Method of determining the antibiotic sensitivity of anaerobic microorganisms using standard disks

Three variants of the procedure for determination of antibiotic sensitivity in anaerobic microorganisms with the use of standard paper discs were developed. According to the first variant the solid nutrient medium is melted at 46 degrees C and mixed with the culture of the microbe being tested. The mixture is added to the cover of a Petri dish. When the medium becomes solid, antibiotic sensitivity discs are placed onto the agar surface. After that one more layer of the medium is added. The medium is allowed to solidify and some more medium is poured near the cover edge. Immediately after that the Petri dish is placed with its flat surface onto the agar layer in its cover. According to the first and second variants the mixture of the medium and culture is added to a Petri dish and immediately a transparent gas-proof polymer film of the dish size is placed onto the agar surface. Previously antibiotic paper discs or solutions are fixed on the films. THe incubation temperature for all three variants is 37 degrees C. The procedure allows one to observe the culture growth and to obtain the results earlier than in case the culture is incubated in an aerostate. The procedure is simple and saves labor and time.     

Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate

The dynamics of a bacterial population exposed to the minimum inhibitory concentration (MIC) of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick's laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antimicrobial agents, and guide clinical treatment.     

Disc Plate Method of Microbiological Antibiotic Assay: I. Factors Influencing Variability and Error 1

Several factors are investigated that normally cause variation in zone diameters in conventional disc plate diffusion assay procedures. Of these factors the most serious is the unequal exposure of the individual plates at top or bottom of stacks to temperatures above and below room temperature. This unequal temperature exposure is avoided by novel handling and incubation procedures. A major variable, but one which can be controlled, is the varying time interval between pouring seeded agar and the time of applying the pads with antibiotic to the plates. This influence of time of setting and the effects of several other sequential operations are combined into a composite variable. This variable is then accounted for and normalized by interposing "external" reference plates set with a reference solution in the sequence of approximately 100 plates. No "internal" reference zones are employed. Such factors as volume of agar poured, wedge shape of agar in a dish, volumetric errors in dilutions, and timing considerations are studied and discussed. The results of this study form the basis for a test protocol which is presented in a following paper.     

Agar Underlay Method for Recovery of Sublethally Heat-Injured Bacteria

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60°C for 1.5 min in buffer or 80°C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.     

Use of sublimation to prepare solid microbial media with water-insoluble substrates

A method was developed to deposit a visible layer of water-insoluble compounds via sublimation onto the surface of solid media. The compound is sublimed from a heated aluminum dish containing the compound onto the surface of an inverted, ice-cooled, inoculated agar petri dish. The method results in the deposition of a thin, even layer on the agar surface without the use of solvent. After incubation, clearing zones around colonies indicate the presence of compound-degrading microorganisms.     

Use of Sublimation To Prepare Solid Microbial Media with Water-Insoluble Substrates

A method was developed to deposit a visible layer of water-insoluble compounds via sublimation onto the surface of solid media. The compound is sublimed from a heated aluminum dish containing the compound onto the surface of an inverted, ice-cooled, inoculated agar petri dish. The method results in the deposition of a thin, even layer on the agar surface without the use of solvent. After incubation, clearing zones around colonies indicate the presence of compound-degrading microorganisms.     

The lytic action of tyrothricin and its derivatives on Staphylococcus aureus

A tyrothricin concentration of 0.01 mg. per ml. causes rapid lysis of Staphylococcus aureus; this lyric action is not enhanced by phage. A concentration of 0.001 mg. per ml. of tyrothricin is likewise capable of lysing staphylococci, and the addition of phage does not appreciably alter the rate of lysis. A concentration of 0.0001 mg. per ml. of tyrothricin is unable to effect lysis though it enhances the lytic activity of phage. The tyrocidine fraction of tyrothricin appears to be responsible for the latter's lytic action.     

Machine for Automatic Bacteriological Pour Plate Preparation

A fully automatic system for preparing poured plates for bacteriological analyses has been constructed and tested. The machine can make decimal dilutions of bacterial suspensions, dispense measured amounts into petri dishes, add molten agar, mix the dish contents, and label the dishes with sample and dilution numbers at the rate of 2,000 dishes per 8-hr day. In addition, the machine can be programmed to select different media so that plates for different types of bacteriological analysis may be made automatically from the same sample. The machine uses only the components of the media and sterile polystyrene petri dishes; requirements for all other materials, such as sterile pipettes and capped bottles of diluents and agar, are eliminated.     

Chemosensory responses by the heterotrophic marine dinoflagellate<Emphasis Type="Italic">Crypthecodinium cohnii</Emphasis>

Chemosensory responses by the colorles inshore marine dinoflagellateCrypthecodinium cohnii were observed in quadrant-divided Petri plates containing an agar layer + liquid overlay. A suspension of organisms in salt solution was poured onto this and allowed to stand 3 hr. A differential tendency of the cells to become firmly attached or embedded in the substratum was observed when various substances were incorporated in the gel. A positive response (tendency to attach) occurred with: α-l-fucose, dimethyl-β-propiothetin, betaine, sucrose, glycine, L-alanine, hemin, and fructose; negative response: formalin, glutathione, acid hydrolyzed agar, protamine SO₄, L-glutamic acid, lactose, glutamine, taurine, L-aspartic acid, putrescine 2 HCl, choline citrate, choline bitartrate, K citrate, and choline HCl. γ-Aminobutyric acid was negative or positive dependeng on concentration. Dead or immotile cells did not become attached. The following compounds elicited no response: α-D-fucose, dimethyl acetothetin chloride, cyclic AMP, and glucose.     

Agar underlay method for recovery of sublethally heat-injured bacteria

A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0. 05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60 degrees C for 1.5 min in buffer or 80 degrees C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.     

Use of Sodium Polyanethol Sulfonate in the Preparation of 5% Sheep Blood Agar Plates

An alternative method to defibrinating sheep blood for use in bacteriological media is described. The new procedure incorporates sodium polyanethol sulfonate in a concentration of 0.05% (vol/vol). In testing 117 bacterial and fungal isolates, no significant differences were found with respect to adequate growth, pigment production, hemolytic reactions, and other physical attributes. Further tests demonstrate that the sodium polyanethol sulfonate in sheep blood agar plates does not cause any aberrations in zone sizes around disks used in antibiotic susceptibility tests. Consequently, the method represents a suitable alternative to the use of defibrinated sheep blood in the preparation of bacteriological media.     

Development of membrane filter holder (MFH) method for recovery of heat-injured Escherichia coli O157:H7 and Salmonella typhimurium

AIMS: A method of recovering sublethally heat-injured bacteria was developed with specific apparatus (membrane filter holder; MFH) which was originally used for Iso-Grid Hydrophobic membrane. filter holder. METHODS AND RESULTS: The procedure used a non-selective agar underlayed with a selective medium with a MFH. A non-selective agar was poured on upper part (compartment A) of MFH, and then injured foodborne pathogens were inoculated on the non-selective medium. After 3-h repair incubation period, selective agar was added to the bottom of the chamber (compartment B) of the MFH and further incubated. By diffusing through the non-selective top agar, selective agents from the underlay medium impart selectivity to the system. CONCLUSIONS: Using the MFH method, recovery of heat-injured foodborne pathogens (Escherichia coli O157:H7 and Salmonella typhimurium) were not different (P > 0.05) from recoveries with non-selective media (TSA). However, the recoveries of foodborne pathogens on MFH were significantly higher (P < 0.05) than those of direct plating on selective medium such as SMAC (MacConkey Sorbitol Agar) or XLD (Xylose Lysine Desoxycholate). SIGNIFICANCE AND IMPACT OF THE STUDY: In conclusion, the MFH method is a simple and convenient method for recovery of heat-injured foodborne pathogens.     

Improved methods for using glass coverslips in cell culture and electron microscopy.

For many applications, cells or tissue must be cultured on an optical surface of high quality. For such applications laboratories often prepare "special dishes," which are made by affixing a glass coverslip beneath a hole in a plastic petri dish bottom. In this report, we offer an improved method, using Parafilm as a dry mount adhesive, for the preparation of special dishes, and show that the resulting dish is non-toxic to neurons in culture. The Parafilm bond is stable at 60 degrees C, permitting electron microscopy resins to be poured directly into the dishes and cured. The glass coverslip can be readily removed from the cured resin mechanically. The techniques we describe offer time-saving and reliable improvements for the use of glass coverslips in cell culture and electron microscopy.     

A SIMPLE PROCEDURE TO DETECT NISIN IN CHEESE

SUMMARY: A suitable amount of agar medium inoculated with a test organism sensitive to nisin ( Lactobacillus lactis or Streptococcus cremoris ) is poured on a cheese cylinder placed in a Petri dish. After a convenient incubation time the presence of a clear zone around the cheese cylinder will indicate the presence of nisin.
The method is applicable both to natural and processed cheese and can also be used for other antibiotics that may be present in cheese.     

The Use of Acenaphthene in Pollen Tube Technic

A procedure is described for growing pollen tubes in such a manner that a large number of clearly analyzed figures can be obtained. The pollen grains are sown on an artificial medium of sugar, agar, gelatin, and water, the proportions of each varying with the species of pollen grain used. The medium is smeared on the slide while still hot to insure a thin covering, and the pollen grains are dusted on when the medium has sufficiently cooled and hardened. The slides are placed in a staining dish provided with slide slots and a cover, the inside of the cover and the bottom of the dish being lined with moist, but not wet, filter paper. Acenapthene crystals are lightly sprinkled on the bottom of the dish. The developing pollen tubes are thus exposed to the fumes given off by these crystals with consequent disturbance to the spindle mechanism. As a result, the chromosomes are not crowded on a metaphase plate but are widely separated in the tube facilitating any observations to be made.     

A method for establishing stable concentration gradients in agar suitable for studying chemotaxis on a solid surface.

A simple technique has been developed for establishing stable gradients of a substance in agar. The technique involves the creation of a spherically symmetric concentration profile in which concentration varies inversely with the distance from the source and is independent of the diffusion coefficient of the substance. It has been shown that the gradients established with this technique are stable for at least 190 h. and, on a theoretical basis, they can be kept stable for more than 1000 h. Time-variant gradients can also be established, if desired, using the same system and limiting either the source or the agar sink. It must be emphasized that a stable gradient cannot be obtained by using a shallow agar layer as a sink. The use of such conditions (e.g. the agar in a standard petri dish) can result only in time-variant gradients. The solution to the diffusion equation in a spherically symmetric system establishes the expected concentration profile, the basis for adjusting it, and the parameters that control the behavior of the system. Some useful applications for examining chemotaxis on a solid surface as well as possible further developments are discussed.     

Agar Diffusion Method for the Assay of Colicins

An agar diffusion method for the assay of colicins A, B, D, E(2), E(3), and K is described. The assays were performed in large, square pyrex dishes that contained an agar layer seeded with an indicator organism sensitive to the colicin. The samples were applied to the agar in steatite beads positioned in a randomized sequence. The plates were stored at 4 C for 24 hr to allow the colicins to diffuse into the agar. After incubation at 37 C, the activity of each colicin preparation was estimated by measuring the diameter of the zone of inhibition of the growth of the indicator strain around each bead. The results of each assay were subjected to a statistical analysis, which included an analysis of variance and calculation of the theoretical regression and the confidence interval of the assay. The size of the inhibition zones, the form of the regression, and the slope of the regression of the responses were affected by the type and concentration of the agar, the depth of the agar layer, the indicator organism, the indicator inoculum density, and the time allowed for prediffusion of the colicins. Optimal conditions for the assay of each colicin were determined. Using a four-point assay design, the relative colicin concentration of unknown preparations was estimated in terms of a standard preparation of the same colicin. The experimental error of these assays (95% confidence interval) was about +/- 10%.     

Detection and isolation of radionuclide-accumulating bacteria by autoradiography

A simple and rapid method for the detection and isolation of radionuclide-accumulating microorganisms is described. Water samples are mixed with a radioisotope solution and peptone agar in Petri dishes and incubated at 25 C. After bacterial colonies have appeared the agar is removed from the dish and placed upside down on X-ray film covered with thin plastic foil. Black spots on the developed radiogram reveal which surface colonies contain radionuclide-accumulating bacteria. From these colonies pure cultures are obtained by standard methods. So far, bacterial strains have been isolated accumulating one of the following nuclides: cobalt-60, strontium-89, ruthenium-106, iodine-131, cesium-134, cerium-144.     

Development of a method for the quantitative evaluation of microorganism sensitivity to antibiotics utilizing disks. The determination of the minimal doxycycline concentration that depresses microorganism growth using disks containing this antibiotic]

A quantitative method for estimation of microbial sensitivity to doxycycline with the use of discs containing 10 gamma of the antibiotic was developed. The antibiotic concentrations in the agar were determined at a distance equal to the radius of the growth inhibition zone with the help of a curve of the dependence of the logarithm of the doxycycline concentration in agar at the period of the average critical time of the zone formation equal to 5 hours and the distance from the disc center. The antibiotic concentration in the agar at the zone edges was almost the same as the MIC of doxycycline against the test-cultures determined with the method of serial dilutions in agar.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.