eBook: TOC and Microbial Detection Monitoring - 32

Biofilm, Endotoxins and Their Relationship

ETTLER TOLEDO White

Figure 1: Biofilm build up occurs in five stages: (1) attachment, (2) formation, (3) development/growth, (4) maturity, and (5) release.

on many variables, such as: organism type, phase
of organism, stress factors, incubation temperature,
humidity, media type and duration of incubation.

tion, surface smoothness, dead legs, sample points,
elbows, gaskets and other factors, organisms will
survive and biofilm will develop.

The majority of plate counts are performed with a
standard media (R2A or TSA) at 30˚ to 35˚ C for 5 to
7 days, but slower growing or sanitant-injured organisms may require a different media and incubation at
a lower temperature for 14 or even 21 days.

Biofilm matures in complex three-dimensional structures or communities. There is the growth of the "fluffy",
shearable biofilm, which forms in low shear, low flow
or stagnant water; and in water systems with high flow,
high shear forces, it develops as a surface-hugging,
tenacious biofilm.

Because of the limitations and shortcomings of the traditional plate count method, the pharmaceutical industry is actively pursuing real-time bioburden detection
technologies for pharmaceutical waters.

More biofilm flocs are sheared from "fluffy" than from
tenacious biofilm. This results in higher counts from
stagnant water and therefore an increase in planktonic bacteria. These flocs are released during sanitization cycles, rinsing of the water loop and during high
demand from production.

Biofilm and water systems
Microbial attachment and biofilm growth will occur in
every water system, regardless of flow rate, material of
construction, turbulent flow and shear forces. Bacteria
is ubiquitous and is everywhere: on surfaces, in water
systems, in the air, on your skin and clothes. Microbial
contamination cannot be avoided but only monitored
and controlled.

Regardless of the characteristics of a water system, if
biofilm develops and becomes established in a water
system it will rarely if ever be completely killed and/
or removed by hot water, chemicals or mechanical
cleaning.
A very important fact about biofilm is that it is hydrophobic and will repel water at a rate exceeding PTFE.
This ability to repel water means that hot water does

In theory, most water borne organisms should be killed
in Hot WFI systems, but depending on the engineering design, age of the system, materials of construc-

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