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Background

Heat Fixation: Heat fixation is a technique used in bacteriology and other biological sciences to affix bacterial cells or other microorganisms to a microscope slide. This is achieved by carefully passing the slide, smeared with the specimen, through a flame. The heat kills the microorganisms, causing them to stick to the slide. Heat fixation helps to preserve the cellular structures and prevent them from being washed away during subsequent staining procedures.

Simple Staining: Simple staining is a basic microbiological technique used to enhance the visibility of microorganisms under a microscope. It involves the application of a single stain (usually a basic dye) to the specimen, which imparts color to all cells, making them easier to see. This technique is called "simple" because it involves only one staining agent. Simple staining provides contrast between the microorganisms and the background, allowing for better visualization and examination of their morphological features. Examples of basic dyes used in simple staining include crystal violet, methylene blue, and safranin.

Question

Is there a reliable method to determine if heat fixation was inadequate in bacteriology before proceeding with further simple staining steps and microscope inspection?

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I work with a teaching group who has looked at this, specifically for the aim of making things easy for undergrad labs in under-resourced countries, where gas and/or Bunsens or alcohol burners might not be available.

It turns out for most commonly used bacteria (E. coli, Staphylococcus, Streptococcus, Bacillus etc.) and on regular glass slides with common stains such as Gram stain, crystal violet, endospore etc. it is the drying that is most important, heat "fixation" just ensures that it is dry enough to not wash off during the subsequent staining steps. The vast majority of stains contain a step (often involving alcohols) that will kill many of the common pathogenic bacteria adequately for most purposes. The step where you are more likely to contaminate yourself is usually during the picking and spreading on the slide, before the heat fixation anyway.

Thickness of film helps here, if you have a thin film, it will dry quickly and adhere, but a thick layer (which you usually don't want anyway) won't dry adequately, and (assuming using the drop technique, not Coplin jars) acts as a barrier for liquid flow which means it is more likely to wash off during wash steps.

Most bacteria are too small to make out a lot of internal structure with basic stain techniques and using 100x oil immersion objectives.

So; let it dry. Instead of a 10 min wait, this might be a 30 min wait, but it works!

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