Question

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.

Answer 1

To ensure reliable Gram stain controls, regularly prepare fresh subcultures from master stocks without immediate refrigeration or freezing before Gram staining, observing proper growth conditions for accurate results.

Step-by-step explanation:

To create sustainable and readily available Gram stain controls, it would be most efficient to prepare fresh subcultures regularly from well-maintained master stocks.

This can be done by inoculating new slant cultures from the master stocks of a known Gram-positive bacterium (e.g., Staphylococcus aureus) and a known Gram-negative bacterium (e.g., Escherichia coli) periodically and ensuring they are not refrigerated or frozen immediately before Gram staining, in line with Ref #4, which advises against using refrigerated/frozen cultures for slide preparation due to potential cell wall damage.

These slant cultures should be prepared and then left at room temperature for a short period to ensure cellular viability and accurate Gram staining results. The fresh subcultures should be incubated under optimal conditions until they reach a suitable growth phase for Gram staining, generally 18-24 hours, and used to prepare smears for the controls.