How MGM Scientists Prevent Mycoplasma (Part 1)

This Article Was Written By Kaustubh Kishor Jadhav, Who Shared His Previous Paragraph About The Causes Of Mycoplasma Contamination And How To Detect It. He Is A Research Assistant Of MGM Institute Of Biological Science And Technology& Nbsp; If You Are Reading This Article, Do Not Be Surprised If You Have Mycoplasma In Your Laboratory. Because They Exist In Most Cell Culture Facilities And Tissue Culture Laboratories, They Are Problems That Every Cell Culture Worker Must Solve. It Is Estimated That Up To 60% Of Cell Culture Contamination Is Mycoplasma Contamination (Uphoff, 2002)& Nbsp; Mycoplasma Is Considered To Be One Of The Simplest And Smallest Bacteria. The Lack Of Hard Cell Walls Makes Them Resistant To Antibiotics And Antibiotics Such As Penicillin And Streptomycin. Mycoplasma Can Pass Through The Filter Because It Can Become Any Shape To Adhere To The Cell Wall& Nbsp; The Cause Of Mycoplasma Contamination Mycoplasma Enters Cell Culture Through Various Sources That Are Difficult To Trace. These Include Laboratory Personnel, Serum, Cell Culture Medium, Water Bath, Incubator, Etc. Human Mycoplasma Pollution Is The Largest Source Of The Above Pollution. The Pollutants Can Be Spread Through Dirty Clothes, Experimental Clothes, And Speech, Dandruff, Sneezing, Coughing, Etc. Near The Laminar Airflow. In Addition, No Matter Where Cell Cultures Are Stored, The Constant Flow Of Individuals Increases The Risk Of Contamination& Nbsp; Due To Backward Laboratory Equipment And Cost Saving, Mycoplasma Can Be Transmitted From These Sources Through Cross Contamination, Including The Reuse Of Pipettes For Multiple Cell Lines Rather Than The Use Of Disposable Pipettes Or Gloves. Serum Is Also The Source Of Mycoplasma Contamination. Because Of Its Turbid Nature, Contamination Is Difficult To Detect In Serum, Which Is The Most Commonly Used For Each Cell Culture. Repeated Use Of The Same Bottle Of Serum Can Promote The Growth Of Mycoplasma. Serum Is Rich In Nutrients, And Is Also The Best Source Of Mycoplasma Proliferation. In Addition, It Is Added To The Culture Medium After Autoclaving, So It Cannot Ensure That The Serum Is Free Of Pollution& Nbsp; The Aerosol Produced When Working In The Laminar Air Flow Bin (talking Or Moving Liquid) Is Also One Of The Main Causes Of Pollution. During Passage Culture, These Aerosols Will Enter The Culture Medium. If The Cell Line Is Exposed For A Long Time, These Aerosols Will Enter The Cell From The Air. These Aerosols Are Invisible To The Naked Eye And Cannot Be Detected Until They Cause Contamination& Nbsp; Another Source Of The Problem Is The Medium Used For Passage Culture, Which Has Two Forms: Liquid And Powder. Due To Improper Treatment Or Bad Laboratory Environment, Mycoplasma Can Be Transmitted Through This Powder Culture Medium. Filtration Does Not Ensure Complete Sterilization, Because Mycoplasma Can Even Escape The 0.2 Micron Filter& Nbsp; Mycoplasma Can Be Kept In A Dry State For A Long Time. When They Come Into Contact With Nutrient Sources, They Will Soon Begin To Proliferate. Once They Appear In The Laboratory Environment, It Is Difficult To Eradicate Them. You Can Inhibit The Growth Of Mycoplasma, But You Cannot Completely Eradicate It. When Mycoplasma Appears In The Culture Medium, It Is A Good Choice To Discard The Culture Source (unless The Culture Source Is Irreplaceable Or Expensive)& Nbsp; Detection Of Mycoplasma Contamination; It Is Very Difficult To Detect Mycoplasma With Naked Eye Or Light Microscope, So How Do You Know Where It Is? You Can Use PCR Detection, DNA Staining, Fluorescent Labeling, Agar Plate, Etc. Choose Any Of The Techniques Listed Below Wisely And Always Use The Second Analysis To Determine. Before Detecting Mycoplasma, The Cells Should Be Cultured Continuously For At Least Two Weeks To Allow Time For Low Concentrations Of Pollutants To Grow. For Tests, The Medium Should Not Be Changed At Least Two Or Three Days Before Sampling (Uphoff And Drexler, 2011). Soft Agar Culture Is Considered As The "gold Standard" For Detecting Mycoplasma. In This Method, The Supernatant Of Cell Culture Is Added To A Liquid Or Semi-solid Medium Containing Nutrients Necessary For The Growth Of Mycoplasma. The Infected Cells Will Grow On The Agar Plate Containing The Culture Medium. Mycoplasma Colony Can Be Easily Seen On The Agar Plate. Except For A Few Mycoplasmas, This Method Can Detect Most Mycoplasmas. Another Method For Mycoplasma Detection Is PCR. In This Technique, PCR Primers For 16sRNA Genes Of Various Common Mycoplasma Infections Were Used. A Wide Range Of Primers Contain Almost All Infectious Mycoplasmas. The Primers Used In The Process Are Either Species/gene Specific Or Generic. This Method Is Fast, Efficient, Reliable And Cost-effective (Sung, 2006). Mycoplasma Cells Were Detected By Chromogenic Method. Once Mycoplasma Is Detected, The Reagent Will Trigger A Series Of Chemical Reactions, Which Will Cause Obvious Color Changes. This Method Has High Sensitivity And Low Specificity (Degeling, 2012). Bioluminescence Detection Kits Can Be Obtained From Different Biotechnology Companies, Which Are Enzyme Based Mycoplasma Detection Kits. Mycoplasma Enzymes Detected By These Kits Are Not Synthesized By Eukaryotic Cells. First, The Components Of The Kit Break The Mycoplasma, Then Use Specific Enzymes To Trigger The Cell Mechanism That Generates Light, And Then Detect It Through The Photometer. Non Specific DNA Stain Can Be Added To The Infection Medium To Detect Mycoplasma. When Observed Under The Fluorescence Microscope, Mycoplasma DNA Appears In Small Clusters In Addition To Cell DNA. Fluorescent DNA Staining Is Another Method Of DNA Staining. This Method Uses DNA Staining Agents Such As DAPI And Hoechst 33258 To Stain All DNA. In This Process, Some Professional Knowledge Is Required, Because The Interpretation Of The Results May Be Difficult. The Low Density Of Mycoplasma, The Contamination Of Other Bacteria Or The Extracellular Fluorescence Signals Produced By These Bacteria Will Hinder The Correct Interpretation Of The Results. In Addition, Signals From The Nuclear Region Make It Difficult To Detect Mycoplasma (Ligasov á, 2019). This Method Is Generally Not Recommended.