Monitoring growthMonitoring growth

Learning objective:Learning objective:To be able to describe ways of To be able to describe ways of growing bacteria and ways of growing bacteria and ways of

monitoring their growthmonitoring their growth

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sampling time following innoculation

viab

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late

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glucose only

lactose only

lactose and glucose

SimilaritiesSimilarities• Lag phase. Lag phase.

– Little increase in cell number;Little increase in cell number;– Individual bacteria may be increasing in size;Individual bacteria may be increasing in size;– Enzymes may be being synthesised to utilise the Enzymes may be being synthesised to utilise the

nutrient medium;max.2nutrient medium;max.2• Log phase.Log phase.

– The population shows exponential phase;The population shows exponential phase;– Nutrient supply is not a limiting factor;Nutrient supply is not a limiting factor;

• Stationary phase.Stationary phase.– The population growth slows down;The population growth slows down;– The number of new cells formed is balanced by The number of new cells formed is balanced by

the number of cells dying;the number of cells dying;– Limiting factors, such a nutrient supply and Limiting factors, such a nutrient supply and

metabolic wastes have started to influence further metabolic wastes have started to influence further increase in population size; max 2increase in population size; max 2

DifferencesDifferences• Lag phase for lactose alone longer; nutrient Lag phase for lactose alone longer; nutrient

less readily available;less readily available;• Growth of population less than with glucose; Growth of population less than with glucose;

fewer cells and longer to increase;max.2fewer cells and longer to increase;max.2• Growth with lactose and glucose gives a Growth with lactose and glucose gives a

greater population;greater population;• Has a second lag; around 100-120 minute Has a second lag; around 100-120 minute

and then increases again;and then increases again;• Uses glucose first as growth curve similar to Uses glucose first as growth curve similar to

glucose alone; and then uses lactose;max 3glucose alone; and then uses lactose;max 3• Death phase is occurring for glucose alone Death phase is occurring for glucose alone

as all the energy source/glucose has run as all the energy source/glucose has run out.1out.1

Growing MicrobesGrowing Microbes

• Microbes for experiments can be Microbes for experiments can be obtained from natural sources (e.g. obtained from natural sources (e.g. soil, food, skin, water, air), or bought soil, food, skin, water, air), or bought from suppliers in agar slopes. from suppliers in agar slopes.

• You don't need much, since a dot may You don't need much, since a dot may contain millions of viable cells, each of contain millions of viable cells, each of which could grow into a whole colony.which could grow into a whole colony.

MediumMedium• The mixture that the microbes are grown (cultured) The mixture that the microbes are grown (cultured)

on. on. • The medium must contain all the nutrients (sugars, The medium must contain all the nutrients (sugars,

minerals, proteins) needed for the microbes to grow.minerals, proteins) needed for the microbes to grow.• By adjusting these nutrients, the medium can be By adjusting these nutrients, the medium can be

made selective for one type of microbe.made selective for one type of microbe.• Culture media can be made up by mixing together Culture media can be made up by mixing together

known amounts of specific chemicals (a defined or known amounts of specific chemicals (a defined or synthetic medium), or they can be made from a synthetic medium), or they can be made from a natural source such as boiled meat or yeast extract, natural source such as boiled meat or yeast extract, which generally contains the nutrients required by which generally contains the nutrients required by most microbes (an undefined or complex medium).most microbes (an undefined or complex medium).

Nutrient mediumNutrient medium

• A cheap general-purpose complex A cheap general-purpose complex medium used for most school medium used for most school experiments.experiments.

BrothBroth

• A liquid medium (i.e. without agar).A liquid medium (i.e. without agar).

Agar Agar

• Agar is mixed with a liquid medium to make a solid Agar is mixed with a liquid medium to make a solid medium, which is very useful to observe, separate medium, which is very useful to observe, separate and store bacteria cultures.and store bacteria cultures.

• A solid medium in a petri dish is known as an agar A solid medium in a petri dish is known as an agar plate, while a solid medium in a Universal bottle is plate, while a solid medium in a Universal bottle is called an agar slope.called an agar slope.

• Agar is actually a polysaccharide extracted from Agar is actually a polysaccharide extracted from seaweed. seaweed.

• It melts at 41°C (so can be incubated at 37°C without It melts at 41°C (so can be incubated at 37°C without melting), is reasonably transparent, and is not melting), is reasonably transparent, and is not broken down by microbes, so it remains solid.broken down by microbes, so it remains solid.

Aseptic transferAseptic transfer

• Also called aseptic technique. Also called aseptic technique. • The transfer of a sample of bacteria from one vessel The transfer of a sample of bacteria from one vessel

to another. to another. • This is the most common and basic technique and is This is the most common and basic technique and is

used in almost all micro-biological experiments. used in almost all micro-biological experiments. • The bacteria are usually transferred using a wire or The bacteria are usually transferred using a wire or

glass inoculating loop, which can carry a tiny glass inoculating loop, which can carry a tiny volume of culture (10 µl) or a scraping of cells from volume of culture (10 µl) or a scraping of cells from an agar plate. an agar plate.

• Larger volumes are transferred using a sterile Larger volumes are transferred using a sterile syringe or pipette.syringe or pipette.

Key wordsKey words• Inoculate -Inoculate -

• Incubate -Incubate -

• Culture - Culture -

• Colony - Colony -

• Streak Plate –Streak Plate –

• Lawn -Lawn -

• To add few cells to a medium, so that To add few cells to a medium, so that they may grow. they may grow.

• To leave a culture to grow under To leave a culture to grow under defined conditions. defined conditions.

• A growth of microbes in a medium. The A growth of microbes in a medium. The culture can be pure (one species of culture can be pure (one species of microbe) or mixed (many species).microbe) or mixed (many species).

• A visible growth of bacteria on an agar A visible growth of bacteria on an agar plate containing many millions of cells. plate containing many millions of cells.

• A method of inoculating an agar plate A method of inoculating an agar plate with bacteria so that the bacteria are with bacteria so that the bacteria are gradually diluted.gradually diluted.

• A layer of bacteria growing on the A layer of bacteria growing on the surface of an agar plate. surface of an agar plate.

Measuring the Growth of Measuring the Growth of MicrobesMicrobes

• Growth of cells in a liquid culture is Growth of cells in a liquid culture is generally measured by simply counting generally measured by simply counting the number of cells.the number of cells.

• There are various techniques for doing There are various techniques for doing this. Some give total cell counts, which this. Some give total cell counts, which include both living and dead cells, include both living and dead cells, while others give viable cell counts, while others give viable cell counts, which only include living cells.which only include living cells.

HaemocytometerHaemocytometer• This counts the total cells by observing the individual This counts the total cells by observing the individual

cells under the microscope. cells under the microscope. • This is reasonably easy for large cells like yeast, but is This is reasonably easy for large cells like yeast, but is

more difficult for bacterial cells, since they are so small. more difficult for bacterial cells, since they are so small. • The cell counter (or haemocytometer) is a large The cell counter (or haemocytometer) is a large

microscope slide with a very accurate grid drawn in the microscope slide with a very accurate grid drawn in the centre. centre.

• The grid marks out squares with 1 mm, 0.2 mm and 0.05 The grid marks out squares with 1 mm, 0.2 mm and 0.05 mm sides. mm sides.

• There is an accurate gap of 0.1 mm between the grid There is an accurate gap of 0.1 mm between the grid and the thick coverslip, so the volume of liquid above and the thick coverslip, so the volume of liquid above the grid is known. the grid is known.

• The number of cells in a known small volume can thus The number of cells in a known small volume can thus be counted, and so scaled up. The units are cells per be counted, and so scaled up. The units are cells per cmcm33 . .

For example:For example:

• A 0.2 mm square has an average of 80 A 0.2 mm square has an average of 80 cells in a 1000x dilution cells in a 1000x dilution

• Volume above square = 0.2 x 0.2 x 0.1 = Volume above square = 0.2 x 0.2 x 0.1 = 0.004 mm³0.004 mm³

• 80 cells in 0.004 mm³ = 20 000 cells per 80 cells in 0.004 mm³ = 20 000 cells per mm³ in the diluted suspensionmm³ in the diluted suspension

• which is 20 000 x 1000 = 2 x 10which is 20 000 x 1000 = 2 x 1077 cells cells per mm³ of undiluted suspensionper mm³ of undiluted suspension

• or 2 x 10or 2 x 101010 cells per cm³ cells per cm³

TurbidometryTurbidometry• This technique also counts the total cells. This technique also counts the total cells. • It is quicker than using a haemocytometer, but less accurate.It is quicker than using a haemocytometer, but less accurate.• A sample of the liquid culture is placed in a cuvette in a A sample of the liquid culture is placed in a cuvette in a

colorimeter, and the absorbance of light is measured. colorimeter, and the absorbance of light is measured. • The greater the concentration of the cells, the more cloudy or The greater the concentration of the cells, the more cloudy or

turbid the liquid is, so the more light it scatters, so the less turbid the liquid is, so the more light it scatters, so the less light is transmitted to the detector. light is transmitted to the detector.

• A wavelength of 600nm is normally used. A wavelength of 600nm is normally used. • Although the absorbance scale of the colorimeter is used, Although the absorbance scale of the colorimeter is used,

light is not actually absorbed by the cells (as it is by pigment light is not actually absorbed by the cells (as it is by pigment molecules), but scattered. molecules), but scattered.

• If the same sample is counted in a haemocytometer and its If the same sample is counted in a haemocytometer and its absorbance measured, than a calibration curve can be absorbance measured, than a calibration curve can be plotted. plotted.

• From this calibration curve the concentration of cells can be From this calibration curve the concentration of cells can be read off for any absorbance.read off for any absorbance.

Dilution PlatingDilution Plating• This technique counts viable cells. This technique counts viable cells. • A sequence of ten-fold dilutions is taken from the A sequence of ten-fold dilutions is taken from the

original culture flask, using sterile medium. original culture flask, using sterile medium. • This is called a serial dilution, and allows large This is called a serial dilution, and allows large

dilutions to be made using small volumes. dilutions to be made using small volumes. • From each dilution a 1 cm³ sample is taken and From each dilution a 1 cm³ sample is taken and

spread evenly onto an agar plate. spread evenly onto an agar plate. • Each viable cell in the sample will multiply and grow Each viable cell in the sample will multiply and grow

into a colony. into a colony. • In most of the samples there will be too many In most of the samples there will be too many

colonies to count, but in one of the dilutions there colonies to count, but in one of the dilutions there will be a good number (20-200) of individual will be a good number (20-200) of individual colonies. colonies.

• From this we can calculate the concentration of From this we can calculate the concentration of viable cells in the original culture.viable cells in the original culture.

For example:For example:

• Suppose there were 83 colonies in the Suppose there were 83 colonies in the x10 000 dilution agar plate.x10 000 dilution agar plate.

• How many viable cells would there have How many viable cells would there have been per been per cm³ in the original culture?cm³ in the original culture?

• There were 83 viable cells in the 1 cm³ There were 83 viable cells in the 1 cm³ sample of the x10 000 dilutionsample of the x10 000 dilution

• So there were So there were 83 x 10 000 = 8.3 x 1083 x 10 000 = 8.3 x 1055 cells cells per cm³ in the original cultureper cm³ in the original culture

QuestionsQuestions