Dye Complexes Produced in Gram Staining

Complexes of crystal violet and iodine containing (a) one molecule and four atoms, and (b) one molecule and two atoms respectively, were prepared and their solubility in alcohol (95%) determined. Both complexes were only slightly soluble in alcohol, the former being less soluble than the latter. The solubility decreased with decreasing concentration of alcohol, and also on storage. Complexes of malachite green, basic fuchsin and Victoria blue B with iodine were also prepared, and complexes of each dye with picric acid. Two complexes of different composition could be obtained from crystal violet and picric acid, and from Victoria blue and iodine. Complexes with basic fuchsin were much more soluble in alcohol (95%) than complexes with the other dyes tested. Dye-mordant complexes have some of the properties of organic charge-transfer complexes.     

Iodine131 Uptake in the Gram Staining Technic

The Hucker modification of the Gram staining technic, in which NaI¹³¹ was incorporated with the Gram's iodine solution, was performed as the basic procedure. The Gram positive test-bacteria were Staphylococcus aureus and Bacillus megaterium; the Gram negative were Escherichia coli and Pseudomonas aeruginosa. The uptake of I¹³¹ was measured after the addition of the Gram's iodine solution (NaI¹³¹) to the test-bacteria dried on a glass slide, after the decolorization process and after counterstaining. Radiation was measured by placing the slide under a GM-TGC-2 end-window counting tube after each procedure. The Gram positive test-bacteria retained approximately twice as much I¹³¹ after decolorization and counterstaining as did the Gram negative bacteria. In this, the basic technic, the uptake of I¹³¹ by the test bacteria appeared to be directly related to the crystal violet concentration in the primary staining solution. The uptake of I¹³¹ was not significantly altered by the time of application of the Hucker crystal violet staining solution (15-180 sec), or of the Gram's iodine (NaI¹³¹) solution (30-120 sec) or by the duration of the alcohol decolorization process (30-120 sec).

Variations (herein referred to as variations 2 and 3) of the basic procedure were carried out in which the primary staining solution contained crystal violet combined with NaI¹³¹ or Gram's iodine solution (NaI¹³¹). In variations 4 and 5 the effect of the order of application of the various staining reagents was investigated. In these variations (2-5) all test-bacteria were stained Gram negative. The initial uptake of I¹³¹ was decreased, though in variations 4 and 5 the percent retention of I¹³¹ was increased. In the staining of bacterial spores by different methods (variation 6), it was noted that the initial uptake and percent retention of I¹³¹ was greater than with the vegetative forms. When ovalbumin was stained by the Hucker technic and variations thereof, it was noted that the initial uptake of I¹³¹ was directly related to the protein (ovalbumin) concentration up to an ovalbumin concentration of 1%.     

Variations in the Gram Staining Results Caused by Air Moisture

The exposure of heat-fixed bacterial smears to relative humidities of 0, 52 and 98%, following the iodine step in a dry Gram stain procedure, markedly influenced the rate of decolorization upon exposure to 95% ethyl alcohol. If a single decolorization time were used to give a proper Gram differentiation after exposure to 98% relative humidity, this decolorization time might not result in proper Gram differentiation following exposure to 0% relative humidity. Different organisms varied in the degree of their response to changes in humidity. Hence the “degree of Gram-positivity,” as compared with other organisms, differed with changes in relative humidity. When Neisseria catarrhalis was compared with strongly Gram-positive and Gram-negative organisms, it was always found to be in an intermediate position in its Gram characteristics regardless of the relative humidity used. Because of the intermediate position of this organism, its proper Gram differentiation would require a precise definition of both the decolorization time and the decolorization procedure to be used. The results for all organisms studied clearly indicated that the control of wetness or dryness of the smear before decolorization would be essential in any Gram procedure where a single decolorization time is to be used.     

Simultaneous Fluorescent Gram Staining and Activity Assessment of Activated Sludge Bacteria

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.     

A Novel Improved Gram Staining Method Based on the Capillary Tube

In this work, an exploratory study was conducted to examine Gram staining based on the capillary tube. Each Gram staining step for all bacterial strains tested was completed in capillary tubes. The results showed that different Gram staining morphologies were clearly visible in the capillary tubes. The results presented here demonstrated that the improved method could effectively distinguish between Gram-positive and Gram-negative bacteria, and only small volumes of reagents were required in this method. Collectively, this efficient method could rapidly and accurately identify the types of bacteria. Therefore, our findings could be used as a useful reference study for other staining methods.Key words: Gram staining, capillary tube, bacteria, and glass slide

Since Hans Christian Joachim Gram reported a staining method in 1884 (Gram 1884), such a technique has experienced more than a century of development and has become frequently used in bacteriology. From 1940 to 1960, Gram staining’s clinical application reaches its peak (Kass 1987). In recent years, several automated instruments for Gram staining have also been applied for microbiological analysis (Baron et al. 2010; Li et al. 2020). With the development of modern science and technology, some new technologies are expected to replace Gram staining. For example, Sizemore et al. (1990) have developed an alternative Gram staining technique using a fluorescent lectin. Later on, several fluorescent Gram staining methods have been established, and some Gram staining techniques suitable for live bacterial suspension have been described (Mason et al. 1998; Fife et al. 2000; Forster et al. 2002; Kwon et al. 2019). Sharma et al. (2020) have found that acridine orange fluorescent staining is more sensitive than the Gram staining. Besides, Berezin et al. (2017) have established a method for detecting Gram-negative bacteria based on enhanced Raman spectroscopy. Lemozerskii et al. (2020) have also reported a method of bacterial discrimination using an acoustic resonator. However, Gram staining is still an vital detection method in practical application for many microbiologists and clinicians due to its rapidity and simplicity (Thompson et al. 2017; Jahangiri et al. 2018; Li et al. 2018a).Over the years, Gram staining has been modified for many times, such as the Brown-Hopps method, Brown-Brenn method, and Gram-Twort method (Brown and Brenn 1931; Brown and Hopps 1973; Peck and Badrick 2017), and these approaches as mentioned earlier are widely used in anatomical pathology laboratories. Through the comparison of various improved methods, it is found that Gram’s original four-step method is still used, and some researchers have adopted the three-step method, while its basic principle has not been changed. As reported by Huang and Cui (1996), the three-step Gram staining method combines the two steps of alcohol decolorization and re-staining procedure in one step. Although Gram staining is one of the most commonly used detection methods in clinical microbiology laboratories, many clinicians are skeptical of its results due to differences in operators, low control, and standardization (Samuel et al. 2016; Thomson 2016). Researchers have made efforts to improve the Gram staining’s accuracy and reliability over the past few years, such as repeated training and standardization of the staining procedure (Thomson 2016; Siguenza et al. 2019). In this study, we developed a standardized Gram staining procedure for bacterial identification using a capillary tube. A modified Gram staining method based on the capillary tube has not yet been reported to the best of our knowledge. Therefore, we proposed a novel improved Gram staining method to improve the accuracy of the detection results and Gram staining efficiency.Eight bacterial strains, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Bacillus Licheniformis, Serratia marcescens, Vibrio parahaemolyticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus were provided by the Laboratory of Microbial Engineering, College of Life Science, Luoyang Normal University. L. bulgaricus and S. thermophilus were inoculated into skim milk culture medium and maintained at 37°C for 12 h. S. marcescens, B. Licheniformis, E. coli, B. subtilis, V. parahaemolyticus, and S. aureus were inoculated into beef peptone agar slants and maintained at 37°C for 16 h.Capillary tubes with an internal diameter of 0.5 mm and a length of 100 mm were purchased from the Instrument Factory of West China University of Medical Sciences. Gram staining reagent was obtained from the Anhui Chaohuhongci Medical Equipment Co., Ltd.Procedure: (1) One or two drops of sterile water were placed in the center of a clean glass slide. An inoculating loop was hold in a flame until it was red-hot and then allowed to cool approximately 30 seconds. Subsequently, a loop of culture was transferred to the center of the slide. The sample was spread onto the slide using the inoculating loop, and a small volume of bacterial suspension was automatically transferred into the capillary tube.(2) The capillary tube was then heated by passing over a flame for several times until the liquid was completely evaporated. The capillary tube was naturally cooled in the air for several seconds.(3) One end of the capillary tube was hold upward, and the crystal violet solution was automatically transferred to the capillary tube, followed by standing for 1 minute. The remaining crystal violet solution of the capillary tube was then transferred to absorbent paper. The capillary tube was washed in a gentle and indirect stream of tap water for a few seconds, and samples were dried on absorbent paper.(4) One end of the capillary tube was hold upward, and Gram’s iodine solution was automatically transferred to the capillary tube, followed by standing for 1 minute. Subsequently, the capillary tube was washed using the same procedure as described above.(5) One end of the capillary tube was hold upward, and 95% ethanol was automatically transferred to the capillary tube, followed by standing for 30 seconds. Subsequently, the capillary tube was washed using the same procedure as described above.(6) One end of the capillary tube was hold upward, and the Safranin solution was automatically transferred to the capillary tube, followed by standing for 30 seconds to 1 minute. The subsequent procedure was the same as described above. Besides, conventional Gram staining was carried out according to the instructions from the reagent kit. According to the instructions, Gram-negative cells are in pink to red, and Gram-positive cells show a purple or blue color when observed under a microscope.The Gram staining is always the “first-stage criteria” in the preliminary identification of bacterial species according to their cell walls (Li et al. 2018b). Eight different bacterial species were examined to investigate our approach, and the strains were selected according to the Gram staining pattern. Gram-negative bacteria E. coli, V. parahaemolyticus, and S. marcescens were examined. Gram-positive bacteria S. thermophilus, L. bulgaricus, S. aureus, B. licheniformis and B. subtilis were also assessed. Fig. ​Fig.1,1, ​,2,2, and ​and33 illustrate the results of Gram staining of E. coli, V. parahaemolyticus, and S. marcescens, respectively. Fig. ​Fig.4,4, ​,5,5, ​,6,6, ​,7,7, and ​and88 show the Gram staining results of S. thermophilus, L. bulgaricus, S. aureus, B. subtilis, and B. licheniformis, respectively. These results were compared with those obtained using a glass slide for Gram staining. No matter spherical or rod-shaped or not, all bacterial strains could be differentiated into two classifications: Gram-positive and Gram-negative. Comparing these results, we found that the results obtained by the capillary tube method were consistent with the conventional Gram staining approach. It was worth mentioning that in contrast to direct heat fixation of bacteria on glass slides, heat fixation by passing the capillary tube over a flame should be carried out quickly and carefully. If the capillary tube was overheated, it might cause the capillary tube to rupture, and it is easy to blur the field of vision, making it challenging to observe the staining result (Fig. ​(Fig.9).9). Therefore, before the experiments, it is better to conduct a preliminary experiment and achieve the desired results.Open in a separate windowFig. 1.The Gram staining results of E. coli. A – Capillary sample, B – Glass slide sample.Open in a separate windowFig. 2.The Gram staining results of V. parahaemolyticus. A – Capillary sample, B – Glass slide sample.Open in a separate windowFig. 3.The Gram staining results of S. marcescens. A – Capillary sample, B – Glass slide sample.Open in a separate windowFig. 4.The Gram staining results of S. thermophiles. A – Capillary sample, B – Glass slide sample.Open in a separate windowFig. 5.The Gram staining results of L. bulgaricus. A – Capillary sample, B – Glass slide sample.Open in a separate windowFig. 6.The Gram staining results of S. aureus. A – Capillary sample, B – Glass slide sample.Open in a separate windowFig. 7.The Gram staining results of B. subtilis. A – Capillary sample, B – Glass slide sample.Open in a separate windowFig. 8.The Gram staining results of B. Licheniformis. A – Capillary sample, B – Glass slide sample.Open in a separate windowFig. 9.The microstructure of the overheated capillary tube.Several studies (Chimento et al. 1996; Wada et al. 2012; Li Zhu 2018b) have already pointed out that the property of the bacterial cell wall determines whether the organism will be Gram-positive or Gram-negative, and it plays a role in the choice of antibiotics when infection occurs. Since it has frequently been observed that not all bacteria react in the same manner to such staining procedure (Hale and Bisset 1956), it is necessary to make more tests upon a representative selection of Gram-positive and Gram-negative bacteria in future studies.Molecular biology techniques and high-precision measurement systems have been successfully developed, and they can distinguish bacterial types in clinical samples and improve microbial detection (Klaschik et al. 2002; Dolch et al. 2016; Kim et al. 2018). However, it is still urgently needed to develop a rapid and straightforward Gram staining approach to detect bacteria, especially for those who have only primary experimental conditions. Our results indicated a promising method for bacterial differentiation using the capillary tube as a carrier. Successful differentiation required only small volumes of reagents, and the results were achieved within a few minutes by applying an optical microscope. In addition, the method proposed in this paper had reference value to other staining methods requiring expensive reagents.In the present study, the improved Gram staining method was developed based on the pure cultures, and it was only a comparison of the staining results between known Gram-negative and Gram-positive bacteria in a glass slide and capillary tube. In order to improve the accuracy and stability of the results, future study is necessary to detect more bacterial species. In addition, the modified method was not applicable for direct Gram staining of clinical samples. In the future, it may have a positive effect by developing a special method for processing clinical samples.The experimental results demonstrated that an improved Gram staining method was suitable for differentiating the strains tested in our laboratory. The method could rapidly discriminate Gram-positive and Gram-negative bacteria. Besides, the method only required small volumes of reagents. A much more comfortable and reproducible Gram staining approach can be developed for microbiology research based on our studies.     

Some Staining Technics for Guayule Studies

For staining tissues and secretions in guayule studies the following dyes and dye combinations have been found satisfactory: rubber in plant sections, Sudan IV; plant sections (rubber and tissues) and ground tissues, Poirrier's blue, Sudan IV and titan yellow; rubber extraction films (with tissue contamination), oil blue NA, safranin O and titan yellow; resin extraction films, Bismark brown and oil blue NA; chopped plants (milling studies), safranin O and Sudan IV. For microscopic studies, sections and ground tissues are pretreated with KOH and a bleaching agent prior to staining.     

Gram Staining Applied to Human Spermatozoa: a Simple Method for Studying Chromatin Condensation Status

Gram staining applied to human spermatozoa from fertile donors is described. The stain revealed populations of Gram-positive and Gram-negative spermatozoa. Data showed a significant and progressive decrease in the percentage of Gram-positive spermatozoa at different times during the chromatin decondensation procedure (SDS-BSA and SDS-EDTA). No significant correlation could be found between Gram staining and other functional tests used for spermatozoa; only the aniline blue staining test showed a poor correlation. Our study demonstrates that normal spermatozoa with regular chromatin condensation appear Gram-positive, while spermatozoa with altered chromatin condensation appear Gramnegative.     

Further Studies on a Modification of the Gram Stain

In perfecting the modification of the Gram-stain previously proposed, the following points are of interest:

1. Acetone is too strong a decolorizer for Gram-positive organisms and alcohol too weak for Gram-negative organisms. Consequently, it is now recommended that equal parts of acetone (100% c.p.) and ethyl alcohol (95%) be used as a decolorizing agent. The time of application should not ordinarily exceed 10 seconds.

2. Aqueous basic fuchsin (0.1%) serves as a strongly contrasting counterstain. Prolonged application renders Gram-positive organisms doubtful or Gram-negative, while short application renders Gram-negative organisms doubtful or Gram-positive. Twenty (20) seconds is therefore recommended as the time of application of the counterstain.

3. The method here described, with due regard for its limitations, is of value in Gram-staining pure or mixed cultures as well as for organic materials, such as Acidophilus milk, feces, etc., either for research purposes or classroom use. The method is as follows:

Air-dry film and fix with least amount of heat necessary.

Flood with dye for 5 minutes. Previously mix 30 drops of a 1% aqueous solution of crystal violet or methyl violet 6B with 8 drops of a 5% solution of sodium bicarbonate. Allow the mixture to remain for 5 minutes or more.

Flush with iodine solution for 2 minutes. Two grams iodine dissolved in 10 cc. normal sodium hydroxide solution and 90 cc. water added.

Drain without blotting but do not allow film to dry.

Add a mixture of equal parts of acetone and alcohol drop by drop until the drippings are colorless. (10 seconds or less.)

Air-dry slide.

Counterstain for 20 seconds with 0.1% aqueous solution of basic fuchsin.

Wash off excess stain by short exposure to tap water and air-dry. If slide is not clear immersion in xylol is recommended.     

The Gram Staining Mechanism of Cat Tongue Keratin. II. The Role of Potassium Iodide-Iodine Solution

The aniline-xylene decolorizer of the Gram-Weigert staining procedure failed to remove crystal violet dye from stained sections of cat tongue without prior treatment of the sections with potassium iodide-iodine solution. The potassium iodide of the iodide-iodine solution was found to release the major part of the crystal violet dye bound by the tongue sections. Iodine appeared also to play a role in dye release, but only to a slight degree. The amount of Gram-positive staining was increased both by alkaline treatment of the tissue prior to staining, and by increasing the pH of the iodide-iodine solution.     

标记抗体染色病毒空斑计数技术的研究

用细胞病变阳性（ｐｏｓｉｔｉｖｅ ｃｙｔｏｐａｔｈｏｇｅｎｉｃ ｅｆｆｅｃｔ,ＣＰＥ＋ ）病毒马立克氏病毒（Ｍａｒｅｋ＇ｓｄｉｓｅａｓｅ ｖｉｒｕｓ, ＭＤＶ）血清１,２,３ 型以及细胞病变阴性（ｎｅｇａｔｉｖｅ ｃｙｔｏｐａｔｈｏｇｅｎｉｃ ｅｆｆｅｃｔ,ＣＰＥ－ ）病毒猪瘟病毒（Ｈｏｇ ｃｈｏｌｅｒａ ｖｉｒｕｓ,ＨＣＶ）强毒与弱毒和鸡新城疫病毒（Ｎｅｗ ｃａｓｔｌｅ ｄｉｓｅａｓｅｖｉｒｕｓ． ＮＤＶ）Ｌａｓｏｔａ 毒株及其对应的异硫氢酸荧光素（ＦＩＴＣ）标记的特异抗体为试验材料,以免疫荧光抗体技术（ＦＡ）为基础、并加以改进,建立了标记抗体染色病毒空斑计数技术． 该技术不仅能克服常规病毒空斑计数技术不能计数细胞病变阴性病毒和一种样品含有两种或两种以上病毒的各自空斑数的缺点,能迅速准确计数出ＣＰＥ－ 病毒和多病毒样品中病毒各自空斑数及其空斑总数,具较高敏感性、良好的可重复性．     

Variables Influencing Results, and the Precise Definition of Steps in Gram Staining as a Means of Standardizing the Results Obtained

Results of a Gram staining procedure varied with modifications of each of the steps involved. The best Gram differentiation was obtained when crystal violet and iodine solutions of high concentrations were used, and when n-propyl alcohol was used as the decolorizer. The decolorization step must be carefully quantitated, and one of the most important variables observed was whether a slide was brought into the decolorizer wet, or dry. Dry slides took 6 to 12 times as long to decolorize as wet. Wash steps, following crystal violet, and following the decolorizer, also greatly influence results by causing Gram-positive organisms to appear to be Gram-negative. The results indicated that Gram-stain procedures should not be varied to suit the whims of individual operators, and that each step could be specifically defined both as to the reagent used, and the procedure to be followed.

The followng Gram procedure is recommended for heat-fixed bacterial smears on glass slides. Flood the slide with Hucker's crystal violet for 1 ruin. Wash for 5 sec by dipping into tap water running into a 250 ml beaker at a rate of 30 ml per sec Rinse off the excess water with Burke's iodine, flood the slide with this solution for 1 min, then wash 5 sec in tap water as above. Decolorize by passing the wet slide through 3 (75 × 25 mm) Coplin dishes containing n-propyl alcohol, decolorize 1 min in each dish for a total of 3 min. Wash 5 sec in tap water as above, rinse off the excess water with 0.25% safranin, then flood the slide with this solution for 1 min. Wash as above, blot dry, and examine. An alternate procedure for decolorization would be to use either 95% n-propyl alcohol or 95% ethyl alcohol, but shorten the decolorization time to 30 sec per dish for a total of 1.5 min. After 10 slides, the decolorizer in the first dish should be replaced by fresh. This dish is then placed last in the sequence, with dish No. 2 moved to the No. 1 position.     

Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 10⁸ cfu/ml of Escherichia coli and 10⁵ cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.     

Staining of Muscle Tissue for Quantitative Studies and Automated Morphometry

Seventy-six staining tests were carried out on paraffin sections of human and animal muscle to find a suitable staining method for quantitative morphometry of muscle fibers. The results were evaluated under the light microscope, on black and white photomicrographs and on an image analysing computer, the Quantimet 720. A brilliant scarlet-phosphotungstic acid-tartrazine method is described and recommended for automated morphometry after additional testing on 140 sections of developing human muscle using the Quantimet 720.