Disc Plate Method of Microbiological Antibiotic Assay: II. Novel Procedure Offering Improved Accuracy 1

A detailed disc plate procedure is introduced for assay of antibiotics. The procedure is based on a previous study by the authors and deviates from conventional procedures in several respects: selected plastic petri dishes are employed; critical temperature control is simply provided at all stages of the test with refrigeration of the plates never used; all dilution is done with displacement microburettes; six pads (6.3 mm diameter) per dish are employed, all filled with the same unknown or reference solution; the sequence of all plates handled on 1 day is made a part of the protocol which allows accounting for the influence of the order of pouring and setting the plates; external reference plates are set at specified locations in the sequence; and, by averaging the diameters of all zones on a plate, most of the consequence of wedge shape of agar in plates, which is common and almost unavoidable, is removed. The present method is economical, uses simple facilities, and provides good accuracy of test results. Bacillus subtilis was most commonly employed, but other organisms may be employed in the present procedure.     

Inactivation of bacterial opportunistic skin pathogens by nonthermal DC-operated afterglow atmospheric plasma

Aims: Multidrug‐resistant opportunistic pathogens are clinically significant and require the development of new antimicrobial methods. In this study, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus cells were exposed to atmospheric plasma on agar plates and in vitro on porcine skin for the purpose of testing bacterial inactivation. Methods and Results: Microbial inactivation at varying exposure durations was tested using a nonthermal plasma jet generated with a DC voltage from ambient air. The observed reduction in colony forming units was quantified as log₁₀ reductions. Conclusions: Direct plasma exposure significantly inactivated seeded bacterial cells by approx. 6 log₁₀ on agar plates and 2–3 log₁₀ on porcine skin. On agar plates, an indirect ‘bystander’ inactivation outside the plasma delivery area was also observed. The reduced inactivation observed on the skin surface was most likely due to cell protection by the variable surface architecture. Significance and Impact of Study: Atmospheric plasma has potential for clinical application as a disinfectant of patient skin and medically relevant surfaces.     

Description of conidia from submerged cultivation of Thermomyces lanuginosus for use as a uniform inoculum.

Conidia produced by submerged cultivation of the thermophilic fungus Thermomyces lanuginosus were superior to conidia from agar plates when used as inoculum, due to a faster and more synchronous germination. With conidia derived from submerged liquid culture at 40-45 degrees C more than 90% germination was achieved at 50 degrees C within 3 h whereas the same percentage germination was only achieved after 5 h incubation of conidia produced on agar plates. The temperature during conidial formation, and conidial age at the time of harvesting, were factors influencing germination of the conidia.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37 degrees C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37 degrees C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37 degrees C for 48 h is recommended.     

Studies on the Heat Resistance of Bacteria, with Particular Reference to the Genus Microbacterium: I. A New Technique using Solid Media

Summary: The new technique depends upon spreading bacterial cells over the surface of an agar medium in a Petri dish. The cells are heated, cooled, grown and counted in situ . The plates are preheated on the surface of a water bath and held for the required time on the surface of a second bath at the desired temperature. They are cooled by cold water jetted on to the base. The experimental control of factors affecting the accuracy of the method are discussed and applications suggested.     

Isothiocyanates inhibit fungal pathogens of potato in in vitro assays

Aims

This study aimed to examine the effects of seven different isothiocyanates against the growth and development of three important soil borne potato pathogens, (Colletotrichum coccodes, Rhizoctonia solani and Helminthosporium solani).

Methods

The study was carried out using an agar diffusion assay. The radial growth of fungal pathogens grown on agar containing different ITCs at a range of concentrations was compared to that of growth on control agar plates that did not contain ITCs.

Results

Results varied depending on the specific isothiocyanate incorporated into the agar. They ranged from those which showed a significant effect on fungal growth to those which appeared to have little or no effect. Where a suppressive effect was observed, due to the presence of the isothiocyanate, fungal colony growth decreased as the concentration of the incorporated isothiocyanate increased.

Conclusions

Results from this study indicate that fungal growth can be inhibited by exposure to ITCs. However the results observed are specific to the ITC structure and exposure concentration.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

Modified Penicillin Enrichment Procedure for the Selection of Bacterial Mutants

Penicillin enrichment and selection of biochemical mutants was performed on agar plates. With this technique, there is an optimum penicillin exposure time for the greatest yield of a particular auxotroph.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and Group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37°C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37°C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37°C for 48 h is recommended.     

Rapid Confirmation of Suspect Salmonella Colonies by Use of the Minitek System in Conjunction with Serological Tests

Colonies of 40 members of the Enterobacteriaceae family (26 Salmonella serotypes and 14 other organisms) were picked from selective agar plates and inoculated into Minitek inoculum broth (BBL) and then onto Minitek discs of dextrose, lactose, sucrose, mannitol, maltose, dulcitol, lysine and H₂S. After incubation for 6 h, the inoculum broth was tested with salmonella Poly O and after 24 h with salmonella Poly H antisera. The results of the biochemical tests were read after 24 h incubation. With this procedure, all the salmonella cultures used in this study were confirmed as salmonella and differentiated from all the other organisms, which were rejected. This procedure provides an alternative to the time consuming conventional procedures for the biochemical and serological confirmation of suspect salmonella colonies on selective agar plates.     

Factors Affecting Comparative Resistance of Naturally Occurring and Subcultured Pseudomonas aeruginosa to Disinfectants

A strain of Pseudomonas aeruginosa was isolated in pure culture from the reservoir of a hospital mist therapy unit by an extinction-dilution technique; its natural distilled water environment was used as a growth and maintenance medium. After a single subculture on Trypticase soy agar, the strain showed a marked decrease in resistance to inactivation by acetic acid, glutaraldehyde, chlorine dioxide, and a quaternary ammonium compound when compared with naturally occurring cells grown in mist therapy unit water. The following factors were observed to affect the relative resistances of naturally occurring and subcultured cells of the P. aeruginosa strain: (i) temperature at which the cultures were incubated prior to exposure to disinfectants, (ii) growth phase of the cultures at the time of exposure to disinfectants, (iii) nature of the suspending menstruum for disinfectants, and (iv) exposure to fluorescent light during incubation of inocula prior to testing. The applied significance of these findings may alter the present concepts of disinfectant testing as well as routine control procedures in the hospital environment.     

Image analysis method for evaluation of specific and non-specific hand contamination

AIMS: To evaluate a quantifying image analysis method for assessing the degree of hand contamination and efficacy of hand washing procedures. METHODS AND RESULTS: Two types of experimental design were used. In one, different concentrations of pure cultures of Escherichia coli, Listeria innocua and Pseudomonas flourescens were applied to hands. In the other, hands were contaminated by handling various raw foods. Imprints of the contaminated palms were made on 24.5 x 24.5 cm agar plates using appropriate agars. After incubation, digital photographs of the plates were analysed using image analysis. In pure culture studies with selective agars, levels from 1 to 10(6) CFU cm(-2) palm could be monitored. For aerobic, mesophilic organisms from raw chicken, levels from 10(3) to 10(6) CFU cm(-2) palm were correlated linearly to image analysis data. CONCLUSIONS: The image analysis of palm imprints made on agar plates was suitable for assessing the degree of contamination from foods on the palms. Sensitivity and specificity depended on the agar used and the type of contamination encountered. SIGNIFICANCE AND IMPACT OF THE STUDY: Data capture by the image analysis method is simple and can be partly automated. Sampling time is short for the person to be tested, which makes it an attractive method for assessing hand hygiene status in larger field trials.     

Trace amounts of furan-2-carboxylic acids determine the quality of solid agar plates for bacterial culture

Background

Many investigators have recognised that a significant proportion of environmental bacteria exist in a viable but non-culturable state on agar plates, and some researchers have also noticed that some of such bacteria clearly recover their growth on matrices other than agar. However, the reason why agar is unsuitable for the growth of some bacteria has not been addressed.

Methodology/Principal Findings

According to the guide of a bioassay for swarming inhibition, we identified 5-hydroxymethylfuran-2-carboxylic acid (5-HMFA) and furan-2-carboxylic acid (FA) as factors that inhibit bacterial swarming and likely inhibit extracellular polysaccharide production on agar. The furan-2-carboxylic acids 5-HMFA and FA effectively inhibited the swarming and swimming of several environmental bacteria at concentrations of 1.8 and 2.3 µg L⁻¹ (13 and 21 nmol L⁻¹), respectively, which are equivalent to the concentrations of these compounds in 0.3% agar. On Luria-Bertani (LB) plates containing 1.0% agar that had been previously washed with MeOH, a mixture of 5-HMFA and FA in amounts equivalent to their original concentrations in the unwashed agar repressed the swarming of Escherichia coli K12 strain W3110, a representative swarming bacterium.

Conclusions/Significance

Agar that contains trace amounts of 5-HMFA and FA inhibits the proliferation of some slow-growing or difficult-to-culture bacteria on the plates, but it is useful for single colony isolation due to the ease of identification of swarmable bacteria as the non-swarmed colonies.     

Effect of Storage of Mueller-Hinton Agar Plates on Zone Sizes for Antimicrobial Testing

The length of time Mueller-Hinton agar plates can be stored at 4 C without affecting the size of zones of inhibition in susceptibility testing by the Bauer-Kirby method was studied. It was found that these plates can be stored for 3 weeks at 4 C without an appreciable affect on zone sizes. Storage of plates in sealed plastic bags did not alter the results significantly. The findings indicate that commercially prepared Mueller-Hinton agar plates, which may be several days old when received at the laboratory, are suitable for use in routine susceptibility tests by the Bauer-Kirby method.     

A microdilution method for drug sensitivity testing of fish associated bacteria at 22°C

When applied to the psychrophilic and psychrotropic bacteria associated with diseases of fish, the disc-diffusion method generally fails to provide correct estimates of minimal inhibitory concentrations (MIC). A microdilution method using a medium containing agar (15 g/l) and 10 selected drugs has been developed. Serial four-fold dilutions of the drugs were performed in specially designed dilution plates, which were prepared and stored at −30° or −70°C. Although its reliability sometimes appeared to be influenced by the temperature at the time of testing, the temperature and duration of freezing and the nature of the drugs, the microtest provided results as accurate as other reference methods. In repeated experiments very rare major discrepancies were noted and minor variations of one dilution step were below 10%, except with tetracyclines and the sulphonamide-trimethoprim mixture. The advantages of the method and the optimum conditions for use in fish disease diagnosis are specified.     

Toxicity of Irradiated Medium for Repair-Deficient Strains of Escherichia coli

Nutrient agar medium is made highly toxic to certain repair-deficient strains of Escherichia coli by exposure of the uninoculated plates to radiation from cool-white fluorescent lamps or black-light fluorescent lamps. This sensitivity is associated with the genetic deficiencies, fil, phr, and recA. Repair-sufficient and uvr strains are only slightly affected by the irradiated media. The poor growth and reduced plating efficiency frequently associated with BPhr and recA strains are very likely caused by inadvertent exposure of the medium to fluorescent light.     

Characterization of Esterases Produced by a Ruminal Bacterium Identified as Butyrivibrio fibrisolvens

A method of obtaining clones of Tetrahymena pyriformis on solid medium has been developed. The medium consists of a basal layer of 1.5% agar topped with 2 ml of 0.3% agar in sterile, plastic petri plates (100 by 15 mm). Both agar layers contain either 2% proteose peptone and 0.1% liver extract (complex medium) or defined medium supplemented with proteose peptone. After drying, 0.5 ml of liquid culture is spread evenly over the top agar, and the plates are then sprinkled lightly and evenly with autoclaved dry Sephadex G-25 (fine). Cell colonies can be observed after 5 days of incubation either by viewing with a microscope or without the aid of a microscope after staining. Plating efficiency is high on either complex or defined medium with a number of strains of Tetrahymena, both micronucleate and amicronucleate. Colonies can be picked and transferred to liquid culture for further growth. The existence of clones was demonstrated by plating a mixture of two different drug-resistant mutants. The method should prove useful in selective procedures for the isolation of mutants and for determining survival after treatments such as ultraviolet irradiation.     

Automated Counting of Microbial Colonies

The time required for direct counting of colonies on agar plates for estimating population density of viable microorganisms has precluded studies requiring measurement of such a parameter. Bowman, Blume, and Vurek have described a photo-electrical capillary tube scanner which automates the counting. Results obtained with an instrument similar to that of Bowman et al. have been intensively analyzed with respect to precision and accuracy. The sources of "errors" have been ascertained, and the instrument's potentialities and limitations are discussed.     

The effects of miracidal aging and dilution of snail-conditioned water on responses of miracidia of Megalodiscus temperatus.

Miracidia of Megalodiscus temperatus from newly hatched until 10 hr old were tested for their ability to react to Helisoma trivolvis snail-conditioned water (SCW) by contact with return (CR) to agar blocks and by percentage of miracidia reacting to a point inoculation of SCW as determined by a photographic time exposure method. CR to agar blocks containing 1:50 SCW was greatest during the first 6 hr after hatching but declined thereafter. The reaction during the first hour to a point inoculation was lower than during the 2nd and 3rd hr. Results were variable from 4 to 10 hr after hatching with the lowest response recorded from 9 to 10 hr. Miracidial responses to dilutions of SCW were assessed by the same two methods. CR to agar blocks containing decreasing concentrations of SCW declined until at a dilution of 1:500 CR was only slightly above the controls. On the other hand, miracidial reactions to point inoculations of SCW as determined by the photographic method were still apparent at a dilution of 1:25,000, when 12% of the miracidia tested reacted. Thus, the photographic time exposure method gives a sensitive means for detecting altered miracidial behavior to various intrinsic and extrinsic factors.