10ml R2O Pep 10ml R2O Pep 90ml H2O Pep Diluciones adicionales 1ml x placa (Agar Plate Count) Incubar 37 ± 0.22°C x 48 hrs Contar colonias
(-1) (-2) (-3) 25g of 25 ml de muestra 10ml 10ml 10ml 225 ml H2O Pep 10ml R2O Pep 10ml R2O Pep 90ml H2O Pep Diluciones adicionales 1ml x placa (Agar Plate Count) Incubar 37 ± 0.22°C x 48 hrs Contar colonias
Corrected Text from the Diagram
- 25 g o 25 ml de muestra (25 g or 25 ml of sample)
- (-1): 225 ml H₂O pep (225 ml of peptone water)
- (-2): 90 ml H₂O pep (90 ml of peptone water)
- (-3): 90 ml H₂O pep (90 ml of peptone water)
- Transfers: 10 ml
- Diluciones adicionales (Additional dilutions)
- 1 ml x placa (Agar Plate Count) (1 ml per plate)
- Incubar 37 ± 0,2°C x 48 hrs (Incubate at 37 ± 0.2°C for 48 hrs)
- Contar colonias (Count colonies)
Explanation of the Microbiological Procedure
The diagram illustrates the standard protocol for the quantitative enumeration of microorganisms in a sample using the serial dilution and agar plate count method. This technique is fundamental in food microbiology, environmental science, and clinical diagnostics to determine the concentration of viable bacteria, yeasts, or molds.
The procedure begins with the preparation of the initial sample. A measured quantity, either 25 grams of a solid sample or 25 milliliters of a liquid sample, is added to 225 ml of a sterile diluent, in this case, peptone water (H₂O pep). This creates a total volume of 250 ml, resulting in an initial 1:10 (or 10⁻¹) dilution. Peptone water is used as a diluent because it is isotonic and non-nutritive, preventing microbial death due to osmotic shock while limiting multiplication during the procedure.
From this initial suspension, a series of subsequent dilutions is prepared. The diagram shows a tenfold serial dilution process, where 10 ml of the previous dilution is transferred into a new tube containing 90 ml of fresh peptone water. This step is repeated to create dilutions of 10⁻², 10⁻³, and so on, as indicated by “Diluciones adicionales.” This logarithmic reduction in concentration is crucial for obtaining a microbial density low enough to yield a countable number of colonies on an agar plate.
Next, a precise volume—typically 1 ml, as specified—from selected dilutions is transferred onto sterile Petri dishes containing a solid growth medium (Agar Plate Count). The diagram indicates that each dilution is plated in duplicate to ensure the reliability and accuracy of the results.
The inoculated plates are then incubated under controlled conditions, specified here as 37 ± 0.2°C for 48 hours. These conditions are selected to facilitate the growth of the target microorganisms. During incubation, each viable microbial cell, or Colony Forming Unit (CFU), multiplies to form a single, visible colony.
The final step is the enumeration of these colonies. After the incubation period, the colonies on each plate are counted. For statistical validity, plates with colony counts between 30 and 300 are typically selected. The final microbial concentration in the original sample is then calculated by multiplying the average colony count by the reciprocal of the dilution factor of the counted plate.
