Bacterial Culture Discussion Example | Colony Counts, Dilution Factors, CFU Calculations, Growth Curves, and Sources of Error

A safety-confirmed nonpathogenic educational strain was serially diluted and inoculated onto general-purpose bacterial agar medium. Three plates were used for each dilution, and the number and morphology of the colonies formed were observed.

At the 10^-2 dilution, colonies were too dense to distinguish individually. At the 10^-3 dilution, 286, 301, and 294 colonies were observed, with a mean of 293.7. At the 10^-4 dilution, 31, 28, and 34 colonies were observed, with a mean of 31.0. At the 10^-5 dilution, the mean was 3.0 colonies.

Based on inoculation of 0.1 mL of the 10^-4 dilution, the bacterial concentration in the sample was calculated as 3.1 × 10⁶ CFU/mL. The value calculated from the 10^-3 dilution was 2.94 × 10⁶ CFU/mL.

The colonies were milky white, circular, entire, convex, smooth-surfaced, and opaque, with a mean diameter of approximately 2.1 mm. No colonies were observed in the control.

The absorbance of the liquid culture increased from 0.05 initially to 0.67 after 5 hours and reached 1.08 after 8 hours. No substantial increase was observed thereafter.

In this experiment, a safety-confirmed nonpathogenic educational strain was serially diluted and inoculated onto agar medium, and the bacterial concentration in the sample was estimated from the number of colonies formed.

At the 10^-2 dilution, colonies were densely packed and could not be distinguished individually. This was likely because too many bacteria were inoculated onto the plate, causing neighboring colonies to merge.

At the 10^-3 dilution, an average of 293.7 colonies formed, while at the 10^-4 dilution, the average was 31.0 colonies. When the dilution increased tenfold, the colony count decreased to approximately one-tenth, suggesting that the serial dilution was performed reasonably correctly.

Based on inoculation of 0.1 mL of the 10^-4 dilution, the bacterial concentration was calculated as 3.1 × 10⁶ CFU/mL. In contrast, the value obtained from the 10^-3 dilution was 2.94 × 10⁶ CFU/mL.

The similarity of the values obtained from the two dilution levels suggests that there was no major inconsistency in the dilution procedure, inoculation volume, or colony counting. However, the values were not identical, possibly because of insufficient mixing during dilution, errors in liquid-volume measurement, bacterial aggregation, uneven distribution on the medium, or differences in counting judgment.

CFU represents the number of units capable of forming colonies on the medium rather than the actual number of individual viable bacterial cells. If multiple bacteria are inoculated as an aggregate, they may form a single colony, so CFU does not necessarily equal the actual cell count.

At the 10^-5 dilution, the mean was only 3.0 colonies. When colony numbers are low, a change of one colony represents a large proportion of the total, increasing the effect of random error. Therefore, bacterial concentration should be calculated using a dilution that is neither overcrowded nor too sparse.

The observed colonies were milky white, circular, entire, convex, smooth-surfaced, and opaque, showing relatively uniform morphology. This result is consistent with the predominant growth of a single strain.

However, bacterial species cannot be identified from colony morphology alone. The same species can show different morphologies depending on the medium and growth state, while different species may have similar appearances.

No colonies were observed in the control. This indicates that there was no obvious contamination from the medium, equipment, or work environment and supports the possibility that the colonies observed in the sample plates originated from the inoculated sample.

In the liquid culture, absorbance increased from 0.05 initially to 0.67 after 5 hours. This was likely due to increased light scattering as bacterial biomass increased.

The period in which absorbance increased only slightly early in the culture can be interpreted as the lag phase, during which bacteria adapted to the new environment. The subsequent period of rapid increase in absorbance corresponds to the logarithmic growth phase, when bacterial cells were actively dividing.

The nearly constant absorbance after 8 hours likely reflects a transition to the stationary phase as growth slowed because of factors such as nutrient depletion, accumulation of metabolic products, and limitations in oxygen or space.

The fold growth calculated from OD values was 13.4, the number of generations was approximately 3.74, and the generation time was approximately 1.34 hours. However, absorbance includes light scattering not only from viable cells but also from dead cells and cell debris, so it does not perfectly correspond to viable counts determined by CFU.

In the comparison of medium conditions, both colony count and mean colony diameter decreased under low-nutrient, high-salt, and unsuitable-pH conditions compared with the standard condition.

Under low-nutrient conditions, substances required for cell growth and colony expansion were likely insufficient. Under high-salt conditions, osmotic effects may have disrupted cellular water balance and metabolism, suppressing growth.

Under unsuitable pH conditions, enzyme activity, membrane transport, protein structure, and other cellular processes may have been affected, creating an environment unfavorable for bacterial growth and thereby reducing colony number and size.

Among the most important sources of error in this experiment are dilution and mixing. In serial dilution, transferring an aliquot before the sample is homogeneous can cause the number of bacteria transferred to the next dilution to vary.

In addition, if the inoculation volume is greater than the set value, the colony count increases, while a smaller volume decreases it; the final CFU/mL is affected in the same direction.

To improve reproducibility, it is useful to use multiple plates at the same dilution and calculate the mean, standard deviation, and coefficient of variation. The coefficient of variation for the 10^-3 dilution in this example was approximately 2.6%, indicating relatively little variation among the three plates.

Overall, serial dilution provided a concentration range in which colonies could be counted, allowing the bacterial concentration in the sample to be estimated at approximately 3.0 × 10⁶ CFU/mL. Bacterial growth was also shown to be influenced by culture time, nutrient availability, salt concentration, pH, and other conditions, as reflected in changes in colony count, colony diameter, and absorbance.

Possible reasons for the absence of colonies include a viable bacterial count below the detection limit, excessive dilution, too small an inoculation volume, medium conditions unsuitable for growth, or lack of viable bacteria in the sample.

If all plates were overcrowded, possible causes include insufficient dilution, a bacterial concentration higher than expected, or an excessive inoculation volume. Measurements should include higher dilution levels.

If colonies formed in the control, microorganisms may have entered from the medium, equipment, work environment, or contact during handling. Because colonies originating from the intended sample cannot be distinguished from contaminants, the reliability of the experimental results is reduced.

Possible reasons why colony counts did not correspond to dilution factors include insufficient mixing at each dilution step, errors in transferred volume, sample mix-ups, bacterial aggregation, uneven distribution on the medium, or counting errors.

If colonies with different colors, shapes, or sizes are observed, multiple bacteria may have been present or contamination may have occurred during handling. However, even a single strain can vary in morphology depending on its position on the medium or nutritional conditions, so colony morphology alone is not sufficient for a definitive conclusion.