eBook: TOC and Microbial Detection Monitoring - 31

Biofilm, Endotoxins and Their Relationship

TTLER TOLEDO White

Attribute

USP

EP

JP

ChP

IP

US, EU, Japan,
WHO drinking
water

Human
consumption

JP water
specification

Potable water
or
Purified Water

Potable water
or
Purified Water

Distillation or
suitable
process

Distillation
or RO plus
additional
technology

Distillation or
RO with UF,
from Water or
Purified Water

Distillation

Distillation

10

10

10

10

10

1.3 (3 stage)

1.3 (3 stage)

1.3 on-line or
2.1 off-line

1.3 (3 stage)

1.3 (3 stage)

TOC (mg/L)

0.5

0.5

0.5 (0.3 for
control)

0.5

0.5

Bacterial Endotoxins
(EU/mL)

0.25

0.25

0.25

0.25

0.25

0.6

0.2

0.1

Required by PW

Source Water for PW
and WFI
WATER FOR INJECTION
Production Method

Total Aerobic (microbial)
(cfu/100 mL)
Conductivity
(μS/cm at 25°C)

Nitrates (ppm)

0.2

Heavy Metals
Acidity/Alkalinity

Indicators

pH

5.0 - 7.0

Table 1: Requirements for Water for Injection by pharmacopeia. Shaded boxes indicated that no test is required.

Benjamin Franklin, a founding father of the United
States, is credited with a quotation that is probably the
best response regarding the presence of bacteria and
describes the reality of water systems: "In wine there
is wisdom, in beer there is freedom, in water there is
bacteria."

This method only provides snapshots of a limited
volume and does not allow real-time control of a water
system or provide a true picture of bioburden in the
system. The test method, developed by Dr. Koch and
Dr. Petri in 1887, is based on the visual appearance
of a colony of cells and cannot be used to determine
if a colony arose from one cell, a hundred cells or a
thousand.

The compendial pharmacopeia tests for WFI are conductivity, TOC, microbial and Limulus Amebocyte
Lysate (LAL) test (for endotoxins). This white paper
focuses on the relationship between traditional plate
count method of microbial testing and the LAL test.

A colony forming unit (CFU) is the measurement used
to estimate the number of viable bacteria or fungal
cells in a sample. Counting CFUs requires culturing the
microbes and then counting only viable cells, i.e., cells
that are healthy and capable of undergoing cell division. Development of CFUs requires culturing a mass,
a chain or a clump of cells, and only the viable cells
will grow. In addition, colony formation is dependent

Limitations of the traditional plate count method
and CFUs
The current test for microbes requires the culturing of
grab samples from pharmaceutical water systems.

31
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