I'm trying to inspect simple stained bacterial smears. But my smear suddenly disappears after a successful inspection with the oil immersion lenses.

The background can become too red (the color of the stain) and the oil residue coming from the oil immersion objective when it's wiped, is red colored.

Red background Usual background

Also when I try to wipe the oil from the slide, the smear is removed, even with a gentle wipe.

I'm suspecting three possible causes, which may include dependent causes:

  1. The immersion oil I use makes the smear easy to wipe: I don't have a dropper that releases just a small drop, but I have used another oil with the same type of dropper that releases more than the needed quantity and it didn't wipe my smear.

  2. The back-and-forth movement that eliminates air bubbles: I hear the spring sound, is this correct?

  3. Smear fixation may not be adequate: could it be possible that the smear can be fixed in an incomplete way so that it resists being wiped from the staining procedure, but not from further manipulation?

My specifications

  • Immersion oil: Non-drying, non-hardening Cedarwood oil

  • Stain: Carbol fuchsin (20%)

  • Smear fixation: heat fixation by passing the slide three times through the flame

  • 5
    $\begingroup$ Anyone else concerned about the fact it says "Ceder wood oil" instead of "Cedar wood oil"? (There doesn't appear to be a "Ceder Grove, N.J.") Also, "Conforms to ISO-8036/1 Specofication (sic)"? -- I might recommend trying an immersion oil from a reputable supplier, one whose quality control for materials is reflected in the labeling. $\endgroup$
    – R.M.
    Commented Oct 13, 2023 at 9:23
  • 1
    $\begingroup$ @R.M. A quick image search on the internet appears to indicate that the labeling shown here is trying to imitate the general appearance of labels on genuine products of Cargille Laboratories in Cedar Grove, NJ. $\endgroup$
    – njuffa
    Commented Oct 13, 2023 at 11:14
  • $\begingroup$ @R.M. - That in itself is an answer. Their slides are mucking up because this is a knockoff product that's probably contaminated or simply contains something like cheap vegetable oil. $\endgroup$
    – Valorum
    Commented Oct 13, 2023 at 18:55

1 Answer 1


Bacteriological smears are a one-and-done scenario. Very few of them are intended for repeat use. Basically, all you can expect is that you observe it and then discard the slide. Wiping the oil off will also remove the bacteria as they are not firmly fixed to the slide, despite the "heat fixation" name, as I will explain below.

If you do want to reuse a slide I would recommend that you get a coverslip and cover your slides, sealing the edges with nail-polish or glue will make these more or less permanent. However, this is tricky because you may need to change your high power objective lens (assuming 100x objective) to cope with the thickness of the coverslip. To do this you will need to look for a longer focal-length on the objective lens. Some higher-quality lenses come with an adjuster for focal lengths.

Heat fixation in bacteriological slides is about two things. Primarily, the heating allows drying out of the specimen, which adheres it to the slide enough that it can be further manipulated. For most stains, simply drying the slide without heating will work perfectly well for adherence. Secondarily, it is about killing the bacteria. This is the fixation part and the name is a hang-over from classical biology where chemicals such as formaldehyde or ethanol were used to "fix" the specimen in an unchanging state (fixed state as opposed to changing).

It should be noted that even with passing the slide through the flame 3x, the slide rarely reaches above about 50 C, which isn't really enough to kill most bacterial species with certainty (food safety tells us at least 65 C for 10 min...), and is certainly not enough for tough bacteria like Mycobacterium species - it's the dehydration from the heating that does most of the killing. In addition, most stains have a fixative of some sort in them, usually ethanol, which will work against most of the common bacterial species (though also not Mycobacterium), again by dehydration of the bacterium, but also protein denaturation.

With the colour coming off, I think you have two problems:

First - staining, make sure that you wash the slide with decolourizer until there is no residual red from the Fuchsin. It's a delicate balance, so takes some experience to get it right. Also, make sure your slide is completely dry after your counterstain and wash step.

Second - I think that cedarwood oil is the wrong choice for immersion oil here. The cedrol and cedrene components strike me as chemically similar to phenol, which is one of the major components of Carbol Fuchsin (Ziehl-Neelson) stain (PDF with components). This means it might well solubilize the stain.

Cedar oil also hardens on the lenses and can easily dissolve the glues that hold the lens together, so be very careful using it with modern lenses.

  • $\begingroup$ I'm not doing an acid-fast stain but rather a simple stain if that would make a difference. $\endgroup$ Commented Oct 13, 2023 at 17:37
  • $\begingroup$ @FreezingSoul I don't know off the top of my head, but I suspect you won't get proper clearance of the slide without the acid wash, so will always have some residual red that might come off in the immersion oil. $\endgroup$
    – bob1
    Commented Oct 13, 2023 at 19:37

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