Actually the S500 Standard and Reference Guide for Professional Water Damage Restoration (Second Edition) does not refer to 72 hours in any instance. There is one reference to 48 hours in the Standard section 5.2 that says, "Time and temperature aggravate category 2 water contamination levels significantly. Gray water in flooded structures that remains untreated for longer than 48 hours may change to category 3 – black water..." I would like to point out that it refers to gray water and not to clean water. In addition, the standard says "may change." The newly released Third Edition of S500 makes the following statement in the Standard section at 9.6, "The cleanliness of Category 2 water can deteriorate for many reasons including but not limited to: contact with building materials, systems, and contents; mixing with soils and other contaminants. Factors that influence the potential organic and inorganic load in a structure include the age and history of the structure, previous water losses, general housekeeping, the type of use of the structure, and elapsed time or elevated temperature." There is also a reference to 72 hours at 9.10.8.2 that states, "The need for cushion replacement should be determined. Cushion (e.g., pad, underlay) should be replaced under the following conditions:

• cushion has remained saturated for approximately 72 hours or more, depending on temperature;
• cushion is saturated with either Category 2 or 3 water;"

The graphic at the end of the S500 Third Edition has been changed and the following clarifying language has been added.

In this document the use of the term microorganisms primarily refers to bacteria and molds. It is true that some molds can colonize in 72 hours and bacteria can start to multiply in just a few hours. These time frames are based upon ideal growing conditions and not necessarily the conditions that you find in the indoor environment. In some circumstances these microorganisms may take considerably longer to grow due to less favorable conditions. It was not intended for the time frames mentioned here or reflected in this graphic to be absolutes, but a starting point at which you might want to be concerned. The terms Clean, Gray and Black are used in this illustration to aid the reader in applying the concepts presented. This document has defined and uses the terms Category 1, 2, and 3 to describe the range of contamination in water.

It is true that some molds can colonize in 72 hours. Bacteria can start to multiply in just a few hours. However, there is more to the story. Most of the references that deal with the time frame for molds to germinate or to colonize are based upon ideal growing conditions and not necessarily the conditions that you find in the indoor environment.

The intent of the S500 and S520 is to provide the restorer or remediator with a general time frame that allows for an evaluation for the indoor environment. If the time frame is greater than 48 hours, then there may be some reason for concern. Then again, there may not be a reason for concern.

Mold spores take up water at varying rates. When mold spores germinate in an environment that is conducive to growth, their vegetative hyphae will grow into the substrate at which time it can be said that the mold has colonized the substrate. At this point, all that has happened is that there has been a microscopic colony established that will not change the appearance of the substrate surface. Could it be said that the mold has colonized? Yes! In some cases the term “colonized” means that the mold spore has not only germinated and become established, but it has also developed additional spores that have germinated, become established and the amount of growth is such that the colony is visible without the aid of magnification. This process can take many more days. You may not see visual evidence of mold growth for 6 to 14 days. In some cases, the growth environment may not be favorable and you will not see any evidence of mold growth for weeks, if ever.

Microbial organisms (e.g., molds and bacteria) go through the following 4 phases of growth.

• Lag Phase
  Once the growing conditions become favorable, mold spores can germinate. This growth starts slowly and accelerates gradually. This phase is referred to as the lag phase.
• Exponential or the Log Phase
  Exponential growth occurs for a brief period as the hyphae starts to develop. The new hypha extends into the substrate. The mass of hyphae is referred to as mycelium. As long as the growing conditions are favorable and there are nutrients available, the growth remains constant. This phase is referred to as the exponential phase.
• Stationary Phase
  When the nutrients are depleted or the growing environment is not longer favorable, growth slows down or is completely stopped. During the stationary phase, hyphal growth stops. In some cases, nutrients are transferred to developing spores as a propagation mechanism. The spores are dispersed by air movement. This phase is referred to as the stationary phase.
• The Death Phase
  During the death phase, the mycelium eventually dies off.

The lag and exponential phases vary in length of time depending upon what molds are present. Generally those molds that are xerophilic (capable of growth in relatively dry conditions) germinate and grow first. They are also referred to as primary colonizers. Xerophilics can germinate in conditions where the water activity (Aw) is below 0.80 and perhaps as low as 0.61. Penicillium, Aspergillus and Eurotium can germinate and colonize in these conditions. In ideal growing conditions, micro-colonies can develop in 48 to 72 hours. Secondary colonizers (mesophilic) can germinate and grow where the Aw is between 0.8 and 0.9. Secondary colonizers include species of Cladosporium, Ulocladium, Alternaria and some species of Aspergillus. Tertiary colonizers (hydrophilic) can germinate in conditions where the Aw is above 0.9. The presence of tertiary colonizers in a building is an indication that there has been a very wet condition for extended periods of time. Stachybotrys, Chaetomium, Trichoderma, Aureobasidium, actinomycetes and other bacteria require very wet conditions. Tertiary colonizers (molds) can take 7 to 14 days to develop micro-colonies. In all cases, it will take more time for the colonies to grow sufficiently to become visible.

Keep in mind that in any environment, bacteria are also present. When you have a very wet surface, bacteria can multiply very rapidly. In some cases, the odors that you might detect in wet buildings are actually bacteria. Bacteria go through the same phase of development and death as molds. As mentioned above, generally bacteria require a very high Aw in order to multiply. The lag phase for bacteria may only be 1 to 3 hours before it starts multiplying exponentially. The cells divide at a constant rate, doubling the population. In ideal growing conditions, E. coli bacteria will double in population every 17 to 20 minutes. At this growth rate, one E. coli bacterium could become a billion (1,000,000,000) bacteria in just 10 hours. In the case of Clostridium tetani which can result in Tetanus, the generation rate is even faster, perhaps doubling as quickly as every 6 minutes.

The S500 deals with bacteria primarily and molds as a secondary consideration. When reading the S500, keep in mind that the time frames mentioned are for microbial organisms that include more than just mold. Also keep in mind that these time frames are for ideal growing conditions. It was not intended for the time frames to absolutes, but a starting point at which we might want to be concerned. If you are living in a hot-humid climate, certain microorganisms will grow faster than others. There are molds and bacteria that prefer warmer or hot environments. Others will not grow as quickly.

— Jim Holland