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

M anafi , M. & K neifel , W. 1990. Rapid methods for differentiating Gram-positive from Gram-negative aerobic and facultative anaerobic bacteria. Journal of Applied Bacteriology 69 , 822–827.
Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy-β-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido-4-methylcoumarin (AAMC); 8-anilino-l-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 μg/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 μg/ml, and led to inhibition of all bacteria at 200 μ/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocoiitica, 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.     

Nonstaining (KOH) method for determination of gram reactions of marine bacteria.

A rapid nonstaining (KOH) method for the determination of the Gram reactions of bacteria is described, and its application to marine isolates is discussed. All gram-positive and gram-negative results obtained by Gram staining were confirmed by the KOH method. Gram-variable bacteria produced equivocal results.     

Nonstaining (KOH) method for determination of gram reactions of marine bacteria

A rapid nonstaining (KOH) method for the determination of the Gram reactions of bacteria is described, and its application to marine isolates is discussed. All gram-positive and gram-negative results obtained by Gram staining were confirmed by the KOH method. Gram-variable bacteria produced equivocal results.     

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.     

A Fluorescent Gram Stain for Flow Cytometry and Epifluorescence Microscopy

The fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13 were used in combination as a Gram stain for unfixed organisms in suspension. HI penetrated gram-positive but not gram-negative organisms, whereas SYTO 13 penetrated both. When the dyes were used together, gram-negative organisms were rendered green fluorescent by SYTO 13; conversely, gram-positive organisms were rendered red-orange fluorescent by HI, which simultaneously quenched SYTO 13 green fluorescence. The technique correctly predicted the Gram status of 45 strains of clinically relevant organisms, including several known to be gram variable. In addition, representative strains of gram-positive anaerobic organisms, normally decolorized during the traditional Gram stain procedure, were classified correctly by this method.Gram’s staining method is considered fundamental in bacterial taxonomy. The outcome of the Gram reaction reflects major differences in the chemical composition and ultrastructure of bacterial cell walls. The Gram stain involves staining a heat-fixed smear of cells with a rosaniline dye such as crystal or methyl violet in the presence of iodine, with subsequent exposure to alcohol or acetone. Organisms that are decolorized by the alcohol or acetone are designated gram negative.Alternative Gram staining techniques have recently been proposed. Sizemore et al. (19) reported on the use of fluorescently labeled wheat germ agglutinin. This lectin binds specifically to N-acetylglucosamine in the peptidoglycan layer of gram-positive bacteria, whereas gram-negative organisms contain an outer membrane that prevents lectin binding. Although simpler and faster than the traditional Gram stain, this method requires heat fixation of organisms.Other Gram stain techniques suitable for live bacteria in suspension have been described. Allman et al. (1) demonstrated that rhodamine 123 (a lipophilic cationic dye) rendered gram-positive bacteria fluorescent, but its uptake by gram-negative organisms was poor. This reduced uptake by gram-negative bacteria was attributed to their outer membranes. The outer membrane can be made more permeable to lipophilic cations by exposure to the chelator EDTA (4). Shapiro (18) took advantage of this fact to form the basis of another Gram stain, one which involved comparing the uptake of a carbocyanine dye before and after permeabilizing organisms with EDTA. All of these methods, however, rely on one-color fluorescence, making analysis of mixed bacterial populations difficult.An alternative to the use of stains is the potassium hydroxide (KOH) test. The method categorizes organisms on the basis of differences in KOH solubility. After exposure to KOH, gram-negative bacteria are more easily disrupted than gram-positive organisms. This technique has been used to classify both aerobic and facultatively anaerobic bacteria, including gram-variable organisms (8). In a study by Halebian et al. (9), however, this technique incorrectly classified several anaerobic strains, giving rise to the recommendation that the method should only be used in conjunction with the traditional Gram stain.In this study we demonstrate a Gram staining technique for unfixed organisms in suspension, by using clinically relevant bacterial strains and organisms notorious for their gram variability. The method uses two fluorescent nucleic acid binding dyes, hexidium iodide (HI) and SYTO 13. Sales literature (11) published by the manufacturers of HI (Molecular Probes, Inc., Eugene, Oreg.), which displays a red fluorescence, suggests that the dye selectively stains gram-positive bacteria. SYTO 13 is one of a group of cell-permeating nucleic acid stains and fluoresces green (11). These dyes have been found to stain DNA and RNA in live or dead eukaryotic cells (16). Both dyes are excited at 490 nm, permitting their use in fluorescence instruments equipped with the most commonly available light sources. We reasoned that a combination of these two dyes applied to mixed bacterial populations would result in all bacteria being labeled, with differential labeling of gram-positive bacteria (HI and SYTO 13) and gram-negative bacteria (SYTO 13 only). The different fluorescence emission wavelengths of the two dyes would ensure differentiation of gram-positive from gram-negative bacteria by either epifluorescence microscopy or flow cytometry when equipped with the appropriate excitation and emission filters. While a commercial Gram stain kit produced by Molecular Probes includes HI and an alternative SYTO dye, SYTO 9, we are unaware of any peer-reviewed publications regarding either its use or its effectiveness with traditionally gram-variable 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.     

Brevinin-1 and -2, unique antimicrobial peptides from the skin of the frog, Rana brevipoda porsa.

Two unique antimicrobial peptides named brevinin-1 and -2 were isolated from the skin of the frog, Rana brevipoda porsa. Both of the peptides did not have any structural homology with bombinin nor magainin; the frog skin derived-antimicrobial peptides isolated from Bombina and Xenopus, nor even with other known antimicrobial peptides of non-amphibian origin. The minimum inhibitory concentration of brevinin-1 against the growth of St. aureus and E. coli was determined to be 8 micrograms/ml and 34 micrograms/ml while that of brevinin-2 was 8 micrograms/ml and 4 micrograms/ml, respectively, indicating the difference of the two peptides in the antimicrobial selectively on Gram-positive and Gram-negative bacteria.     

Studies on the aminopeptidase test for the distinction of gram-negative from gram-positive bacteria

Summary The aminopeptidase test was performed with representatives of gram-negative, gram-positive, and gram-variable bacteria. All gram-negative bacteria tested gave a positive test reaction with L-alanine-4-nitroanilide as test substrate. Representatives of the coryneform bacteria and some streptococci showed aminopeptidase activity after prolonged reaction times. A correlation between aminopeptidase activity and distinct interpeptide bridge composition in the peptidoglycan of many strains was demonstrated. The influence of growth conditions on aminopeptidase activity of intact bacterial cells is shown.     

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.     

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.     

Gram staining and lectin binding properties of Myxosporea and Sporozoea

The staining method developed by Christian Gram was introduced as a simple and highly selective tool for demonstrating myxosporean and coccidian sporogonic stages. When using standard blood staining procedures for those enigmatic parasites it is sometimes difficult to distinguish them from fish host tissue. They clearly exhibit a partial Gram-positive reaction in histological sections, but staining is variable in air dried fish organ imprints. To visualize the Gram-negative background of different host tissue components in histological sections, the conventional safranin counterstain of the Gram protocol may be modified as follows: after application of 2% crystal violet (basic violet 3) and Lugol's solution, sections are stained with 0.1% nuclear fast red-5% aluminum sulfate and 0.35% aniline blue (acid blue 22) dissolved in saturated aqueous picric acid. Replacement of the Gram-specific dye crystal violet with 2% malachite green gave similar results in organ imprints containing myxospores or coccidia, but only in sections containing myxosporea. Staining for 1 min with an aqueous solution of 0.5% malachite green and followed 1 min washing was sufficient for rapidly demonstrating the parasite spores in organ imprints of both myxosores and oocysts. With regard to the role of acid mucopolysaccharides and other carbohydrates in the Gram reaction of spores, alcian blue 8GX staining was compared to the binding of FITC-labeled WGA, GS I and GS II. Each lectin was applied at 20 μl/ml PBS, HEPES for 1 hr. Whereas WGA yielded a nonspecific pattern like the alcian blue staining, GS II resulted in a pattern similar to the Gram staining results. This binding was weak in untreated specimens, but was significantly enhanced when digested first within trypsin overnight in a humid chamber at 37 °C. The binding of GS II to both myxosporidian and coccidian spores suggests that they are both composed of polymers containing N-acetyl-D-glucosamine residues. Furthermore, the results suggest that this hexosamine plays a key role in the Gram reaction.     

Gluconeogenesis and the peroxisome

The interaction between hydroperoxides, cytochrome P450 and 8-anilino-1-naphthalenesulfonic acid (ANS) has been investigated. The addition of ANS to the cytochrome P450 solution did not effect the P450 Soret absorption peak or the reduced CO difference spectrum, suggesting that ANS may not bind to P450 heme directly. H2O2 or CuOOH alone did not effect ANS fluorescence and absorption spectra indicating that no detectable reaction occurs between hydroperoxide and ANS in the absence of P450. The reconstituted system of cytochrome P450, P450 reductase, lipid and NADPH did not mediate ANS metabolism. In the presence of P450, the addition of either H2O2 or CuOOH, however, leads to a decrease in ANS absorption around 258 nm and 350 nm indicating possible destruction of ANS. ANS destruction was confirmed with the disappearance of the ANS elution peak in the reverse phase HPLC profiles and with the changes in P450-bound ANS fluorescence intensity and the shift of max of ANS. Moreover , a very sensitive method to detect trace fluorescent products of ANS by thin layer chromatography has been developed based on the fact that ANS fluorescence is enhanced more than 1000-fold by the organic solvent butanol. A UV-sensitive fluorescent product was detected on thin layer chromatography profiles of the reaction mixtures. P450 was also observed to be modified by a fluorescent derivative of ANS, when the fluorescence was enhanced by butanol. These results also show that an organic compound which can not be metabolized by the reconstituted system of cytochrome P450 and NADPH-P450 reductase is metabolized by the reconstituted system of P450 and hydroperoxide, suggesting the activities of these two systems may not be completely comparable. (Mol Cell Biochem 167: 159-168, 1997)     

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.     

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.     

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.     

1株副溶血性弧菌Bh-06的分离、鉴定及其药敏试验

从患病的南美白对虾分离出1株副溶血性弧菌Bh-06,通过革兰染色和Biolog全自动细菌鉴定仪鉴定,并对健康南美白对虾进行攻毒试验和对该菌株进行药物敏感试验。试验结果表明:Bh-06菌株为革兰阴性弧菌,通过细菌自动鉴定仪鉴定为副溶血性弧菌;该菌在培养第5小时进入对数早期,对数期一直延续到25小时;该菌在1.0×106cfu/mL浓度时可引起南美白对虾发病,而且浓度越高,病症越严重;该菌对氧哌嗪青霉素产生高度的敏感性。     

荧光染色法与KOH法在皮肤真菌检查中的效果比较

目的探讨真菌荧光法在皮肤浅部真菌筛查的敏感性。方法收集皮肤科门诊疑似真菌感染的患者标本,同时用KOH湿片法和真菌荧光染色法进行检测,并对检验结果进行对比。结果 1 209例患者中真菌荧光染色法阳性的阳性率为42.76%,KOH湿片法的阳性率为24.81%,结果差异有统计学意义(χ^2=87.094,P<0.05)。荧光染色法和KOH法在患者不同部位的阳性率:头面部为55.59%、7.90%,体股部为46.93%、30.06%,手足部为27.37%、26.25%,外阴部为31.85%、14.07%,指甲(趾甲)部为51.64%、48.36%;在不同疾病中的阳性率:马拉色毛囊炎为77.00%、8.50%,手足癣为48.04%、46.08%,体癣为50.31%、33.44%,甲癣为51.64%、48.36%,花斑癣为66.67%、48.00%,湿疹为10.24%、5.42%,皮炎为48.11%、9.43%。结论荧光染色法是一种简单、快速、准确的真菌镜检方法,值得在临床上推广使用。其总体的阳性率比KOH湿片法高,毛囊虫、疥疮等真菌荧光染色法在镜下更容易辨认。头面部、外阴等油脂分泌旺盛的部位以及马拉色毛囊炎、湿疹和皮炎等疾病推荐使用荧光染色法来排除是否有真菌的感染。     

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.     

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives.

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.