False identification ofNeisseria gonorrhoeae in lower respiratory tract secretions—Result of mixed culture

A Gram-negative diplococcus obtained by transtracheal aspiration was initially identified asNeisseria gonorrhoeae by three different laboratories. Further examination indicated that the culture did not containN. gonorrhoeae, but did contain a mixture ofBranhamella catarrhalis andHaemophilus influenzae. B. catarrhalis had provided growth factors that permittedH. influenzae to grow in cystine tryptophan peptone agar with resultant production of acid from glucose and misidentification of the diplococcus. When all isolates from respiratory secretions are fully identified, erroneous conclusions because of bacterial interactions could be avoided if tests were performed from pure culture plates rather than from the primary inoculum plate.     

Fatty acid composition of membranes of L-forms ofStreptococcus faecalis andProteus mirabilis cultured at different osmolalities

The fatty acid composition of membranes of L-forms ofStreptococcus faecalis andProteus mirabilis cultured at different osmolalities and in different osmotic stabilizers was examined.S. faecalis L-forms cultured with sucrose in the medium showed a decrease in the unsaturated fatty acid C₁₈₁ and an increase in the C₁₈ fatty acid and C₁₉ cyclopropane fatty acid. Fatty acid composition ofS. faecalis L-forms cultured in medium containing 1.8% NaCl was similar to the fatty acid composition of L-forms cultured in brain-heart infusion broth (BHI) without osmotic stabilizer and was between the composition of fatty acids of L-forms in BHI with sucrose and that in BHI without 0.5 M sucrose. InProteus mirabilis L-forms, there were differences between L-forms cultured with and without sucrose, but these differences were not comparable to the changes observed inS. faecalis L-forms.P. mirabilis L-forms cultured with and without NaCl in the medium had similar fatty acid compositions.     

Hyphal and mycelial interactions betweenAgaricus bisporus andScytalidium thermophilum on agar media

The interaction betweenAgaricus bisporus andScytalidium thermophilum on agar media was studied by differential interference contrast and phase contrast microscopy.A. bisporus combatively replacesS. thermophilum in culture on agar media. The antagonistic effect ofA. bisporus is transmissible through a cellophane membrane and causes irreversible disintegration ofS. thermophilum protoplasm, resulting in a total loss of viability after prolonged interaction between the two fungi. On compost extract agar, but not on other media, the growth rate ofA. bisporus increased from 2.7 to 5.3 mm·d^–1 following contact withS. thermophilum mycelium.     

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.     

Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

Inhibition of fungi,actinomycetes and bacteria by Stachybotrys atra

The effect ofStachybotrys atra on fungi, actinomycetes and bacteria was studied in agar culture. Of the 27 genera and 52 species of fungi, 6 species of actinomycetes and 5 genera and 10 species of bacteria tested,S. atra was found to inhibit 95.7 % fungi, all the actinomycetes and 83.3 % of bacteria.Aspergillus ustus, 2 unidentified species ofFusarium, Rhizobium trifolii andPseudomonas spp. were however not inhibited. In experiments in soil,S. atra inhibited the growth ofTrichoderma viride. S atra was ineffective whereT. viride andS. atra were inoculated simultaneously or whereT. viride was added 7 days earlier. Besides,S. atra was not found to controlMacrophomina phaseoli infection on cotton. Soil culture ofS. atra showed phytotoxic effect on cotton seedlings.S. atra produced a dark brown to black thermostable toxic metabolite in the medium. The culture filtrate retarded the growth ofM. phaseoli in agar culture. Despite the importance ofS. atra as a fungistatic agent with wide antimicrobial spectrum, the use ofS. atra as a protectant against plant pathogens seems to be limited because of its phytotoxicity on cotton.     

Effect of selective media on loss of congo red binding inShigella flexneri

Summary The effect of growth ofShigella flexneri on various selective media on retention of congo red (CR) binding ability was determined to evaluate the effectiveness of isolation techniques regarding maintenance of the virulence plasmid. WhenS. flexneri was surface-plated onto selective agars and the resulting colonies replica plated onto CR plates, no white colonies indicative of loss of virulence were found despite repeated trials. However, whenS. flexneri was grown in liquid media (agar was removed from agar-containing media by centrifugation), white colonies were found upon plating onto CR plates. Most common selective media for shigellae produced fewer than 5–10 white colonies/1000 red colonies. However, growth in broth prepared from violet red bile agar, desoxycholate citrate agar, and SS agar gave more than 100 white colonies/1000 red colonies. Loss of CR binding was demonstrated whenS. flexneri was grown in broth containing tergitol 7, sodium dodecyl sulfate, bile salts #3, crystal violet, eosin y or methylen blue. However, concentrations of selective agents that led to loss of CR binding were much higher than those used in selective media. Results indicate that under usual conditions of isolation ofS. flexneri from food and clinical specimens, CR binding appears to be a relatively stable character with most selective media; however, use of violet red bile agar, desoxycholate citrate agar, and SS agar may lead to substantial loss of congo red binding indicating that the isolates may not be virulent.     

Inhibition of growth ofLegionella species by heterotrophic plate count bacteria isolated from chlorinated drinking water

The ability of heterotrophic plate count bacterial strains isolated from chlorinated drinking water on low-nutrient media to inhibit the growth ofLegionella species was examined. Between 16% and 32% of these strains were able to inhibit the growth ofLegionella species when tested on buffered charcoal yeast extract agar. The exact proportion of inhibiting strains varied with the individualLegionella species. Two strains that inhibited the growth of severalLegionella species could also stimulate the growth of the same species when both the test strain and theLegionella species were grown on buffered charcoal yeast extract agar that lacked the essential amino acidl-cysteine.     

Alterations of the L-forms of a sporebearing bacillus

A large pleomorphic gram-negative bacillus developed as a contaminant on blood-agar. Spores were formed in one culture. L-forms were produced with penicillin on blood-agar with 2.5% NaCl; they grew well when transplanted to agar with 0.5% NaCl. After several transplants and long incubation of the L-forms without penicillin, in three transplants small gram-negative pleomorphic bacilli grew, but no L-forms. This occurred once on blood-agar and twice on 30% gelatin. The growth obtained from these small bacilli was similar in morphology and in the physical properties of the organisms to the altered L-forms of Proteus and Salmonella. Multiplication of the pleomorphic organisms and development of branching filaments from the round forms was apparent. The original large gram-negative bacillus was regularly recovered from the L-forms, and was recovered several times from the descendants of the small bacilli. These observations are essentially similar to those made with L-forms of Proteus and with an L-form studied in 1952, indicating alterations in L-forms of bacteria which do not produce B type L-forms.     

Bacterial Interference with L-Forms

During the course of studying the effect of normal nasal flora on the growth of L-forms, a clear zone of inhibition was observed around colonies of many coagulase-negative staphylococci. Subsequent investigation demonstrated that Staphylococcus aureus and some S. albus strains produce a substance which is capable of markedly inhibiting the growth of stable staphylococcal and streptococcal L-forms. This interfering substance is separable from the staphylococcal organism and is diffusible through 1.5% agar, but not through a dialysis membrane. It is heat-stable.     

Growth and surface binding of proteins byNeisseria meningitidis in normal human serum

The addition of normal human serum to a chemically defined medium stimulated the growth ofNeisseria meningitidis strain M1011 greatly. Both lag times and mean generation times were reduced. Growth in human serum alone was rapid but quickly became nutrientlimited. Iron was not a limiting factor for growth in human serum. One major limiting factor of serum was cysteine.N. meningitidis grown in serum also bound serum proteins, including transferrin and immunoglobulin, as demonstrated by direct and indirect fluorescent-antibody techniques.     

Antigenic and deoxyribonucleic acid base composition of aMycoplasma and associated diphtheroids

Three diphtheroids isolated from cultures ofMycoplasma arthritidis (Campo strain) were shown to cross-react serologically with theMycoplasma. Stable L-forms prepared from these bacteria were shown to have still greater antigenic similarity to theMycoplasma. But the three diphtheroids isolated on three separate occasions were shown not to be identical antigenically. The buoyant densities of the DNA's of the L-forms were similar to those of the diphtheroids from which they were derived. The caesium chloride gradient method indicated a significant difference in nucleotide base composition between the purified deoxyribonucleic acid of the diphtheroids and of theMycoplasma.Junior Research Fellow supported by USPH training grant 5 ROI AI 00232.     

The effect of agar on the inhibitory activity of phenols

Nineteen phenols were tested for their effectiveness in inhibiting the growth ofStaphylococcus aureus in both broth and agar media. The presence of agar in the test medium interfered with the action of eight of the compounds tested.The results of this investigation show that no general statement can be made as to the effect of agar on the phenols as a group. It appears, rather, that this effect is dependent upon the individual compound.     

A peculiar example of bacterial antagonism

Summary An example of antagonism has been described. The properties of the growth inhibiting bacterium have been investigated more closely. These appear to agree nearly fully with those of the genusGaffkya.The development ofPasteurella avicida, Bacillus orpheus andStaphylococcus was strongly inhibited by this bacterium. Other species are less sensitive or insensitive.After the theoretical problems have been discussed, which such investigations give rise to, it has been studied under which conditions Staphylococci are inhibited.The occurrence of the inhibition effect depends on the stage of development either bacterium is in;Staphylococcus having developed completely it will not notably be acted upon. Initially inhibition of growth occurs along with dying off ofStaphylococcus. In a somewhat later stage the vitality remains unimpaired.The inhibiting bacteria produce inhibiting substances merely in the first 24 hours. In the filtrate of a broth culture these are hard to detect. Their behaviour in agar plates makes it apparent that their action depends strongly on the concentration of the antagonist.The durability of the inhibiting substances in the agar medium is not great. In all probability this depends on a volatibility of these substances.     

Interactions of soil fungi with Macrophomina phaseoli (Maubl.) ashby,the cause of root rot of cotton

Summary Interactions of 46 isolates of fungi with an isolate ofM. phaseoli from cotton, was studied in agar culture. They were grouped into 4 different types of reactions.T. viride was found to inhibit the growth ofM. phaseoli and grow over its colony. The hyphae ofT. viride were observed to coil around the hyphae ofM. phaseoli. A majority of the isolates tested had no effect on the growth ofM. phaseoli. M. phaseoli, however, was found to overgrow the test fungi.     

Cultural and physiological studies of phytophthora parasitica dast. var. macrospora ashby,causing fruit rot of anona squamosa L.

Summary This paper gives an account of some cultural and physiological studies of an isolate ofPhytophthora parasitica Dast. var.macrospora Ashby, the causal agent of fruit rot ofAnona squamosa L. Among the various culture media studied, non-synthetic ones like oat meal agar, corn meal agar, lima-bean agar, carrot extract agar, soya-bean extract agar and steamed rice agar were the best, on which the organism showed marked growth and sporulation. Non-synthetic types were poor in this direction. Regarding the effect of various carbon sources, sucrose, lactose, maltose, raffinose, inulin, dextrin, dulcitol, glycogen, rhamnose and xylose supported growth and sporulation of the organism. Sodium nitrate, ammonium nitrate, magnesium nitrate, potassium nitrate, ammonium lactate and asparagin were the best sources of nitrogen. 6.5 was found to be the best pH for the growth and sporulation of the organism.A portion of Senior Author's M.Sc. Agric. Thesis, University of Poona, India.     

Effect of some antibiotics on Sclerotium cepivorum Berk., the cause of white rot of onion

The effect of some antibiotics onSclerotium cepivorum, the cause of white rot of onion was studied in agar culture and soil. The growth ofS. cepivorum was inhibited in Czapek Dox yeast agar containing 50µg of gliotoxin, viridin, actidione and 100µg of patulin per ml of the medium. Lower concentrations of the antibiotics retarded the growth of the fungus. In soil, patulin had no effect in the control ofS. cepivorum infection of onion seedlings. Concentration of actidione of 5µg/g of soil completely controlled white rot infection but severely stunted the growth of onion seedlings; 40µg/g of actidione killed the seedlings. Despite the importance of actidione as a fungistatic agent its use on onion is limited by its phytotoxicity.     

Thiamine pyrophosphate,a growth factor forTreponema vincentii andTreponema denticola

Growth ofTreponema vincentii N-9 in complex media was stimulated by culture filtrates ofT. minutum. Thiamine pyrophosphate (TPP) in nanogram quantities could substitute for the stimulatory factor inT. minutum culture filtrates. Characterization of the filtrates indicated that the stimulatory factor was similar to TPP in heat stability and ultrafiltration properties. The oral treponemesT. vincentii andT. denticola required TPP for maximum growth, whereas the genital treponemesT. minutum, T. phagedenis, andT. refringens do not require TPP. The presence of TPP activity in culture filtrates of the genital treponemes was confirmed by using a strain ofNeisseria gonorrhoeae auxotrophic for TPP. TPP activity was not found in culture filtrates of oral treponemes. Culture filtrates of 13 of 14 species representing eight genera found in the microbial flora of the oral cavity also contained TPP activity.     

Fungal colonization of computer diskettes and other magnetic media

Computer diskettes can be colonized by saprophytic fungi, especially in the humid tropics. Fungi of the generaAlternaria, Aspergillus, Epicoccum, Paecilomyces, Penicillium, andTrichoderma were observed on diskettes from several tropical countries. Common saprophytic fungi from Minnesota colonized clean standard and high density diskettes in growth chambers, indicating that fungal contamination could occur wherever temperature and humidity were adequate.Fusarium species infested diskettes buried in garden soil in Minnesota. Audiotapes, videotapes, and computer magnetic tapes chemically resemble diskettes and also can be colonized by fungi, as can photographic film. The Mylar core of these magnetic media did not support the growth ofPenicillium glabrum, the most aggressive fungus in diskette inoculation studies. However, growth of several fungal species was enhanced when the common plasticizer, lecithin, was added in powdered form to nutrient agar, suggesting that this ingredient of the diskettes may be metabolized by the fungi.Abbreviations DD double density - HD high density - MA malt agar - NA nutrient agar - PDA potato dextrose agar - SEM scanning electron microscopy     

Effects on winter wheat seedling growth by toxin-producing rhizobacteria

Summary Root-colonizing pseudomonads capable of inhibiting seedling winter wheat (Triticum aestivum L.) root growth in an agar seedling bioassay also significantly inhibited wheat root growth in vermiculite; however, the inhibitory trait is quite labile in laboratory culturing. The extent of inhibition in both the agar and vermiculite medium depended on inoculum level. These pseudomonads were found to produce a toxin capable of inhibiting growth ofEscherichia coli C-la andBacillus subtilis. Field isolates that strongly inhibit growth of indicator bacteria also inhibited root growth. Toxin production by the bacteria appeared necessary for inhibition of root growth and indicator bacteria as toxin-negative (TOX^–) mutants no longer inhibited either. Antibiosis towardsE. coli as well as wheat seedling root inhibition in agar was reversed by L-methionine, providing further evidence that a toxin, produced by these organisms, is involved in growth retardation.Contribution in cooperation with the College of Agric. Res. Center, Washington State Univ., Pullman, WA 99164. Scientific Paper No. 6837.