An Introduction to Culturing Bacteria

Bacteria are an essential part of the ecosystem. They are crucial to our health and the environment, have an important role in food production and provide bioengineers with tools to harness their properties and manufacture compounds. However, they can also be harmful, causing damage and disease. The ability to grow these microbes is therefore an essential step in being able to utilize their power, identify harmful culprits and advance our understanding and capabilities. In this article, we consider what bacterial culture is, factors affecting culture conditions, common problems and some of the numerous applications.

What is bacterial culture?

Bacterial culture is a method that allows the multiplication of bacterial cells in or on a culture medium under controlled laboratory conditions. The exact conditions required for optimal replication will depend on the target bacterial species.

What's the difference between aerobic culture vs anaerobic culture?

Most bacteria can grow to some extent in the presence of oxygen, known as aerobic culture. But for optimal growth, the conditions should be adjusted to suit the target bacterium. Species found in atmospheric conditions, such as on the skin surface or in the upper respiratory tract, will typically grow well in the presence of oxygen. Species that are naturally found in low oxygen environments, such as in deep wounds or abscesses or the deep ocean, will typically grow best in the absence of oxygen – anaerobic culture. Some cannot grow in the presence of oxygen at all, and these are called obligate anaerobes. Examples include Fusobacterium and Bacteroides.¹ Likewise, those that cannot grow in the absence of oxygen are called obligate aerobes. Examples for the purposes of culture include the Gram negative Pseudomonas aeruginosa² and Mycobacterium tuberculosis,³ the causative agent of tuberculosis. However, studies suggest that both can undertake anaerobic respiration in certain circumstances. Bacteria that can grow in either aerobic or anerobic conditions, switching from aerobic respiration to fermentation or anaerobic respiration if oxygen is absent, are called facultative anaerobes. Examples include the Gram positive staphylococci,⁴ Escherichia coli (E. coli),⁵ Salmonella⁶ and Listeria spp..⁷

Bacterial culture methods

To be cultured successfully, bacteria require the provision of nutrients in the culture medium. There are many different formulations available to suit the differing nutritional needs of bacterial species. The type of medium you choose will depend on the purpose of the culture. Rich, nutrient or complete media can be helpful when trying to bulk up a pure culture and get the bacterial cells in good condition. Minimal media on the other hand will supply only the bare necessities for survival and can be useful in manipulating which pathways are turned on in the bacterium.

Media may also be classed as defined or undefined. As the names suggests, in a defined media, all the ingredients are known. Undefined media tend to contain complex mixtures of nutrients and chemical species in unknown proportions, such as yeast extract.

Whichever medium is chosen, this may be in liquid form as a broth culture, or agar may be added to set the media and allow bacterial cells to be grown on a solid surface.

Culture broth

Culture in liquid media, also known as a broth culture, gives the bacteria present easy access to the available nutrients compared to static bacterial colonies. Gentle agitation to keep the bacteria dispersed through the medium during incubation can aid this access further. Liquid media will also dilute out waste products as they are formed, distributing them through the culture. Consequently, a greater mass of bacteria may be obtained for an equivalent volume of liquid as opposed to solid media.

You may therefore want to use broth cultures when aiming to bulk up your culture, e.g., when using bacteria to produce a desirable compound, in food production or to extract DNA or plasmids from.

When looking to store bacterial strains long term, they may be grown up in liquid media. Glycerol is then added, which will prevent complete freezing and consequent lysis of the bacterial cells, permitting their storage at -80 °C. Long-term storage in this manner preserves strains – helpful when collecting strains over a long period of time – prevents the loss of valuable strains and also reduces the risk of mutations that may occur from repeated passaging.

Nutrient agar

Adding agar to liquid media enables it to be set in petri dishes, as slopes or in plugs for example. Solid media is useful when you wish to select individual colonies from a mixed culture, for example when purifying a diagnostic sample. If you wish to enumerate the number of colony forming units (cfu) within a given volume of liquid sample, plating and incubation on solid media also permits this. Inoculation onto slopes or in stab cultures can also be a convenient method for transporting strains from lab to lab without the danger of spillage of potentially infectious materials.

Selective and differential media

Selective media⁸ are also available that promote or suppress the growth of certain species, groups of species or strains with particular properties. This may be based on a strain’s ability to utilize specific nutrients, produce certain byproducts or resistance to certain antibiotics. Selection may be used in both broth and solid media.

The ability of the strain to grow or not may be indicated by color changes in differential media and is often used to identify bacterial species or subtypes. For example, with analytical profile index (API) test strips, bacteria are cultured with a range of substrates, producing differing color change patterns based on their metabolism, thus enabling identification.

Where strains are hemolytic, growth on blood agar enables the type of hemolysis to be evaluated, helping to identify the species present (Figure 1).

Figure 1: Blood agar culture showing alpha (left), beta (center) and gamma (right) hemolysis. Credit: Mibilehre, reproduced under the Creative Commons Attribution-Share Alike 4.0 International license.

Addition of antibiotics to liquid media will prevent the growth of non-resistant strains. This may be helpful when culturing an engineered strain into which an antibiotic resistance gene has been added as a marker. Growth of contaminating species or colonies in which the engineering has been unsuccessful will therefore be selected against.

Antibiotics may be added to solid media during preparation, fulfilling a similar role to that in liquid media. Alternatively, antibiotic-infused disks may be placed onto solid media onto which a stain of interest has been inoculated. Where the strain is sensitive to the antibiotic, a clear zone of no growth will then be visible around the disc as the bacterial lawn grows, enabling, for example, a suitable antibiotic to be chosen for treatment of an infection (Figure 2).

Figure 2: Antibiotic resistance tests; the bacteria in the culture on the left are sensitive to the antibiotics contained in the white paper discs. The bacteria on the right are resistant to most of the antibiotics. Credit: Dr Graham Beards, reproduced under the Creative Commons Attribution-Share Alike 4.0 International license.

As well as the oxygen conditions and nutrient requirements already discussed, the temperature and humidity at which different species will grow optimally varies too, reflecting their natural habitat. Species typically found deep in the body, such as the gut or lower respiratory tract, are likely to grow best at 37 °C – body temperature. Conversely, species found, for example, in soil are likely to require cooler temperatures. When performing genetic manipulations on bacteria, temperature can be used as a switch to control the integration of temperature-sensitive plasmids and consequently promote the desired outcome.

What's a bacterial growth curve?

While rates of division will vary between bacterial species, they will normally follow the same general growth pattern in broth culture. The number of bacterial cells in a culture can be estimated by various means including plating and colony counting or by measuring the turbidity of the culture with UV-visible spectroscopy. When this is plotted (typically on a logarithmic scale) against time, it is known as a growth curve,⁹ as depicted in Figure 3.

Figure 3: Example of a bacterial growth curve showing the 1) lag phase, 2) exponential/log phase, 3) stationary phase and 4) death phase.

1.       Lag phase – Bacteria are adjusting to their new growth conditions. The length of this phase will depend on how similar these are to their previous conditions and the condition of the cells. The bacteria may need to repair themselves, produce enzymes and RNA for replication or synthesize molecules that are lacking in their surroundings.

2.       Exponential or log phase – Once the cells have adjusted to their conditions and have the molecules they require, cell division begins in earnest. This follows a predictable doubling pattern, the duration of which will depend on how well suited the conditions are to the bacterial species. Rapid growth, and consequently a steep slope, will occur where conditions are close to optimal. This is the point at which the bacterial cells are healthiest and so typically the phase from which cells are utilized for other experiments.

3.       Stationary phase - Nutrients become exhausted, waste products build up and space may run short, slowing further division such that the number of new cells produced equals the number dying. This is seen as a flattening out of the growth curve. New bacterial cells undergo physiological changes in an attempt to adapt to starvation conditions. For spore-producing species, sporulation may also begin.

4.       Death or decline phase – As conditions no longer favor growth, a steady deterioration in the condition of the cells present is observed leading to a decline in the growth curve. Non-viable cells may still contribute to turbidity measures where they are used to estimate cell numbers, keeping values higher than the number of truly viable cells. Usually, some cells will always remain viable as they mutate or enter a dormant state to survive.
 

Obtaining a pure culture

A pure culture is one that only contains the bacterial species you wish to grow. The ease with which this can be achieved is likely to depend very much on the source of your sample, the abundance of the target species compared to other species and the target species itself. If your source is another pure culture or a strain that has been isolated and stored in the freezer, then the culture may already be pure. If, however, the source is a clinical or environmental sample, there are likely to be many other bacterial species and potentially fungi present too that will also grow happily in your culture conditions. Selective media and restricted growth conditions (e.g., aerobic vs anerobic culture) can help to eliminate non-target species and narrow the field. Streaking the sample onto solid media rather than into broth culture will allow visual identification of colonies of interest from the general background. It may be necessary to pick and re-streak bacterial colonies of interest onto fresh agar plates a few times before a pure culture can be obtained. Once this is achieved, they may then be grown in liquid culture if desired. If the target species is present only in low numbers, it may be necessary to streak multiple plates from the original sample in order to isolate them. Some species grow more rapidly and vigorously than others, so this too is a factor to consider.

It may not always be necessary to obtain a pure culture depending on the purpose of your experiment. If it is possible to identify the target species among a background of others and this is sufficient for your purpose, then obtaining a pure culture may not be required. However, if you wish, for example, to perform further targeted assays or the bacteria are being cultured for production or food purposes, then obtaining and maintaining a pure culture may be essential.

Common problems with bacterial culture

•  Contamination – Contamination of bacterial cultures can be very problematic, particularly if it goes undetected. At best, it can mean the need to reisolate a pure culture, but at worst could lead to illness and very costly remedial work if it were to occur in a food or production setting. Culture contamination can come from many sources, right from the original sample itself through the process of culturing and even storage. Good aseptic technique can help to avoid contamination of bacterial cultures.
  
  
• Overgrowth of some species – Some bacterial species grow easily and vigorously. When attempting to isolate a species from a mixed sample, these vigorous species may overgrow and mask the presence of slower-growing target species. Using selective media and optimal growth conditions for your target species (if known) can help to mitigate this. Try to culture the sample as soon as possible after it has been taken to ensure it is as representative as possible.
  
  
• Antibiotic treatment prior to sampling – In a diagnostic setting, it is important to know if antibiotic treatment has been administered prior to sampling. If this is the case, failure to culture a particular species may not indicate that it was not the cause of the infection.
  
  
• Incorrect growth conditions – The use of inappropriate or suboptimal growth conditions may impede or completely prevent the growth of your target strain. Be sure to double check growth requirements or if using antibiotic selection ensure the correct antibiotic has been chosen for the resistance gene present.
  
  
• Non-culturable and slow-growing organisms – Some bacterial species, even now, cannot be grown in the lab.¹⁰ Others, such as mycobacteria,¹¹ are very slow growing and can take months to culture successfully, which is particularly problematic when trying to diagnose infections.

Applications of bacterial culture

There are many reasons why it may be necessary or desirable to culture bacterial cells. Here, we consider some of the common purposes.

Diagnose infection

Despite the length of time it can take to isolate and identify bacterial species from a sample, bacterial culture remains an important diagnostic tool.¹² While PCR may rapidly identify the presence of a specific pathogen, isolating the culprit will confirm that it is alive, alerting analysts to potential transmission risks and informing treatment. It also means the bacterial strain can be interrogated further for information like antibiotic sensitivity, directing treatment choices. Strains may also be stored down for future reference, for example for disease monitoring purposes.

Genetic manipulation

It may be desirable to manipulate the genome of bacterial strains for a number of reasons; trying to understand the basic biology, to attenuate it when creating vaccine strains, to overproduce proteins and to create a reference strain with a detectable marker to name just a few. Whether mutating, deleting or inserting genetic material, there is a fundamental need to culture the strain of interest¹³ before, during and after the genetic engineering process.

Epidemiological study

Culturing and characterizing bacterial strains can be vital for epidemiological studies.¹⁴ This enables scientists to study how bacterial populations change over time – which can inform therapeutic, vaccine and diagnostic design and updates – and study transmission events which can in turn inform things like public health policy and advice. The Gonococcal Isolate Surveillance Project (GISP) is one such project that monitors strains for antibiotic resistance, helping to inform drug therapy recommendations. The Centers for Disease Control and Prevention (CDC) also runs the Active Bacterial Core surveillance (ABCs) system, providing laboratory- and population-based surveillance of invasive bacterial pathogens of public health importance.

Scale up to enable omics studies

While sequencing of DNA and RNA can be performed with tiny amounts of genetic material, even on the single-cell level, for many studies, next-generation sequencing (NGS) is still performed on material from many bacterial cells and as such, the bacteria often need to be cultured prior to DNA or RNA extraction.¹⁵ If you are interested in a specific strain (unlike microbiome studies which will contain a mix) then this will likely be derived from a pure culture.

Develop vaccines and therapeutics

In order to fight a bacterial pathogen, you typically need to be able to culture that pathogen too. During the development of vaccines,¹⁶ it may be necessary to culture strains to understand their genomes, amplify their genes or manipulate them. Equally, in order to test out candidate vaccines or therapies, it is often necessary to perform challenge experiments¹⁷ in which individuals are challenged with the pathogen to see if the therapy is effective. To do this, the bacterial strain is normally grown and, in a defined challenge model, enumerated to control and determine the dose subjects receive.

Food and beverage production

Bacteria are an important part of the production of many foods and are broadly split into probiotics and starter cultures.

Probiotics are generally cultured for their benefit to human health,¹⁸ often through our gut microbiome. While probiotics may contain many different bacterial species, Lactobacillus and Bifidobacterium are common choices for culture.

Starter cultures on the other hand are typically used as part of a food production process, to develop flavor, texture, nutritional value or improve preservation. Examples include sourdough breads, salami,¹⁹ pepperoni and dried ham. Lactic acid bacteria (LAB) are commonly found among starter cultures. Some foods and drinks could however, arguably sit in both camps, such as yogurt and the increasingly popular kimchi²⁰ and kombucha where products are consumed for their flavor as well as their probiotic benefits.

Regardless of the purpose a culture is intended for, maintaining a healthy, contaminant-free culture is vital for optimal production and consumer safety.

Detecting food contaminants

While some bacteria may be desirable in food production, they may also be present as a contaminant and have the potential to cause serious foodborne illnesses. Common causes include Salmonella sp., Listeria monocytogenes, Campylobacter jejuni and E. coli. It is therefore important that analysts are able to culture any potentially hazardous bacteria from food samples, even if they are present in low numbers.

References

1. Garrett WS, Onderdonk AB. 249 - Bacteroides, Prevotella, Porphyromonas, and Fusobacterium Species (and other medically important anaerobic Gram negative bacilli). In: Bennett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases (Eighth Edition). W.B. Saunders; 2015:2773-2780. doi:10.1016/B978-1-4557-4801-3.00249-6

2. Crespo A, Pedraz L, Astola J, Torrents E. Pseudomonas aeruginosa exhibits deficient biofilm formation in the absence of class II and III ribonucleotide reductases due to hindered anaerobic growth. Front Microbiol. 2016;7:688. doi:10.3389/fmicb.2016.00688

3. Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev. 2003;16(3):463-496. doi:10.1128/CMR.16.3.463-496.2003

4. Harris L, Foster S, Richards R. An introduction to Staphylococcus aureus, and techniques for identifying and quantifying S. aureus adhesins in relation to adhesion to biomaterials: review. eCM. 2002;4:39-60. doi:10.22203/eCM.v004a04

5. von Wulffen J, Sawodny O, Feuer R. Transition of an anaerobic Escherichia coli culture to aerobiosis: Balancing mRNA and protein levels in a demand-directed dynamic flux balance analysis. PLoS One. 2016;11(7):e0158711. doi:10.1371/journal.pone.0158711

6. Parkin A, Bowman L, Roessler MM, et al. How Salmonella oxidises H2 under aerobic conditions. FEBS Letters. 2012;586(5):536-544. doi:10.1016/j.febslet.2011.07.044

7. Lungu B, Ricke SC, Johnson MG. Growth, survival, proliferation and pathogenesis of Listeria monocytogenes under low oxygen or anaerobic conditions: a review. Anaerobe. 2009;15(1-2):7-17. doi:10.1016/j.anaerobe.2008.08.001

8. Bonnet M, Lagier JC, Raoult D, Khelaifia S. Bacterial culture through selective and non-selective conditions: the evolution of culture media in clinical microbiology. New Microbes New Infect. 2020;34:100622. doi:10.1016/j.nmni.2019.100622

9. Maier RM. Chapter 3: Bacterial growth. In: Maier RM, Pepper IL, Gerba CP Environmental Microbiology. Academic Press. 2000: 37-54. ISBN:978-0-12-497570-5

10. Bodor A, Bounedjoum N, Vincze GE et al. Challenges of unculturable bacteria: environmental perspectives. Rev Environ Sci Biotechnol 19, 1–22 (2020). doi:10.1007/s11157-020-09522-4

11. Pfyffer GE, Wittwer F. Incubation time of mycobacterial cultures: How long is long enough to issue a final negative report to the clinician? J Clin Microbiol. 2012;50(12):4188-4189. doi:10.1128/JCM.02283-12

12. Giuliano C, Patel CR, Kale-Pradhan PB. A guide to bacterial culture identification and results interpretation. P T. 2019;44(4):192-200. PMC6428495

13. Fels U, Gevaert K, Van Damme P. Bacterial genetic engineering by means of recombineering for reverse genetics. Front. Microbiol. 2020;11:2161. doi:10.3389/fmicb.2020.548410

14. Parkhill J, Wren BW. Bacterial epidemiology and biology - lessons from genome sequencing. Genome Biol. 2011;12(10):230. doi:10.1186/gb-2011-12-10-230

15. Gautam SS, KC R, Leong KW, Mac Aogáin M, O’Toole RF. A step-by-step beginner’s protocol for whole genome sequencing of human bacterial pathogens. J Biol Methods. 2019;6(1):e110. doi:10.14440/jbm.2019.276

16. Giesker K, Hensel M. Bacterial vaccines. In: Reference Module in Biomedical Sciences. Elsevier; 2014. doi:10.1016/B978-0-12-801238-3.00141-0

17. Osowicki J, Azzopardi KI, Fabri L, et al. A controlled human infection model of Streptococcus pyogenes pharyngitis (CHIVAS-M75): an observational, dose-finding study. Lancet Micro. 2021;2(7):e291-e299. doi:10.1016/S2666-5247(20)30240-8

18. Bazireh H, Shariati P, Azimzadeh Jamalkandi S, Ahmadi A, Boroumand MA. Isolation of novel probiotic Lactobacillus and Enterococcus strains from human salivary and fecal sources. Front Microbiol. 2020;11:3064. doi:10.3389/fmicb.2020.597946

19. Yoon JY, Kim D, Kim EB, Lee SK, Lee M, Jang A. Quality and lactic acid bacteria diversity of pork salami containing kimchi powder. Korean J Food Sci Anim Resour. 2018;38(5):912-926. doi:10.5851/kosfa.2018.e24

20. Song HS, Whon TW, Kim J, et al. Microbial niches in raw ingredients determine microbial community assembly during kimchi fermentation. Food Chem. 2020;318:126481. doi:10.1016/j.foodchem.2020.126481