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Site author Richard Steane
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Culturing microorganisms

I have put in a few (explanatory?) comments on terms in green text - mouseover for dropdown box (mouse only).
And you can test your own comprehension by mouseover or tapping at the > prompts.


The term microorganism refers to bacteria, fungi and other small organisms which are usually so small that they can only be seen using a microscope.

Bacterial reproduction

Bacterial cells increase in number as a result of individual cells 'simply' dividing to produce two - a process known as binary fission.

It may be said that bacteria multiply by division, which are opposite mathematically!

Each bacterial cell produced after division quickly grows to full size, and can divide again in a short time: every 20 minutes or 30 minutes are generation times often expected in optimal growth conditions.

What do you understand by optimal growth conditions?

> best environment
> containing enough (i.e. excess) nutrients - carbon and nitrogen sources
> suitable temperature - probably close to 37 °C.
> presence of air/oxygen for aerobic bacteria OR absence of air/oxygen for anaerobic bacteria
> no buildup of toxic wastes

Culture media

Bacteria can be grown - 'cultured' - on specially produced microbiological media.

Nutrient broth is a liquid medium : a fairly clear solution of carbohydrates e.g. glucose, and digested protein products. It is normally contained in a small (glass) tube.

Nutrient agar is a semi-solid medium - a gel - and it also contains a jelly-like extract obtained from seaweeds. It is normally contained in a Petri dish or 'plate'.

Aseptic technique

The prepar­ation of micro­biological media involves a lot of background work, so show your gratitude to the lab technician!
From a microbiological perspective, it is always assumed that bacteria and fungi of various sorts are widely distributed in the environment, and that includes the classroom and laboratories as well as the home and kitchen.

Media and their containers need to be sterilised before use. This is achieved using heat: 120° C is usually the temperature in an autoclave or pressure cooker. A period of 15 minutes at this temperature and pressure (15 PSI) kills all bacteria, including spores, but it takes time to heat up and cool down as the pressure reduces. In fact when the pressure is back to atmospheric it is still at 100 °C. After it has cooled more, molten agar can be poured out of bottles and into Petri dishes, then left to set. In fact Petri dishes are made sterile by heat used in the manufacturing process so they just need to be handled carefully before the agar is poured.

Special procedures are necessary when we are trying to encourage the growth of our own cultures of bacteria, and not the other microorganisms which can be regarded as contaminants. This is called aseptic technique - meaning without (unwanted) growth of micro-organisms.

This includes

Inoculation

[This term is also used in vaccination - which originally involved the introduction of (deactivated) disease-causing microorganisms into the body.]

How to prepare an uncontaminated culture using aseptic technique

Inoculation of broth or agar
Stage 2 grey colour shows a bacterial culture to be subcultured

Bacteria can be transferred into broth or onto agar plates using a mounted wire loop, which holds a small drop of liquid or a thin film of bacteria.
This may be taken from a broth culture, or a colony on another plate. Alternatively it may be from a food item or an environmental sample.
Sterile pipettes (disposable plastic or re-usable glass) can be used to transfer (known) slightly larger quantities of liquids.
Sterilised L-shaped glass or plastic 'spreaders', or even cotton swabs may be used to spread bacterial cultures evenly across the surface of agar.

After use, wire loops must be heated again to kill and burn away the remaining bacteria.
Other apparatus contaminated by bacteria should be placed into a beaker of disinfectant.

In the school environment, special precautions are necessary from this stage:
After an appropriate time, the bacterial cells produced build up next to one another to form 'colonies' which are easily visible, whereas bacteria in broth float apart and are not seen, but the broth becomes cloudy.

And as they grow, bacteria produce waste products which have a distinctive smell.

After incubated Petri dishes have been examined, they must be placed into a special autoclavable bag and disposed of, after heat treatment - back in the autoclave/pressure cooker.

Explanations required


Why must culture media be sterilised before use?
> to kill any contaminating microorganisms

Why must inoculating loops used to transfer microorganisms to the media be sterilised by strong heating using a flame?
> to kill any contaminating microorganisms, notably from its previous use

Why must the lid of the Petri dish be secured with adhesive tape?
> to keep the lid on! - for security

Why must they not be sealed all round the dges of the Petri dish, however?
> To prevent development of anaerobic conditions which favour the growth of some pathogenic bacteria

Why must Petri dishes be kept upside down in the incubator?
>To prevent condensation of water which could wash off bacteria and drip onto the hands when cultures are being examined

Why must cultures in school laboratories generally be incubated at 25 °C?
> Pathogenic bacteria are adapted to human body temperature (37° C) so they do not grow so well at temperatures below this

Bacterial population growth

How many bacteria will be present after a single cell is left to divide for one hour (60 minutes):
if they divide once every 20 minutes? Clue: > This is after 3 divisions >      8     
if they divide once every 30 minutes? Clue: > This is after 2 divisions >      4    

How many bacteria will be present after a single cell is left to divide for two hours (120 minutes):
if they divide once every 20 minutes? >      64     
if they divide once every 30 minutes? >      16    

As bacteria increase exponentially in numbers, it soon becomes necessary to express this using standard form: A × 10B, where A is a number between 1 and 10, and B is the power of 10 it is multiplied by.
So if B is 3, it is a number in the thousands, and if it is a 6 then it is millions. And of course it is soon a matter of reducing A to a sensible (low) number of significant figures.

You should be able to calculate the number of bacteria in a population after a certain time T if given the mean division time D.

Number of divisions N = T/D (Make sure you use the same units)

Number of bacterial cells = 2N

It is probably easiest to use ⅓ and ½ (hrs) for 20 and 30 minutes. For 24 hours N=(24 × 3) or (24 × 2).

How many bacteria will be present after a single cell is left to divide for a whole day (24 hours)):
if they divide every 20 minutes? >      4.72 × 10 21   (272)  
if they divide every 30 minutes? >      2.81 × 10 14   (248)  

The actual number of bacteria in a nutrient broth culture (or another liquid, or even solid e.g.food item) can also be calculated directly by following the technique of dilution plating (reference below).

Medical microbiology

Usually, there are no bacteria within most parts of the human body, but there are large numbers of bacteria in the colon (large intestine). Bacteria can enter the body via the mouth and nose (and other 'openings'), but they usually do not last long as they are trapped by digestive juices and mucus, and usually inactivated by the body's defence systems. Some bacteria do not present much of a risk to the body, whereas others may cause diseases. These are called pathogenic. There are different species of bacteria, as well as different strains, with different characteristics.

Bacteria may enter wounds or become attached to some parts of the body . . .

For instance, if someone has a throat infection, bacteria may grow on the surface of the skin there.
These bacteria may be sampled using a (sterile) throat swab - like a long cotton wool bud, kept in a tube.

Sterile throat swab

swab (41K)



This will be re-sealed into its tube, taken to the laboratory and used to spread any bacteria that have been picked up from the throat onto the surface of an agar plate. This is called streaking, and there are a number of spreading techniques aiming to separate individual bacterial cells so that they can each form distinct colonies. Sometimes these colonies are distinctively different, which shows there are a number of different bacterial species or strains present in the sample, or that the source is contaminated with other bacteria.

A streak plate on blood agar
streak (101K)
Individual colonies can be seen.


A ring containing different antibiotic discs

antibiotics_ring (45K)
More detail here
In medical/pathology labs, it is normal to include ingredients such as (animal or human ) blood in agar to simulate conditions within the body, and to see whether the bacterial strain has the ability to break open red cells. 'Haemolytic streps' (Streptococci) can cause painful sore throats.

Sometimes it is considered worthwhile to test the susceptibility of a particular strain of bacteria to antibiotics - so that a patient will be prescribed an appropriate antibiotic to treat an infection. It is obviously necessary to have a pure or uncontaminated bacterial culture before proceding with this.

In order to do this, it is necessary to choose a bacterial colony which is well separated from any others on the streak plate, and pick up a sample of bacterial cells from this, probably using a sterilised loop.
This is then cultured in broth or diluted in a small amount of (sterile) liquid, then spread on the surface of an agar plate, forming a 'bacterial lawn'. Paper discs containing a variety of antibiotics are placed on top of this, and the antibiotic will diffuse out in all directions, becoming more dilute as it moves.

This plate is then incubated for a few hours. As the bacteria grow, they will form a whitish film on the agar but if they are killed by one or more of the antibiotics this will result in fairly circular clear 'zones of inhibition'. The size of these zones will give a measure of the susceptibility of this bacterium to the various antibiotics. The patient may then be treated with the most effective antibiotic.

Required practical activity

This technique may be simulated in the school or college laboratory as an exercise to investigate the effect of antiseptics or antibiotics on bacterial growth using agar plates and measuring zones of inhibition. A non-pathogenic bacterium such as Escherichia coli or the yellow Micrococcus luteus may be used

Using all the appropriate aseptic technique, a broth culture of the bacteria can be spread on the surface of a plate, and discs of paper, impregnated with the antibacterial substances, are also placed on the surface, then it is incubated for some time.

After the bacteria have had a chance to grow to produce a bacterial lawn, the diameter of the resulting clear areas where bacteria have not grown - zones of inhibition - can be measured using a ruler. It is best to do this twice at right angles, and average the results, then halve this to get the radius r, and using π=3.14, calculate the area ('cross-sectional area') - πr2.


zones_of_inhibition (36K)

Obviously the antibacterial substance showing the largest zone of inhibition is the most effective.

A measurement exercise

Mouseover for another angle

plate-plus_ruller (23K)

What is the cross-sectional area for substance A above?

Diameter = > 2.1 cm
Radius r = > 1.05 cm
π r2 = > 3.46 cm2




This topic has connections with other units on this site:

Microbiological techniques - All very close to this topic
Experiments to show the growth of bacteria - basic techniques - I am rather proud of these graphics
Streaking - Keep your clothes on for this
Dilution plating- A procedure used to find the number of viable micro-organisms in a fixed amount of a liquid
Testing bacteria for sensitivity to antibiotics - Rather similar to the required practical task!
Production of Penicillin in Industrial Fermenters (Bioreactors) - The technology behind the pills!


Cell biology
Eukaryotes and prokaryotes - The two different groups of organisms
Bacterial cell structure - Details at the microscopic level
Bacterial Reproduction - Part of a series on asexual reproduction - but surprisingly similar calculation

Other sources of information

GCSE Biology: Required practical activities - DRAFT outline of several practicals from AQA

Investigating antibiotic resistance - advice from Philip Harris about GCSE Biology required practicals (and opportunities to obtain all the products required for the practical!)

Sore throat - from the NHS - reminding us that sore throats do not always merit medical attention

Throat swab (pharyngitis tonsillitis) - Thanks to NHS Hospitals Gloucester

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