Revolutionizing Microbial Cultivation: CO2 Incubators for the Growth of Obligate Anaerobes and Microaerophiles Listen with ReadSpeaker White paper

Microbial growth is a crucial factor in many industries such as medicine, food production, and environmental science. The environmental factors controlling this growth hold the key to establishing optimal conditions for culture and obtaining accurate research results. Oxygen, making up to 21 percent of the earth's atmosphere, is an important factor in how microbes grow. Nevertheless, some organisms have distinct requirements for oxygen, ranging from being necessary to for toxic in higher concentrations.

Bacteria, which encompass a large number of diverse microorganisms bearing ecological and biotechnological significance, have varying oxygen requirements. These bacteria can be categorized into three groups: aerobes, facultative anaerobes, microaerophiles, and obligate anaerobes, each of which is affected by different levels of oxygen availability. 

Aerobes are organisms that require oxygen for their survival and growth. While the facultative anaerobes exhibit an adaptability of metabolism. They thrive in conditions both aerobic and anaerobic. Microaerophiles, on the contrary, like environments where oxygen is low. Such conditions are typically, 2% to 10% oxygen—often with some carbon dioxide. Obligate anaerobes, which are sensitive to oxygen, will die if exposed to air.  

Obligate anaerobes can be further categorized into three groups. The strict varieties tolerate ≤ 0.5% oxygen, while moderate anaerobes can withstand oxygen levels from 2 to 8%. Aerotolerant anaerobes are anaerobic, but they can endure atmospheric oxygen for short periods.  

Considering the strict oxygen requirements of anaerobic bacteria, precise oxygen control and customizable gas mixing is essential for optimal growth. This highlights the need for advanced systems like CO2 incubators, capable of providing tailored environments to support specific organisms. 

Traditional Methods for Cultivating Obligate Anaerobes and Microaerophiles

Obligate anaerobes and microaerophiles have traditionally been cultured using anaerobic chambers or jars fitted with gas-generating systems. These systems maintain controlled atmospheres that are conducive to the growth of anaerobic or microaerophilic organisms by removing oxygen and providing appropriate amounts of carbon dioxide.  

To create an anaerobic condition, water is added to the GasPak generator envelope, triggering the evolution of H2 and CO2. The hydrogen reacts with oxygen on the surface of the palladium catalyst, forming water and establishing anaerobic conditions, while the carbon dioxide aids growth of the anaerobes. Furthermore, the presence of an anaerobic indicator strip saturated with methylene blue helps in the visual indication of anaerobiosis. When oxygen is absent, the strip changes color from blue to colorless, indicating the existence of anaerobic conditions.

In order to support the microaerophilic bacteria's nutrient needs, environments contain oxygen concentrations of typically 2 to 10%. Nevertheless, organisms such as Campylobacter species require more precise gas compositions for optimal growth – 5% oxygen, 10% carbon dioxide, and 85% nitrogen. Specialized systems like CampyGen Compact are designed to generate the microaerophilic environments required for Campylobacter species. They achieve this by controlling the amount of oxygen in the air and adding CO2: perfect conditions under which microaerophilic anaerobes will thrive.  

User Challenges From Using Traditional Method

Although widely used, the traditional method has several challenges. The huge per-test costs, like anaerobic sachets and GasPak systems, are obstacles to moving forward. And because these consumables are costly, this has financial implications, especially in high-throughput test environments or resource-constrained ones. The chamber capacity for anaerobes is limited by the number of culture plates that can be incubated at a time, all of which can lead to flow inefficacies and experimental project delays. 

The workflow also poses a challenge due to the fact that it often requires manual calculations to figure out the appropriate number of anaerobic jars and sachets to use for each experiment. On top of that, the steps of assembling jars, flushing gas, and maintaining them drive up the demand for skilled individuals. It takes considerable expertise and skill to be able to carry out these tasks accurately, adding to the operational complexity of the traditional method. 

Above all, the traditional method is short in terms of systems capable of monitoring and controlling oxygen concentrations inside the anaerobic chambers. Although an anaerobic indicator strip is present to indicate an oxygen-free environment, unintentional penetrations of oxygen are often encountered which pose threats to the anaerobic environment's integrity and impact experimental results. The lack of real-time oxygen monitoring ability and traceability makes it impossible to detect and respond to variations such as unpleasant changes in environmental conditions, undermining the reliability and reproducibility of experimental results. 

Solving Day-to-Day Challenges with CO2 Incubators

CO2 incubators have emerged to resolve the problems encountered when cultivating obligate anaerobes and microaerophiles by traditional methods. Through these advanced incubation systems, environmental factors can be strictly controlled. These include parameters such as temperature, humidity, carbon dioxide concentration and oxygen limitation, which can tailor environmental conditions for an array of microorganisms in precise detail. 

An important factor in the cultivation of obligate anaerobes is temperature, and that is because it directly affects microbial metabolic rates and growth rates. Being able to get temperature right or wrong can be a matter of life and death for microorganisms. CO2 incubators allow for precise control over temperature. Users can maintain the optimum growth temperature for a given strain of microorganism. These systems maintain stability of temperature throughout the incubation chamber, so that temperature fluctuations are minimized and microbes grow consistently throughout an experiment. 

Another critical factor in microbial cultivation is the regulation of humidity, especially in the case of microorganisms susceptible to moisture. CO2 incubators have built-in mechanisms for controlling humidity that prevent the media from drying out and promote the growth of moisture-loving organisms. These incubator systems, by maintaining proper humidity levels as well, provide a medium for healthy microbial growth. They minimize the risk of culture desiccation and contamination. 

Microaerophilic organisms thrive in a CO2-enriched environment. In order for the gas to be delivered in precise concentrations at the right time, some CO2 incubators incorporate gas injection systems right into their incubation chambers. This is done to induce growth as required by microaerophiles. With their ability to create controlled CO2-enriched atmospheres, these systems cater to a wide variety of microbial species in which specific gases are needed. 

Prevention of contamination is an important consideration when cultivating microorganisms. Microbial contamination may spoil an experiment so that the results obtained may be inaccurate. 

In the environmental atmosphere that an incubator—CO2 manufactures feature the most advanced designs of contamination prevention. HEPA filters and seamless chamber walls all reduce the risk of outside contaminants entering the incubator environment. These systems ensure that microbial cultures are free from outside contamination and that experimental results can be replicated with certainty by keeping asepsis conditions within the chamber. 

In order to grow obligate anaerobes, these CO2 incubators lack oxygen. This prevents the atmospheric oxygen from coming in and so maintains the anaerobic conditions necessary for their growth. With oxygen flushing and oxygen sensors, these incubation systems use advanced oxygen control techniques to monitor and regulate oxygen levels within the chamber. By actively removing residual oxygen and maintaining low oxygen concentrations, CO2 incubators create a truly anaerobic environment conducive to the growth of obligate anaerobes. 

Applications in Research and Industry

CO2 incubators have multiple applications in research and industry, including: 

  1. Microbial genetics research: Help in studying the metabolic pathways and growth rates of obligate anaerobes and microaerophiles. 
  2. Biotechnology: Making microbial enzymes, metabolites, and biofuels under strictly controlled conditions. 
  3. Medical research: Cultivating pathogenic anaerobic bacteria for studies of infectious diseases and the development of antimicrobial therapies. 
  4. Environmental monitoring: Help in monitoring the microbial communities in anaerobic environments, such as soil and sewage. 

Specifications of Our Incubators

Temperature Control: 

It is designed to keep the temperature of a microbial culture in the range of 35-37°C, which is widely used. An accuracy of +/-0.1°C is controlled precisely. The fluctuation of temperature is maintained within 0.1°C, which promotes uniformity throughout the chamber. Through the addition of a direct heat system controlled by PT100 sensor, it is possible to maintain exactly the temperatures that are required for growth of microbes. 

Oxygen Monitoring and Control: 

We incorporate ZrO2 sensor technology to measure oxygen concentration levels. The control range of hypoxia is 0.2%-20%, and oxygen levels vary as much as 0.1%. To create particular atmospheric conditions depending on the growth characteristics of microorganisms, precise levels of oxygen are necessary. 

CO2-Enriched Atmosphere Control:

The CO2 sensors employ infrared (I.R.) to provide accurate measuring up to 20% CO2 with fluctuations held within very close tolerances (0.1%). Injecting atmospheric CO2 is particularly effective at stimulating microbial growth by offering an optimal environment for the growth of numerous microorganisms. 

Prevention of Contamination: 

Our incubator has adopted a robust contamination prevention design, which includes a mode for hot air sterilization with 180°C temperature. This sterilization method is deemed most efficient in decontaminating the chamber and creating a sterile environment for cultivating microbes. Moreover, the "less is more" design model makes an effort to avoid unnecessary features that can lead to contamination. 

Natural Convection:

The incubator uses natural convection with no fan, allowing for all shelves to have uniform conditions for cultivation. The air currents are also reduced, which minimizes water evaporation and airborne transmission of contaminants, helping to create a pure growth environment. 


The Gas Tank Changer:

Each incubator has three gas tank changers to combine the CO2, O2, and N2 tanks into one unit, so the hypoxia and hyperoxia cultures can be maintained with oxygen concentrations from 0.2 - 20% and 70 - 95% respectively. 


CO2/O2 Incubator Options:

There are two models available in our incubator: the CB and CBF. Both models offer controlled oxygen of varying intensities. Along with a digital setting and readout for oxygen concentration, experience safe and reliable operation from the many pin connectors that make for easy assembly or replacement. They use a sterilizable zirconium oxide sensor. 


Hot Air Sterilization at 180°C:

An automatic sterilization routine of high reliability eliminates microbial contamination and makes reproduction results more reliable. Experimenters can plan with ease as the countdown display facilitates scheduling, leaving the incubator ready for loading once sterilization is done. 


Compliance with International Standards:

Our CO2 incubator meets stringent ISO 7937, ISO 15213, and ISO 10272 standards, ensuring quality and reliability in microbial culture applications. 

Key Features

The incubators are specifically developed to accommodate the needs of anaerobic and microaerophilic bacteria, in food microbiology, with an open and close system that tracks operation. An easy-to-use interface is supported by high-performance gas generation conditions, with gas sensors to maintain traceability, for optimal growth conditions of wide-ranging microbial species. 

Beyond operational benefits, adoption of CO2 incubators supports sustainability initiatives, reducing consumption of GasPak, chemicals, plastics, and paper over time. Users that have sustainability policies in place for their company may improve their environmental footprint over time while at the same time realizing significant cost savings. Tools like the R01 calculator enable informed decision-making that allows users to quantify the economic benefits of transitioning to CO2 incubators. 

In conclusion, CO2 incubators have a wide range of advantages for growth of obligate anaerobes and microaerophiles employing these incubators as indispensable for microbiological research and testing. With a "less is more" approach, these incubators simplify and streamline design, maximizing efficiency while reducing surface area, eliminating extraneous components such as fans and plastics, and with an interior constructed entirely from stainless steel, they facilitate more effective monitoring, improve clean-ability and sterilization, providing a more sterile environment in which to culture microorganisms. And they eliminate re-occurring costs associated with traditional methods such as HEPA filters, UV lamps, H2O2 vaporizers. Their every 180°C sterilization process, mandated by international guidelines, maximizes ease and reliability in loading cultures immediately post-cycle. 

CO2 incubators provide a paradigm shift in the cultivation of microorganisms. They are unmatched in their performance, simplicity, and sustainability. Through their innovative features and economic operation, CO2 incubators enable researchers and laboratory professionals to explore scientific breakthroughs without enormous environmental impact and cost of operation.