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My investigation on the matter

Ref #1

Staining known Gram-positive and Gram-negative organisms on either side of your unknown organism act as positive controls for your technique.

(Microbiology Laboratory Theory and Application (Michael J. Leboffe, Burton E. Pierce)

Ref #2

Similarly, cultures should undergo evaluation while they are still fresh. Old cultures tend to lose the peptidoglycan cell walls, which predisposes gram-positive cells to be gram-negative or gram variable.

(Tripathi N, Sapra A. Gram Staining. 2023 Aug 14. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan–. PMID: 32965827.)

Ref #3

Refrigeration impedes further growth of the bacteria, so you will have "fresh" cultures to work with during the next laboratory session.

(Laboratory Exercises in Microbiology By Robert A. Pollack, Lorraine Findlay, Walter Mondschein, R. Ronald Modesto)

Ref #4

Remember the following points concerning your [refrigerated or frozen] slant or agar stab stock cultures: -Do not use stored stock culture for making slides or routine inoculations.

(Laboratory Practices in Microbiology By Osman Erkmen)

Question

How do I create a sustainable readily available pair of stock cultures to act as Gram stain controls (as Ref #1), in the least time-consuming and tedious way?

From Ref #2 and #3, I can prepare two slant cultures, one is of a Gram-positive bacteria and the other is of a Gram-negative bacteria, then store them in the refrigerator or freezer. Then I can retrieve them daily and use them. After that, they are returned to storage.

However, Ref #4 argues against using refrigerated/frozen cultures for making slides.

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    $\begingroup$ Just spitballing, but what if you made a stack of slides and heat fixed smears of your controls onto each slide, so that you could just grab one out of the slide box and have your controls already fixed to the slide. It would probably take some verification testing against fresh controls to determine the quality/stability, but I know I have used commercially available QC slides like this in the past. $\endgroup$
    – MikeyC
    Oct 20, 2023 at 21:07
  • $\begingroup$ It's not about the mean, but the end. Anything that would substitute as a Gram stain control would do. So this is an answer. $\endgroup$ Oct 21, 2023 at 6:09

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Aside from MikeyC's commented suggestion, which I think is a good idea and they should write it up as an answer.

In my experience, most species of bacteria you might use as controls for Gram staining will easily last 2-4 weeks in the fridge on a plate and still be usable as controls. These will be genera like Staphylococcus and Escherichia, well known, readily available and well characterized.

For fairly minimal effort, one can simply take a plate of the organism wanted and re-streak/sub-culture once a week or once every two weeks. The streaking itself should only take you a minute or two, adding in time for labelling of plates and general fluffing around in the lab (opening incubator, placing plate in incubator etc.), maybe a maximum of 5-10 min per plate. I know that with pre-labelled plates and disposable loops, I can sub ~100 plates in about an hour.

For longer term storage of species, it is best to keep a glycerol stock. These are stored at -80 C (ultracold freezer) and can be used indefinitely. If you wanted to work from glycerol stock (to be sure of getting the right species), add maybe another 5 min to look up location in the freezer and get them out for streaking. There is no need to thaw a glycerol stock (in fact it is detrimental to thaw them) - just use a cooled flamed loop, or a clean disposable loop to scrape a tiny amount of the stock from the top of the tube and streak this out.

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