This document contains summarised notes for Topic 6 Microbiology and pathogens, taken using the Pearson Edexcel B biology activate textbook. Notes taken with referencing to specification.
TOPIC 6 – MICROBIOLOGY AND PATHOGENS
4 groups of microorganisms are:
6.1 – BACTERIA AND DISEASE Viruses
Bacteria
PATHOGENS – microorganisms that cause disease Fungi
Protozoa
CULTURING MICROORGANISMS
~ Culturing is used to investigate microorganisms as they’re too small to see with a
naked eye.
~ Culturing involves growing large numbers of the microorganisms so they can be
measured in some way.
~ They need to be provided with the right level of nutrients and oxygen along with the
ideal pH and temp for them to grow.
RISKS OF CULTURING
~ Mutant strain arising that may be pathogenic
~ Contamination of the culture by pathogenic microorganisms from the environment.
~ Entry of microorganisms from the air or your skin into the culture will contaminate it
The culture must be sterile before the culture is started.
ASEPTIC CULTURE TECHNIQUES
1. Decide which microorganisms to culture
2. Provide microorganisms with the right nutrients for growth
~ The NUTRIENT MEDIUM can be made in the form of nutrient broth or in the
solid form, nutrient agar.
~ Agar – jelly extracted from seaweed
3. Solid and liquid media must be kept sterile until ready for use.
~ Some microorganisms will grow on a pure agar but most need added nutrients.
~ Most microorganisms grow on or in a medium enriched with good protein
sources such as blood, yeast extract or meat extract.
~ Some need a precise balance of nutrients.
4. SELECTIVE MEDIUM – a medium in or on which a select group of microorganisms will
grow. You can identify microorganisms that have been genetically modified.
5. Introduce microorganisms
e.g. bacteria
~ INOCULATION – getting the bacteria onto your agar/broth. This is done with an
inoculating loop, scraping of bacteria from the solid media surface either into a
liquid medium, or streaking across another solid medium plate.
~ INCOCULATING BROTH – involves making a suspension of the bacteria to be
grown and mixing a known volume with the sterile nutrient broth in the flask.
6. Flask is then stoppered quickly with cotton wool to prevent contamination from the
air.
7. Flask is incubated and often shaken regularly, allowing oxygen to the growing
bacteria.
,GROWING A PURE CULTURE
~ To achieve a pure culture, the desired microorganism needs to be isolated.
~ Growing a culture under anaerobic conditions will ensure that only anaerobic
bacteria will survive.
~ Growing aerobic conditions, in oxygen means only aerobic organisms can survive.
~ Some bacteria will grow in both conditions.
~ You can produce a medium that will favour the growth of the organism you want to
culture and inhibit the growth of others.
~ Allows you to identify the colony you want and then re-inoculate to produce a single
culture (pure).
~ May need to control range of nutrients, or introduce selective growth inhibitors,
antibiotics or antifungal chemicals.
INDICATOR MEDIA – cause certain types of bacteria to change colour.
~ Colonies that change colour or don’t change colour can’t be isolated and cultured.
MEASURING THE GROWTH OF BACTERIAL CULTURES
CELL COUNTS
~ Bacteria and single-celled fungi cultures in nutrient broth can be counted directly
using a microscope and a haemocytometer.
HAEMOCYTOMETER – consists of a specialised thick microscope slide with a rectangular
chamber that holds a standard volume of liquid.
~ The chamber is engraved with a grid of lines.
PROCESS
1. Sample of nutrient broth is diluted by half with an equal volume of trypan blue.
TRYPAN BLUE – a dye that stains dead cells blue so you can identify and count only
the living cells.
2. Cells are viewed using a microscope and counted.
3. Each corner of the haemocytometer grid has a square divided into 16 smaller
squares.
4. The number of ells in each of these four sets of 16 squares is counted and the mean
calculated.
5. The haemocytometer is calibrated so that the number of bacterial or fungal cells in
one set of 16 squares equated to the number of cells x 10-4 per cm3 of broth.
6. By taking measurements at regular intervals throughout the life of a bacterial colony,
a picture of the changing cell numbers can be built up.
OPTICAL METHODS
TRUBIDIOMETRY – a special form of colorimetry.
~ As the number of bacterial cells in a culture increase it becomes cloudier looking /
turbid.
~ As the solution gets more turbid, it absorbs more light, so less light passes through it.
ADV – quick
DISADV – expensive
, COLORIMETER – measures how much light passes through a sample, showing how much
light is absorbed. It indirectly shows how many microorganisms are present.
A CALIBRATION CURVE is produced by growing a control culture, take samples at regular
time intervals. You can measure the number of microorganisms using turbidimetry.
~ The turbidity of each sample is measured and the cell count using a haemocytometer
is made for each sample.
~ This shows the relationship between the turbidity of the culture and the number of
bacterial cells present.
DILUTION PLATING
~ It’s another way to count microorganisms in a culture.
~ It’s used to find the total viable cell count.
~ Each colony on an agar plate has grown from a single, viable microorganism on the
plate.
PROBLEM – a solid mass of microbial growth is often present after culturing and it’s not
possible to work out the number of individual colonies.
SOLUTION – Dilute the original culture in stages until a point is reached when the colonies
can be counted.
~ If the number of colonies is multiplied by the dilution factor, then a total viable cell
count can be determined.
AREA AND MASS OF FUNGI
When fungi is being cultured, to assess growth measure the diameter of the patches of
mycelium.
This can be used to compare growth rates in different conditions.
To discover the best concentration for nutrients or optimum pH at which to grow fungi test
the DRY MASS OF THE MICROORGANISM.
PROCESS
1. Best done using a liquid growth medium
2. Samples of broth can be removed at regular intervals and the fungi separated from
the liquid by CENTRIFUGATION or FILTERING.
3. Material is dried thoroughly (for an example in an oven overnight) – 100 ℃
4. This gives a measure of dry mass
5. An increase or decrease in the dry mass gives an indication of the increase or
decrease in the mycelial mass
6. Greatest dry mass is at optimum conditions
DIFFERENT PHASES OF A BACTERIAL GROWTH CURVE
LAG PHASE – when bacteria are adapting to their new
environment and aren’t get reproducing at their max
rate.
LOG PHASE / EXPONENTIAL PHASE – when the rate of
bacterial reproduction is close to or at its theoretical
maximum, repeatedly doubling in a given time period.
STATIONARY PHASE – when the total growth rate is
zero as the number of new cells formed by binary fission is equal to number of cells dying.
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