When performing experiments with microorganisms, there are certain practices you should follow. These are known as aseptic techniques. Aseptic techniques allow scientists to study a specific bacteria of interest. They can use different culture media to grow different bacterial species and count the number of bacteria in a culture using both scientific and mathematical methods.

Culture media

Definition

Culture media are mixtures of specific nutrients and other substances that allow microorganisms such as bacteria to grow. They are sometimes called growth media.

Culture media can either be liquid or solidified.

Type	Method	Advantage(S)
Liquid	This is known as broth culture and will be maintained in a conical flask. This creates a large surface area that is in contact with the air. The broth will be stirred and if anaerobic organisms are being cultured, the broth will need sterile air.	Broth cultures ensure the culture does not die so active cells and metabolic products can always be harvested.
Solidified	This usually involves a gelling agent such as agar being added to a Petri dish, a glass flat-sided bottle or a test tube.	There is little risk of spillage so solid mediums are useful for storage. This method provides a large surface area for growth and gas exchange. Bacterial cells in the Petri dish will form visible colonies that can be selected. Test tubes and glass flat-sided bottles provide more depth than a Petri dish which reduces the chance of dehydration and salt crystallisation.

Batch vs continuous

Liquid culture allows two types of culture to be carried out. These are known as batch and continuous.

In batch culture, microorganisms are inoculated into a sterile container that has a fixed volume of growth media.
In continuous culture, microorganisms are inoculated into a container with the growth media but every so often some media is removed and replaced with fresh, sterile medium.

Note: Batch culture can also be performed with solidified culture media as it contains a fixed amount of agar.

Broad spectrum media

Broad spectrum media are types of media that contain many basic nutrients needed by most microorganisms for growth. They often contain yeast extracts which are a mixture of soluble amino acids, peptides, sugars, vitamins, bases, inorganic ions and/or peptones.

Example

Nutrient agar is a broad spectrum medium.

Narrow spectrum media

These are also called selective media and they are limited as they can only grow a select few species of microorganism. This ensures scientists can grow the specific bacteria they are interested in.

Example

MacConkey agar is a selective medium that only allows the growth of Gram negative bacteria.

Incubation

After the microorganisms have been inoculated in culture media, they must be incubated. This means they are placed in a temperature-controlled environment for a specific period of time. The temperature must be appropriate for the microorganism you are culturing.

Sterilisation methods

Sterilisation methods are techniques you can use to ensure surfaces and equipment in the lab are free of microorganisms. These prevent contamination and are an important part of the aseptic technique required when handle microorganisms.

Method	Description	Aseptic technique
Chemical agents	Disinfectants are chemicals that can inhibit or slow the growth of bacteria.	Wipe down surfaces before and after working with microorganisms. Treat any spillages with disinfectant. Equipment should be cleaned thoroughly with chemical agents.
Heat treatment	Use a naked flame, such as a Bunsen burner, to sterilise equipment as no microorganism will be able to survive.	The inoculating loop is 'flamed', this means it is held in Bunsen flame until glows. Needles, forceps and glass equipment (that is dipped in $70\%$ ethanol) can be sterilised in this way also. The neck of a glass bottle or flask that contains the microorganisms culture should be flamed also.
	Using dry heat, an object can be placed in a hot air oven at $160\degree C$ for $1\ hour$ .	Glassware is often sterilised using this technique.
	Using moist heat, an object can be placed in an autoclave at $121\degree C$ for around $15\ minutes$ .	This is the preferred technique for the sterilisation of laboratory equipment and culture media that are not heat sensitive. It can also be used to sterilise old cultures and spent media before they are discarded.
Filtration	The liquid culture can be passed through a filtration device that has been sterilised using another technique. A filter with pore size $0.2\ \mu m$ will removed bacteria.	This is suitable for filtering only small volumes of liquid due to the small size of the pores.
Radiation	Objects could be exposed to UV or ionising radiation.	These techniques are only used in a specialist lab as they can be very dangerous and require industrial facilities.

Streak plating

To isolate a microorganism from a mixed culture, you can perform a technique known as streak plating. Before you perform any plating, you should ensure the Petri dish is labelled with your initials and the date. The sample should be removed from the mixed culture using a sterile wire loop and following aseptic techniques.

Biology; Microbiology and pathogens; KS5 Year 12; Culturing microorganisms and growth curves

Procedure

A	Open the lid of the Petri dish as little as possible and hold the loop parallel with the agar surface. Smear the inoculum backwards and forwards across a small area shown in blue and replace the lid. Flame the wire loop and allow it to cool.
B	Turn the Petri dish and open the lid as little as possible. Using the loop, streak the inoculum from the small area across in three parallel lines. Replace the lid of the Petri dish and flame the loop.
C	Turn the Petri dish again and repeat step B.
D	Turn the Petri dish one last time and repeat step B.

Measuring growth of bacterial cultures

To measure the the growth of bacterial populations, the number of bacterial cells needs to be known at repeated time intervals. There are two ways to do this: total count and viable count. Total count involves counting all of the cells in the culture and viable counts involves counting only the living cells in the culture.

Total count

Measuring total count can be done directly, or indirectly. You must first perform a serial dilution as it it likely your original sample will contain too many bacteria to count. You can then use your dilution factor to estimate the number of cells in undiluted culture.

Direct or indirect	Method	Description
Direct	Haemocytometer and light microscope	This is a special type of microscope slide, it has a depth so you know the volume of liquid you are adding. Using a light microscope you can count the number of bacterial cells in the sample and use this to work out the total count.
Indirect	Measuring dry mass	The culture must first be filtered to removed bacterial cells, the filter membrane containing bacteria are heating to $100\degree C$ until the mass is constant. The mass of the filter membrane can be subtracted from the total mass to work out the dry mass of bacteria.
Indirect	Measuring turbidity	As more bacteria grow in a culture, it becomes cloudier. This cloudiness, or turbidity can be measured using a colorimeter. The colorimeter will measure the amount of light that passes through the sample, this can be used to estimate total count.

Viable cell count

Viable cell count can be estimated by plating bacteria as only the growing bacteria will form colonies. This also requires a serial dilution to be performed.

Procedure

1.	Pipette a small, known volume of each dilution onto a Petri dish.
2.	Use a L-shaped spreader to spread the bacterial cells over the whole surface of the Petri dish.
3.	Flame the speader.
4.	The Petri dishes will be incubated and viable cells will form colonies on the plate that can be counted.

Bacterial growth curve

If you plot the number of bacteria against time you can produce a curve showing exponential growth. This is known as a bacterial growth curve.

1.	Lag	During the lag phase, there is no increase in the number of bacteria. The bacteria are alive but they are adapting to their new environment by switching on genes and synthesising important molecules.
2.	Log/Exponential	Bacterial cells are rapidly dividing by binary fission at their maximum rate.
3.	Stationary	As the nutrients in the media are beginning to be used up, the conditions are no longer optimal for bacterial growth so the cells begin to die. Here, the rate at which new cells are formed is equivalent to the death rate.
4.	Death	During the death phase, more cells begin to die as the media becomes a less suitable environment. Total cell count might fall.

The log phase

You can quantitatively analyse the number of cells during the log phase using three techniques.

1.	Finding the number of cells	$N= N_0 \times 2^n$
2.	The exponential growth rate constant	$\mu = {{2.303(logN_x - logN_0)}\over (t_x-t_0)}$
3.	Generation time	$g = {{0.301(t_x-t_0)}\over logN_x-logN_0}$

Symbol	Description
$N_0$	The number of original cells in the inoculum (when time $=t_0$ )
$N$	Number of cells
$n$	Number of generations
$\mu$	Exponential growth rate constant
$N_x$	Number of cells when time $=t_x$
$t_0$	Time when the number of cells $= N_0$
$t_x$	Time when the number of cells = $N_x$
$g$	Generation time

Example

A cell culture starts with $10,000$ cells, how many cells does it have after three generations?

=10,000\times 2^3

80,000

\underline{Therefore,\ after\ three\ generations\ there\ will\ be\ 80,000\ cells}

Example

A scientist samples her bacterial culture at $9\ am$ and counts $4\times 10^4$ cells in the sample. At $12\ pm$ she counts $6.2\times 10^5$ cells. Calculate the exponential growth constant.

The initial number of cells:

N_0=4 \times 10^4

Log this number:

logN_0=4.60

The number of cells at $t_x$ :

N_x =6.2 \times 10^5

Log this number:

logN_x= 5.79

Work out the time:

t_x-t_0= 3\ hours

Put into formula:

\mu = {2.303(5.79-4.60)\over 3}

Simplify:

= 0.914\ hour^{-1}

\underline{Therefore,\ \mu = 0.914\ hour^{-1}}

Example

A scientist samples her bacterial culture at $10\ pm$  and counts $3\times 10^2$  cells in the sample. At $9\ am$  she counts $5\times 10^6$  cells. Calculate the generation time.

The initial number of cells:

N_0=3 \times 10^2

Log this number:

logN_0=2.48

The number of cells at $t_x$ :

N_x =5 \times 10^6

Log this number:

logN_x=6.70

Work out the time:

t_x-t_0= 11\ hours

Put into formula:

g = {0.301\ \times\ 11 \over 6.70-2.48}

Simplify:

= 0.784\ hours

\underline{Therefore,\ the\ generation\ time\ is\ 0.784\ hours\ or\ 47\ minutes.}

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