The Actinomyces of Potato Scab Demonstrated by Fluorescence Microscopy

The potato scab Actinomyces, like other acid-fast organisms, can be selectively impregnated with carbol-auramin and when exposed to ultraviolet radiation fluoresces bright yellow. The marked contrast of the bright yellow filaments permits ready localization and study of the micropathology of the tissue with a simple fluorescence microscope. The staining technic is done at room temperature and no counterstain is used. The fluorescence technic confirms Lutman's conclusion that the filaments are intercellular and grow within the middle lamellae. After complete removal of the paraffin, the sections are stained 4 minutes in carbol-auramin, (distilled water 97 ml., liquified phenol 3 ml., certified auramin 0.1 g.), washed, destained in a 0.5% solution of NaCl in 70% alcohol with 0.5 ml, HCl (conc.) per 100 ml., washed, and mounted in glycerin.     

Detailed Differentiation of Bacteria by Means of A Mixture of Acid and Basic Dyes at Different pH-Values

As previously reported by the author (1927), a mixture of methylene blue and eosin Y can be used for the differential staining of bacteria. It gives a fairly deep staining of bacteria at about pH 3 and above. Below pH 3 the eosin Y stains bacteria only a very pale pink; at such high H-ion concentration, the eosin is present as undissociated color acid, and for this reason not enough eosin is in solution to stain bacteria. To improve the staining at such reactions, the eosin was replaced by a stronger acid dye, namely acid fuchsin. The mixture of methylene blue medicinal Merck and acid fuchsin can be successfully used at a pH-value as low as 0.8. The method of staining by this new mixture is entirely the same as with the old mixture. It is sensitive enough to detect the difference in the isoelectric points: (1) of the single bacteria from the same pure culture, (2) of different strains of the colon and typhoid organisms. Some strains of the colon organism were found by this method with an isoelectric point at a pH-value as low as that of the Staphylococcus. Others, on the contrary, have their isoelectric point as high in the pH-scale as that of the typhoid organism. The new mixture can also be used for the study of the chemical composition of the different parts of bacterial body. Applying it at a definite pH-value, the author was able to stain differentially polar bodies of the typhoid group and of the diphtheria organism. This new mixture can be recommended in staining of B. diphtheriae as a substitute for Neisser's stain. It is interesting to note that polar bodies of the colon group consist of more alkaline protein than the body of the bacteria itself, i. e., they are stained by acid fuchsin. The polar bodies of the B. diphtheriae on the contrary are composed of more acid protein than the bacterial body; i. e., they are stained by methylene blue. The impossibility of detecting the above mentioned variations in the isoelectric points of bacteria using the Gram method is explained by the absence of pH variations in the latter technic. The differentiation of bacteria by the Gram stain depends chiefly on the varying stability of the compound formed (Gram-positive or Gram-negative bacteria plus gentian violet and iodine) in the presence of organic solvents, such as alcohol, acetone, etc.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30-90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30–90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.     

A Modification of the Fite Formaldehyde (Fite I) Method for Staining Acid-Fast Bacilli in Paraffin Sections

For maximal demonstration of acid-fast bacilli in tissue sections it is necessary to avoid sequences of reagents which, first, affect the integrity of the complex upon which acid-fastness depends, and, second, extracts it from those bacillary elements which have been made vulnerable by age or other factors. The greatest damage occurs during dewaxing of paraffin sections by xylene and alcohols; the older and more decrepit bacilli being especially affected. In the technic presented, which is a modified combination of two processes devised by Fite, sections are deparaffinized by a protective mixture of rectified turpentine and heavy liquid petrolatum (2:1) and blotted to water. Staining is with Fite's new fuchsin (magenta III) solution, overnight at room temperature. The sections are then treated with reagent grade formaldehyde, which turns the color of the bacilli deep blue-black, followed by an aqueous sulfuric acid decolorizer, the potassium permanganate-oxalic acid sequence, and a modified Van Gieson counterstain, nuclear staining with hematoxylin being omitted. For total demonstration of all stainable bacilli, restorative treatment in the turpentine-oil mixture before staining is sometimes required, most frequently with leprosy material but also with some tuberculosis lesions.     

Acid-Fastness of pine pollen

This is a study of the acid-fast staining characteristics of pine pollen and an investigation of factors causing loss of acid-fastness after pine pollen has been in contact with tisues or mucous membranes. Intact loblolly pine pollen was readily stained with cold carbol-fuchsin, and retained its acid-fastness after decolorization with 3% HC1 in 95% ethyl alcohol for 2 min, followed by methylene blue counterstain. Pine pollen resembles spermatozoa in ease of staining and resistance to decolorization. Acid-fastness was destroyed by crushing or by germination of the pollen grain, and by contact for several hours with serum or saline solutions, but was unchanged by exposure to 0.1% solution of streptomycin in water. Nonviable pine pollen did not lose acid-fastness after suspension for several days in serum or water. When counterstain was omitted, crushed or germinated pollen appeared red to pink after staining with carbol-fuchsin and decolorization with acid alcohol, thus indicating that lipids of an acid-fast nature were still present. The large size and biologic properties of pine pollen provide a unique means of studying the chemical and physical aspects of the acid-fast phenomenon.     

Comparison of auramine-rhodamine B and acridine orange for staining of acid-fast bacteria.

In cooperation of 6 laboratories in Czechoslovakia and in the GDR, the efficiency of auramine-rhodamine B (AR) and acridine orange (AO) (short-time method) for staining of acid-fast bacilli was compared. Whereas a former comparison of AR and AO (original method) pointed out the superiority of AR, the investigation of both methods used as short-time procedures showed significantly more acid-fast rods after using AO. The number of "false positive" results was somewhat higher on AR staining. However the results depend not only on the method used but also on the procedure of staining and the optical equipment, and they are essentially influenced by the experience and proficiency of the microscopist. Taking into account the results of both studies both auramine-rhodamine B and acridine orange can be proposed for the staining of slides for microscopical detection of acid-fast rods. In case of AO, the short-time method is superior to the original long-time procedure.     

Phloxine As An Histologic Stain, Especially In Combination With Hematoxylin

A staining schedule employing phloxine as a counter-stain to Erlich's acid hematoxylin is presented. Fixation is best with Zenker's fluid, although formalin can be used. The technic is similar to the standard hematoxylin-eosin formulae but because of the staining advantages of phloxine over eosin, the technic is simpler, and quicker, resulting in clearly differentiated sections which do not fade as soon as do eosin-stained slides. A brief summary of the uses of phloxine as a biological stain is given and its advantages over eosin are discussed.     

Ziehl-Neelsen's stain for skin sections to show wool and hair follicles

Ziehl-Neelsen's carbol-fuchsin stain differentiates between the keratin of the medulla and the keratin of the cortex of hair shafts in sections of skin biopsies. Deparaffinized, hydrated sections of Bouin-fixed or formalin-fixed skin of sheep and goats are stained 20-21 hr at about 25° C in carbol-fuchsin. They are rinsed and decolorized in acid alcohol, washed and then counterstained in Harris' haematoxylin. If no additional counterstain is used, the medullary keratin will appear colorless against a dark red acid-fast cortex. In case orange G, as a saturated solution in 95% ethyl alcohol, is applied after the haematoxylin, the medulla will be orange against the red cortex.     

革兰氏染色三步法与质量控制

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,自从1884年微生物学家Gram氏发明著名的革兰氏染色法以后,100多年来虽然经过后来学者的几次改进,但都仍然沿用着Gram氏原来的四步法,基本原理也没有改变。最近Allen氏对Ziehl-Neelsen抗酸菌染色法的改进,是一个良好的启示,使我们开始了革兰氏染色三步法的研究并取得了成功。现将我们建立的革兰氏染色三步法与质量控制报告如下。 1 材料和方法 1.1 结晶紫染色液 甲液:结晶紫2g;95％乙醇20ml。 乙液:草酸铵0.8g;蒸馏水80ml。 甲乙二液先分别溶解,然后混合在一起,过滤除去残渣后装入滴瓶中备用。     

Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin?

Abstract

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining depending on the differentiator used. Selective nuclear staining was obtained by differentiation in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH difference facilitated control of differentiation. When used with an eosin B counterstain, results were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selectively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear counterstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques. With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used for both cytological and histological samples, and was suitable for use in automated tissue stainers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too acidic.     

Combination of Gomori and Ziehl-Neelsen Stains for Showing Acid Phosphatase Activity and Mycobacterium tuberculosis in Macrophages

Tubercle bacillus-infected macrophage monolayer cultures, after fixation in glutaraldehyde or without fixation, are stained by a Gomori method to show acid phosphatase activity. The method includes H,S to convert lead phosphate to sulphide. Only a minority of the mycobacteria is outlined by the black or brown stain; the similarity to the surrounding stained cytoplasmic particles makes identification difficult. The Gomori staining procedure is followed by a Ziehl-Neelsen method to stain acid-fast microorganisms; the temperature of the carbol fuchsin is just high enough to produce steaming, and the time of decolorisation is short. To avoid loss of the Gomori stain from the acid-fast procedure it is essential to repeat the exposure to H₂S between the decolorisation and the light counterstaining. This combined method preserves the Gomori stain, against which the red acid-fast bacilli stand out sharply, so that acid phosphatase activity and bacteria can be located easily in the cell.     

A new method for the demonstration of the Bordetella bronchiseptica capsule in electron microscopy

A new and rapid working method is presented for electronmicroscopic preparation of capsules from gram negative bacteria, e.g. Bordetella bronchiseptica and Pasteurella multocida. The advantage of the new technique is the availability of the results within 30 min after starting the preparation. The staining of the capsule by alcian blue has to be done as a first step together with glutaraldehyde fixation, before staining the bacterial cell with phosphotungstic acid. The new staining technic also reveals structural details of the capsule. The described procedure was found to be useful in controlling the development of the bacterial capsule depending on culture media for propagation and maintenance of the above mentioned bacteria.     

The Negative Staining of Bacteria

The author does not propose new methods but calls attention to the value of Burri's India ink technic and Fischer's use of nigrosin for the negative staining of bacteria. The latter is particularly useful. The technic is as follows: Boil 10 g. nigrosin in 100 cc. water about 30 minutes. Filter several times thru the same filter paper, adding 10 drops of formalin before the last filtration as a preservative. Place a small loopful of this solution on a clean slide and add the bacteria with a needle or loop. After mixing spread the mixture a little irregularly on the slide and dry either at room temperature or slowly over a low flame. The preparation may be examined in oil immersion without the use of a cover slip.     

Ethidium bromide counterstain for differentiation of quinacrine stained interphase bodies and brilliant metaphase bands.

Visual perception of quinacrine mustard stained brilliant bands and interphase fluorescent bodies is enhanced by a staining procedure employing ethidium bromide as a counterstain.     

金胺O荧光染色在石蜡组织切片麻风病诊断中的应用

目的为了验证金胺O荧光染色法应用于石蜡组织切片麻风杆菌检测的可行性。方法用金胺O荧光法对6例确诊为麻风病的病理组织切片进行染色,并与抗酸染色结果进行对比。结果荧光染色法6例结果均为阳性,在暗背景下麻风杆菌显示明亮淡绿色荧光;在菌量较少荧光染色片中寻找单根麻风杆菌,较抗酸染色片更为容易。结论金胺O荧光染色法可用于石蜡组织切片麻风病的诊断,麻风杆菌单根散在时比抗酸染色法有一定优势。     

Lipoarabinomannan localization and abundance during growth of Mycobacterium smegmatis

Lipoarabinomannan (LAM) is a structurally heterogeneous amphipathic lipoglycan present in Mycobacterium spp. and other actinomycetes, which constitutes a major component of the cell wall and exhibits a wide spectrum of immunomodulatory effects. Analysis of Mycobacterium smegmatis subcellular fractions and spheroplasts showed that LAM and lipomannan (LM) were primarily found in a cell wall-enriched subcellular fraction and correlated with the presence (or absence) of the mycolic acids in spheroplast preparations, suggesting that LAM and LM are primarily associated with the putative outer membrane of mycobacteria. During the course of these studies significant changes in the LAM/LM content of the cell wall were noted relative to the age of the culture. The LAM content of the M. smegmatis cell wall was dramatically reduced as the bacilli approached stationary phase, whereas LM, mycolic acid, and arabinogalactan content appeared to be unchanged. In addition, cell morphology and acid-fast staining characteristics showed variations with growth phase of the bacteria. In the logarithmic phase, the bacteria were found to be classic rod-shaped acid-fast bacilli, while in the stationary phase M. smegmatis lost the characteristic rod shape and developed a punctate acid-fast staining pattern with carbolfuchsin. The number of viable bacteria was independent of LAM content and phenotype. Taken together, the results presented here suggest that LAM is primarily localized with the mycolic acids in the cell wall and that the cellular concentration of LAM in M. smegmatis is selectively modulated with the growth phase.     

Differential Staining of Yeast for Purified Cell Walls, Broken Cells, And Whole Cells

Intact yeast cells are Gram positive but broken or disrupted cells are Gram negative. A counterstain with methyl green provides differential staining between cell wall and cytoplasm. The cells and cell fragments are dried on a slide and stained by a standard Gram stain. The preparation is then treated for 5 min with 1% phosphomolybdic acid, washed, and stained 0.5 min with 1% aqueous methyl green (unpurified by CHCl₃ extraction). Under these conditions whole, intact cells are dark purple or black, walls of broken cells and purified walls are light green, and the exposed cytoplasm stains light purple. All fractions can be easily differentiated.     

Demonstration of Acid-Fast Bacteria by Reacting Chromogenic Amines with Their Capsular Lipids

Coupling a chromogenic amine to carboxyl groups of the lipids in the capsules of acid-fast bacteria was accomplished by reaction with benzidine, diazotization and using sodium beta naphthylate as the chromogen. By these means, acid-fast bacteria can be differentiated from non-acid-fast, and the intensity of the staining correlates well with the amount of lipid found in their capsules. By comparing the intensity of staining before and after treatment with 5% HCl, it is possible to demonstrate that some acidic capsular components are combined with calcium. Similarly, by comparing the intensity of staining before and after treatment with petroleum ether, the presence of oxyfatty acids (insoluble therein) can be demonstrated.     

Demonstration of Acid-Fast Bacteria by Reacting Chromogenic Amines with Their Capsular Lipids

Coupling a chromogenic amine to carboxyl groups of the lipids in the capsules of acid-fast bacteria was accomplished by reaction with benzidine, diazotization and using sodium beta naphthylate as the chromogen. By these means, acid-fast bacteria can be differentiated from non-acid-fast, and the intensity of the staining correlates well with the amount of lipid found in their capsules. By comparing the intensity of staining before and after treatment with 5% HCl, it is possible to demonstrate that some acidic capsular components are combined with calcium. Similarly, by comparing the intensity of staining before and after treatment with petroleum ether, the presence of oxyfatty acids (insoluble therein) can be demonstrated.