Transcript pure culture

Outline of Lectures
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Review of the work on contagious disease by Koch.
Epidemiology and Public Health.
Microbial interactions with higher animals.
Examination of the normal flora of animals.
Entry of the pathogen into the host. Colonisation
and growth.
Transmission of pathogens. Bacterial respiratory
infections and sexually transmitted bacterial
diseases.
Insect transmitted diseases.
Food-borne and water-borne bacterial diseases.
Course text: Brock Biology
of Microorganisms
Michael T. Madigan
John M. Martinko
Paul V. Dunlap
David P. Clark
Tenth/Eleventh/Twelfth
(international) Edition
Publisher: Pearson Education
18. Review of the work on contagious
disease by Robert Koch.
Dr. Çiğdem Williams
([email protected])
Microorganisms as disease agents
•In the U.S., the major causes of death at the beginning
of the 20th century were infectious agents called
pathogens.
•The elderly and the young were the most susceptible to
the infectious diseases caused by these pathogens.
•Today, in developed countries the incidence of death is
greatly reduced through understanding how these
pathogens cause disease and the disease process itself,
improved sanitary and public health practices and the
development and use of antimicrobial agents.
•Instead non-microbial diseases (e.g. heart disease,
cancer, etc.) are the major causes of death in the U.S.
according to the data obtained in 2008.
Microorganisms and Disease in
the USA
2008
1900
Influenza and
pneumonia
Tuberculosis
Heart disease
280
Cancer
205
Stroke
Pulmonary
disease
Accidents
Influenza and
pneumonia
Diabetes
Gastroenteritis
Heart disease
Stroke
Kidney disease
Accidents
AIDS
Cancer
Suicide
Infant diseases
Cirrhosis of
the liver
Homicide
Diphtheria
0
100
Deaths per 100,000 population
200
0
100
200
Deaths per 100,000 population
• Red: microbial disease
Brock 12th ed. Fig. 1.8
• Green: non-microbial disease
Microorganisms as disease agents
•Although, medical and microbial science have been
successful in controlling many microorganisms and even
eradicating them (smallpox), there are still microbial
infections (malaria, tuberculosis, cholera, measles,
pneumonia to count some) that cause death.
•Furthermore, diseases that could emerge suddenly
[severe acute respiratory syndrome (SARS), bird flu,
swine flu] and have the potential to infect different
species including humans, in addition to exotic and rare
diseases such as ebola haemorrhagic fever can also
spread across the world due to the ease of global travel.
Some early history of
microbiology
Brock 12th ed. Table 1.1
Some early history of microbiology
(a) Drawing of a bluish-coloured mold by
Robert Hooke. This is the first drawing
describing a microorganism. (b) Antoni
van Leeuwenhoek’s drawings of bacteria.
Rod-shaped: A,C,F and G; spherical or
coccus-shaped: E; cocci packets: H. (c)
Photomicrograph of a blood smear taken
through a van Leeuwenhoek microscope.
Red blood cells are visible and a single
cell is about 6 μm in diameter.
Some early history of microbiology
Ferdinand Cohn’s drawing of the large filamentous sulfur-oxidizing bacterium
Beggiatoa mirabilis.
Robert Koch’s Germ Theory of
Disease
"Bring out your dead," from scene 2 of the film,
Monty Python and the Holy Grail
It was recognized in the 16th
century that diseases would
spread in populations.
Concept of contagious disease
understood.
Work by Pasteur (1864) and
subsequent discovery of
sanitization (Lister 1867) was
indirect evidence that
microorganisms were responsible
for disease – spontaneous
generation theory finally
quashed.
Robert Koch provided firm proof
that microbes were responsible
for certain diseases.
http://www.microbiologyplace.com – Spontaneous generation theory quashed.
Steam forced
out open end
Nonsterile liquid
poured into flask
Neck of flask
drawn out in flame
Dust and microorganisms
trapped in bend
Liquid sterilized
by extensive heating
Open end
Long time
Liquid remains
sterile indefinitely
Liquid cooled slowly
Short time
Flask tipped so
microorganism-laden dust
contacts sterile liquid
Microorganisms
grow in liquid
Earlier developments made Koch’s
work possible
• Development of the light
microscope.
• Development of conditions
for growth of
microorganisms in pure
culture on solid media.
• Prevention of contamination
(Ferdinand Cohn)
• Animal models of disease.
Robert Koch (1843-1910)
•
1862: studies medicine at University of
Göttingen, Germany.
•
He studied anthrax, a disease of the cattle
and occasionally humans, caused by the
endospore forming bacterium Bacillus
anthracis. He noticed by careful microscopy
that blood of diseased animals contained
the bacterium.
•
This association does not prove that
anthrax caused the disease – maybe growth
of the bacterium is a result of the disease?
•
During his “spare time” he worked at home
inoculating healthy mice with the
“unpurified” anthrax from (blood of)
spleens of diseased (infected) farm animals
-the newly infected mice died of anthrax.
•
He then isolated the anthrax bacillus and
showed this organism still caused anthrax.
He thus proved that it was the anthrax
bacterium and not the “bad” blood that
caused the disease.
KOCH’S POSTULATES
The Postulates:
Tools:
1. The suspected pathogenic
organism should be
present in all cases of the
disease and absent from
healthy animals.
Microscopy, staining
2. The suspected organism
should be grown in pure
culture.
Laboratory culture
Diseased
animal
Red
blood
cells
Suspected
pathogen
Colonies of
suspected
pathogen
Observe
blood/tissue
under the
microscope
Streak agar plate
with sample
from either
diseased or
healthy animal
Inoculate healthy animal with
cells of suspected pathogen
Healthy
animal
Red
blood
cell
No
organisms
present
KOCH’S POSTULATES
The Postulates:
Tools:
3. Cells from a pure culture
of the suspected organism
should cause disease in a
healthy animal.
Experimental animal
Diseased
animal
Healthy
animal
Diseased animal
Remove blood or tissue sample
and observe by microscopy
4. The organism should be
reisolated and shown to be
the same as the original.
Laboratory reisolation Suspected
pathogen
Laboratory
culture
Pure culture
(must be
same
organism
as before)
Robert Koch’s work on Tuberculosis (TB)
• In 1881, 1/7th of all human deaths were caused by TB – a
wasting disease (consumption)
• Until Koch’s work, the causal agent had not been
identified.
• He used microscopy, purified culture, tissue staining and
animal inoculation.
• Koch developed a specific stain for Mycobacterium
tuberculosis cells.
• Importantly he developed conditions for growth of M.
tuberculosis – blood serum agar.
• He used Guinea pigs to examine infection (animal model).
• Again he satisfied his four criteria that M. tuberculosis
caused TB.
Koch’s work on TB
• Koch used all his methods as he set out to
identify the causative agent of TB.
–
–
–
–
Microscopy
Staining
Pure culture
Animal models
• Growth of M. tuberculosis in pure culture
was very challenging – but he never gave up!
• Once pure culture growth was achieved his
four postulates could be readily satisfied.
Studies of Mycobacterium tuberculosis
• (a) Section through a tubercle
from lung tissue. Cells of M.
tuberculosis stain blue, whereas
the lung tissue stains brown.
• (b) Cells of M. tuberculosis in a
sputum sample of a tuberculous
patient.
• (c, d) Growth of M. tuberculosis
in pure culture.
• (c) Growth of M. tuberculosis on
a glass plate of coagulated
blood serum inside a glass box
(with lid open).
• (d) A colony of M. tuberculosis
cells taken from the plate in (c)
and observed microscopically at
700×; cells appear as long
Brock 12th ed. Fig. 1.16
"cordlike" forms.
Robert Koch (1843-1910)
• 1882: Koch publishes his
work on tuberculosis.
• Studied cholera in India.
• Did further work on pure
cultures on solid media in
flat dishes designed by
Petri.
• 1905: Awarded Nobel Prize
in Physiology or medicine
for his decisive work on
tuberculosis.
http://nobelprize.org/medicine/laureates/1905/koch-bio.html
Mycobacterium tuberculosis
• Gram positive rod shaped
bacterium.
• Grows very slowly.
• Cell wall has high lipid
content.
• Primary infection in lung
but can spread to other
tissues.
• Increasing prevalence
today due to AIDS and
antibiotic resistance.
• Genome sequenced
See Brock Chapter 34.5
Diagnosis of Tuberculosis today
Tuberculosis X-rays. (a) Normal chest X-ray.
(b) Advanced case of pulmonary TB chest X-ray.
A century later:
Peptic Ulcers in the 1980’s
Peptic Ulcers in the 1980’s
• Prevailing view (pharmaceutical
industry vested interest?): “ulcers
were caused by acid, lifestyle stress,
spicy foods, and should be treated
by drugs blocking acid production”
• Tagamet™ and Zantac™ two of best
ever selling drugs (Histamine H2
receptor blockers).
• These drugs did not “cure” disease
but managed it - so perfect drugs
for maximum profit!
• At the time the stomach was
considered sterile -acid environment
(pH 2)
Barry Marshall
Dr. Barry J. Marshall and Dr J.
Robin Warren
• Warren was convinced that he
could visualize bacteria in
stomach ulcer biopsies (Koch’s
postulate number 1)
• Marshall developed this work.
• Hypothesis – “bacteria might be
responsible for some stomach
ulcers”
• However – it was difficult to
grow these bacteria in the lab
(just like M. tuberculosis)
• Growth of pure cultures (Koch’s
postulate number 2) was
achieved only when cultures
incubated accidentally for
longer than normal.
Endoscopy of stomach
Dr Marshall was originally
ridiculed for his idea
• He failed to infect a pig animal
model (Koch’s 3rd postulate failed).
• 1998 quoted as saying: "Everyone
was against me, but I knew I was
right".
• Marshall then took the rather
drastic step of swallowing the
purified bacterium (Koch’s 3rd?)
• His wife, Adrienne, was not
impressed but she thought it
typical of him!
• He soon became very ill.
• He was eventually cured by use of
antibiotics.
• Did not even attempt Koch’s
postulate number 4 (re-isolation
from himself)
Helicobacter in the stomach mucosa
• Stomach protected by
gastric juices by a
thick mucus lining
• Helicobacter
neutralizes acid by
excreting a urease.
• Immune system is
activated but cannot
reach infection
• Inflammation leads to
an ulcer
Helicobacter pylori
• Gram negative, highly
motile, spiral shaped
bacterium.
• Size 2.5-3.5 m long and
0.5-1 m in diameter
• 80% of gastric ulcer
patients are infected
• Also implicated in gastric
cancers
Koch’s postulates are as
relevant today as ever
Press Release: The 2005 Nobel Prize in
Physiology or Medicine
3 October 2005The Nobel Prize in Physiology or
Medicine for 2005
jointly to
Barry J. Marshall and J. Robin Warren
for their discovery of
"the bacterium Helicobacter pylori and its role in
gastritis and peptic ulcer disease"
Further reading
• Brock, Chapter 1, Work of Robert Koch and
work of Louis Pasteur (spontaneous
generation, pasteurization, swan necked flask)
• http://nobelprize.org/medicine/laureates/1905/press.html
• http://nobelprize.org/medicine/laureates/2005/press.html