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Effect of temperature on inoculum as a potential source of error in agar dilution plate count bactericidal
B F Woolfrey1, M E Gresser-Burns, R T Lally
1Department of Anatomic and Clinical Pathology, St. Paul-Ramsey Medical Center, Minnesota 55101.
Abstract:
The effect of increased temperature on Staphylococcus aureus during the inoculation step of the agar dilution plate count method was investigated as a possible cause of artificially high persister counts. For some isolates, exposure of the inoculum to increased temperature resulted in higher persister counts and diminution or loss of the paradoxical effect. The persister patterns for three representative S. aureus isolates are presented to illustrate the strain- and temperature-dependent nature of the phenomenon. For any isolate, the net effect appears to be caused by an interplay of temperature-induced inoculum loss and temperature-induced cell division cycle blockage. A modification of the agar dilution plate count inoculation step to circumvent such problems is described.
Insights
Increased temperature during Staphylococcus aureus inoculation can artificially elevate persister cell counts. This temperature-dependent effect, influenced by inoculum loss and cell division blockage, impacts bacterial enumeration methods.
Area of Science:
- Microbiology
- Bacterial Physiology
- Analytical Methods
Background:
- The agar dilution plate count method is a standard technique for enumerating bacterial populations.
- Persister cells in Staphylococcus aureus populations can exhibit complex behaviors under varying conditions.
- Potential artifacts in enumeration methods can lead to inaccurate assessments of bacterial viability.
Purpose of the Study:
- To investigate the impact of elevated temperatures during the inoculation step on Staphylococcus aureus persister cell counts.
- To identify the mechanisms underlying temperature-induced alterations in persister cell enumeration.
- To propose a modification to the agar dilution method to mitigate observed artifacts.
Main Methods:
- Exposure of Staphylococcus aureus inoculum to increased temperatures prior to plating.
- Comparison of persister cell counts between temperature-exposed and control inocula.
- Analysis of strain-specific responses and correlation with cell division and viability.
- Description of a modified inoculation procedure.
Main Results:
- Increased temperature during inoculation led to higher persister cell counts in some Staphylococcus aureus isolates.
- The paradoxical effect, where persister cells are more numerous than expected, was diminished or lost.
- Observed effects were strain- and temperature-dependent, attributed to inoculum loss and cell division cycle blockage.
Conclusions:
- Elevated temperatures during the inoculation phase can cause artificial overestimation of Staphylococcus aureus persister cells.
- Temperature-induced inoculum loss and cell division cycle blockage are key factors influencing persister patterns.
- A modified inoculation technique is recommended to ensure accurate enumeration of Staphylococcus aureus persister cells.