Rapid method of determining the dry mass of whooping cough microbes

A colorimetric method for the rapid determination of the quantitative content of microbial mass in B. pertussis suspensions has been developed. The method is based on the indirect determination of carbon in microbial suspensions by its oxidation with the mixture consisting of potassium bichromate in concentrated sulfuric acid and the subsequent colorimetric analysis of the products of this reaction. The method ensures sufficient accuracy, the determination procedure is simple, takes not more than 2 hours and requires no complex reagents. The results thus obtained are well comparable with those obtained by the classical gravimetric method. The new method permits the determination of microbial mass in B. pertussis suspensions with a minimum concentration of 0.5 mg/ml. The method is recommended for the determination of dry microbial mass in B. pertussis suspensions.     

A cassette micromethod of determining the sensitivity of anaerobic microorganisms to antimicrobial drugs]

A micromethod based on the use of plates with wells and microquantities of microbial suspensions is described. It provides determination of MICs of antibacterial drugs and sensitivity of clinical strains of anaerobes of 2 or 3 species predominating in pathological materials as well as to preliminarily identify some anaerobic bacteria by their antibiotic sensitivity.     

用管碟法测定碱性蛋白酶活力

参照抗生素效价生物测定法,用管碟法测定碱性蛋白酶活力,可大大提高菌种筛选的工作效率。待测样品在２％酪蛋白平板上的水解圈直径被效正后,从标准曲线上可查得相应的酶活。管碟法测得的酶活与Ｆｏｌｉｎ法测得的酶活相差一般小于２０００ｕ／ｍｌ。管碟法测定酶活时,操作要求认真仔细,操作误差可使一样品的水解圈直径相差２．９ｍｍ,但一般误差在０．２～０．３ｍｍ,相应酶活误差小于１０００ｕ／ｍｌ。     

Action of the alkyl derivatives of amphotericin B on the conductivity of bilayer lipid membranes

Amphotericin B alkyl derivatives increased conductivity of bilayer membranes by formation of channels in them. The properties of such channels were studied. A new method for determination of the polyene antibiotic toxicity is described. The method is based on measurement of the constant of the relaxation time on the antibiotic removal from the membrane solution. It was shown that the amphotericin B alkyl derivatives had very low toxicity for the mammalian cells and were highly toxic for the fungal cells. These antibiotics may be used as new effective antifungal compounds.     

Development of an accelerated method of determining the antibiotic sensitivity of Cl. perfringens type A]

An express method for determination of antibiotic sensitivity in the strains of Cl. perfringens of type A using Soviet dry nutrient media and antibiotics is proposed. The criteria for estimation of the level of the antibiotic sensitivity of the causative agent of gas gangrene in short periods on the basis of comparison of the data of the antibiotic agar diffusion procedure and the antibiotic MIC were worked out. Twelve antibiotics and 45 collection strains of Cl. perfringens of type A were used in the experiment. The antibiotic agar diffusion method with the use of the nutrient media, microbial load and cultivation conditions developed by the authors is recommended for tentative determination of the antibiotic sensitivity in Cl. perfringens of type A for 4 hours. The use of the agar diffusion method and determination of the antibiotic MIC provided complete estimation of tha antibiotic sensitivity of Cl. perfringens of type A within not more than 24 hours.     

Microbial fuel cell-based diagnostic platform to reveal antibacterial effect of beta-lactam antibiotics

Beta-lactam antibiotics comprise the largest group of antibacterial agents. Due to their bactericidal properties and limited toxicity to humans they are preferred in antimicrobial therapy. In most cases, therapy is empiric since susceptibility testing in diagnostic laboratories takes a relatively long time. This paper presents a novel platform that is based on the microbial fuel cell (MFC) technology and focuses on the early antibiogram determination of isolates against a series of beta-lactam antibiotics. An advantage of the system is that it can be integrated into traditional microbiological diagnostic laboratory procedures. Tested bacterium suspensions are uploaded into the anodic chambers of each miniaturized MFC unit integrated into a panel system, containing different antibiotic solutions. Electronic signals gained in each MFC unit are continuously monitored and are proportional to the metabolic activity of the presenting test bacterium. Using this method, antibiotic susceptibility can be evaluated in 2–4 h after inoculation. Hereby we demonstrate the efficacy of the platform in antibiogram determination by testing the susceptibilities of Escherichia coli strain ATCC 25922 and Staphylococcus aureus strain ATCC 29213 against 10 beta-lactam antibiotics (penicillin, ampicillin, ticarcillin, cefazolin, cefuroxime, cefoperazone, cefepime, cefoxitin, cefaclor, imipenem). This paper also presents the construction of the background instrumentation and the panel system into which a printed circuit board (PCB) based electrode was integrated. Our results suggest that MFC based biosensors have the potential to be used in diagnostics for antibiogram determination.     

A photometer for the extinctiometric determination of the density of suspensions of microorganisms with minimal deviation from the law of Beer-Lambert

Summary The theory and construction of a photometer for the extinctiometric determination of the density of suspensions of bacteria are given. The application of the principle of directional selectivity and the use of sodium light leads to a satisfactory accuracy in the determination of the density of suspensions of bacteria. The results are in conformity with the law ofBeer-Lambert. The advantages of the proposed method are discussed.     

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.     

Tetracycline inactivation by magnesium sulfate in testing for microbial contamination by the membrane filtration method]

It was shown that the residual amounts of tetracyclines adsorbed on membrane filters may be inactivated by addition of 2M magnesium sulphate solution to the agar for placing the filters after filtration. The antibiotic inactivation increases the possibilities of the test for antibiotic sensitivity of the microflora present in the drugs and may be used in determination of microbial dissmination of non-injection tetracyclines.     

THE USE OF STORED SUSPENSIONS OF ESCHERICHIA COLI I IN THE EVALUATION OF BACTERICIDAL ACTION

SUMMARY: Suspensions of Escherichia coli I, consisting of washed cells suspended in a phosphate buffer solution, maintained a higher viability and resistance to phenol than suspensions either of unwashed cells or of washed cells suspended in water. When stored for 5 weeks at room temperature, variations in their extinction times on exposure to aqueous phenol solutions were not significantly greater than variations with suspensions freshly prepared for each determination. Loss of resistance of a stored suspension to phenol, roxenol, lysol and potassium laurate was roughly parallel. Conditions of culture of the bacteria influenced the survival of suspensions, but the results indicated that pronounced differences may only be found in suspensions prepared from young cultures. The use of stored suspensions in the routine evaluation of bactericides is recommended.     

Comparative study of the biological activity of tetracycline hydrochloride and morphocycline by using reverse turbidimetry]

The method of back turbidimetry was used for determination of the biological activity of the antibiotics, since high turbidity of the nutrient medium with Staph. aureus as the testculture prevented from direct measurements. Broth containing phosphate buffer, Staph. aureus and definite concentrations of the antibiotic was used as the reference solution. The experiments showed that the differences in the biological activities of tetracycline hydrochloride and morphocycline may be found with the method of back turbidimetry 6-8 hours after the microbe cultivation on media with the antibiotics.     

Use of a polarographic method for determining trichothecin]

The polarographic behaviour of trichothecin was studied. It was shown that the antibiotic could be detected in solutions at concentrations of 7.10(-7) moles with the help of the polarographic method. Conditions for the polarographic determination of trichothecin in fermentation broth were developed. The error was not more than 3 per cent. The reliability of the results was shown by statistical treatment of data performed in accordance with the requirement of the USSR State Pharmacopeia, X ed., prescribing that the precision of the assay is such that the fiducial limits at p = 95 per cent deviate from the average value by not more than 5 per cent. Comparison of the results of trichothecin determination in the fermentation broth with the polarographic and biological methods showed no significant difference. Therefore, the polarographic method may be recommended for trichothecin determination in the fermentation broth.     

A rapid method for the determination of antibiotic resistance in bacterial pathogens within diseased specimens

A technique was developed, which permitted the rapid determination of antibiotic resistance of bacterial pathogens in diseased material. The method involved use of an antibody-based antigen capturing system, exposure to antibiotic solutions, and thence the determination of viability by reduction of thiazolyl blue.     

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.     

Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro.

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.     

Electronic sizing and counting of bacteria

An electronic apparatus is described that permits rapid determination of the concentration and size distribution of bacteria in electrolyte suspensions by a resistance method. The resulting size-concentration distribution may be displayed on an oscilloscope and recorded with an X–Y plotter and an electric typewriter-tape punch unit. The paper tape is analyzed with a computer program. Comparisons are made between electronic measurements of bacterial cell concentration and size distribution and values obtained by other methods. Effects of heat-killing and disruption of the cell membrane on the electrical counting characteristics of the organisms are discussed.     

Effect of different grades of agar-agar on the nature of the test microbe growth inhibition zones in controlling antibiotic activity by means of agar diffusion]

The effect of various agar grades on the size and margin character of the inhibition growth zones in assay of antibiotic activity by the agar diffusion method was studied. It was shown that not all the agar grades could be used in antibiotic activity assay. Depending on the agar type the size of the inhibition growth zones produced by the same antibiotic concentration significantly varied. The variations in the size of the inhibition growth zones depended on the agar ability to bind antibiotics and were mainly defined by the agar purity. The agars with low content of nitrogen admixtures bound the antibiotics to a low extent. The commerical grades of the agars of the South-Sea and Korsakov Plants, the experimental grade of the TINRO agar with additional purification, as well as the agars imported from Argentina and France proved to be most useful for determination of the antibiotic activity by the agar diffusion method.     

Immunochromatographic technique for express determination of ampicillin in milk and dairy products

An immunochromatographic method for determination of β-lactam antibiotic ampicillin has been developed. The method is based on the competitive interaction between antibiotic molecules contained in the sample and protein conjugate of penicillin immobilized on a membrane for binding with specific antibodies labeled with colloidal gold, which occurs during movement of the sample to be tested and reagents along the membrane. The completion of the test system ensures control of exceeding the maximum permissible content of the antibiotic in milk and dairy products (10 ng/mL). The possibility of testing milk, raw milk, and dairy products for 10 minutes at room temperature without sample pretreatment has been demonstrated.     

Determination of the heat resistance of spores using a solid heating block system

A simple and accurate technique for the determination of the heat resistance of spores is described. The technique combines a modified capillary tube method with a solid heating block. The come-up time of spore suspensions was found to be short and simple and accurate technique is suggested for the correction of the come-up times. Experimental results are presented for the destruction of spores of Bacillus stearothermophilus at 120 degrees which indicates the accuracy and reproducibility of the new method.     

Relationship between growth of parsley and soybean cells in suspension cultures and changes in the conductivity of the culture medium

Summary Changes in conductivity and pH during the growth cycle of cell suspensions derived from parsley (Petroselinum hortense) and soybean (Glycine max) have been investigated. Measurement of the conductivity of the medium represents a simple, rapid, and reliable method for the precise determination of the growth phase of a culture. The accuracy of this method has been tested by using phenylalanine ammonia-lyase, an enzyme that has a characteristically short, distinct period of activity during the growth cycle of soybean cell suspensions. It is suggested that an automatic regulation of the conductivity of the medium might be employed for growing plant cells in a continuous culture at a defined stage of growth.