Rapid methods for differentiating gram-positive from gram-negative aerobic and facultative anaerobic bacteria

Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy- beta-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido- 4-methylcoumarin (AAMC); 8-anilino-1-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 micrograms/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 micrograms/ml, and led to inhibition of all bacteria at 200 micrograms/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocolitica, Bacillus cereus, and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test. With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.     

Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium

Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5μg/ml crystal violet, 5.0 μg/ml daptomycin, and 5.0μg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.     

Antibacterial effect of protamine assayed by impedimetry

Impedimetric measurements were used to assay the antibacterial effect of protamine. A good linear correlation between the impedance detection time and the initial cell counts was obtained ( r = 0.99, n = 2). As basic peptides may cause clumping of cells, this correlation curve was used when estimating the cell number after protamine treatment, rather than colony counts.
Protamine from salmon killed growing Gram-positive bacteria and significantly inhibited growth of Gram-negative bacteria in Tryptone Soy Broth (TSB) at 25°C. In general Gram-positive bacteria were more sensitive to protamine than Gram-negative bacteria; the minimum inhibitory concentrations (MIC) determined for Gram-positive strains varied from 20 to 1000 μ ml^-1 and for Gram-negative strains from 500 μ ml^-1 to more than 4000 μ ml^-1.
The effect of protamine on non-growing Listeria monocytogenes Scott A suspended in buffer was not lethal as was the effect on growing cells; however, protamine (50–500 μg ml^-1) killed the Gram-negative fish spoilage bacteria Shewanella putrefaciens when the live cells were suspended in buffer.     

Antibacterial activities of mono-, di- and tri-substituted triphenylamine-based phosphonium ionic liquids

We report the synthesis of new mono, di and tri phosphonium ionic liquids and the evaluation of their antibacterial activities on both Gram-positive and Gram-negative bacteria from the ESKAPE-group. Among the molecules synthesized some of them reveal a strong bactericidal activity (MIC?=?0.5?mg/L) for Gram-positive bacteria (including resistant strains) comparable to that of standard antibiotics. A comparative Gram positive and Gram negative antibacterial activities shows that the nature of counter-ion has no significant effects. Interestingly, the increase of phosphonium lateral chains (from 4 to 8 carbons) results in a decrease of antibacterial activities. However, the increase of the spacer length has a positive influence on the activity on both Gram-positive and Gram-negative bacteria except for E. aerogenes. Finally, the increased charge density has no effect on the Gram-positive antibacterial activities (MIC between 2 and 4?mg/L) but seems to attenuate (except for P. aeruginosa) the discrimination between Gram-positive and Gram-negative. Overall these results suggest a unique mechanism of action of these triphenylamine-phosphonium ionic liquid derivatives.     

Efficacy of the Ryu nonstaining KOH technique for rapidly determining gram reactions of food-borne and waterborne bacteria and yeasts.

A simple and rapid (< 60 s) nonstaining technique with 3% potassium hydroxide to determine Gram reactions was tested with 495 food-borne and waterborne bacteria and yeasts. In KOH, suspensions of gram-negative bacteria become viscous and string out. Gram-positive bacteria are not affected. There was 100% correlation between the KOH string test results and gram-positive and gram-negative strains.     

Assessment of resistomycin,as an anticancer compound isolated and characterized from <Emphasis Type="Italic">Streptomyces aurantiacus</Emphasis> AAA5

A new actinomycete strain, isolated from humus soils in the Western Ghats, was found to be an efficient pigment producer. The strain, designated AAA5, was identified as a putative Streptomyces aurantiacus strain based on cultural properties, morphology, carbon source utilization, and analysis of the 16S rRNA gene. The strain produced a reddish-brown pigmented compound during the secondary metabolites phase. A yellow compound was derived from the extracted pigment and was identified as the quinone-related antibiotic resistomycin based on ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, liquid chromatography and mass spectroscopy, and nuclear magnetic resonance analyses. The AAA5 strain was found to produce large quantities of resistomycin (52.5 mg/L). It showed potent cytotoxic activity against cell lines viz. HepG2 (hepatic carcinoma) and HeLa (cervical carcinoma) in vitro, with growth inhibition (GI₅₀) of 0.006 and 0.005 μg/ml, respectively. The strain also exhibited broad antimicrobial activities against both Gram-positive and Gram-negative bacteria. Therefore, AAA5 may have great potential as an industrial resistomycin-producing strain.     

Degradation of mimosine, 2,3-dihydroxy pyridine and 3-hydroxy-4(1H)-pyridine by bacteria from the rumen of sheep in Venezuela

Abstract The addition of Leucaena leucocephala herbage did not diet of sheep in Venezuela did not affect the concentration of volatile fatty acids (VFA) in the rumen, the degradation of rice straw incubated in sacco, or the numbers of rumen fungi or bacteria. However, feeding Leucaena increased the concentration of ammonia in the rumen. In addition, two products of the degradation of the toxic amino acid mimosine were detected in the rumen when Leucaena was fed. One of these products, 2,3-dihydroxy pyridine (2,3-DHP), was detected at concentrations of up to 1.1 μmol/ml. The other, 3-hydroxy-4(1H)-pyridone (3,4-DHP) was found at concentrations of up to 0.96 μmol/ml. The examination of bacterial cultures isolated from the rumen of the sheep under investigation showed that feeding Leucaena increased the relative proportions of short Gram-negative rods and decreased the proportion of long roads and coccobacilli present. Although the animals fed Leucaena showed a small loss in weight during the feeding trial, no evidence of Leucaena toxicity was seen. A total of 18 cultures capable of degrading 2,3-DHP or 3,4-DHP were isolated from the rumen of the sheep before Leucaena was fed. These included both Gram-positive and Gram-negative bacteria, and a Gram-positive sporeformer. It seems that 2,3-DHP and 3,4-DHP may be degraded by a much wider range of bacteria than has been recognised previously. The detection of these bacteria before Leucaena was fed suggests that they were indigenous members of the rumen microflora of sheep in Venezuela.     

Effect of unsaturated fatty acids in growth inhibition of some penicillin-resistant and sensitive bacteria

The growth of two penicillin-resistant Gram-positive bacteria, Bacillus licheniformis (749/C, penicillin G-resistant) and Staphylococcus aureus (metR 18, methicillin-resistant) and one Gram-negative strain, Escherichia coli (cloxacillin-resistant) as well as that of their wild counterparts was inhibited by the long-chain unsaturated fatty acids, linoleic, linolenic and arachidonic acid. The minimum inhibitory concentrations (MIC) of all the fatty acids were found to be 4–6 μg/ml for Staph. aureus (metR 18 & wild), 8–30 μg/ml for B. licheniformis (749/C & wild) and 70–90 μg/ml for E. coli (cloxacillin-resistant & wild). The inhibitory activity increased as the number of double bonds in the fatty acids increased. In most instances the concentrations of fatty acids required to inhibit the growth of the penicillin-resistant strains were lower than that required for their sensitive counterparts. This inhibition of growth in the presence of fatty acids may be due to an increase in permeability of the membrane as evidenced by the measurement of the leakage of 260 nm absorbing material and fluidity.     

A new bacterial staining method involving Gram stain with theoretical considerations of the staining mechanism.

In order to investigate the mechanism of Gram staining of bacteria, tests with anionic dyes followed by treatment with cationic octyltrimethylammonium (OTMA) were carried out. The study revealed that tetrabromophenolphthalein ethylester (TBPE) gave the most reliable staining of Gram-negative bacteria with negative staining of Gram-positive bacteria. Tests on many species of bacteria showed that TBPE positive bacteria were Gram-negative and vice versa, without exception.     

Bacteriocins with anti-MRSA activity produced by water and soil isolated bacteria

Among a thousand of bacteria isolated from forty-three samples, ten isolated bacteria strain WARY1-6, WARY9-1, WARY9-2, WARY6-6, SOPB1, WARY9-10, WARY7-4, WASM9-25, SOPB8-91 and WAS14 with antimicrobial activity against methicillin resistantStaphylococcus aureus (MRSA) were selected for further study. The activity of crude active supernatant (CAS) from these isolated bacteria was completely lost after treated with pronase E, chymotrypsin and trypsin demonstrating its proteinaceous nature. These isolated bacteria could be regarded as bacteriocin producing bacteria (BAC). It was also found that CAS from five Gram-positive isolated bacteria strain WARY1-6, WARY9-1, WARY9-2, WARY6-6 and WASM9-25 showed a broad range of inhibition as they can inhibit at least five Gram-positive and two Gram-negative test microorganisms. Two Gram-negative bacteria can be regarded as BAC with a broad range against both Gram-positive and Gram-negative test bacteria. These seven isolated bacteria can be regarded as BAC with a broad range of antagonistic activity. One isolated bacteria strain SOPB1 harboured a single large plasmid name pSOPB1-19. Its bacteriocin production was associated with plasmid as analysed by plasmid extraction and curing experiment. The strain SOPB1 was identified asBacillus sphaericus according to its 16s rRNA gene sequence. Its bacteriocin was heat stable up to 121 °C, 15 min and active within the pH range of 6–9.     

Variability of the Gram Reaction Report of Committee on Bacteriological Technic,of the Society of American Bacteriologists

The results of a cooperative investigation on the Gram stain are reported. One hundred and twenty slides were made by a single technician in one laboratory and distributed to ten collaborators. Each of these slides bore smears of six organisms, which were known to differ considerably from one another in their behavior to the Gram reaction. Identical directions were sent to all those taking part in the work as to how to perform the staining technic.

In regard to four of the six cultures fairly consistent reports were received from all those taking part in the tests. The other two cultures, however, proved so variable in their reaction toward the staining method that it is impossible to consider them either Gram-positive or Gram-negative. Such organisms must be regarded as belonging to an intermediate group, and should be called Gram-variable.

It is pointed out that these results agree with recent work, such as that of Churchman and of Steam and Steam; also that according to the theory of the latter investigators as to the relation between Gram reaction and the isolectric point of the bacteria, no sharp distinction between Gram-positive and Gram-negative organisms could be expected.

These considerations are very important when interpreting results of the Gram technic in the study of pure cultures; but they do not invalidate its use in diagnostic work where it is ordinarily employed to distinguish strongly positive from strongly negative organisms.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be ''Gram-positive,'' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be ''Gram-negative.'' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

Variability of the Gram Reaction Report of Committee on Bacteriological Technic, of the Society of American Bacteriologists

The results of a cooperative investigation on the Gram stain are reported. One hundred and twenty slides were made by a single technician in one laboratory and distributed to ten collaborators. Each of these slides bore smears of six organisms, which were known to differ considerably from one another in their behavior to the Gram reaction. Identical directions were sent to all those taking part in the work as to how to perform the staining technic.

In regard to four of the six cultures fairly consistent reports were received from all those taking part in the tests. The other two cultures, however, proved so variable in their reaction toward the staining method that it is impossible to consider them either Gram-positive or Gram-negative. Such organisms must be regarded as belonging to an intermediate group, and should be called Gram-variable.

It is pointed out that these results agree with recent work, such as that of Churchman and of Steam and Steam; also that according to the theory of the latter investigators as to the relation between Gram reaction and the isolectric point of the bacteria, no sharp distinction between Gram-positive and Gram-negative organisms could be expected.

These considerations are very important when interpreting results of the Gram technic in the study of pure cultures; but they do not invalidate its use in diagnostic work where it is ordinarily employed to distinguish strongly positive from strongly negative organisms.     

Use of magnetic beads for Gram staining of bacteria in aqueous suspension.

A Gram staining technique was developed using monodisperse magnetic beads in concentrating bacteria in suspension for downstream application. The technique does not require heat fixation of organisms, electrical power, or a microscope. Gram-negative and Gram-positive bacteria were identified macroscopically based on the colour of the suspension. The bacteria concentrated on magnetic beads may also be identified microscopically.     

Studies in Gram Staining

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuchsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal viokt-stained organisms with alcoholic safranin (0.25%) for 15 scc will distinguish Gram-positive bacteria (viokt) from Gram-negative bacteria (pink).

Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily dccolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Sublethal injury makes Gram-negative and resistant Gram-positive bacteria sensitive to the bacteriocins, pediocin AcH and nisin

Gram-negative and some Gram-positive bacteria that are resistant to bacteriocins of lactic acid bacteria (LAB) were subjected to sublethal stresses and treated with nisin and pediocin AcH. Both bacteriocins reduced the viability of cells surviving sublethal stresses. The results explain the possible mechanisms by which bacteriocins of LAB enter through the walls (or outer membranes) to destabilize the cytoplasmic (or inner) membranes and kill cells of sensitive Gram-positive and resistant, but injured, Gram-negative and Gram-positive bacteria.     

In vitro antibacterial activity of laboratory grown culture of Spirulina platensis

The hexane, ethyl acetate, dichloromethane, methanol extracts and spent media (extracellular substances) were tested in vitro for their antibacterial activity for which one Gram-positive bacterium (Staphylococcus aureus) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae) were used as test organisms. The methanol extract showed more potent activity than other organic extracts, spent medium of the culture exhibited little activity against E. coli only. No inhibitory effect was found against Klebsiella pneumoniae.The broth microdilution assay gave minimum inhibitory concentrations (MIC) values ranging from 1 to 512 μg/ml. The MIC of methanol extract against S. aureus and E. coli were 128 μg/ml and 256 μg/ml, respectively.     

塔里木河天然胡杨林中病灶微生物的分离与形态学初探

目的:从塔里木河中下游废弃上百年的河道周围选取2处样点,采集具有不同病症胡杨的茎叶,从中进行菌种分离。方法:显微镜镜检,革兰氏染色及生理生化检测。结果:通过形态观察,得到13株细菌、4株真菌。其中的7株细菌分离于病叶,其余10株分离于病变树皮。对13株细菌革兰氏染色结果表明,2株为革兰氏阴性菌,11株为革兰氏阳性菌。对真菌的孢子形态观察表明,4株均为霉菌。结论:实验首次对胡杨特殊病灶微生物进行分类统计,为解决由微生物感染引起的胡杨林退化问题积累数据。     

Occurrence of pydixaminephosphate oxidase and pyridoxal kinase in Gram-negative and Gram-positive bacteria

Pyridoxamine-phosphate oxidase and pyridoxal kinase were detected in Gram-negative bacteria. In Gram-positive bacteria, generally only pyridoxal kinase is detectable.