What are the different steps in culturing microorganisms?

Sample poured onto the medium. Sample spread evenly on medium. Microorganisms are then incubated in their culture medium.

What are the five basic techniques used to culture microorganisms?

Inoculation Isolation Incubation Inspection Identification

Culturing Microorganisms

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In order to study microorganisms like bacteria or fungi, scientists need to be able to grow or culture them in an 'artificial' laboratory setting. This allows us to better understand them and their possible applications in our lives, as well as what they are sensitive to and how to destroy them if needed. It's vital to learn how to handle and culture microorganisms in a lab safely because they can multiply quickly and some of them can be very harmful to us!

Microorganisms are microscopic organisms like bacteria, fungi or algae. There are both prokaryote and eukaryote microorganisms and all require specific conditions to be successfully grown in laboratories.

Read our article Eukaryotes and Prokaryotes to find out more about these two types of organisms!

What is the importance of culturing microorganisms and what are they used for?

Essentially, by cultivating and studying microorganisms we can learn more about them. In medicine, we can identify what microorganisms are causing an infection in a sick patient by isolating and culturing any microorganism present in a patient sample. Bacteria culture tests are often used for this purpose to help find harmful bacteria in the body and diagnose bacterial infections. In fact, bacteria are the most common microorganism cultured in a lab. Patient samples can be taken for example of blood, urine, stool, mucus, skin or even spinal fluid for microorganism culturing and investigation. This allows doctors to identify the microorganism (i.e. the bacteria) and decide the best way to treat the infection. When culturing bacteria we can test their antibiotic susceptibility and treat infections based on this information.

We can use bacterial cultures to measure the effectiveness of antibiotics, antiseptics or disinfectants by calculating the size of the zone of inhibition around the substance being tested. The zone of inhibition is the area that is not colonised by bacteria due to the presence of antibiotics. To calculate this area, you can simply use the equation πr². (area of the circle where r is its radius)

Read our article Uses of Antibiotics to find out more about these drugs and how they can be used to treat bacterial infections!

Besides infection diagnosis, culturing microorganisms is also important for example for research purposes that involve genetic manipulation of the microorganism, for epidemiological studies or to develop vaccines and other therapeutics.

Culturing microorganisms is essential to detect any possible food contaminants. Harmful bacteria like Salmonella and Campylobacter can contaminate our food and be responsible for food poisoning. Food microbiology plays an important role in public health.

Lastly, culturing microorganisms has several important applications in biotechnology. Microbial biotechnology plays an important role in the health, agriculture, chemical, energy, environment and food and beverage industries.

Bacteria and yeast (fungi) cultures are very important in the production of many foods and beverages.

Culturing yeast is important in the making of products like beer, bread and wine while culturing bacteria is used in the making of cheese and yoghurt. These microorganisms are used by these industries because of their ability to perform fermentation. In this process, some yeast and bacteria convert sugars into relevant byproducts (ethanol, lactic acid) that confer the characteristic texture and flavour of these products and improve their preservation.

Culturing Microorganisms Figure 1: Fermentation | StudySmarter Figure 1 . Wine fermentation.

Broadly, microorganisms have several characteristics that explain why they have so many uses. These include

High adaptability
Fast growth
Easy to culture

Cultures of microorganisms

Culturing involves multiplying microorganisms in a controlled way and under laboratory conditions. In order for culturing to work, scientists must be able to mimic the environmental conditions needed for these microorganisms to not only survive but also grow in a lab setting. Everything the microorganism needs must be provided and controlled to optimized standards. Conditions that must be controlled for successful microbial cultures include:

Temperature
pH
Sterility
Nutrients

In microbiology, sterility refers to the absence of contaminating viable microorganisms.

The growth of each type of microorganisms can be optimised by providing the best possible range in each of these conditions. This ensures our microbial culture is able to thrive. We can provide nutrients to the microorganism by using a culture medium. This medium can be in either liquid or solid (gel) form. It provides the surface for the microorganisms to grow on and contains all the essential nutrients. These usually include minerals, a nitrogen source (for protein production) and carbohydrates (energy source) and other chemicals that the microorganisms would usually find in nature.

Microorganisms usually also require warmth and oxygen to be able to grow, but not all have the same temperature requirements and some may even be able to grow in the absence of oxygen! pH is also important as it affects the structure of macromolecules so it must be kept to the microorganisms' preferable range.

Culture methods and media in microbiology

The two most common methods of growing cultures are using nutrient broths in sterile flasks or agar gel plates. Nutrient broth and agar gel are different in the way that to make the gel media you simply add agar to nutrient broth, melt it and it solidifies in a petri dish.

Nutrient Broth Solution: A solution that contains carbohydrates for energy, nitrogen for protein synthesis as well as other minerals to provide optimum conditions for microorganism growth.

Agar plate: Bacteria are spread on plates made by pouring hot molten agar into sterile Petri dishes, which then sets. The bacteria form colonies on the agar surface.

Culturing Microorganisms Figure 1: Petri Dish | StudySmarter Figure 2. Scientist holding petri dish.

Steps in colonising bacteria

When culturing bacteria in agar plates the following general steps are followed:

The sample is poured onto the culture medium.
The sample is spread evenly on the culture medium.
The plate is incubated (kept warm) until bacteria colonies grow on the surface of the medium/agar.

In microbiology, it's very important that pure microbial cultures don't get contaminated with other microorganisms. This is for a few reasons:

You don't want bacteria to be competing with the contaminating bacteria for nutrients.
Some microorganisms such as pathogens are very harmful and you wouldn't want them to be cultured.
It's important that the results are as reliable and valid as possible when testing for example the efficacy of certain antibiotics or other chemicals. To achieve uncontaminated cultures of microorganisms, scientists use aseptic techniques.

Aseptic techniques are laboratory procedures carried out to prevent the contamination of pure cultures of microorganisms.

Aseptic techniques

Several standard laboratory aseptic techniques and how they help keep equipment sterile and microbial cultures pure are described below.

Technique	Why do we use it?
Work is carried out in front of a bunsen burner with a yellow flame.	A convection current is created above the bench meaning that the air isn't contaminated.
The Petri dishes and agar gel must be sterilised before use using an autoclave or by purchasing pre-sterilised plastic Petri dishes.	Any potential microorganisms that could compromise the experiment are killed.
The inoculating loop is heated to red hot in a bunsen flame, before and after use.	Microorganisms on the loop are killed to prevent contamination.
The lid of the agar plate is taped at intervals.	This allows oxygen in, so harmful anaerobic bacteria (bacteria that do not require oxygen to grow) can't grow.
The neck of the bottle of microorganisms is placed in the flame.	This kills microorganisms that could be on the neck of the bottle.
Store the petri dish upside down during incubation.	This prevents condensation from dripping onto the agar.
The cultures should not be incubated above 25ºC in schools.	Harmful pathogens which grow at higher temperatures cannot grow.
Swirl the bacterial suspension rather than shaking it to ensure the culture is well mixed.	To make ensure the bacteria aren't all at the bottom of the container.

Figure 2 - Aseptic techniques

General laboratory rules like using gloves and lab coats, clearing the workspace of any non-essential items and cleaning the lab surfaces often, not only help keep scientists safe but help prevent contamination of their cultures. Contamination of microbial cultures can originate from bacteria present on our skin, the air, soil or water present, as microorganisms especially bacteria are everywhere!

Autoclaves are heated vessels, a type of special oven, used to sterilise equipment using steam at high pressure.

Bunsen Burners are gas burners typically used as heat sources in laboratory benches.

Inoculating loops are used for inoculating or spreading a sample like bacteria on the agar plate.

5 I's of culturing microorganisms

Culturing microorganisms (bacteria) follows the 5 I's principle:

Inoculation: Introducing the sample in a culture medium using aseptic techniques.
Incubation: Growing microbes under proper favourable conditions like the right temperature.
Isolation: Separating different species of microbes.
Inspection: Observation of microbial characteristics.
Identification: Use of data collected in the inspection stage to identify the microorganism.

Characteristics of cultures

Individual bacterial cells are too small to be viewed without a microscope but in the ideal conditions of an agar plate, the cells divide many times to form a colony which is easily seen. This is because bacteria rapidly divide by binary fission.

Binary fission is a simple form of cell division in which bacteria replicate their DNA and divide. It's a form of asexual reproduction that can be carried out as fast as every 20 minutes (however this time can vary and is called the mean division time). Starting with 1 bacteria, at 20mins there are 2, at 40mins there are 4, at one hour there are 8 and so on!

Asexual reproduction: A type of reproduction that does not involve the fusion of gametes or change in the number of chromosomes.

There is a very simple way to calculate the number of bacteria in a population:

1. Calculate how many times the bacteria will divide in a given time period:

Assuming the division time is 20mins, divide this period by 20.

For example, in 8 hours of bacterial growth, the bacteria will divide 3 times every hour (8 $\times$ 3 = 24 divisions).

2. Calculate the total number of bacteria by using the formula:

Number of bacteria at the start $\times$ 2^{(number of divisions)}

Following the previous example, 1 $\times$ 2²⁴= 16, 777, 216 bacteria are present after 8 hours.

Culturing Microorganisms - Key takeaways

Microorganisms are microscopic organisms like bacteria, fungi or algae. They require specific conditions to be grown in laboratories.
By culturing and studying microorganisms, we can learn more about them. For example, their antibiotic susceptibility and how we can treat diseases caused by them.
Culturing involves multiplying microorganisms in a controlled way and under laboratory conditions.
The two most common methods of growing cultures are using nutrient broths in sterile flasks or agar gel plates.
Microorganisms have so many uses because:
- They are highly adaptable
- They grow fast
- They are easy to cultivate

References

Figure 2: Petri Dish (https://commons.wikimedia.org/wiki/File:Petri_dish_at_the_Pacific_Northwest_National_Laboratory_high_res.tif) by Pacific Northwest National Laboratory, US Department of Energy. Public Domain.
Figure 1: Wine Fermentation (https://www.flickr.com/photos/20721230@N00/4003299453) by Ian Brown (https://www.flickr.com/photos/igb/). Licensed by CC BY 2.0 (https://creativecommons.org/licenses/by/2.0/deed.en).

Frequently Asked Questions about Culturing Microorganisms

Sample poured onto the medium.
Sample spread evenly on medium.
Microorganisms are then incubated in their culture medium.

Inoculation
Isolation
Incubation
Inspection
Identification

Inoculation
Isolation
Incubation
Inspection
Identification

By cultivating and studying micro-organisms, we can learn more about them. For example, their antibiotic susceptibility and how we can treat diseases caused by them. Microorganisms have so many uses because:

They are highly adaptable
They grow fast
They are easy to cultivate
They produce many products

Culturing involves multiplying microorganisms in a controlled way and under laboratory conditions.

Flashcards in Culturing Microorganisms5 Start learning

What is an agar plate?

Bacteria are spread on plates made from pouring hot molten agar into sterile Petri dishes which then sets. The bacteria form colonies on the agar.

Why are petri dishes stored at a maximum of 25 degrees in schools?

Harmful pathogens which grow at higher temperatures cannot grow.

What is an autoclave?

A heated vessel used to sterilise equipment.

What is asexual reproduction?

A type of reproduction that does not involve the fusion of gametes or change in the number of chromosomes.

What are the 5 I's of culturing microorganisms?

Inoculation
Isolation
Incubation
Inspection
Identification

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