Medical Microbiology I - Lecture2
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Transcript of Medical Microbiology I - Lecture2
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MEDICAL MICROBIOLOGY I
LECTURE 2LECTURE 2
Culture of Bacteria and
Bacterial Growth
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Culture Media
Microbiology depends on the ability to GROWand MAINTAIN microorganisms in the laboratory - use of suitable culture media
A culture medium is a solid or liquid preparation A culture medium is a solid or liquid preparation used to grow, transport, and store microorganisms.
They must contain water and sources of nitrogen, carbon, mineral salts and essential vitamins.
Some bacteria may require additional specific substances which may be added to the medium.
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Culture Media
Two categories of culture medium:
1. Chemically defined media
Concentration of each ingredient is known
Made of highly purified inorganic salts and Made of highly purified inorganic salts and simple organic compounds such as glucose or purified amino acids
Little or no difference in composition between batches
Expensive and not for routine use
Used to determine specific growth requirements of bacteria
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Culture Media
2. Complex Media
Media prepared using natural products such
as meat extract or vegetable infusions.
Natural products contain essential bacterial Natural products contain essential bacterial
nutrients
Exact concentration of nutrients are unknown
Used routinely
Easy to prepare, relatively cheap and able to
support the growth of many bacteria
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Types of Media
1. Liquid media or broth
Bacteria can move freely in them
Growth in liquid medium is shown by turbidity
Some organism show surface growth Some organism show surface growth
Used for biochemical testing, blood culture,
testing for motility and as enrichment broth
Disadvantage: purity of growth cannot be
guaranteed
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Types of Media
2. Solid Media
Microorganism grown on solid media will grow and
multiply at the site of inoculation and form visible
colonies
Liquid agar is made solid by adding a solidifying agent Liquid agar is made solid by adding a solidifying agent
which does not alter the nutritional content of the
medium - agar
Agar is an inert carbohydrate extract obtained from a
type of seaweed found in Japan, New Zealand, and
California.
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Types of Media
Properties of agar:
1. Melts at 98C,
2. Sets at around 40C,
3. Easily soluble 3. Easily soluble
4. Remains clear
5. Concentration of 1% to be gel.
6. It is solid at 37C
7. Once solidified, it can be remelted
8. Bacteriologically inert (will not be degraded by
most bacteria)
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Types of Media
3. Basal Media
Simple media that will support the growth of most
microorganisms that do not need special
nutritional requirementsnutritional requirements
Contain basic nutrients: peptone, mineral salts
and water
Normally called nutrient broth
2 types: infusion broth and digest broth
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Types of Media
4. Enriched media
Culture media that are enriched with whole or
lysed blood, serum, special extracts or nutrients
Support growth of bacteria that cannot grow on Support growth of bacteria that cannot grow on
basal media
Nutrient broth + agar = nutrient agar
Nutrient agar + blood = blood agar
Blood agar + heat = chocolate agar
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Types of Media
5. Selective media
Solid media has substances that prevent, slow
down or inhibit the growth of microorganisms
other than those for which the media are other than those for which the media are
devised e.g.
tellurite medium for diptheria organism
Deoxycholate citrate agar (DCA) for Salmonella
and Shigella groups
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Types of Media
6. Enrichment media
Liquid media, similar in function to selective
media
Difference: selective media is broth Difference: selective media is broth
e.g. selenite F broth for the isolation of
Salmonella group
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Types of Media
7. Differential media
Contain substances or indicators that will
differentiate one organism from another
e.g. MacConkey agar - differentiate lactose e.g. MacConkey agar - differentiate lactose
fermenting bacteria from non-lactose fermenting
bacteria; blood agar - differentiate haemolytic
bacteria from non-haemolytic ones.
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Types of Media
8. Transport media
Usually semi-solid
Transportation of clinical specimens containing
delicate microorganisms, if there is to be a delay in
their delivery to the laboratory or in processingtheir delivery to the laboratory or in processing
Contain substances that can prevent the
overgrowth of commensals and prevent bacteria
from dying as a result of pH change or enzyme
action, e.g.
Amies transport medium for Neisseria gonorrhoeae
Stuarts transport medium for delicate organisms
including anaerobes
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Isolation of Pure Cultures
In their natural habitats, microorganisms
usually grow in complex, mixed populations
with many other species.
Pure culture - a population of cells arising from Pure culture - a population of cells arising from
a single cell to characterise an individual species
Approaches:
1. Spread plate and streak plate
2. Pour plate
3. Microbial growth on agar surfaces
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Mixed Bacterial Growth
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Isolation of Pure Cultures
1. Spread plate and streak plate
Mixture of cells is spread out on agar surface at a
relatively low density.
Every cell grows into a completely separate Every cell grows into a completely separate
colony, a macroscopically visible growth of
clusters of microorganism on a solid medium.
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Isolation of Pure Cultures
I. Spread plate
Small volume of dilute microbial mixture
containing around 30 - 300 cells is transferred to
the center of agar plate and spread evenly over the center of agar plate and spread evenly over
the surface with a sterile bent-glass rod.
Dispersed cells develop into isolated colonies
The number of colonies should equal the
number of viable organism in the sample.
Spread plates can be used for colony count.
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Isolation of Pure Cultures
II. Streak plate
The microbial mixture is transferred to the edge of
an agar plate with an inoculating loop or swab and
then streaked out over the surface in one of several
pattern.pattern.
After the first quarter is streaked, the inoculating
loop is sterilised and an inoculum for the second
quarter is obtained from the first quarter.
Similar process is repeated for the subsequent
quarters.
This is essentially a dilution process to separate
individual colonies.
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Streak Plate
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Isolation of Pure Cultures
2. Pour plate
After the agar solidifies, each cell is fixed in place
and forms an individual colony
Plates containing 30 -300 colonies are counted Plates containing 30 -300 colonies are counted
The colonies equal the number of viable
microorganism in the sample that are capable of
growing in the medium used
Colonies growing on the surface also can be used
to inoculate fresh medium and prepare pure
cultures
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Pour Plate
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Isolation of Pure Cultures
3. Bacterial growth on agar surfaces
Colony development on agar surfaces aids
microbiologists in identifying microorganisms
because individual species often form colonies of because individual species often form colonies of
characteristic size and appearance
In nature, microorganisms often grow on
surfaces in biofilms - slime-encased aggregations
of microbes
Sometimes they form discrete colonies
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Isolation of Pure Cultures
Generally the most rapid cell growth occurs at
the colony edge
Growth is much slower in the center, and cell
autolysis takes place in the older central autolysis takes place in the older central
portions of some colonies.
These differences in growth are due to
gradients of oxygen, nutrients, and toxic
products within the colony.
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Isolation of Pure Cultures
At the colony edge, oxygen and nutrients are
plentiful
The colony center is much thicker than the
edgeedge
Oxygen and nutrients do not diffuse readily
into the center, toxic metabolic products
cannot be quickly eliminated, and growth in
the colony center is slowed or stopped
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Isolation of Pure Cultures
Cells on the periphery can be growing at
maximum rate while cells in the center are
dying
The bacteria growing on solid surfaces vary The bacteria growing on solid surfaces vary
with nutrient diffusion and availability, the
hardness of the agar surface, bacterial
chemotaxis, and the presence of liquid on the
surface
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Isolation of Pure Culture
Tube culture methods
Used for specific identification tests
Only small volumes of media are required for
the teststhe tests
Not used for colonial morphology study
e.g. are:
1. Slope cultures
2. Deep cultures
3. Stab cultures
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Isolation of Pure Culture
Slope (slant) cultures are tubes containing
small quantity of medium that has been
allowed to set in sloped position.
Aka slopes or slants Aka slopes or slants
Used for maintenance of isolated bacteria or for
performing biochemical tests
e.g. Loeffler's serum agar, Dorset egg medium and
Lowestein-Jensen medium
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Isolation of Pure Culture
Deep culture media are prepared in tubes of
about 150 x 20 mm to a depth of about 60-70
mm.
Aka shake
Cultivation of anaerobic bacteria and can be used for Cultivation of anaerobic bacteria and can be used for
viable count
Stab cultures refer to the method of inoculation,
where the inoculating needle is stabbed through
the center of the medium.
e.g. Kohns II medium, motility medium, indole urea
medium
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Isolation of Pure Culture
Inoculation into broth
Carried out using inoculating loop, inoculating
needle or Pasteur pipette
Depends on whether the inoculum is colonial Depends on whether the inoculum is colonial
growth, liquid culture or specimen
Pick colonies with sterile inoculating loop, hold
the tube at an angle, and rub the loop against the
inner side of the container below the level of the
broth
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Sub-culturing
Sub-culturing means to transfer
microorganism from one medium to another.
Basically, it is starting new bacterial cultures
from the old cultures.from the old cultures.
Usually performed to maintain continuous
growth of microbes
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Measurement of Microbial Growth
To determine growth rates and generation
times
Either population number or mass may be
followed because growth leads to increases in followed because growth leads to increases in
both
No single technique is always best, the most
appropriate approach will depend on the
experimental situation
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Measurement of Microbial Growth
Classified into 2 methods:
1. Cell number
i. Direct counting
ii. Coulter counter and flow cytometerii. Coulter counter and flow cytometer
iii. Membrane filter technique
iv. Plating methods (visible counting methods, spread
plate and pour plate methods)
2. Cell mass
i. Microbial dry weight
ii. Spectrophotometry
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Measurement of Microbial Growth
1. Direct counting
Using counting chamber e.g. Petroff-Hausser
counting chamber, haemocytometers
Prokaryotes are more easily counted when stained,
or if phase contrast or flourescence microscope is or if phase contrast or flourescence microscope is
used
Advantage: easy, inexpensive, and relatively quick; it
gives information about the size and morphology of
organisms
Disadvantage: the microbial population must be
fairly large for accuracy because only a small volume
is sampled
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Petroff-Hausser Counter
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Measurement of Microbial Growth
2. Coulter chamber and flow cytometer
Large microorganisms like protists and yeasts can
be directly counted with electronic counters,
Coulter chamber and flow cytometer
The microbial suspension is forced through a small
hole or orifice in the Coulter counter
An electrical current flows through the hole, and
electrodes placed on both sides of the orifices
measure its electrical resistance
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Coulter Counter
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Measurement of Microbial Growth
Every time a microbial cell passes through the orifice,
electrical resistance increase (or conductivity drops)
and the cell is counted
Advantage: accurate with large cells and extensively
used
Disadvantage: interference by small particles, the
formation of filaments and other problems
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Measurement of Microbial Growth
3. Membrane filter techniques
The number of bacteria in aquatic samples is
frequently determined from direct counts after
the bacteria have been trapped on special the bacteria have been trapped on special
membrane filters
Sample is first filtered through a black
polycarbonate membrane filter.
Then, the bacteria are stained with a fluorescent
dye and observed microscopically
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Measurement of Microbial Growth
Stained cells are easily observed against the black
background of the membrane filter and can be
counted when viewed with an epifluorescence
microscope
Traditional counting methods do not distinguish
dead cells from live cells
New methods (e.g. commercial kits) make this
possible
First traps bacteria in aquatic samples on a
membrane filter
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Measurement of Microbial Growth
The filter is then placed on an agar medium or on
a pad soaked with liquid media and incubated
until each cell forms a separated colony
A colony count gives the number of
microorganisms in the filtered sample, and special
media can be used for specific microorganisms
Specially useful technique in analysing water
purity
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Measurement of Microbial Growth
4. Plating methods
To determine the number of viable microbes
Viable counting methods - count only those cells
that are alive and able to reproducethat are alive and able to reproduce
Pour plate and spread plate methods - diluted
samples of bacteria or other microorganisms is
dispersed over a solid agar surface
Simple, sensitive, and widely used for viable
counts
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Measurement of Microbial Growth
Low counts will result if clumps of cells are not
broken up and microorganisms not well dispersed
Results expressed in colony forming units (CFU) -
30 - 300 colonies for most accurate counting
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Measurement of Microbial Growth
5. Microbial dry weight
Cells growing in liquid medium are collected by
centrifugation, washed, dried in an oven and
weighedweighed
Especially useful technique for measuring the
growth of filamentous fungi
Time-consuming and not very sensitive
Bacteria weigh so little, it may be necessary to
centrifuge several hundred millimeters of culture
to collect a sufficient quantity
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Measurement of Microbial Growth
6. Spectrophotometry
More rapid and sensitive
Depend on the fact that microbial cells scatter
light that strikes themlight that strikes them
Microbial cells in a population are of roughly
constant size
The amount of scattering is directly proportional
to the biomass of cells present and indirectly
related to cell number
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Measurement of Microbial Growth
When the concentration of bacteria reaches
about 107/mL, the medium appears slightly
cloudy or turbid
The population growth can be easily measured
as long as the population is high enough to give as long as the population is high enough to give
detectable turbidity
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Measurement of Microbial Growth
If the amount of a substance in each cell is constant,
the total quantity of that cell constituent is directly
related to the total microbial cell mass
Example, a sample of washed cells collected from a Example, a sample of washed cells collected from a
known volume of medium can be analysed for total
protein or nitrogen
An increase in the microbial population will be
reflected in higher total protein levels.
ATP can be used to estimate the amount of living
microbial mass