three of the seven specimens in the clinical trials yielded colonies of only the B. fragilis group on BFAG agar plates. Also BFAG agar plates inoculated with human feces and contents of the alimentary tract (stomach, small intestine, cecum and colon) of mice gave rise to colonies of only the B. fragilis group. The high selectivity and good plating efficiency of BFAG agar enabled us to isolate the B. fragilis group rapidly from various clinical specimens.     

Selective Medium for the Isolation of Streptococci from Clinical Specimens

Incorporating neomycin and nalidixic acid into a blood-agar base resulted in a medium highly selective for beta-hemolytic streptococci under conditions in which detection of streptococcal colonies by conventional means would have been very difficult.     

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.     

Modified Selective and Differential Isolation Medium for Vibrio parahaemolyticus

The semiselective salt-starch-agar formulation of Baross and Liston was modified as the result of a systematic study of the effect of each constituent on the growth of Vibrio parahaemolyticus and competitive species characteristic of the marine environment. The selection of nutrient constituents depended on an analysis of their effect on generation time. The addition of inhibitors depended on an analysis of minimal inhibitory concentrations. The modified formulation included: peptone, 2.0%; yeast extract, 0.2%; corn starch, 0.5%; NaCl, 3.0%; agar, 1.5% (pH 8.0). Penicillin at 2 to 5 units/ml increased selectivity without significantly inhibiting Vibrio in pure suspensions. Over 62% of the most sensitive strain (YM-K33) was recovered at a concentration of 5 units of penicillin per ml. The per cent recovery of V. parahaemolyticus from fish homogenate compared favorably with other selective formulations. At an initial concentration of 10(5) cells/ml, recovery varied with the strain used from 60 to 119%, whereas at 10(2) cells/ml the range was 36 to 94%. Applications of the medium for Vibrio quantification are discussed.     

Selective Broth Medium for Isolation of Group B Streptococci

A selective medium containing Todd-Hewitt broth, sheep blood, nalidixic acid, and gentamicin sulfate was found to enhance significantly the isolation of group B streptococci from vaginal cultures. Preparation of the medium, which is stable for up to 4 weeks at 4 C, is simple and inexpensive. Use of such a medium should facilitate identification of vaginal colonization with group B streptococci.     

Development of a Selective Medium for the Isolation of Clostridium sporogenes and Related Organisms

An attempt was made to develop a selective isolation medium for Clostridium sporogenes and related organisms based on the ability of these organisms to obtain their energy for growth by means of coupled oxidation-reduction reactions between appropriate pairs of amino acids (Stickland reaction). Using a semi-defined basal medium containing various combinations of amino acids, it was found that Cl. sporogenes utilized a wider range of amino acid pairs than strains of five other species of clostridia known to carry out a Stickland-type fermentation.
With alanine and proline as the principal energy sources and the medium solidified with agar. it was shown that reference strains of Cl. sporogenes and proteolytic Cl. botulinum types A, B and F could be recovered almost quantitatively, with or without prior heating at 80 °C for 10 min. By contrast, growth of test strains of Streptococcus faecalis, Strep. faecium , 'saccharolytic' Cl. botulinum types B, C, D, E and F and 'proteolytic' strains of types C and D was suppressed on this medium, as were strains of 26 other species of clostridia.
Addition of 50 μg/ml of polymyxin to the agar medium had no detectable effect on the recovery of Cl. sporogenes or Cl. botulinum. When samples of soil and mud were plated on the antibiotic-containing medium, 63.1% of 225 isolates thus obtained were identified as Cl. sporogenes/botulinum.     

Development of a Novel Selective and Differential Medium for the Isolation of Listeria monocytogenes

A new medium (lecithin and levofloxacin [LL] medium) is described for the isolation of Listeria monocytogenes from food samples. LL medium includes lecithin from soybeans for the detection of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) produced by L. monocytogenes. Levofloxacin is incorporated to inhibit the growth of microorganisms other than L. monocytogenes, especially Bacillus cereus, shown to possess PI-PLC and PC-PLC activities. L. monocyogenes produced white colonies with a halo on LL medium, whereas Listeria innocua appeared as white colonies without a halo. Levofloxacin at 0.20 mg/liter completely inhibited the growth of B. cereus, while the growth of L. monocytogenes was unaffected. In the second phase of the study, the sensitivity and the specificity of LL medium were compared to those of modified Oxford agar (MOX) and two chromogenic media (Brilliance Listeria agar and CHROMagar Listeria), using a total of 250 food samples. From 200 unspiked food samples, the specificity of LL medium (96.0%) was superior to that of MOX (72.0%) and similar to the specificities of Brilliance Listeria agar (96.5%) and CHROMagar Listeria (94.5%). From 50 spiked food samples, LL medium and CHROMagar Listeria represented the highest sensitivities (96.0%), followed by Brilliance Listeria agar (92.0%) and MOX (54.0%). Also, LL medium showed the highest confirmation rate (98.8%), followed by Brilliance Listeria agar (98.7%), CHROMagar Listeria (98.3%), and MOX (52.0%). On the basis of its good specificity and cost effectiveness, LL medium is useful for the isolation of L. monocytogenes from food samples.     

A Selective Medium for Mycoplasma Suipneumoniae

The isolation of Mycoplasma suipneumoniae (M. snip.) has not hitherto been possible in broth culture if the material also harboured Mycoplasma hyorhinis (M. hyor.), since the latter organism would outgrow M. suip. The present paper reports an effort to solve this problem.     

Medium for the Selective Isolation of Members of the Genus Pseudomonas from Natural Habitats

A new medium was developed that was superior to four others tested in selecting for members of the genus Pseudomonas.