Transcript monitoring_growth

Monitoring growth
Learning objective:
To be able to describe ways of
growing bacteria and ways of
monitoring their growth
viable plate count per mm3 medium
Questions
250
200
150
glucose only
lactose only
lactose and glucose
100
50
0
0
50
100
150
200
250
sampling time following innoculation
300
Similarities
• Lag phase.
– Little increase in cell number;
– Individual bacteria may be increasing in size;
– Enzymes may be being synthesised to utilise the
nutrient medium;max.2
• Log phase.
– The population shows exponential phase;
– Nutrient supply is not a limiting factor;
• Stationary phase.
– The population growth slows down;
– The number of new cells formed is balanced by
the number of cells dying;
– Limiting factors, such a nutrient supply and
metabolic wastes have started to influence further
increase in population size; max 2
Differences
• Lag phase for lactose alone longer; nutrient
less readily available;
• Growth of population less than with glucose;
fewer cells and longer to increase;max.2
• Growth with lactose and glucose gives a
greater population;
• Has a second lag; around 100-120 minute
and then increases again;
• Uses glucose first as growth curve similar to
glucose alone; and then uses lactose;max 3
• Death phase is occurring for glucose alone
as all the energy source/glucose has run
out.1
Growing Microbes
• Microbes for experiments can be
obtained from natural sources (e.g.
soil, food, skin, water, air), or bought
from suppliers in agar slopes.
• You don't need much, since a dot may
contain millions of viable cells, each of
which could grow into a whole colony.
Medium
• The mixture that the microbes are grown (cultured)
on.
• The medium must contain all the nutrients (sugars,
minerals, proteins) needed for the microbes to grow.
• By adjusting these nutrients, the medium can be
made selective for one type of microbe.
• Culture media can be made up by mixing together
known amounts of specific chemicals (a defined or
synthetic medium), or they can be made from a
natural source such as boiled meat or yeast extract,
which generally contains the nutrients required by
most microbes (an undefined or complex medium).
Nutrient medium
• A cheap general-purpose complex
medium used for most school
experiments.
Broth
• A liquid medium (i.e. without agar).
Agar
• Agar is mixed with a liquid medium to make a solid
medium, which is very useful to observe, separate
and store bacteria cultures.
• A solid medium in a petri dish is known as an agar
plate, while a solid medium in a Universal bottle is
called an agar slope.
• Agar is actually a polysaccharide extracted from
seaweed.
• It melts at 41°C (so can be incubated at 37°C without
melting), is reasonably transparent, and is not
broken down by microbes, so it remains solid.
Aseptic transfer
• Also called aseptic technique.
• The transfer of a sample of bacteria from one vessel
to another.
• This is the most common and basic technique and is
used in almost all micro-biological experiments.
• The bacteria are usually transferred using a wire or
glass inoculating loop, which can carry a tiny
volume of culture (10 µl) or a scraping of cells from
an agar plate.
• Larger volumes are transferred using a sterile
syringe or pipette.
Key words
• Inoculate • Incubate • Culture • Colony • Streak Plate –
• Lawn -
• To add few cells to a medium, so
that they may grow.
• To leave a culture to grow under
defined conditions.
• A growth of microbes in a
medium. The culture can be pure
(one species of microbe) or mixed
(many species).
• A visible growth of bacteria on an
agar plate containing many
millions of cells.
• A method of inoculating an agar
plate with bacteria so that the
bacteria are gradually diluted.
• A layer of bacteria growing on the
surface of an agar plate.
Measuring the Growth of
Microbes
• Growth of cells in a liquid culture is
generally measured by simply counting
the number of cells.
• There are various techniques for doing
this. Some give total cell counts, which
include both living and dead cells,
while others give viable cell counts,
which only include living cells.
Haemocytometer
• This counts the total cells by observing the
individual cells under the microscope.
• This is reasonably easy for large cells like yeast, but
is more difficult for bacterial cells, since they are so
small.
• The cell counter (or haemocytometer) is a large
microscope slide with a very accurate grid drawn in
the centre.
• The grid marks out squares with 1 mm, 0.2 mm and
0.05 mm sides.
• There is an accurate gap of 0.1 mm between the grid
and the thick coverslip, so the volume of liquid
above the grid is known.
• The number of cells in a known small volume can
thus be counted, and so scaled up. The units are
cells per cm3 .
For example:
• A 0.2 mm square has an average of 80
cells in a 1000x dilution
• Volume above square = 0.2 x 0.2 x 0.1 =
0.004 mm³
• 80 cells in 0.004 mm³ = 20 000 cells per
mm³ in the diluted suspension
• which is 20 000 x 1000 = 2 x 107 cells
per mm³ of undiluted suspension
• or 2 x 1010 cells per cm³
Turbidometry
• This technique also counts the total cells.
• It is quicker than using a haemocytometer, but less
accurate.
• A sample of the liquid culture is placed in a cuvette in a
colorimeter, and the absorbance of light is measured.
• The greater the concentration of the cells, the more
cloudy or turbid the liquid is, so the more light it scatters,
so the less light is transmitted to the detector.
• A wavelength of 600nm is normally used.
• Although the absorbance scale of the colorimeter is used,
light is not actually absorbed by the cells (as it is by
pigment molecules), but scattered.
• If the same sample is counted in a haemocytometer and
its absorbance measured, than a calibration curve can be
plotted.
• From this calibration curve the concentration of cells can
be read off for any absorbance.
Dilution Plating
• This technique counts viable cells.
• A sequence of ten-fold dilutions is taken from the
original culture flask, using sterile medium.
• This is called a serial dilution, and allows large
dilutions to be made using small volumes.
• From each dilution a 1 cm³ sample is taken and
spread evenly onto an agar plate.
• Each viable cell in the sample will multiply and grow
into a colony.
• In most of the samples there will be too many
colonies to count, but in one of the dilutions there
will be a good number (20-200) of individual
colonies.
• From this we can calculate the concentration of
viable cells in the original culture.
For example:
• Suppose there were 83 colonies in the
x10 000 dilution agar plate.
• How many viable cells would there have
been per cm³ in the original culture?
• There were 83 viable cells in the 1 cm³
sample of the x10 000 dilution
• So there were 83 x 10 000 = 8.3 x 105 cells
per cm³ in the original culture
Questions