教学中革兰氏染色两种方法的比较研究

微生物染色技术是观察微生物形态结构的重要手段 ,而革兰氏染色 ( Gram stain)是细菌学中一个经常使用和十分重要的方法 ,微生物学家 Gram氏于 1884年发明著名的革兰氏染色法后 ,10 0多年来一直使用着Gram氏发明的四步法。革兰氏染色四步法 ,就是第 1步用草酸铵结晶紫进行初染 ,第 2步用碘液进行媒染 ,使细菌着色 ,第 3步用乙醇脱色 ,第 4步用蕃红复染。革兰氏染色反应是细菌分类和鉴定的重要依据。革兰氏染色的原理为 :革兰氏阴性细菌 ( G-)细胞壁中脂类物质含量较高 ,肽聚糖含量较低 ,因而用乙醇处理时 ,溶解了脂类物质 ,使细胞壁通透性…     

一种简易革兰氏染色法

我们用简易G染色法,对细菌进行染色,实践证明该方法稳定性好。简单快速,特介绍如下:一、试剂:(一)甲液:结晶紫0.25g,蒸留水加至100ml(二)乙液:碱性品红0.3g,碘化钾0.5g,碘0.2g,用95％酒精加至100ml溶解(可以水浴溶解)。二、方法:     

一种光学显微镜下观察原生质体的染色方法

Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ ｌａｃｔｏｆｅｒｍｅｎｔｕｍ菌液经溶菌酶处理后分别与４种微生物染色液混合,在光学显微镜下比较观察原生质体形态的效果。结果表明;染色样品中的原生质体比未经染色的形态清晰易观察,而且显微照相效果好;其中使用草酸铵结晶紫和复红染色液的效果更佳。该方法程序简单、操作方便、效果明显,还适用于悬滴法观察菌体形态和细菌运动方式。     

HACCP体系在细菌形态学检测之革兰氏染色中的应用

细菌形态学检测方法是细菌检验中极为重要的鉴定手段之一,革兰氏染色法是细菌中最常见的一种鉴别染色法,是细菌分类鉴定中的重要标志。革兰氏染色法常因各种因素造成误差,造成反应结果的不稳定。因此,我们需要依靠有效的质量控制措施来消除影响染色结果的各种误差。本文将HACCP体系应用到革兰氏染色中,确定革兰氏染色的关键控制点和关键限值,规范了试验操作,以便更好地保证染色结果的准确性。     

Mallory氏胰岛细胞染色法的改良

目的:为了更清晰地显示胰岛的A细胞、B细胞和D细胞。方法:zenker液固定,分别用苏木精-伊红和改良的Mallory氏法染色。结果:苏木精-伊红染色法,不易区分胰岛内三种细胞,而采用改良的Mallory氏染色方法,能够清晰地显示A细胞、B细胞和D细胞。结论:改良的Mallory氏染色法能够清楚的显示胰岛内三种细胞结构,为实验教学提供了优良的切片标本。     

不同固定液和染色方法显示山羊子宫内肥大细胞效果的比较

目的探讨用不同固定液和染色方法对显示处于间情期山羊子宫肥大细胞的影响。方法用四种不同的固定方法,应用改良甲苯胺蓝（MTB）染色法和阿尔辛蓝-番红花红（AB-S）染色法显示处于间情期山羊子宫肥大细胞。结果山羊子宫组织采用Carnoy氏液固定,MTB和AB-S染色对所有的肥大细胞均可获得良好的染色反应,但10％中性福尔马林,4％多聚甲醛,Bouin氏液固定的组织仅有少量肥大细胞着染。结论MTB和AB-S染色法均是山羊子宫肥大细胞良好的染色方法。     

Holzer氏神经胶质纤维染色方法的改良

Ｈｏｌｚｅｒ氏神经胶质纤维染色方法的改良北京军区总医院病理科田玉旺,丁华野北京卫戍区２９２医院病理科李景仁在神经系统疾病的诊断中,观察神经胶质纤维的变化是鉴别诊断神经胶质细胞瘤和其它肿瘤的可靠依据。其中Ｈｏｌｚｅｒ氏磷钼酸结晶紫染色是常用的方法,但在...     

教学用细菌染色方法的改良

常用细菌染色有革兰氏染色,抗酸染色及检测细菌特殊结构的特殊染色法,本文对改良了的染色法进行比较和进一步改进,实验结果证明有操作简单,染色效果好等优点。     

结晶紫溶液鞭毛染色法 郭恒彬

细菌的鞭毛是细菌的一种特殊结构,鞭毛染色常用硷性复红乙醇饱和液或结晶紫乙醇饱和液染色。对后者反复试验未能使鞭毛着色,分析染料的溶解度及配制时各染液混合后出现大量结晶紫沉淀物,考虑染液配制过程中损耗大量媒染剂使鞭毛     

诺卡氏菌科分类的研究II．诺卡氏菌属中的两个新种和一个新变种

从我国各地的土壤中分离得到百余株诺卡氏菌形放线菌,其基丝形成横隔并断裂成秆状或球状体,细胞壁化学组份IV型,属于诺卡氏菌科诺卡氏菌属(Nocardia)。经形态、培养特征及生理生化特性等鉴定为15个种。本文只报道其中两个新种及一个新变种：念球状诺卡氏菌(Noeaedia nostocoidea n. sp．);紫褐诺卡氏菌(Nocardta violaccofusca n. sp.);鲑色诺卡氏菌桔橙变种(Nocardia salmontcolor var. aurantiaca n. var.)。     

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.     

A Note on the Gram Stain

A modification of the Gram stain in which iodine-alcohol is substituted for 95% alcohol as a decolorizing agent has been found particularly useful in staining Gram-positive organisms in tissues and also for smears. The technic for tissue sections follows:

1. Apply nuclear stain.
2. Wash.
3. Stain in Hucker's gentian violet 2 to 3 minutes (i. e. 1 part Sat. Alc. Sol. crystal violet to 4 parts 1% Aqu. Sol. ammonium oxalte).
4. Wash in water.
5. Stain in Gram's iodine 5 minutes.
6. Wash in water.
7. Decolorize in 95% alcohol to which enough tincture of iodine has been added to give a mahogany color.
8. Counterstain.
9. Dehydrate and mount.

     

Staining Scab Actinomyces in Potato Tuber Tissues

Actinomyces hyphae imbedded in the middle lamellae of potato tuber cells may be stained in sections by the use of a modified Gram's stain. The modifications are: a very strong (5%) solution of crystal violet in anilin oil; a 24-hour exposure to both the dye and the iodine solution; and a slow decolorization in absolute alcohol until no more color flows.     

Simplified Aldehyde-Fuchsin Staining of Neurosecretory Cells

Gomori's original aldehyde-fuchsin method has been modified by the combination of Halmi's counter stain with Gabe's preparation, consisting of basic fuchsin, 1 gm; boiling water, 200 ml; with HC1, 2 ml and paraldehyde, 2 ml added after cooling and filtering. The solution so made was allowed to ripen 3-4 days at room temperature, and the precipitate which formed was filtered off and dried at 55-60°C. The staining solution consisted of 0.5 gm of the dry precipitate dissolved in 100 ml of 70% alcohol. The staining follows original procedures except that it is very important to bring slides from water to 70% alcohol before placing them in the aldehyde-fuchsin solution and also to remove all excess staining solution by rinsing in 95% alcohol after staining. The staining solution is stable for at least 6 mo.     

The Mechanism of the Gram Reaction. II. The Function of Iodine in the Gram Stain

Fifty-five reagents were studied as to their ability to replace iodine in the Gram stain. None gave results as good as iodine. Eight gave usable Gram preparations, and forty-seven gave negative results. Omission of the counterstain resulted in increasing to thirty-three the number of reagents giving differentiation, but this, was not considered a true Gram differentiation. Many oxidizing agents were shown not to be substitutes for iodine; therefore the function of iodine must be more than to serve as an oxidizing agent. Many reagents which formed precipitates with the dye could not replace iodine; therefore factors other than precipitate formation must be involved. However, all agents which were good substitutes for iodine were both good oxidizing and dye precipitating agents. Experiments involving the study of cell membrane permeability showed that Gram-positive cells were less permeable to iodine in alcoholic solution than Gram-negative cells. This difference could not be demonstrated for iodine in aqueous solution. It was concluded that iodine served to form a dye-iodine precipitate (or complex) in the cell. Since Gram-positive cells were less permeable to iodine in alcohol than Gram-negative cells, this resulted in a slower dissolving out of this complex from Gram-positive cells during de-colorization and hence a slower decolorization time. The relative solubilities of dye precipitates in alcohol and in aqueous safranin solution were also indicated as an important factor influencing decolorization. Dyes which formed highly soluble precipitates with iodine could not be used in the Gram stain. It is not proposed that the mechanism of the Gram stain is entirely one of membrane permeability; chemical factors are undoubtedly important and will be discussed in a later paper. However, it is proposed that the chemical and physical factors are closely interrelated in the Gram stain mechanism.     

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.     

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.     

Comparison of the quick Gram stain method to the B&M modified and favor methods

The Gram stain is an established method for bacterial identification, but the time needed to carry out this stain is 2-3 min. We attempted to shorten this time and stained a total of 70 clinical specimens isolated from using the Bartholomew & Mittwer (B&M) modified or Favor methods with a 3 s duration for washing and staining steps. Results were plotted and analyzed using a Hue Saturation Intensity (HSI) model. The range based on a plot of the two methods with the HSI model was presented as a reference interval. Our results indicated that 100% (35/35) of strains were Gram positive and 97.1% (34/35) were Gram negative for the quick B&M modified method. In the quick Favor method, 80.0% (28/35) were Gram positive and 68.6% (24/35) of strains were Gram negative. We propose that the quick B&M modified method is equivalent to the standard Gram staining method and is superior to the quick Favor method.     

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.