Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory

Summary

This protocol presents essential cell culture techniques and practices to be used in the research cell culture laboratory to avoid contamination by fungi and bacteria. Within the category of bacteria, special emphasis will be placed on preventing mycoplasma contamination.  

Abstract

Cell culture is a delicate skill necessary for growing human, animal, and insect cells, or other tissues, in a controlled environment. The goal of the protocol is to emphasize the correct techniques used in a research laboratory to prevent contamination from fungi and bacteria. Special emphasis is placed on avoiding mycoplasma contamination, a major concern in the cell culture room due to its small size and resistance to most antibiotics used for cell culture. These same techniques ensure continuous growth and maintain healthy cells. For new and experienced cell culture users alike, it’s important to consistently adhere to these best practices to mitigate the risk of contamination. Once a year, laboratories should review cell culture best practices and follow-up with a discussion or additional training if needed. Taking early action to prevent contamination in the first place will save time and money, as compared to cleaning up after contamination occurs. Universal best practices keep cell cultures healthy, thereby reducing the need to constantly thaw new cells, purchase expensive cell culture media, and reducing the amount of incubator decontamination and downtime. 

Introduction

Cell culture has many uses in the research laboratory. Since the origins of cell culture in the early 20th century, cell lines have helped advance science. Cell lines have several advantages; various cell lines can help researchers study cell biology, produce baculovirus for further studies, or produce large quantities of a protein of interest, to name a few¹. Some additional uses include studying tissue growth, helping to advance vaccine development, toxicology research, studying the role of genes in healthy organisms and diseased models, and the production of hybrid cell lines^2,3. Cel....

Protocol

1. Preparations

1. General
   1. Wear a clean lab coat designated to be worn only in the cell culture room and no other parts of the laboratory.
      NOTE: The lab coat does not need to be sterile.
   2. Wear new gloves that have not touched any other surfaces. Make sure the gloves fit tightly. Nitrile, powder-free gloves are best.
      NOTE: The gloves do not need to be sterile.
   3. To prepare for work, spray the gloves, lab coat sleeves, and interior of the biological .......

Discussion

While contamination is one of the primary concerns when performing cell culture work, the practices and techniques outlined in this manuscript will help mitigate the risks. The critical steps include wearing a clean lab coat, which is only used in the cell culture room, using clean, powder-free gloves that are sprayed with 70% EtOH often and which are changed when switching between cell lines, encouraging each individual to not share media bottles, cleaning the cabinet thoroughly prior to and after finishing work, neatly.......

Materials

Name	Company	Catalog Number	Comments
DPBS	Gibco	14-190-144
DMEM F-12 Media	ATCC	30-2006
Glass Baffled Flask	Pyrex	09-552-40
Glass Pipettes	Fisher	13-678-6B
Pipette Aid	Drummond	13-681-15A
Serological Pipette	Corning	07-200-573
T75 flask	Corning	07-202-004
Trypsin	Gibco	25-300-054
*Items may vary because this video is about general cell culture techniques