Inoculation of culture,positive and negative control
Mycoplasma culture requires high sterility standards, so care must be taken both before and during the procedure. Negative culture is designed to easily detect color differences, allowing for the initial growth of mycoplasmas to be determined and prompt replacement of the culture medium to prevent excessive growth and subsequent death. However, actual samples may contain inhibitory factors such as antibiotics or enzymes, so adding sufficient culture medium is crucial to supplement nutrients and dilute inhibitory factors.
First, disinfect the biosafety cabinet (or clean bench) with UV light. Determine mycoplasma growth by observing the color difference between the positive and negative controls. Once a clear color change appears compared to the negative control, start the countdown for subculturing.
Growth rates vary significantly between different mycoplasma species:
Slow-growing mycoplasmas (typically human-origin strains with lower nutritional requirements): subculture to new agar plates and fresh liquid medium 2–3 days after the color change appears (the 2–3 days mentioned in the video refer to these slower-changing species).
Difficult-to-culture mycoplasmas (usually animal-origin strains, e.g., ATCC 25204, ATCC 17981): subculture within 12 hours after the color change appears (must not exceed 2 days).
Subculture ratio Old medium : fresh medium = 1 : 10 (recommended). For actual test samples, the sample volume is usually large (typically 10 mL), and the total culture volume is 100 mL. For agar plates: pipette 200 µL of the culture onto the plate, gently swirl to spread evenly. If it cannot be swirled uniformly, use a sterile spreader to gently distribute the inoculum.
After inoculation, place the plates and new liquid cultures back into the incubator and continue incubation.
P.S.: Timing of medium color change is most directly related to medium nutrition, mycoplasma species, and inoculum concentration.
If the color change (compared to negative control) appears on day 4–5, subculture to plates and fresh liquid medium within 2–3 days after the color change.
If the color change appears within 1–2 days, subculture must be performed within 12–24 hours.
Exceeding these time windows can easily lead to nutrient depletion or pH values exceeding the tolerance range of the mycoplasma, resulting in weakened strains or even cell death.
