Experiments Unit 1 When transferring the microorganisms from a Preparation of a streak plate to isolate single colonies The Haemocytometer
Investigating microorganisms using aseptic techniques culture bottle to a petri dish, the lid of the bottle -Using an inoculating loop, spread microorganisms It is an instrument for counting cell numbers - resembles a
should be held in the same hand that holds the over a small section of the agar in the petri dish microscope slide with a grid containing squares of known
Aseptic technique prevents Solid agar or liquid broth can be culture bottle - not allowed to touch the bench, with -With a resterilised inoculating loop, streak several size. The design enables the central area containing the
contamination of both used to culture microorganisms - the other hand holding the inoculating loop. lines of microorganism across the agar at an angle grid to be slightly lower by a fixed distance (0.1mm) than
microbe culture and of care needed when transferring Immediately after opening the culture bottle the taking care to not allow separate 'lines' to overlap the cover slip. Ensures it represents not only a known area
individuals. microorganisms to petri dishes. neck should be passed through the Bunsen flame to -Repeat stage 2 once or twice making sure that a but volume of liquid.
sterilise the lid region and repeated before replacing If there are too many cells then a serial
Method sterile loop is used on each occasion Type A square = 1mm2 x 0.1mm = 0.1mm3 dilution would be necessary - usually by
If using metal inoculating loop - needs to be 'flamed' in the lid. -Make a final single streak Type B square = 0.04mm2 x 0.1mm =
the hottest part of a Bunsen flame until it becomes red 3
factor of 10 e.g. add 1cm3 of
-Incubate the petri dish at a suitable temperature. 0.004mm suspension added into 9cm3 of isotonic
hot to sterilise it. After this it should air cool since when When transferring the microorganisms to a petri After 24 hours it should be possible to identify Type C square = 0.0025mm2 x 0.1mm =
red-hot it would kill microorganisms it comes into dish, the lid of the dish should be only raised enough buffer and new solution examined.
isolated pure colonies 0.00025mm3
contact with. After transfer of microorganisms, the loop to allow the microorganisms to be added to the
has to be re-sterilised and care has to be taken to not agar. North-west rule - only count cells lying on north and west
Investigating antimicrobial properties of plants sides of a square. It is impossible to
create a microorganism rich aerosol. distinguish between
Disposable plastic loops should be discarded into a A spread plate method can also be used Many plants have a range of defences against If there are few cells then the A square should be use, if
where cells in suspension are used in microorganisms - prevent decay and further loss in there are too many cells that it would take too long to dead or alive cells -
solution of disinfectant. haemocytometer is
inoculation, an L-shaped spreader is used parts of plants which become damaged in natural count a B square then a C square should be used
Petri dishes should be labelled on the to spread it over the surface of the agar. environment. effective during lag
outside of the base and incubated Important points: and log phases but not
Plastic sterile disposable spreaders are an Many common woodland plants produce compounds -Mix the yeast suspension thoroughly before taking a
upside down in an incubator at an alternative to glass spreaders that require that prevent or reduce fungal/and or bacterial stationary and decline
appropriate temperature. sample (agitate it) as counting dead cells
sterilisation before and after use. growth. -Sample from the same depth in the flask
-Grind up leaves of each woodland plant and a small may not suggest
amount of water in separate mortars -If sampling from natural environment, the retrieval of number is falling.
Investigating effect of different antibiotics on bacteria E-strips can also be used which are prepared strips different samples should be done at same depth & same
containing a concentration gradient of antibiotic running -Prepare some agar plates and spread a fungus or
Discs of antibiotics can be placed on agar bacterium culture on the plate using appropriate time of day if taken over a period of time
in a petri dish, the agar is inoculated with the length of the strip. The process involves placing the e- -Avoid getting yeast suspension on top of coverslip or
strip on the agar of a petri dish inoculated with a bacterium. aseptic techniques. (Fungus Pythium debaryanum or
a particular type of bacterium and the bacterium Bacillus are suitable) grooves of haemocytometer
effect of an antibiotic/antibiotics can be Following incubation (18-72 hours) and depending on the e- -Carry out an appropriate number of replicates for
strip/microorganism there should be results. E-strips -Soak a small disk of filter paper with each plant
investigated. extract and place on the agar in the petri dish reliability
contain markings that show antibiotic concentrations and as
such it is possible to work out the minimum antibiotic -Incubate for 24-48 hours and compare results in the Example - number of cells in B
concentration to inhibit bacterial growth. different petri dishes. Volume - 0.04mm2 x 0.1mm = 0.004mm3
1/0.004 x 22 = 5500/mm3
Capture-recapture technique When estimating the pop size by this method there are a few
-A large sample of the species is caught or trapped using an appropriate technique e.g. pitfall assumptions made;
trap or sweep net Population sizes of animals can be harder to estimate as opposed
-No significant gains or losses through immigration/emigration, can be to plants as they move and spend time in places difficult to
-The caught animals are marked in a way that will last over the sampling period (permanent avoided through carrying out sample in a discrete area where mixing
or semi-permanent) observe.
with other populations is less likely After carrying out capture and re-capture there should be a set of
-Should be done in a way that does not harm the animal or make it more likely to be -There are no significant gains or losses through births or deaths
predated than other non-marked animals (can be done on dorsal surface which is out of marked animals. The following formula is used to estimate the
-The trapping process does not affect the animal in any way e.g. making population size:
sight for predators) it more wary of trapping mechanism, reducing its likelihood of being If a population is small then a
-The marked animals are released allowing enough time for them to mix throughout the S1 x S2 large amount of marked animals
recaptured in the re-sample or more likely to be predated r
population -The marked animals have had enough time to mix throughout the will be recaptured, if large then a
-The population is then re-sampled using the same trapping process as before and the size population small amount will be recaptured.
estimated using the Lincoln index formula -The marking does not disadvantage the animal
Biology A2 3 Page 1
Investigating microorganisms using aseptic techniques culture bottle to a petri dish, the lid of the bottle -Using an inoculating loop, spread microorganisms It is an instrument for counting cell numbers - resembles a
should be held in the same hand that holds the over a small section of the agar in the petri dish microscope slide with a grid containing squares of known
Aseptic technique prevents Solid agar or liquid broth can be culture bottle - not allowed to touch the bench, with -With a resterilised inoculating loop, streak several size. The design enables the central area containing the
contamination of both used to culture microorganisms - the other hand holding the inoculating loop. lines of microorganism across the agar at an angle grid to be slightly lower by a fixed distance (0.1mm) than
microbe culture and of care needed when transferring Immediately after opening the culture bottle the taking care to not allow separate 'lines' to overlap the cover slip. Ensures it represents not only a known area
individuals. microorganisms to petri dishes. neck should be passed through the Bunsen flame to -Repeat stage 2 once or twice making sure that a but volume of liquid.
sterilise the lid region and repeated before replacing If there are too many cells then a serial
Method sterile loop is used on each occasion Type A square = 1mm2 x 0.1mm = 0.1mm3 dilution would be necessary - usually by
If using metal inoculating loop - needs to be 'flamed' in the lid. -Make a final single streak Type B square = 0.04mm2 x 0.1mm =
the hottest part of a Bunsen flame until it becomes red 3
factor of 10 e.g. add 1cm3 of
-Incubate the petri dish at a suitable temperature. 0.004mm suspension added into 9cm3 of isotonic
hot to sterilise it. After this it should air cool since when When transferring the microorganisms to a petri After 24 hours it should be possible to identify Type C square = 0.0025mm2 x 0.1mm =
red-hot it would kill microorganisms it comes into dish, the lid of the dish should be only raised enough buffer and new solution examined.
isolated pure colonies 0.00025mm3
contact with. After transfer of microorganisms, the loop to allow the microorganisms to be added to the
has to be re-sterilised and care has to be taken to not agar. North-west rule - only count cells lying on north and west
Investigating antimicrobial properties of plants sides of a square. It is impossible to
create a microorganism rich aerosol. distinguish between
Disposable plastic loops should be discarded into a A spread plate method can also be used Many plants have a range of defences against If there are few cells then the A square should be use, if
where cells in suspension are used in microorganisms - prevent decay and further loss in there are too many cells that it would take too long to dead or alive cells -
solution of disinfectant. haemocytometer is
inoculation, an L-shaped spreader is used parts of plants which become damaged in natural count a B square then a C square should be used
Petri dishes should be labelled on the to spread it over the surface of the agar. environment. effective during lag
outside of the base and incubated Important points: and log phases but not
Plastic sterile disposable spreaders are an Many common woodland plants produce compounds -Mix the yeast suspension thoroughly before taking a
upside down in an incubator at an alternative to glass spreaders that require that prevent or reduce fungal/and or bacterial stationary and decline
appropriate temperature. sample (agitate it) as counting dead cells
sterilisation before and after use. growth. -Sample from the same depth in the flask
-Grind up leaves of each woodland plant and a small may not suggest
amount of water in separate mortars -If sampling from natural environment, the retrieval of number is falling.
Investigating effect of different antibiotics on bacteria E-strips can also be used which are prepared strips different samples should be done at same depth & same
containing a concentration gradient of antibiotic running -Prepare some agar plates and spread a fungus or
Discs of antibiotics can be placed on agar bacterium culture on the plate using appropriate time of day if taken over a period of time
in a petri dish, the agar is inoculated with the length of the strip. The process involves placing the e- -Avoid getting yeast suspension on top of coverslip or
strip on the agar of a petri dish inoculated with a bacterium. aseptic techniques. (Fungus Pythium debaryanum or
a particular type of bacterium and the bacterium Bacillus are suitable) grooves of haemocytometer
effect of an antibiotic/antibiotics can be Following incubation (18-72 hours) and depending on the e- -Carry out an appropriate number of replicates for
strip/microorganism there should be results. E-strips -Soak a small disk of filter paper with each plant
investigated. extract and place on the agar in the petri dish reliability
contain markings that show antibiotic concentrations and as
such it is possible to work out the minimum antibiotic -Incubate for 24-48 hours and compare results in the Example - number of cells in B
concentration to inhibit bacterial growth. different petri dishes. Volume - 0.04mm2 x 0.1mm = 0.004mm3
1/0.004 x 22 = 5500/mm3
Capture-recapture technique When estimating the pop size by this method there are a few
-A large sample of the species is caught or trapped using an appropriate technique e.g. pitfall assumptions made;
trap or sweep net Population sizes of animals can be harder to estimate as opposed
-No significant gains or losses through immigration/emigration, can be to plants as they move and spend time in places difficult to
-The caught animals are marked in a way that will last over the sampling period (permanent avoided through carrying out sample in a discrete area where mixing
or semi-permanent) observe.
with other populations is less likely After carrying out capture and re-capture there should be a set of
-Should be done in a way that does not harm the animal or make it more likely to be -There are no significant gains or losses through births or deaths
predated than other non-marked animals (can be done on dorsal surface which is out of marked animals. The following formula is used to estimate the
-The trapping process does not affect the animal in any way e.g. making population size:
sight for predators) it more wary of trapping mechanism, reducing its likelihood of being If a population is small then a
-The marked animals are released allowing enough time for them to mix throughout the S1 x S2 large amount of marked animals
recaptured in the re-sample or more likely to be predated r
population -The marked animals have had enough time to mix throughout the will be recaptured, if large then a
-The population is then re-sampled using the same trapping process as before and the size population small amount will be recaptured.
estimated using the Lincoln index formula -The marking does not disadvantage the animal
Biology A2 3 Page 1
