Detection of Spore Germination

for Sterilization Processes

by
Florine C. Cleary

freshman at Moses Brown High School

Providence, Rhode Island
 In the Headlines
US$ 9000
• “No population is more vulnerable to
8000 multi drug-resistance than those
7000
admitted to hospital wards”.
per patient

6000
• “Of the resistant organisms now
5000 proliferating around the world, none
carry more potential for
4000 destruction and threaten existing
3000
medical interventions than the
emergence of hospital-acquired
2000 "super-infections".
1000
• “In the United States alone, some
0 14,000 individuals are infected and
1st line 2nd line 3rd line die each year from drug-resistant
microbes picked up in hospital”.
Source: Farmer et al. The Global Impact of
Drug Resistant TB, Harvard Med School and Source: World Health Organization's Report on
Open Society Institute: pp. 168,1999 Infectious Disease
• “So far, current preventive
methods emphasizing hygiene and
aggressive infection-control
measures have reaped only dubious
benefits and at best, only slowed Percentage
the spread of resistant bacteria.” 35

• “This means that commonplace 30

medical procedures once previously
taken for granted – hip 25
replacements, dental surgery and
cyst removals – could conceivably 20
be consigned to medical limbo. The
repercussions are almost 15
unimaginable.”
10

• “An added concern is that hospital- 5

acquired infections rarely stay put.
Ample evidence would suggest that
many resistant infections erupted in
hospital settings before migrating
to the community at large.”
0
89 90 91 92 93 94 95 96 97
Source: ReacherMH at al. BMI 2000,320: 213-216

Source: World Health Organization's Report on

Infectious Disease
 A Global Problem
• “Inadequately cleaned equipment is
also a major determinant in the
spread of infectious disease.”

Hospitals are a breeding • “In one study, researchers surveying

ground for antibiotic health clinics in United Republic of
Tanzania discovered that some 40%
resistant bacteria. of presumed sterile reusable needles
and syringes were contaminated with
Costs (US$) include: bacteria.”
- USA = $10 billion per year
• “Inadequate training, monitoring and
- Mexico = $450 million per year education on basic hygiene has
serious implications, not only for the
- Thailand = $40 million per year hospital population itself, but also
for the community at large.”

Source: World Health Organization/CDS. Data Source: World Health Organization's Report on
from published sources Infectious disease.
 The Reason

With the number of antibiotic-resistant strains rising dramatically,

the emergence of new virulent strains, and concerns about food
contamination, it is clear that something must be done to improve
current methods of determining the presence of bacteria.
The current methods are subject to human error and often require
days to culture samples - days that some facilities don’t have,
nor do they have the ability to keep the equipment sterile while
they wait for the results. So often they don’t.

In view of the risk to human life, this uncertainty is unacceptable.

 Goals

1) To find faster, more reliable methods for

determining bacterial contamination.

3) To develop technology to use these new

methods for easy and quick detection of
bacteria.
 The Science

• All living cells need to perform respiration to produce energy

from food.

• The first step in cellular respiration is glycolysis where glucose

[sugar] is converted to pyruvate and energy is released.

• Once glycolysis has begun, there are ten steps with products
[metabolites] produced along the way.

• Two of these metabolites are NADH (reduced nicotinic

adenine dinucleotide) and FP (oxidized flavoproteins).
 How We Use It
• live cells perform respiration,
producing NADH and FP
input light

• both of these metabolites

fluoresce
fluorescence light is given off at a
different color than the input light
fluorescence
color of input light needed and
color of fluorescence depends on
the chemical composition of the
metabolite
each metabolite fluoresces at a
different color
sample
• metabolite fluorescence can be
used to determine the presence of
bacterial contamination
 Light Microscopy
Bacterial Cells
X 1000

NADH fluorescence flavoproteins fluorescence

white light
Confocal Microscope

• clearer, better-defined image

out-of-focus fluorescence is
eliminated by a shallow depth-of-field

• observe the structure of cells

successive views along the Z-axis
allows construction of 3-D models of
the sample

• ideal tool for quantitative studies

of the fluorescence of cells
laser beam, special optics, and high
signal-to-noise ratio of the
photodetector provide the capability
for sensitive and quantitative
measurements
 Confocal Microscopy
Bacterial Cells
combined

flavoproteins fluorescence 680nm fluorescence

µm
porphyrins fluorescence
 Germination of Bacterial Spores

140

120 flavoproteins

100 porphyrins
8-bit gray scale

80
680nm

60

40

20

0
0 10 20 30 40 50 60 70 80 90
minutes after addition of minimal media
 Applications

This method can be used to determine

• effectiveness of sterilization and decontamination procedures

• presence of infection in spinal fluid and urine

• abnormal cell function

• food and water contamination

• presence of extraterrestrial life and life in extreme

environments
 Future Efforts

• study metabolite behavior during sporulation and cell death

• design and build a device (about the size of a flashlight) which

uses these methods to detect bacteria

• test this device in a variety of conditions where bacterial

contamination is present