detection of spore germination for sterilization processes
DESCRIPTION
2001 IEEE powerpoint i did on work in lab.TRANSCRIPT
Detection of Spore Germination for Sterilization Processes
by Florine C. Cleary
freshman at Moses Brown High School Providence, Rhode Island
In the Headlines
• “No population is more vulnerable to multi drug-resistance than those admitted to hospital wards”.
• “Of the resistant organisms now proliferating around the world, none carry more potential for destruction and threaten existing medical interventions than the emergence of hospital-acquired "super-infections".
• “In the United States alone, some 14,000 individuals are infected and die each year from drug-resistant microbes picked up in hospital”.
Source: World Health Organization's Report on Infectious Disease
US$ 9000
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7000
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0 1st line 2nd line 3rd line
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pati
ent
Source: Farmer et al. The Global Impact of Drug Resistant TB, Harvard Med School and Open Society Institute: pp. 168,1999
• “So far, current preventive methods emphasizing hygiene and aggressive infection-control measures have reaped only dubious benefits and at best, only slowed the spread of resistant bacteria.”
• “This means that commonplace medical procedures once previously taken for granted – hip replacements, dental surgery and cyst removals – could conceivably be consigned to medical limbo. The repercussions are almost unimaginable.”
• “An added concern is that hospital-acquired infections rarely stay put. Ample evidence would suggest that many resistant infections erupted in hospital settings before migrating to the community at large.”
Source: World Health Organization's Report on Infectious Disease
Source: ReacherMH at al. BMI 2000,320: 213-216
89 90 91 92 93 94 95 96 97
35
30
25
20
15
10
5
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Percentage
A Global Problem
• “Inadequately cleaned equipment is also a major determinant in the spread of infectious disease.”
• “In one study, researchers surveying health clinics in United Republic of Tanzania discovered that some 40% of presumed sterile reusable needles and syringes were contaminated with bacteria.”
• “Inadequate training, monitoring and education on basic hygiene has serious implications, not only for the hospital population itself, but also for the community at large.”
Source: World Health Organization's Report on Infectious disease.
Hospitals are a breeding ground for antibiotic resistant bacteria.
Costs (US$) include:
- USA = $10 billion per year
- Mexico = $450 million per year
- Thailand = $40 million per year
Source: World Health Organization/CDS. Data from published sources
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.
2) 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
• both of these metabolites fluoresce
• metabolite fluorescence can be used to determine the presence of bacterial contamination
fluorescence light is given off at a different color than the input light
each metabolite fluoresces at a different color
fluorescence
input light
sample
color of input light needed and color of fluorescence depends on the chemical composition of the metabolite
Light MicroscopyBacterial Cells
X 1000
NADH fluorescence flavoproteins fluorescence
white light
Confocal Microscope
• clearer, better-defined image
• observe the structure of cells
• ideal tool for quantitative studies of the fluorescence of cells
out-of-focus fluorescence is eliminated by a shallow depth-of-field
successive views along the Z-axis allows construction of 3-D models of the sample
laser beam, special optics, and high signal-to-noise ratio of the photodetector provide the capability for sensitive and quantitative measurements
Confocal MicroscopyBacterial Cells
porphyrins fluorescence
680nm fluorescence
µm
flavoproteins fluorescence
combined
Germination of Bacterial Spores
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0 10 20 30 40 50 60 70 80 90
minutes after addition of minimal media
8-bi
t gra
y sc
ale
flavoproteins
porphyrins
680nm
Applications
• 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
This method can be used to determine
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