Aseptic Techniques in Microbiology: Principles and Procedures

Aseptic techniques in microbiology are followed during the handling of microbial cultures, sterile growth media and laboratory instruments. Microorganisms are present in the surrounding air, on laboratory surfaces and on the hands of laboratory workers. They may enter a culture when the materials are opened or handled. Even a sterile growth medium can become contaminated during this work.

In microbiology laboratories, aseptic techniques are required during culture inoculation, transfer of an inoculum (microbial material used to start a culture) from one growth medium to another and handling of agar plates. Unwanted microorganisms may pass from one culture to another. The cultured organisms may also spread into the laboratory environment if they are handled improperly.

A pure culture contains only the microorganism being studied. Contamination of such cultures may affect the observed microbial growth and interfere with the identification of microorganisms or interpretation of laboratory test results.

What is Aseptic Technique in Microbiology?

Aseptic technique in microbiology is a set of laboratory practices used to prevent unwanted microbial contamination during the handling of microbial cultures, sterile growth media and laboratory instruments. It prevents the entry of external microorganisms into the cultures. The accidental transfer of microorganisms from one culture to another or into the laboratory environment is also reduced by following these practices.

The condition in which unwanted microbial contamination is absent is known as asepsis. It is maintained by following proper aseptic practices. Sterilization involves the complete removal or destruction of all microorganisms, including bacterial spores. In disinfection, microorganisms present on non-living surfaces are reduced or eliminated. All microbial spores may not be destroyed. The term sterile technique is also used for aseptic handling where sterile materials and instruments are maintained without contamination. However, the entire laboratory environment does not become completely sterile by following aseptic technique.

Objectives of Aseptic Techniques

• To prevent unwanted microorganisms from entering microbial cultures and sterile growth media.
• To obtain and maintain pure cultures for accurate identification and laboratory test results.
• To prevent cross-contamination between different microbial cultures during inoculation and subculturing.
• To prevent the accidental release of microorganisms into the laboratory environment and reduce the risk of exposure among laboratory workers.

Principles of Aseptic Techniques

The principles of aseptic techniques in microbiology are based on preventing the entry and accidental transfer of microorganisms during laboratory work. Some of the basic principles are as follows-

• The laboratory working area should be clean and properly arranged before handling microbial cultures. Unnecessary air movement and disturbances should be avoided near the working area.
• The laboratory benches are disinfected before and after work to reduce the microorganisms present on their surfaces. Disinfection of the working surface is different from sterilization of laboratory instruments and culture media.
• Only sterile instruments and sterile growth media should be used during microbial culture work. The reusable inoculating loops and needles are sterilized before use and between culture transfers.
• The culture tubes and Petri dishes should be opened only when required. Their exposure to the surrounding air must be kept as short as possible.
• The sterile instruments and culture media should not come in contact with hands, laboratory benches or other nonsterile surfaces. The caps and lids are also handled carefully to avoid contamination of their inner surfaces.
• During aseptic transfer, the microbial cultures are handled carefully to prevent splashing, spilling and the formation of aerosols containing microorganisms.
• Proper hand hygiene should be maintained before and after handling microorganisms. Appropriate protective equipment is used according to the laboratory work being performed.

Common Aseptic Techniques in Microbiology

There are different aseptic techniques used in microbiology laboratories for handling microbial cultures. These include disinfection of work surfaces, hand hygiene, sterilization of instruments and proper handling of culture media.

1. Cleaning and Disinfection of Work Surfaces

Before starting the microbial culture work, the laboratory benches are cleaned to remove visible dirt and other unwanted materials. A suitable disinfectant, such as 70% ethanol, is then applied to the working surface. The surface is also disinfected after completing the work. It is necessary to allow the disinfectant to remain on the surface for the required contact time.

Disinfection reduces the microorganisms present on a surface. It is different from sterilization. In sterilization, microorganisms are completely removed or destroyed from the laboratory instruments and culture media.

2. Hand Hygiene and Protective Clothing

Hands are washed before and after handling microbial cultures. It reduces the transfer of microorganisms from hands to the cultures and other laboratory materials. The laboratory coat is used to protect clothing from contamination.

Gloves are worn depending on the microorganisms and the type of laboratory work being performed. They may also become contaminated during handling. The contaminated gloves should be removed carefully.

Gloves do not replace hand washing. Protective eyewear is used when there is a risk of splashing during laboratory work.

3. Sterilization and Aseptic Handling of Instruments

The instruments such as inoculating loops, needles, pipettes and forceps are commonly used for handling microbial cultures. These instruments must be sterile before use. Reusable metal inoculating loops and needles are sterilized by heating them in a flame at a suitable open-bench workstation. Autoclaving is used for the sterilization of heat-resistant laboratory instruments and culture media.

Some instruments are available as presterilized disposable instruments. They can be used directly without sterilizing them again before their first use.

During handling, the sterile parts of the instruments should not come in contact with hands, laboratory benches or other nonsterile surfaces. Microorganisms may enter the cultures through such contact.

4. Handling of Culture Media and Laboratory Vessels

The sterile culture media are kept in closed culture tubes, bottles and Petri dishes until they are required for use. During inoculation or culture transfer, these containers are opened for a short time. This reduces their exposure to microorganisms present in the surrounding air.

The inner surfaces of culture tube caps and Petri dish lids should not touch nonsterile surfaces. They are handled carefully. Sterile pipettes and other transfer instruments are also prevented from coming in contact with unwanted materials.

After the work, the culture tubes and Petri dishes are closed properly.

5. Aseptic Work in Biological Safety Cabinets

A biological safety cabinet (BSC) is used for laboratory work where additional protection against biological hazards is required. The Class II BSC contains a controlled and filtered airflow system which protects the laboratory workers, microbial cultures and surrounding environment from contamination.

The instruments and other laboratory materials are arranged inside the cabinet without blocking the front and rear air grilles. Sudden hand movements and unnecessary movement of equipment may disturb the airflow. These movements are avoided during work.

A laminar-flow clean bench provides clean air to the materials being handled. It does not protect the laboratory workers or the surrounding environment from infectious microorganisms. For such work, a suitable biological safety cabinet is required.

Open flames such as Bunsen burners are not recommended inside the biological safety cabinets. The heat from the flame may disturb the protective airflow and create a fire hazard. Instead, sterile disposable inoculating loops or suitable electric microincinerators can be used when required.

Procedure for Aseptic Culture Transfer

Aseptic culture transfer involves transferring a small amount of microbial culture (inoculum) from one growth medium to another under aseptic conditions. The following is a general procedure used in microbiology laboratories. The actual procedure and biosafety requirements may vary depending on the microorganisms and laboratory facilities.

Materials Required for Aseptic Culture Transfer

1. Microbial stock culture (broth or agar culture).
2. Sterile growth medium (nutrient broth, agar slant or agar plate).
3. Sterile inoculating loop, needle or suitable transfer pipette.
4. Test tube rack and labeling marker.
5. Suitable disinfectant and cleaning materials.
6. Laboratory coat, gloves and other protective equipment as required.
7. Approved instrument sterilization equipment and biological waste container.

Preparation of the Work Area

1. Before starting, check the laboratory procedure and biosafety requirements for the microorganisms being handled. Use a suitable biological safety cabinet if required by the laboratory risk assessment.
2. Wash hands properly and wear a laboratory coat along with other required protective equipment.
3. Clean the laboratory bench and apply a suitable disinfectant. Allow the disinfectant to remain on the surface for the required contact time.
4. Arrange the microbial stock culture, sterile growth medium and required instruments on the working area. Label the fresh culture medium with the organism name, date and other required details.
5. Select a sterile disposable inoculating instrument or sterilize a reusable instrument using an approved method. The heated instrument is allowed to cool before collecting the inoculum. It should not touch any nonsterile surface.

Aseptic Transfer of Microbial Cultures

1. Take the tube containing the microbial culture and carefully remove its cap. The inner surface of the cap should not touch the laboratory bench or any other nonsterile material.
2. Insert the sterile and cooled inoculating loop into the microbial culture and collect a small amount of inoculum. In the case of an agar culture, the inoculum is taken from the surface of the microbial growth.
3. Remove the inoculating loop containing the inoculum and close the stock culture tube properly.
4. Take the tube containing sterile growth medium and carefully remove its cap. The inoculum is then transferred into the sterile broth or onto the surface of the agar medium, depending on the type of culture being prepared. Avoid touching the sterile instrument to the nonsterile parts of the container. In the case of agar medium, the surface should not be damaged during inoculation.
5. After transferring the inoculum, remove the instrument and close the culture tube or Petri dish immediately. The culture is handled gently to prevent spilling or formation of aerosols.

Completion of the Procedure

1. The stock culture and newly inoculated culture are closed properly and placed in their designated racks. Check that the cultures are labeled with the organism name, medium, date and other required identification details.
2. Reusable inoculating instruments are sterilized using the approved method after completing the transfer. Used disposable instruments are placed in the designated biological waste container.
3. The contaminated materials and culture waste are subjected to appropriate decontamination and disposal procedures according to laboratory biosafety requirements.
4. Clean and disinfect the laboratory workstation after completing the work. Remove the used gloves carefully and wash hands properly.

Advantages of Aseptic Technique

Some of the advantages of aseptic technique in microbiology are as follows-

• Prevention of Contamination- Aseptic technique reduces the entry of unwanted microorganisms into sterile culture media and microbial cultures. During inoculation and subculturing, cross-contamination between different cultures is also prevented.
• Maintenance of Pure Cultures- The isolated microorganisms can be transferred and maintained as pure cultures without introducing other microbial species.
• Accurate Laboratory Results- Contamination of microbial cultures may affect the results of biochemical tests, microbial identification and colony counting. It can produce errors in the observations. The use of aseptic technique reduces these errors and helps in obtaining reliable experimental results.
• Laboratory Safety- While handling pathogenic microorganisms, aseptic practices reduce the chances of accidental exposure among laboratory workers. The spread of cultured microorganisms into the surrounding laboratory environment is also reduced.
• Reduced Wastage- Contaminated culture media and laboratory samples often have to be discarded or prepared again. This requires additional materials and time. Proper aseptic handling reduces the loss of laboratory materials and the need for repeating experiments.

Uses of Aseptic Techniques in Microbiology

The following are some of the common uses of aseptic techniques in microbiology–

• Used for inoculation of culture media, such as nutrient broth, agar slants and agar plates, with the selected microorganisms.
• During subculturing, microorganisms are transferred from an existing culture to fresh growth medium under aseptic conditions. It is used for maintaining laboratory stock cultures.
• To isolate microorganisms from mixed cultures using the streak plate method and obtain pure cultures for further study.
• Aseptic techniques are used during the preparation and dispensing of sterile culture media into test tubes, bottles and Petri dishes to prevent contamination before inoculation.
• Used in microbial enumeration during the preparation of serial dilutions and inoculation of agar plates by spread plate or pour plate methods.
• In biochemical tests and antibiotic susceptibility testing, the selected microbial cultures are transferred to the required test media without introducing other microorganisms.
• Clinical microbiology laboratories use aseptic techniques during the inoculation of patient specimens into suitable culture media for isolation and identification of infectious microorganisms.

Common Errors in Aseptic Technique

Some of the common errors made during aseptic techniques in microbiology are as follows-

• Improper Disinfection- The laboratory work surface is not properly cleaned and disinfected before and after handling microbial cultures. Using an unsuitable disinfectant or not allowing sufficient contact time may leave microorganisms on the surface.
• Using Nonsterile Instruments- Inoculating loops, needles and other instruments are used without proper sterilization. Sometimes the sterile instruments come in contact with hands, clothes or laboratory benches, introducing unwanted microorganisms into the culture. Reusing an instrument between different cultures without sterilization may also cause cross-contamination.
• Using a Hot Inoculating Loop- The inoculating loop is not allowed to cool after heat sterilization. The hot loop may kill the microorganisms collected from the culture.
• Leaving Culture Vessels Open- The culture tubes and Petri dishes are kept open for a longer period during inoculation. This increases the chances of contamination by airborne microorganisms.
• Improper Handling of Caps and Lids- The inner surfaces of culture tube caps and Petri dish lids are touched with hands or placed on nonsterile laboratory surfaces. Microorganisms may enter the cultures when these caps or lids are replaced.
• Poor Hand Hygiene- Working with microbial cultures without proper hand washing or handling sterile materials with contaminated gloves. Gloves may carry microorganisms from one laboratory surface to another.
• Careless Culture Handling- Rapid movements, splashing or spilling of microbial cultures may produce microbial aerosols and increase the risk of exposure among laboratory workers.
• Improper Use of Biological Safety Cabinets- Blocking the air grilles, making unnecessary movements or using open flames inside a biological safety cabinet (BSC) may disturb its protective airflow.
• Improper Waste Disposal- Used inoculating instruments, contaminated culture media and other microbial wastes are not properly decontaminated or disposed of. This may spread microorganisms to the laboratory environment.

Difference Between Aseptic Technique, Sterilization, and Disinfection

References

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