- Why are vegetarians more likely to suffer from depression than meat eaters?
- Small wireless device implanted between skin and skull helps kill cancer cells
- Will the mRNA vaccine that can cure cancer come out near soon?
- Allogeneic T-cell therapy set for landmark first approval
- Boston University denies that the new COVID strain they made has 80% fatality rate
- A new generation of virus-free CAR-T cell therapy
Medicine knowledge: How to carry out bacterial culture?
- First human trial of HIV gene therapy: A one-time cure will be achieved if successful!
- New breakthrough in CAR-T cell therapy: Lupus erythematosus patients achieved treatment-free remission for up to 17 months
- How long can the patient live after heart stent surgery?
- First time: Systemic multi-organ recovery after death
- Where do the bacteria in the human gut come from?
Medicine knowledge: How to carry out bacterial culture?
Bacteria are an important part of the natural ecosystem. They are vital to our health and the environment, play an important role in the production of medicines, and provide bioengineers with tools to use their properties and make compounds.
However, they can also be harmful and can cause human damage and disease. Therefore, cultivating these microorganisms is an important step to be able to use their power, identify harmful culprits, and improve our research understanding and ability.
In this article, we will discuss what bacterial culture is, factors affecting culture conditions, common problems, and some of the many applications.
What is bacterial culture?
Bacterial culture is a method that allows bacterial cells to proliferate in a controlled culture medium under laboratory conditions. The exact conditions required for optimal culture conditions will depend on the target bacteria species.
Aerobic culture and anaerobic culture
Most bacteria can grow and reproduce to a certain extent under aerobic conditions, which is called aerobic culture . But in order to achieve optimal growth, the conditions should be adjusted to suit the target bacteria.
Species found under natural conditions, such as on the surface of the skin or in the upper respiratory tract, usually grow well under aerobic conditions.
Species found naturally in hypoxic environments, such as deep wounds, abscesses or deep seas, usually grow best under hypoxic conditions- anaerobic culture . Some bacteria cannot grow at all under aerobic conditions. These are called obligate anaerobes , such as Fusobacterium and Bacteroides .
Similarly, those that cannot grow without oxygen are called obligate aerobes . Examples of the culture include gram negative Pseudomonas aeruginosa ( the Gram negative Pseudomonas ), and Mycobacterium tuberculosis ( Mycobacterium tuberculosis) , it is the causative agent of tuberculosis. However, studies have shown that both can perform anaerobic respiration under certain conditions.
Bacteria that can grow under aerobic or anaerobic conditions and convert from aerobic respiration to fermentation or perform anaerobic respiration in hypoxia are called facultative anaerobes . Examples include Gram positive staphylococci , E. coli , Salmonella) And Listeria monocytogenes ( Listeria ).
Bacteria culture method
In order to cultivate smoothly, bacteria need to provide nutrients in the culture medium. There are many different formulas available to meet the different nutritional needs of bacterial species. The choice of this type of medium will depend on the purpose of the culture.
When trying to expand a bacterial culture and keep the bacterial cells in good condition, a rich, nutritious or complete medium may be helpful. On the other hand, the minimal medium will only provide the necessities for survival and can be used to manipulate which pathways in the bacteria are turned on.
The culture medium can also be divided into known or unknown components. As the name implies, in a particular medium, all the ingredients are known. Unknown media often contain complex mixtures of nutrients and chemicals in unknown proportions, such as yeast extract.
Whichever medium is selected, it can be a liquid culture in liquid form, or agar can be added to a solid medium and allow bacterial cells to grow on a solid surface.
Compared with static bacterial colonies, culturing in liquid medium, also known as liquid culture, makes it easier for existing bacteria to obtain available nutrients. Gently stirring during the cultivation process to keep the bacteria dispersed in the medium can further help the bacteria have better nutrition.
Liquid media also dilutes waste as they are formed, distributing them in the culture. Therefore, compared with solid medium, a larger quality of bacteria can be obtained for an equal volume of liquid culture.
Therefore, when you plan to expand your culture, you may need to use a liquid medium, for example, when using bacteria to produce required compounds, food production, or extracting DNA or plasmids from it.
When long-term storage of bacterial strains is desired, they also need to be grown in liquid media.
Glycerol is then added, which will prevent the bacterial cells from being completely frozen and subsequent lysis, and allow them to be stored at -80°C. Long-term storage in this way can preserve the strains, help to collect strains for a long time, prevent the loss of valuable strains, and reduce the risk of mutations that may occur during repeated passages.
Nutrient agarose solid medium
adds agar to a liquid medium so that it can be placed in a petri dish, such as a ramp or a test tube. The solid medium is very useful when you want to select a single colony from a mixed culture, such as when purifying diagnostic samples. If you want to calculate the number of colony forming units (cfu) in a given volume of liquid sample, you should do the same for plating and culturing on solid medium. Inoculation to the slope to cultivate inoculation is also a convenient method to transfer strains from the laboratory to the laboratory without the risk of spilling potentially infectious materials.
Selective and differential media
can also use selective media to promote or inhibit the growth of certain species, species groups, or strains with specific characteristics. This may be based on the strain’s ability to utilize specific nutrients, produce certain by-products, or resistance to certain antibiotics. Selection can be used for liquid and solid media.
The ability of a strain to grow or not can be indicated by the color change in the differential medium, and is usually used to identify bacterial species or subtypes.
For example, through the analytical curve index (API) test, bacteria can be cultured with a series of substrates to produce different color change patterns according to their metabolism to realize identification.
In the case of strain hemolysis, growing on blood agar can assess the type of hemolysis and help determine the species present ( Figure 1 below ).
Figure 1: Blood agar culture shows alpha (left), beta (middle) and gamma (right) hemolysis.
Adding antibiotics to the liquid medium will prevent the growth of non-resistant strains. This may be helpful when cultivating engineered strains with antibiotic resistance genes added as markers.
During the preparation process, antibiotics can be added to the solid medium to play a similar role to the liquid medium. Alternatively, the antibiotic-infused plate can be placed on a solid medium that has been inoculated with the stain of interest. Where the strain is sensitive to antibiotics, as the bacteria grow, a clear area without growth will appear around the plate. For example, you can choose a suitable antibiotic to treat the infection (see Figure 2 below ).
Figure 2: Antibiotic resistance test; bacteria in the culture on the left are sensitive to antibiotics in the plate. The bacteria on the right are resistant to most antibiotics.
In addition to the oxygen conditions and nutrient requirements that have been discussed, the temperature and humidity for optimal growth of different species are also different, which reflects their natural growth state. Species that are usually found deep in the body, such as the intestines or lower respiratory tract, may grow best at a body temperature of 37°C . Conversely, for example, species found in the soil may require lower temperatures. In the genetic manipulation of bacteria, temperature can be used as a switch to control the integration of temperature-sensitive plasmids, thereby facilitating the desired results.
Bacterial growth curve
Although the division rate between bacterial species will vary, they usually follow the same growth pattern in liquid culture. The number of bacterial cells in the culture can be estimated by various methods, including plating and colony counting, or by measuring the turbidity of the culture using ultraviolet-visible spectroscopy. When it is plotted against time (usually on a logarithmic scale), it is called a growth curve, as shown in Figure 3 below.
Figure 3: Example of bacterial growth curve, 1) lag phase, 2) exponential/log phase, 3) static phase and 4) death phase.
1. Lag phase – bacteria are adapting to new growth conditions. The growth at this stage will depend on the degree of similarity to the previous culture conditions and cell conditions. Bacteria may need to repair themselves, produce enzymes and RNA for replication, or synthesize molecules that are lacking in the surrounding environment.
2. Exponential or logarithmic growth phase -once cells have adapted to their conditions and acquired the molecules they need, cell division begins. This phase follows a predictable doubling pattern, and its duration will depend on the suitability of the conditions to the bacterial species. Under conditions close to optimal, there will be rapid growth, so logarithmic growth will occur. This is the healthiest stage of bacterial cells, so the cells are usually used in other stages of experiments.
3. The stationary phase -nutrients are exhausted, waste accumulates, and space may be insufficient, slowing down further division, so that the number of new cells is equal to the number of deaths. This is seen as a flattening of the growth curve. The new bacterial cells undergo physiological changes to adapt to starvation conditions. For species that produce spores, sporulation may also begin at this stage.
4. Death or decay stage -because the conditions are no longer conducive to growth, it is observed that the condition of the existing cells continues to deteriorate, resulting in a decline in the growth curve. Non-viable cells may still be useful for turbidity measurements, if they are used to estimate the number of cells, the value obtained will be higher than the number of truly live cells. Generally, certain cells will always remain viable when they mutate or enter a dormant state to survive.
Bacterial culture purpose
A pure culture is a culture that contains only the types of bacteria you wish to cultivate. The difficulty of achieving this goal may largely depend 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 refrigerator, the culture may already be a pure culture.
However, if the source is a clinical or environmental sample, there may be many other bacterial species and potential fungi that will also grow rapidly under your culture conditions. Selective media and restricted growth conditions (for example, aerobic and anaerobic culture) help eliminate non-target species and shrink non-target bacteria.
Streaking the sample onto a solid medium rather than a liquid culture will allow visual identification of the colony of interest from the general background. Before obtaining a pure culture, it may be necessary to pick and re-streak the bacterial colonies of interest several times on a fresh agar plate. Once achieved, they can be grown in liquid culture if needed.
If the number of target species is small, it may be necessary to streak multiple plates from the original sample to separate them. Some species grow faster and more vigorously than others, so this is also a factor to consider.
If desired, they can then be grown in liquid culture. If the number of target species is small, it may be necessary to streak multiple plates from the original sample to separate them.
Depending on the purpose of your experiment, it may not always be necessary to obtain a pure culture. If the target species can be identified in other contexts and this is sufficient for your purposes, you may not need to obtain a pure culture. But, for example, if you want to perform further targeted testing, or grow bacteria for production or food purposes, obtaining and maintaining pure cultures may be essential.
Frequently Asked Questions about Bacterial Culture
Contamination of bacterial cultures can cause big problems, especially if they are undetected. This may mean that new cultivation steps need to be re-established, but in the worst case, if it occurs in a food or production environment, it may cause disease and very expensive remedial work.
Contamination of cultures can come from a variety of sources, from the original sample itself to the process of culture and even storage. Good aseptic technique helps to avoid contamination of bacterial cultures.
Overgrowth of certain species
certain bacterial species grow easily and vigorously. When trying to isolate a species from a mixed sample, these vigorous bacteria 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 alleviate this situation. Try to incubate the sample as soon as possible after sampling to ensure that it is as representative as possible.
Antibiotic treatment before sampling
In a diagnostic setting, it is important to know whether antibiotic treatment was performed before sampling. If this is the case, failure to cultivate a particular species may not indicate that it is not the cause of the infection.
Incorrect growth conditions
The use of inappropriate or suboptimal growth conditions may hinder or completely prevent the growth of the target strain. Be sure to double check the growth requirements, or if antibiotic selection is used, make sure that the correct antibiotic is selected for the resistance genes that are present.
Non-cultivable and slow
growing organisms -even now, certain bacterial species cannot grow in the laboratory. Mycobacteria, for example, grow very slowly and may take several months to successfully culture. This is especially problematic when trying to diagnose an infection.
Application of bacterial culture
There are many reasons why it may be necessary or desirable to cultivate bacterial cells. Here, we consider some common applications.
Although it may take a long time to isolate and identify bacterial species from a sample, bacterial culture is still an important diagnostic tool. Although PCR can quickly identify the presence of a specific pathogen, isolating the culprit will confirm that it is alive, alert analysts to potential transmission risks and inform treatment methods. This also means that the bacterial strains can be further asked to obtain information such as antibiotic susceptibility to guide treatment options. Strains can also be stored for future reference, for example for disease monitoring purposes.
For many reasons, it may be necessary to manipulate the genome of bacterial strains; try to understand the basic biology, weaken it when creating vaccine strains, overproduce proteins and create reference strains with detectable markers. Whether it is mutation, deletion or insertion of genetic material, strains need to be cultivated before, during and after the genetic engineering process.
Cultivation and characterization of bacterial strains are essential for epidemiological research. This allows scientists to study how bacterial populations change over time-which can inform treatment, vaccine and diagnostic design and updates-and study transmission events, which in turn can inform public health policies and recommendations. The Gonococcal Isolation and Surveillance Project (GISP) is one such project, which monitors bacterial resistance to antibiotics and helps to promote drug treatment recommendations. The Centers for Disease Control and Prevention (CDC) also operates the Active Bacteria Core Surveillance (ABC) system, which provides laboratory and population-based surveillance of invasive bacterial pathogens of public health importance.
Scale up to realize omics research
Although DNA and RNA sequencing can be performed with a small amount of genetic material, even at the single-cell level, for many studies, next-generation sequencing (NGS) is still performed on materials from many bacterial cells. Therefore, bacteria usually require DNA Or culture before RNA extraction. If you are interested in a specific strain (unlike microbiome research will involve mixing), then this is most likely from a pure culture.
Development of vaccines and new therapies
In order to fight bacterial pathogens, you usually also need to be able to cultivate the pathogen. During vaccine development, strains may need to be cultivated to understand their genomes, amplify their genes, or manipulate them. Similarly, in order to test candidate vaccines or therapies, a challenge experiment is usually required, in which an individual is challenged by a pathogen to see if the treatment is effective. To this end, bacterial strains are usually cultivated and counted in a defined challenge model to control and determine the dose received by the subject.
Food and beverage production
Bacteria are an important part of the production of many foods and are roughly divided into probiotics and starters.
Probiotics are usually cultivated for the benefit of human health and 18 are usually cultivated through our gut microbiome. Although probiotics may contain many different bacterial species, lactobacilli and bifidobacteria are common choices for culture.
On the other hand, starters are often used as part of the food production process to develop flavor, texture, nutritional value or improve preservation. Examples include sourdough bread, salami, pepperoni, and dry ham. Lactic acid bacteria (LAB) are commonly found in starter cultures. However, some foods and beverages can be said to belong to two camps, such as yogurt and the increasingly popular kimchi and kombucha. Among these products, the products are consumed for their flavor and probiotic benefits.
Regardless of the purpose of the cultivation, maintaining a healthy, pollution-free cultivation is essential for optimal production and consumer safety.
Detection of food contaminants
Although certain bacteria may be suitable for food production, they may also exist as contaminants and may cause serious foodborne illnesses. Common causes include Salmonella, Listeria monocytogenes, Campylobacter jejuni and Escherichia coli. Therefore, it is important that analysts be able to cultivate any potentially dangerous bacteria from food samples, even if their numbers are small.
1. Garrett WS, Onderdonk AB. 249 – Bacteroides, Prevotella,Porphyromonas, and Fusobacterium Species (and other medically importantanaerobic Gram negative bacilli). In: Bennett JE, Dolin R, Blaser MJ, eds. Mandell,Douglas, and Bennett’s Principles and Practice of Infectious Diseases (EighthEdition). 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. Pseudomonasaeruginosa exhibits deficient biofilm formation in the absence of class II andIII ribonucleotide reductases due to hindered anaerobic growth. Front Microbiol.2016;7:688. doi:10.3389/fmicb.2016.00688
3. Smith I. Mycobacterium tuberculosis pathogenesis andmolecular 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 Staphylococcusaureus, and techniques for identifying and quantifying S. aureus adhesins inrelation 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 ananaerobic Escherichia coli culture to aerobiosis: Balancing mRNA and proteinlevels 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 Salmonellaoxidises 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 oranaerobic conditions: a review. Anaerobe. 2009;15(1-2):7-17. doi:10.1016/j.anaerobe.2008.08.001
(source:internet, reference only)