How to Maintain Sterility in Cell Culture Lab? Complete Guide

How to Maintain Sterility in Cell Culture Lab? Complete Guide

How to Maintain Sterility in Cell Culture Lab? Cell culture labs are used to grow cells for research purposes. They provide a sterile environment for cell cultures so that researchers can study the effects of various chemicals on living organisms.

Successful cell culture depends heavily on keeping the cells free from contamination by microorganisms such as bacterial, fungi, and viruses. Nonsterile supplies, media, and reagents, airborne particles laden with microorganisms, unclean incubators, and contaminated instruments can all contribute to contamination.

Keeping your lab clean and sterile is essential to maintaining high-quality cell culture. Follow these steps to ensure that your lab remains clean and safe.

How to Maintain Sterility in Cell Culture Lab?
by NASA is licensed under CC-CC0 1.0

What is Aseptic Technique?

Aseptic technique refers to the practice of using sterilized equipment and materials when working with infectious agents. The most common method of sterilization is steam autoclaving at 121°C (250°F) for 15 minutes. This process kills bacteria, fungi, and other pathogens. However, it does not kill viruses or spores. To prevent the spread of disease, you must also use an techniques to maintain Sterility in Cell Culture Lab.

  • Cleanliness
  • Microbiology
  • Cell Culture
  • Lab Safety
  • Infectious Agents
  • Pasteurization
  • Heat treatment
  • Ultraviolet light
  • Antimicrobial agents
  • Disinfectants
  • Germicidal irradiation
  • Isolation
  • General safety precautions
  • Hand washing
  • Gloves
  • Aprons
  • Gowns

How to Maintain Sterility in Cell Culture Lab?

In order to maintain aseptic conditions in cell culture labs must follow strict guidelines. Here are some tips to ensure that your lab stays clean and safe.

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Practice to Maintain Sterility in Cell Culture

1. Use Cleaning Supplies Only Once

Cleaning supplies are expensive, so it makes sense to only buy them when needed. When cleaning supplies run out, it’s best to replace them rather than buy This will save you money and help keep your lab clean.

2. Wash Hands Before Entering Your Lab

Washing hands is an important part of any laboratory procedure. It helps prevent bacteria from spreading throughout your lab to maintain sterility in cell culture lab. If you don’t wash your hands before entering your lab, you could contaminate your samples with germs.

3. Keep Lab Equipment Clean

If you have a lot of equipment in your lab, make sure to clean it regularly. You should also use cleaning solutions to clean your equipment.

4. Don’t Leave Chemicals Out

Chemicals are extremely dangerous if they aren’t stored properly. Make sure to store all chemicals in their original containers. Also, make sure to label each container clearly to maintain sterility in cell culture lab.

5. Store Chemicals Safely

When storing chemicals, be careful not to spill or mix them together. Mixing different types of chemicals can cause explosions.

6. Label All Reagents

Labeling reagents is very important because it allows you to identify what type of chemical you are using. For example, if you are working with DNA, you need to know whether you are dealing with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

7. Use Proper PPE

Personal protective equipment (PPE) includes gloves, goggles, masks, aprons, etc. These items protect you from harmful substances while performing experiments.

8. Avoid Contaminating Samples

Contaminated samples may lead to inaccurate results. Therefore, it is important to avoid contaminating to maintain sterility in cell culture lab.

9 . Wash Hands Before Working With Cells

Washing hands before working with cells reduces the risk of spreading germs. To wash your hands properly, make sure to scrub all surfaces including under fingernails to maintain sterility in cell culture lab.

10. Don’t Touch Surfaces Without Gloves

It’s also important to wear gloves when handling surfaces like glassware, pipettes, and petri dishes. Wearing gloves prevents bacteria from being transferred onto these items.

What is Contamination in cell culture?

Contamination of cell cultures can occur at any stage during the process. It may be introduced by a technician, or it could be an unintended consequence of the laboratory environment. The most common contaminants are bacteria and fungi. Other types include viruses, mycoplasma, prions, endotoxins, and other chemicals that have been used to grow cells.

The contamination of cell cultures with mycoplasma is a major problem in the field of molecular biology. The presence of mycoplasmas can be detected by many different methods, including direct observation under light microscopy and immunofluorescence staining, as well as by PCR amplification of specific DNA sequences.

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Mycoplasma are small prokaryotic organisms that lack a cell wall and therefore cannot grow on conventional media. They have been isolated from many different species of animals, including humans, but they can also be found in the environment. 

The term “contamination” refers to the introduction of foreign material into a sample. The most common sources of contamination include:

  • Airborne particles
  • Bacteria
  • Fungi
  • Viruses
  • Germs
  • Dust
  • Mold spores
  • Other contaminants

Requirement of Aseptic Techniques 

The requirements for a sterile processing facility depend on the risk associated with the task at hand. In regards to this post, we’ll simply outline some of the materials required for support of a sterile outcome in cell culturing and micro-propagation.

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Aseptic technique is critical when working with cells or tissues that are susceptible to contamination by microbes. This includes all tissue culture work, including cell line establishment, propagation, cryopreservation, and storage.

In addition, aseptic techniques must be used in the preparation of reagents for use in cell-based assays such as ELISA, flow cytometry, immunohistochemistry (IHC), Western blotting, and other molecular biology applications.

Protocol for Aseptic Techniques in Cell Culture Experiment

The following protocol describes how to prepare media, buffers, and solutions for cell culture protocol and molecular biology experiments using aseptic techniques. The procedure described here is designed to minimize the risk of microbial contamination. However, it should not be considered complete because there are always risks associated with working with biological specimens.

Chemical Preparations Cautions

1. Prepare Media

Media preparations should be performed in a laminar flow hood. All equipment should be cleaned thoroughly with 70% ethanol prior to use.

2. Prepare Buffers and Solutions

Buffers and solutions should be prepared in a laminar airflow cabinet. All glassware should be autoclaved prior to use.

3. Prepare Reagents

Reagent preparations should be made in a laminar air flow cabinet. All glassware and plastic containers should be sterilized prior to use.

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4. Prepare Working Solutions

Working solutions should be prepared in the same manner as buffer and solution preparations.

5. Prepare Growth Media

Growth media should be prepared in a biosafety cabinet. All glassware, plastics, and utensils should be autoclaved before use.

6. Prepare Antibiotic Solution

Antibiotics should be added to growth medium immediately after autoclaving.

7. Prepare Trypsin/EDTA Solution

Trypsin/EDTA solution should be prepared in a clean container. It should be stored at room temperature until ready to use.

8. Prepare PBS Buffer

PBS buffer should be prepared in a sterile container. It should be kept refrigerated until needed.

9. Prepare FCS

FCS should be prepared in a separate container from the rest of the components. It should be stored frozen at -20°C.

10. Prepare Hanks Balanced Salt Solution (HBSS)

HBSS should be prepared in an aseptic environment. It should be stored in aliquots at 4°C.

11. Prepare DMEM Medium

DMEM medium should be prepared in aseptic conditions. It should be stored refrigerated until ready to use.

12. Prepare RPMI 1640 Medium

RPMI 1640 medium should be prepared in an incubator set at 37°C. It should be stored under refrigeration.

13. Prepare L-15 Medium

L-15 medium should be prepared in the incubator set at 37 °C. It should be kept under refrigeration.

14. Prepare MEM Medium

MEM medium should be prepared fresh daily. It should be kept at 4°C.

15. Prepare Penicillin Streptomycin Solution

Penicillin streptomycin solution should be prepared in the laboratory. It should be stored on ice until ready to use. Penicillin streptomycine solution can be obtained from GIBABRL Life Technologies.

16. Prepare Fetal Calf Serum (FCS)

Fetal calf serum should be thawed slowly at room temperature. It should then be centrifuged at 3000 rpm for 10 minutes. The supernatant should be removed carefully and discarded. The pellet should be resuspended in 1 ml of Dulbecco’s phosphate buffered saline (DPBS). This suspension should be transferred into a new tube.


The above mentioned protocol is very simple and easy to follow. In addition to this, it has been designed with all the essential steps required for cell culture maintenance. We hope that you will find this article useful.

You need to have a good understanding of microbiology and biochemistry to do this properly. I would suggest getting a book like “Principles of Microbiology” by Daniel E. Koshland or something similar.

You also need to understand how cells work and what they require. For example, if you are growing mammalian cells, you need to know about the different types of nutrients your cells require. If you are growing bacteria, you need to know how to grow them and keep them alive.

If you don’t understand these things, then you won’t be able to maintain sterility in your lab.

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