You Absolutely Don't Know: the Selection, Use, and Maintenance of A Microbial Culture Incubator

A microbial culture incubator is crucial in the process of microbial culture. Whether its quantity, quality, performance, accuracy, and other aspects meet the requirements of culture is related to whether the laboratory can operate normally. Various regulations have relatively high requirements for the temperature of microbial culture, with a general accuracy of ±1℃, and some even higher at ±0.5℃. At the same time, contamination is easy to occur during microbial culture, which requires users to fully understand the quality, performance, accuracy, and other aspects of the incubator when purchasing, in order to choose the most suitable one.

1

Structure and Types of Microbiological Culture Incubators

Microbiological culture incubators are widely used in research fields such as pharmaceutical microbiology, food microbiology, agricultural microbiology, medical microbiology, and have become one of the commonly used instruments in laboratories in these areas. The principle of the incubator is to simulate the growth environment of microorganisms inside a living organism within the incubator chamber, and provide a device for culturing microorganisms outside of their natural habitat.

Structure of Microbiological Culture Incubators

Most modern microbiological culture incubators are made of high-quality steel plates and have a vertical box structure. The inner door is generally made of tempered glass, and stainless steel partitions are placed inside the incubator to hold the culture samples. The partitions are movable and can be adjusted in height. There is a silicone rubber seal between the working chamber and the glass door, and there are hot and cold air ducts inside the incubator for smooth gas circulation and even temperature distribution. The incubator is equipped with an independent temperature-limiting alarm system, which will automatically interrupt the operation when the temperature exceeds the set limit. Fungal culture incubators generally consist of refrigeration system, heating system, ultraviolet disinfection system, culture chamber, air humidifier, control circuit, and operating panel. Temperature and humidity sensors are used to maintain a stable environment inside the incubator.

Classification of Microbiological Culture Incubators

Microbiological culture incubators can be classified according to the heating method as water-jacketed or air-jacketed. Water-jacketed incubators heat the internal chamber by heating the liquid layer surrounding the incubator. This heating method is slower, but can maintain a constant temperature inside the incubator for a longer period of time. Air-jacketed incubators heat the internal chamber by using a heating element in the air jacket layer surrounding the incubator.

Microbiological culture incubators can be classified according to the temperature control method as computer intelligent control (programmable) and automatic constant temperature adjustment (mechanical). Computer intelligent control is the mainstream temperature control method for incubators. Most computer intelligent control systems use microcomputer PID controllers as the control unit, with temperature sensors as the thermal elements. The set and measured values are displayed digitally, forming a complete control system.

Automatic constant temperature adjustment temperature control devices often use a "metal strip" type, which uses a metal strip with a larger thermal expansion coefficient to make a spiral shape. One end of the metal strip is fixed on the inner wall of the incubator, and the other end is fitted with a movable contact point. At normal temperature, the two contact points are closed. After the power is turned on, the temperature inside the incubator rises, causing the fixed metal strip to expand due to heat, changing its curvature and causing the other end of the contact point to move away, cutting off the circuit and stopping the heating. When the temperature drops to a certain level, the spiral metal strip returns to its original shape, the two contact points come into contact, and the circuit is turned on, starting the heating again. In this way, the circuit is turned on and off to maintain a constant temperature inside the incubator.

According to the culture environment  Microbiological culture incubators can be classified  as standard incubators, carbon dioxide incubators, hypoxic incubators, and anaerobic incubators

According to the target organism Microbiological culture incubators can be classified as fungal culture incubators, constant temperature incubators, constant temperature and humidity incubators, and light culture incubators.

According to the level of temperature automation   Microbiological culture incubators can be classified  as fully automatic temperature acquisition incubators, semi-automatic temperature acquisition incubators, and manually temperature acquisition incubators.

2

Contamination Factors in Biological Culture

Wind Speed and Wind Direction

Generally, microbiological culture incubators are equipped with air ducts and circulation systems inside the chamber. Appropriate wind speed and wind direction are beneficial for the uniformity of the incubator's temperature and for the normal growth of microorganisms. However, when the wind speed is too high, it can cause the culture medium to dry up and lead to inaccurate results. Additionally, according to pharmacopoeia requirements, culture dishes should be inverted during incubation. After multiple blank culture dish validations, it was found that, with the same wind speed, if the direction of the airflow in the incubator is opposite to the direction of the culture dish cover, dust and other contaminants in the air can easily pollute the microbial cultures. Therefore, it is best for the direction of the airflow in the incubator to be consistent with the direction of the culture dish cover during operation.

Airtightness or Tightness of the Culture Dish

Culture dishes are composed of a flat circular bottom and a cover, and are mainly made of plastic and glass. The culture dishes used in microbiological laboratories usually have a diameter of 90 mm and are sealed with a cover. There is a certain space between the bottom and the cover of the flat dish, and the two are not completely airtight. This design can meet the oxygen requirements of aerobic microorganisms but also increases the possibility of contamination.

Especially, culture dishes from different manufacturers have different gaps between the bottom and cover due to different molding processes and parameters. Through experimental verification, under the same culture conditions, culture dishes with larger gaps have a higher probability and degree of contamination compared to those with smaller gaps. In addition, the difference in gap size between the bottom and cover of the flat dish can also cause inconsistencies in the level of moisture evaporation from the culture medium in the culture dish, leading to inconsistent culture results.

Humidity Inside the Culture Incubator

Moisture is one of the main conditions for the survival and reproduction of microorganisms. Microbial cells contain 70% to 85% of water and must live in a moist environment. The effect of humidity on microbial growth is through its effect on the water activity (AW) inside microbial cells, thereby affecting metabolism and growth. Microbial growth has an optimum AW, and when AW decreases, microbial growth slows down and stops at a certain level. The minimum AW during microbial development varies, and the optimal humidity for the growth and reproduction of various fungi and microorganisms varies slightly depending on the genus.

Generally speaking, bacteria are the most sensitive, followed by yeast and mold. This means that the AW required for bacterial growth is higher than that required for yeast, and the AW required for yeast growth is higher than that required for mold. In general, bacteria cannot grow when AW<0.90, most yeast is inhibited when AW<0.87, and most mold cannot grow when AW<0.80. Reducing humidity will lower the AW and slow down the growth rate of microorganisms.

Excessively high or low humidity in the culture incubator can cause an imbalance of humidity between the culture medium and the incubator. For example, if the humidity in the incubator is too high, water droplets can form on the culture dish, drip into the culture medium, and promote the growth of bacteria, affecting the experimental results. If the humidity in the incubator is too low, moisture loss from the culture medium can occur, affecting the growth of bacteria on the culture medium. Therefore, appropriate temperature and humidity are beneficial to the growth of bacteria, mold and yeast.

The Sources of Humidity Inside the Culture Incubator Are: 

1) the loss of moisture from the culture medium;

2) the regulation of humidity by the manual or automatic control system of the culture incubator;

3) the environment where the culture incubator is placed, which is usually a clean, dry, and well-ventilated natural environment.

Spillage of the Culture Material

The spillage of the culture material refers to the accidental separation of liquid or solid substances containing biological hazardous materials from the packaging material. Once a biological hazard spills in the culture incubator and microorganisms grow and reproduce, the incubator should be cleaned immediately. Effective disinfectants should be used to disinfect the inner walls of the incubator and all materials that come into contact with the spilled material, or they should be sterilized under high pressure.

If a spillage of a culture containing mold or other pathogenic bacteria is not dealt with promptly and is subsequently used to culture other microorganisms, the residual mold or pathogenic bacteria can contaminate the incubator, leading to cross-contamination and affecting the accuracy of the experimental results. Therefore, spillage of culture material should be avoided as much as possible in daily experiments. If spillage occurs, the incubator should be cleaned and disinfected immediately by a qualified person.

If the spilled material contains broken glass, it should not be removed or discarded directly by hand. Instead, it should be handled with a hard cardboard and forceps, placed in a durable waste container, and the instrument and equipment surfaces should be wiped twice with 75% ethanol for 3 minutes. Finally, cleaning tools should be disinfected.

Environmental Contamination

The culture incubator should be placed in a clean, dry, and well-ventilated natural environment. If the air cleanliness in the environment is poor, it is easy to breed bacteria, fungi, and viruses, contaminating the culture medium through the gap between the bottom and the cover of the petri dish, and affecting the accuracy of the culture results.

3

Selection and Management of Biochemical Incubators

When selecting a biochemical incubator, the first requirement is that it must have precise control over temperature and humidity. Secondly, it should be able to effectively prevent microbial contamination within the incubator, and ideally, be able to regularly eliminate contamination. There are many types of biochemical incubators, and when selecting one, it is important to consider the following factors based on practical needs and laboratory conditions.

Heating Method of Biochemical Incubators

The advantage of water jacket heating is that when there is a power outage, the system can maintain the accuracy and stability of the temperature within the incubator for a longer period of time. The time it maintains a constant temperature is 3-4 times that of an air jacket system. This is beneficial for experiments in an unstable environment that require stable conditions over a long period of time. Water jacket heating requires water to be added, emptied, and cleaned, and the operation of the water tank needs to be monitored regularly. Air jacket heating has the advantage of heating up quickly and recovering temperature more quickly than a water jacket incubator, which is beneficial for short-term culture and frequent opening and closing of the incubator door.

Temperature Control System and Uniformity of Biochemical Incubators

An accurate and reliable temperature control system is an essential part of an incubator. It should have three independent temperature control functions within the incubator for temperature control, over-temperature alarm control, and environmental temperature monitoring. The temperature control system parameters include temperature fluctuation, temperature resolution, and temperature uniformity. The uniformity of the incubator temperature is related to the airflow circulation within the incubator, and an incubator equipped with a fan and air ducts within the enclosure should be selected.

Temperature Range Control of Biochemical Incubators

Select a product with a suitable temperature range based on the desired experimental temperature. The temperature control range of a biochemical incubator can be: room temperature 5℃ to 60℃, 0℃ to 60℃, 4℃ to 60℃, or 5℃ to 50℃. Constant temperature incubators are divided into two types: one with a low-temperature incubator, which maintains a temperature between 0℃ and 35℃, and includes a refrigeration system and heating system, making it more expensive. Generally, the temperature of this type of incubator is set to be constant between 0℃ and 50℃. 

The other type is a room temperature incubator, which maintains a temperature above room temperature. The temperature of this type of incubator is generally set to be constant between room temperature and 65℃. The choice of a low-temperature incubator is relatively simple, as it should be selected to achieve the desired culture temperature below the ambient temperature.

Relative Humidity Control of Biochemical Incubators

Choose an incubator with a large evaporation area for humidity, as a larger evaporation area makes it easier to reach relative humidity saturation, and the recovery time for humidity after opening and closing the door is shorter.

Disinfection and Sterilization System of Biochemical Incubators

The disinfection and sterilization system of an incubator generally has the following methods: UV sterilization, high-temperature sterilization, and HEPA filter sterilization of the air inside the incubator. The UV sterilization ability is inversely proportional to the square of the distance between the UV lamp and the target, and the farther away, the worse the sterilization ability. Therefore, UV sterilization has its limitations and may not achieve thorough sterilization. High-temperature sterilization is divided into two types: dry heat sterilization and moist heat sterilization. Moist heat sterilization has a higher sterilization efficiency than dry heat sterilization because steam has a strong penetration power, and it is easy to cause denaturation or coagulation of proteins. HEPA filters can filter the air inside the incubator, with a filtration efficiency of 99.97% for particles larger than 0.3μm.

Capacity of Biochemical Incubators

If the capacity of the incubator is too small, it may not be enough, and if it is too large, it may take up too much space. The capacity of biochemical incubators ranges from small incubators with a capacity of less than 50L, suitable for laboratories with small cultures, to large incubators with a capacity of over 400L, suitable for large laboratories. The commonly used incubator capacity is between these two ranges, and the capacity should be selected based on practical needs. It is also important to reserve some space to ensure that future needs can be met.

Material of Biochemical Incubators

There are generally two types of materials used for the inner chamber of microbiological incubators available on the market: iron (galvanized material) and stainless steel. Iron chambers are lighter and more convenient for transportation, while stainless steel is more durable. Currently, the most popular material for the inner chamber is 304 stainless steel, which is more corrosion-resistant and durable than traditional cold-rolled steel plates. If the inner chamber has a rounded corner structure, it is easy to clean and leaves no dead corners.

Price Factor When Purchasing A Microbiological Incubator

Incubators with higher configurations such as password protection, high-temperature automatic adjustment and alarm devices, automatic calibration systems, LCD display systems/data output systems, etc. are more convenient to use and have good performance, but they are more expensive due to their comprehensive functions. Therefore, it is important to choose an incubator that fits your budget and main cultivation needs to achieve the best value for money.

4

The use, Monitoring, and Maintenance of Microbiological Incubators

When transporting, repairing, and maintaining the incubator, the maximum inclination angle should be less than 45 degrees. The incubator should be placed in a cool, dry, well-ventilated area, away from heat sources and direct sunlight. The outer shell of the incubator should be reliably grounded and placed steadily to prevent noise due to vibration. The distance between the incubator and the wall should be greater than 10 cm, there should be a 5 cm gap on the side of the incubator, and there should be at least 30 cm of space above the incubator to ensure good heat dissipation of the refrigeration system.

Before using the equipment, carefully check whether the power supply voltage matches the instrument requirements. If the incubator uses a three-pronged plug, the socket should be properly grounded to ensure reliable contact between the incubator ground wire and the power supply ground wire. The culture in the incubator should not be placed too tightly to ensure uniform temperature distribution. Items placed on each layer of the metal grid should not be too heavy to avoid bending or breaking the metal grid and damaging the culture.

Do not place items that are too hot or too cold in the incubator. When taking or placing items, close the incubator door to maintain constant temperature. Do not turn on or off the incubator frequently in a short period of time to avoid continuous start-up of the compressor. When the incubator is working, avoid opening the door frequently to maintain temperature stability and prevent dust and dirt from entering. When the device is not in use, turn off the main power switch and the power switch at the back of the device, and unplug the power plug for long-term storage. When the incubator is cooling, the temperature difference between the inside and outside of the incubator should not exceed 25℃.

During continuous operation, observe whether the incubator is operating normally every day, and perform an annual performance validation on the instrument. After cleaning and disinfecting the incubator, place several blank culture dishes inside, some covered and some uncovered, to test whether the cover affects the test results and how much contamination there is in the uncovered dishes.

When cleaning the incubator, wipe the inner wall of the incubator with gauze soaked in alcohol for disinfection, and then wipe off the alcohol with a dry cloth. If it is a mold incubator, use a disinfectant that can eliminate mold or perform regular UV sterilization to reduce mold contamination. Do not use acidic/alkaline or other corrosive solutions to wipe the outer surface. During incubator monitoring, if abnormal heating or cooling, sudden shutdown, or other anomalies are found, repair should be carried out promptly, and maintenance records should be kept.