Methanol fixation an alternative to heat fixation of smears before staining

Mangels, James I.; Cox, Mike E.; Lindberg, Lois H.

doi:10.1016/0732-8893(84)90008-7

Cited by 15 publications

(8 citation statements)

References 5 publications

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“…Alternatively, lack of material on the slide could also reflect failure to perform adequate fixation prior to staining. Mangels et al noted that use of methanol fixation held significant advantages over heat fixation, including reduced distortion of cellular morphology, reduced background debris, and increased likelihood for detection of microorganisms on smear (8). However, in our study the incidence rate of Gram stain errors in the laboratory did not appear to correlate specifically with method of smear fixation utilized.…”

Section: Discussioncontrasting

confidence: 54%

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Multicenter Assessment of Gram Stain Error Rates

et al. 2016

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Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories. C linical microbiology laboratories have undergone dramatic changes with the implementation of novel technologies, yet traditional techniques, such as the Gram stain, still play key roles throughout the diagnostic process (1-3). Gram stains are initially used as a preanalytical indicator of specimen quality and acceptability for culture. They also give the clinician preliminary information regarding the nature of potential pathogens present in the patient specimen and thus serve to guide empirical therapy. Although the Gram stain has been the staple of clinical microbiology laboratories for over a century, it is still considered a high-complexity procedure by the Clinical Laboratory Improvement Amendments (CLIA) program. The manual nature of the staining process and the subjectivity of Gram stain interpretation contribute to the incidence of errors (4-7). Inappropriate specimen sampling, specimen processing, smear preparation, and prior antibiotic therapy are all factors that can have an adverse impact on Gram stain result. The inherent nature of some organisms may also produce misleading results; for example, Acinetobacter spp. may stain Gram positive, while Bacillus spp. species may appear Gram negative. In addition, staining practices, such as use of cytospin and ...

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Section: Discussioncontrasting

confidence: 54%

“…species may appear Gram negative. In addition, staining practices, such as use of cytospin and fixation methods (heat versus methanol), may vary from one laboratory to another and even within laboratories, with a potentially significant impact on the quality of the result (8).…”

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confidence: 99%

Multicenter Assessment of Gram Stain Error Rates

et al. 2016

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“…This step makes the background of inflammatory cells and mucus appear gray or green rather than red or pink, thus allowing easier visualization of gram-negative organisms (91), but it causes a slight change in the color of organisms compared with that in the routine Gram stain, which initially could cause confusion for the inexperienced observer. With regard to fixation, methanol results in less distortion of bacteria and tissue cells and less interference by background debris than does heat (115), and it provides for the most reliable staining of anaerobes. For optimal identification of anaerobes, performing the entire staining procedure, including methanol fixation, in an anaerobic chamber appears to lessen the tendency of certain gram-positive anaerobes to stain gram negative (87).…”

Section: Direct Detection In Smearsmentioning

confidence: 99%

Detection of infection or infectious agents by use of cytologic and histologic stains

1996

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A wide variety of stains are useful for detection of different organisms or, for viruses, the cytopathologic changes they induce, in smears prepared directly from clinical specimens and in tissue sections. Other types of stains, such as hematoxylin and eosin, are used routinely to stain tissue sections and are most valuable for assessing the immunologic response of the host to the invading pathogen. In many cases, the pattern of inflammation provides important clues to diagnosis and helps to guide the selection of additional "special" stains used predominantly for diagnosis of infectious diseases. A stain may be nonspecific, allowing detection of a spectrum of organisms, as do the Papanicolaou stain and silver impregnation methods, or detection of only a limited group of organisms, as do the different acid-fast techniques. Some nonspecific stains, such as the Gram stain, are differential and provide valuable preliminary information concerning identification. Immunohistochemical stains, on the other hand, are specific for a particular organism, although in some cases cross-reactions with other organisms occur. Despite the wealth of information that can be gleaned from a stained smear or section of tissue, however, the specific etiology of an infection often cannot be determined on the basis of only the morphology of the organisms seen; culture data are essential and must be considered in the final diagnosis.

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“…It is done by passing the bottom of a slide through the tip of the burner flame for several times or using an electric slide warmer. Methanol and ethanol are the most common fixative chemicals [10], [15]. A number of studies have shown that chemical fixation gives more reliable Gram staining results than heat fixation, and it has even been recommended as a standard staining method for automated instruments [15], [16].…”

Section: Discussionmentioning

confidence: 99%

Ultraviolet Light Enhances the Bovine Serum Albumin Fixation for Acid Fast Bacilli Stain

et al. 2014

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The use of a liquid culture system such as MGIT broth has greatly improved the sensitivity of isolating mycobacteria in clinical laboratories. Microscopic visualization of acid fast bacilli (AFB) in the culture positive MGIT broth remains the first routine step for rapidly indicating the presence of mycobacteria. We modified an ultraviolet (UV) light fixation process to increase AFB cells adherence to the slide. The retained haze proportion of a 1-cm circle marked area on the smear slide was quantified after the staining procedure indicating the adherence degree of AFB cells. More AFB cells were preserved on the slide after exposure to UV light of either germicidal lamp or UV crosslinker in a time-dependent manner. We demonstrated both the bovine serum albumin (BSA) in MGIT media and UV light exposure were required for enhancing fixation of AFB cells. While applying to AFB stains for 302 AFB positive MGIT broths in clinics, more AFB cells were retained and observed on smear slides prepared by the modified fixation procedure rather than by the conventional method. The modified fixation procedure was thus recommended for improving the sensitivity of microscopic diagnosis of AFB cells in culture positive MGIT broth.

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Methanol fixation an alternative to heat fixation of smears before staining

Cited by 15 publications

References 5 publications

Multicenter Assessment of Gram Stain Error Rates

Multicenter Assessment of Gram Stain Error Rates

Detection of infection or infectious agents by use of cytologic and histologic stains

Ultraviolet Light Enhances the Bovine Serum Albumin Fixation for Acid Fast Bacilli Stain

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