This document provides an overview of mycoplasma contamination in cell culture and methods for detection and treatment. Mycoplasma are the smallest prokaryotes that can contaminate cell cultures invisibly. They attach to host cell surfaces and can cause adverse effects including altered gene expression, growth inhibition, and cell death. Routine monitoring is important as mycoplasma are commonly introduced from infected cultures or reagents. Detection methods include culture, PCR, staining, and enzymatic assays like MycoAlert. While antibiotics are often used for treatment, MycoZap reagent offers a fast and reliable way to eliminate mycoplasma contamination within 4 days with minimal toxicity to host cells.
Susceptibility testing is used to determine which antimicrobials will inhibit the growth of the bacteria or fungi causing a specific infection. The results from this test will help a healthcare practitioner determine which drugs are likely to be most effective in treating a person's infection.
This document provides information about anti-fungal susceptibility testing. It discusses various fungi and the available anti-fungal drugs that act on different fungal targets like the cell wall, cell membrane, microtubules, RNA/DNA, and protein synthesis. It covers different testing methods like macrodilution, microdilution, and disk diffusion. It provides details on test medium, inoculum preparation, drug dilutions, and incubation conditions. It also discusses interpretive criteria, quality control strains and ranges, emerging resistance, and the need for susceptibility testing.
This document discusses antifungal susceptibility testing. It provides background on the history of antifungal susceptibility testing and why it is needed. It describes different methods for testing including broth dilution and disk diffusion. It discusses various antifungal agents and their mechanisms of action. The document outlines the procedures for broth microdilution and macrodilution testing according to CLSI guidelines, including preparation of inoculum, drug solutions, reading results, and testing of filamentous fungi.
This document discusses bacteriophages (phages), viruses that infect bacteria. It covers the composition and structure of phages, how they infect host cells through adsorption and nucleic acid injection, and their multiplication cycles of either the lytic or lysogenic pathways. The document also discusses phage typing, which uses specific phages to identify and differentiate bacterial pathogens, and applications of phages in areas like diagnostics, therapeutics, biocontrol, and more.
MALDI - TOF MS: Where Mass Spectrometry Meets MicrobiologyICAR-CIFE
Abstract:
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has turned a promising tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics laboratory. MALDI-TOF measures the mass of molecules from a sample that has been embedded in a matrix by using a laser to ablate and desorb the molecules with minimal fragmentation. The mass spectrum is produced from the pattern (i.e., position and relative intensity) of detected m/z peaks, generating a distinct profile for a particular sample. The method is continuously being improved, and new applications for distinguishing strains, identifying metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in host samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research. Nowadays, MALDI-TOF MS technology is most widely used for discovering potential cancer biomarkers. MALDI-TOF MS presents advantages as it is easy to use the technique with high automation and throughput potential and high sensitivity. New fields of application for MALDI-TOF MS, such as Surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF MS) and imaging mass spectrometry (IMS) are promising tools in the field of oncology.
MALDI-TOF mass spectrometry is a technique used to analyze proteins. It works by ionizing protein samples using a laser and then measuring the time it takes for the ions to travel through a flight tube, which allows calculating the mass-to-charge ratio. The sample is mixed with an absorbing matrix and dried on a target plate before being ionized by a laser pulse. Ions are accelerated through a flight tube and reach a detector, with lighter ions traveling faster and reaching it first. The time of flight is converted to a mass spectrum, allowing identification of proteins in the sample. MALDI-TOF provides sensitive, high-throughput protein analysis and is widely used in fields like proteomics, microbiology,
Susceptibility testing is used to determine which antimicrobials will inhibit the growth of the bacteria or fungi causing a specific infection. The results from this test will help a healthcare practitioner determine which drugs are likely to be most effective in treating a person's infection.
This document provides information about anti-fungal susceptibility testing. It discusses various fungi and the available anti-fungal drugs that act on different fungal targets like the cell wall, cell membrane, microtubules, RNA/DNA, and protein synthesis. It covers different testing methods like macrodilution, microdilution, and disk diffusion. It provides details on test medium, inoculum preparation, drug dilutions, and incubation conditions. It also discusses interpretive criteria, quality control strains and ranges, emerging resistance, and the need for susceptibility testing.
This document discusses antifungal susceptibility testing. It provides background on the history of antifungal susceptibility testing and why it is needed. It describes different methods for testing including broth dilution and disk diffusion. It discusses various antifungal agents and their mechanisms of action. The document outlines the procedures for broth microdilution and macrodilution testing according to CLSI guidelines, including preparation of inoculum, drug solutions, reading results, and testing of filamentous fungi.
This document discusses bacteriophages (phages), viruses that infect bacteria. It covers the composition and structure of phages, how they infect host cells through adsorption and nucleic acid injection, and their multiplication cycles of either the lytic or lysogenic pathways. The document also discusses phage typing, which uses specific phages to identify and differentiate bacterial pathogens, and applications of phages in areas like diagnostics, therapeutics, biocontrol, and more.
MALDI - TOF MS: Where Mass Spectrometry Meets MicrobiologyICAR-CIFE
Abstract:
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has turned a promising tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics laboratory. MALDI-TOF measures the mass of molecules from a sample that has been embedded in a matrix by using a laser to ablate and desorb the molecules with minimal fragmentation. The mass spectrum is produced from the pattern (i.e., position and relative intensity) of detected m/z peaks, generating a distinct profile for a particular sample. The method is continuously being improved, and new applications for distinguishing strains, identifying metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in host samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research. Nowadays, MALDI-TOF MS technology is most widely used for discovering potential cancer biomarkers. MALDI-TOF MS presents advantages as it is easy to use the technique with high automation and throughput potential and high sensitivity. New fields of application for MALDI-TOF MS, such as Surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF MS) and imaging mass spectrometry (IMS) are promising tools in the field of oncology.
MALDI-TOF mass spectrometry is a technique used to analyze proteins. It works by ionizing protein samples using a laser and then measuring the time it takes for the ions to travel through a flight tube, which allows calculating the mass-to-charge ratio. The sample is mixed with an absorbing matrix and dried on a target plate before being ionized by a laser pulse. Ions are accelerated through a flight tube and reach a detector, with lighter ions traveling faster and reaching it first. The time of flight is converted to a mass spectrum, allowing identification of proteins in the sample. MALDI-TOF provides sensitive, high-throughput protein analysis and is widely used in fields like proteomics, microbiology,
Antimicrobial susceptibility testing determines which drugs will inhibit bacterial or fungal growth causing an infection. The results help identify the most effective treatment and monitor resistance patterns. Standardized methods involve using Mueller Hinton agar or broth with standardized inoculum and controls under uniform conditions. Common testing methods include disk diffusion, broth dilution, and agar dilution. Minimum inhibitory concentration (MIC) identifies the lowest drug concentration inhibiting growth, with lower MIC indicating greater effectiveness. Qualitative and quantitative results guide appropriate drug use and prevent resistance.
This document discusses methods for detecting Methicillin-Resistant Staphylococcus aureus (MRSA). MRSA is any strain of S. aureus that is resistant to beta-lactam antibiotics due to the mecA gene. Rapid detection of MRSA is important for optimal treatment and reducing costs. The document describes several screening methods, focusing on the oxacillin salt agar screening test which involves growing bacterial samples on agar containing oxacillin and 4% NaCl. Growth of more than one colony indicates oxacillin resistance and identifies the strain as MRSA.
Molecular methods and clinical microbiologyimprovemed
This document discusses various molecular diagnostic methods used in clinical microbiology. It describes techniques such as nucleic acid hybridization, polymerase chain reaction (PCR), and real-time PCR which can directly detect microorganisms, identify pathogens, and detect antibiotic resistance genes. The advantages of these molecular methods include speed, ability to detect fastidious or non-culturable bacteria, and quantitative analysis. However, limitations include possible false negatives from inhibitors and inability to determine bacterial viability. Overall, molecular diagnostics provide crucial information to improve patient treatment and conduct epidemiological analyses.
The document discusses conventional microbiological techniques used in diagnostic microbiology laboratories. It describes how Robert Koch and Ronald Ross helped develop culturing pathogens and the discovery that specific microbes cause diseases. It also discusses how conventional techniques like growing bacteria in broth or on solid media, staining, and microscopy are still important today, but that molecular biology techniques may revolutionize disease diagnosis in the future. Gram staining remains one of the most rapid diagnostic methods for identifying bacteria in clinical specimens.
This document discusses antifungal susceptibility testing and the disk diffusion method. It describes how the increased incidence of fungal infections has led to greater attention on antifungal resistance testing. The disk diffusion method involves inoculating agar plates with fungal cultures, applying antifungal disks, incubating the plates, and measuring inhibition zones to determine antifungal susceptibility. Interpretation of zone diameters provides clinicians with guidance on optimal antifungal therapy.
Bacillus is a genus of rod-shaped, Gram-positive bacteria that can form dormant endospores. The document focuses on Bacillus anthracis, which causes anthrax. It describes the morphology, cultural characteristics, virulence factors, and methods of diagnosis and prevention of B. anthracis. Key points include that B. anthracis forms encapsulated, non-motile rods and terminal spores. The anthrax toxins are composed of lethal factor, edema factor, and protective antigen, which combine to cause disease. Diagnosis involves microscopy, culture, and serology. Prevention for humans involves vaccination with anthrax toxoid and occupational hygiene, while animals are vaccinated with attenuated spore
The use of a machine designed to follow repeatedly and automatically a predetermined sequence of individual operations.
AUTOMATED WASHING
AUTOMATED MEDIA PREPARATORS
AUTOMATED COLLECTION AND
PROCESSING OF SAMPLES
CYTOSPIN
AUTOMATED GRAM STAINING
AUTOMATED STREAKING
SPIRAL PLATER
AUTOMATED ANTIBIOTIC -
SENSITIVITY SYSTEM
AUTOMATIC COLONY COUNTER
AUTOMATED URINE MICROSCOPY -
ANALYSER
Antimicrobial susceptibility testing determines how effective antibiotic therapy is against bacterial infections. There are several methods for testing, including disk diffusion, dilution tests, and automated systems. Disk diffusion involves placing disks impregnated with antibiotics onto inoculated agar plates and measuring inhibition zones. Mueller-Hinton agar is commonly used as the growth medium. Dilution tests determine the minimum inhibitory concentration (MIC) of antibiotics using serial dilutions in broth or agar. Automated systems streamline the testing process. Proper technique and controls are important for accurate and reproducible results.
1. Mycobacterium is a genus of bacteria that can cause tuberculosis and leprosy. It has a waxy lipid cell wall and grows slowly.
2. The tuberculosis-causing bacteria include M. tuberculosis, M. bovis, M. africanum, and M. microti. Lepra bacilli that cause leprosy include M. leprae.
3. Diagnosis involves microscopy, culture, nucleic acid tests, tuberculin skin tests, chest x-rays, and analysis of symptoms. Treatment uses several first-line and second-line drugs in combination over 6-8 months to prevent drug resistance.
Oxidase Test Microbiology - Principle, Procedure, Limitations, Results, QC - in lab #Oxidase Test
As the channel name suggests, our channel will be a perfect lounge for the malayali medicos..we wil be covering videos which will be like lecture classes related to the subjects biochemistry and microbiology in which we are specialised.. It will be a better learning experience for the students especially for those who are not able to understand and follow the normal classes in college..we assure the students that you will get a basic idea regarding the topic and extra reading can be done from the reference textbooks...
If you like my video
#like
#comment
#subscribe my channel
don't forget to subscribe my channel
Qualification
Maneesha M Joseph
MSc MLT (Microbiology)
Assistant Professor
Baby memorial college of allied Health science
Kozhikode
Our Partner Channel
Health & Voyage channel link - https://youtu.be/nzKqRVjlwc0
#Oxidase Test
#Medical
#Microbiology
# malayalam lecturer
#Mallu Medicos Lounge
#MalluMedicosLounge
#MLT
The document discusses nested PCR, which is a modification of polymerase chain reaction (PCR) that improves specificity. It involves two rounds of PCR where the product of the first reaction is used as a template for the second reaction with a nested primer set. This increases specificity by reducing non-specific binding. Some key advantages are improved accuracy and sensitivity for low abundance targets or difficult templates. However, it is more time-consuming and costly than standard PCR due to the extra reagents and steps required. Nested PCR has applications in microbial detection, genetic analysis, and other areas where high specificity is needed.
Recent advances in cultivation & identification of anaerobicabhishek yadav
This document discusses recent advances in cultivating and identifying clinically significant anaerobic bacteria. It covers:
1. The challenges in detecting anaerobic infections due to their slow growth and long turnaround times for identification.
2. Methods for classifying, isolating, and identifying anaerobic bacteria including their oxygen tolerance levels, suitable specimens for culture, transport methods, and culture techniques.
3. The major infections caused by different anaerobic bacteria like Clostridium, Bacteroides, Fusobacterium species.
4. Clues that suggest anaerobic infections and approaches to the presumptive identification of isolated organisms.
Multiplex PCR is a technique whereby PCR is used to amplify several different DNA sequences simultaneously. It is a type of target enrichment approach. It was first described in 1988 as a method to detect deletion mutations in the dystrophin gene – the largest known human gene
Automated system for bacterial identificationDEEKSHANT KUMAR
[DOWNLOAD IT OPEN IT WITH MICROSOFT POWERPOINT THEN YOU WILL BE ABLE TO UNDERSTAND THE TOPIC COVERED.]
1. WHOLE TEXT IS RELIABLE.
2. TEXT HAS BEEN TAKEN FROM STANDARD TEXT BOOK FOR MEDICAL MICROBIOLOGY.
3. SOME PICTURE HAS BEEN TAKEN FROM JOURNAL.
This document discusses different types of polymerase chain reaction (PCR) techniques. It begins by providing background on PCR and its development. It then describes several types of PCR including multiplex PCR, which allows for simultaneous detection of multiple pathogens; nested PCR, which increases specificity; reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR), which are used to detect RNA; quantitative PCR, which measures specific target DNA/RNA amounts; and other variants like hot-start PCR, touchdown PCR, and methylation-specific PCR. Each type is briefly explained along with its uses and applications in medical research.
This document discusses Rickettsia and Coxiella bacteria. Rickettsia species are gram-negative, obligate intracellular parasites transmitted by arthropod vectors like ticks and mites. They cause diseases like epidemic typhus, murine typhus, and Rocky Mountain spotted fever by infecting endothelial cells. Coxiella burnetii causes Q fever, a typhoid-like illness transmitted through inhalation of aerosols from infected animals. Both are diagnosed serologically and treated with doxycycline.
Rickettsia are tiny, obligate intracellular bacteria that can cause diseases like Rocky Mountain spotted fever when transmitted to humans via arthropod vectors like ticks and mites. They grow within the cells of vertebrates and can be cultured in embryonated eggs or tissue cell culture. Rickettsia have cell walls, undergo binary fission, and are sensitive to antibiotics unlike viruses. Chlamydia are also obligate intracellular bacteria with two forms - the infectious elementary body and reproductive reticulate body. They have a characteristic developmental cycle but are unable to produce their own ATP. Mycoplasmas are the smallest bacteria that can be grown on artificial media and lack cell walls, taking on various shapes. Their colonies in
Antimicrobial susceptibility testing determines which drugs will inhibit bacterial or fungal growth causing an infection. The results help identify the most effective treatment and monitor resistance patterns. Standardized methods involve using Mueller Hinton agar or broth with standardized inoculum and controls under uniform conditions. Common testing methods include disk diffusion, broth dilution, and agar dilution. Minimum inhibitory concentration (MIC) identifies the lowest drug concentration inhibiting growth, with lower MIC indicating greater effectiveness. Qualitative and quantitative results guide appropriate drug use and prevent resistance.
This document discusses methods for detecting Methicillin-Resistant Staphylococcus aureus (MRSA). MRSA is any strain of S. aureus that is resistant to beta-lactam antibiotics due to the mecA gene. Rapid detection of MRSA is important for optimal treatment and reducing costs. The document describes several screening methods, focusing on the oxacillin salt agar screening test which involves growing bacterial samples on agar containing oxacillin and 4% NaCl. Growth of more than one colony indicates oxacillin resistance and identifies the strain as MRSA.
Molecular methods and clinical microbiologyimprovemed
This document discusses various molecular diagnostic methods used in clinical microbiology. It describes techniques such as nucleic acid hybridization, polymerase chain reaction (PCR), and real-time PCR which can directly detect microorganisms, identify pathogens, and detect antibiotic resistance genes. The advantages of these molecular methods include speed, ability to detect fastidious or non-culturable bacteria, and quantitative analysis. However, limitations include possible false negatives from inhibitors and inability to determine bacterial viability. Overall, molecular diagnostics provide crucial information to improve patient treatment and conduct epidemiological analyses.
The document discusses conventional microbiological techniques used in diagnostic microbiology laboratories. It describes how Robert Koch and Ronald Ross helped develop culturing pathogens and the discovery that specific microbes cause diseases. It also discusses how conventional techniques like growing bacteria in broth or on solid media, staining, and microscopy are still important today, but that molecular biology techniques may revolutionize disease diagnosis in the future. Gram staining remains one of the most rapid diagnostic methods for identifying bacteria in clinical specimens.
This document discusses antifungal susceptibility testing and the disk diffusion method. It describes how the increased incidence of fungal infections has led to greater attention on antifungal resistance testing. The disk diffusion method involves inoculating agar plates with fungal cultures, applying antifungal disks, incubating the plates, and measuring inhibition zones to determine antifungal susceptibility. Interpretation of zone diameters provides clinicians with guidance on optimal antifungal therapy.
Bacillus is a genus of rod-shaped, Gram-positive bacteria that can form dormant endospores. The document focuses on Bacillus anthracis, which causes anthrax. It describes the morphology, cultural characteristics, virulence factors, and methods of diagnosis and prevention of B. anthracis. Key points include that B. anthracis forms encapsulated, non-motile rods and terminal spores. The anthrax toxins are composed of lethal factor, edema factor, and protective antigen, which combine to cause disease. Diagnosis involves microscopy, culture, and serology. Prevention for humans involves vaccination with anthrax toxoid and occupational hygiene, while animals are vaccinated with attenuated spore
The use of a machine designed to follow repeatedly and automatically a predetermined sequence of individual operations.
AUTOMATED WASHING
AUTOMATED MEDIA PREPARATORS
AUTOMATED COLLECTION AND
PROCESSING OF SAMPLES
CYTOSPIN
AUTOMATED GRAM STAINING
AUTOMATED STREAKING
SPIRAL PLATER
AUTOMATED ANTIBIOTIC -
SENSITIVITY SYSTEM
AUTOMATIC COLONY COUNTER
AUTOMATED URINE MICROSCOPY -
ANALYSER
Antimicrobial susceptibility testing determines how effective antibiotic therapy is against bacterial infections. There are several methods for testing, including disk diffusion, dilution tests, and automated systems. Disk diffusion involves placing disks impregnated with antibiotics onto inoculated agar plates and measuring inhibition zones. Mueller-Hinton agar is commonly used as the growth medium. Dilution tests determine the minimum inhibitory concentration (MIC) of antibiotics using serial dilutions in broth or agar. Automated systems streamline the testing process. Proper technique and controls are important for accurate and reproducible results.
1. Mycobacterium is a genus of bacteria that can cause tuberculosis and leprosy. It has a waxy lipid cell wall and grows slowly.
2. The tuberculosis-causing bacteria include M. tuberculosis, M. bovis, M. africanum, and M. microti. Lepra bacilli that cause leprosy include M. leprae.
3. Diagnosis involves microscopy, culture, nucleic acid tests, tuberculin skin tests, chest x-rays, and analysis of symptoms. Treatment uses several first-line and second-line drugs in combination over 6-8 months to prevent drug resistance.
Oxidase Test Microbiology - Principle, Procedure, Limitations, Results, QC - in lab #Oxidase Test
As the channel name suggests, our channel will be a perfect lounge for the malayali medicos..we wil be covering videos which will be like lecture classes related to the subjects biochemistry and microbiology in which we are specialised.. It will be a better learning experience for the students especially for those who are not able to understand and follow the normal classes in college..we assure the students that you will get a basic idea regarding the topic and extra reading can be done from the reference textbooks...
If you like my video
#like
#comment
#subscribe my channel
don't forget to subscribe my channel
Qualification
Maneesha M Joseph
MSc MLT (Microbiology)
Assistant Professor
Baby memorial college of allied Health science
Kozhikode
Our Partner Channel
Health & Voyage channel link - https://youtu.be/nzKqRVjlwc0
#Oxidase Test
#Medical
#Microbiology
# malayalam lecturer
#Mallu Medicos Lounge
#MalluMedicosLounge
#MLT
The document discusses nested PCR, which is a modification of polymerase chain reaction (PCR) that improves specificity. It involves two rounds of PCR where the product of the first reaction is used as a template for the second reaction with a nested primer set. This increases specificity by reducing non-specific binding. Some key advantages are improved accuracy and sensitivity for low abundance targets or difficult templates. However, it is more time-consuming and costly than standard PCR due to the extra reagents and steps required. Nested PCR has applications in microbial detection, genetic analysis, and other areas where high specificity is needed.
Recent advances in cultivation & identification of anaerobicabhishek yadav
This document discusses recent advances in cultivating and identifying clinically significant anaerobic bacteria. It covers:
1. The challenges in detecting anaerobic infections due to their slow growth and long turnaround times for identification.
2. Methods for classifying, isolating, and identifying anaerobic bacteria including their oxygen tolerance levels, suitable specimens for culture, transport methods, and culture techniques.
3. The major infections caused by different anaerobic bacteria like Clostridium, Bacteroides, Fusobacterium species.
4. Clues that suggest anaerobic infections and approaches to the presumptive identification of isolated organisms.
Multiplex PCR is a technique whereby PCR is used to amplify several different DNA sequences simultaneously. It is a type of target enrichment approach. It was first described in 1988 as a method to detect deletion mutations in the dystrophin gene – the largest known human gene
Automated system for bacterial identificationDEEKSHANT KUMAR
[DOWNLOAD IT OPEN IT WITH MICROSOFT POWERPOINT THEN YOU WILL BE ABLE TO UNDERSTAND THE TOPIC COVERED.]
1. WHOLE TEXT IS RELIABLE.
2. TEXT HAS BEEN TAKEN FROM STANDARD TEXT BOOK FOR MEDICAL MICROBIOLOGY.
3. SOME PICTURE HAS BEEN TAKEN FROM JOURNAL.
This document discusses different types of polymerase chain reaction (PCR) techniques. It begins by providing background on PCR and its development. It then describes several types of PCR including multiplex PCR, which allows for simultaneous detection of multiple pathogens; nested PCR, which increases specificity; reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR), which are used to detect RNA; quantitative PCR, which measures specific target DNA/RNA amounts; and other variants like hot-start PCR, touchdown PCR, and methylation-specific PCR. Each type is briefly explained along with its uses and applications in medical research.
This document discusses Rickettsia and Coxiella bacteria. Rickettsia species are gram-negative, obligate intracellular parasites transmitted by arthropod vectors like ticks and mites. They cause diseases like epidemic typhus, murine typhus, and Rocky Mountain spotted fever by infecting endothelial cells. Coxiella burnetii causes Q fever, a typhoid-like illness transmitted through inhalation of aerosols from infected animals. Both are diagnosed serologically and treated with doxycycline.
Rickettsia are tiny, obligate intracellular bacteria that can cause diseases like Rocky Mountain spotted fever when transmitted to humans via arthropod vectors like ticks and mites. They grow within the cells of vertebrates and can be cultured in embryonated eggs or tissue cell culture. Rickettsia have cell walls, undergo binary fission, and are sensitive to antibiotics unlike viruses. Chlamydia are also obligate intracellular bacteria with two forms - the infectious elementary body and reproductive reticulate body. They have a characteristic developmental cycle but are unable to produce their own ATP. Mycoplasmas are the smallest bacteria that can be grown on artificial media and lack cell walls, taking on various shapes. Their colonies in
This document discusses Rickettsia and Chlamydia. It provides details on their classification and characteristics. Both are obligate intracellular parasites, but Rickettsia are transmitted by arthropods while Chlamydia have two distinct forms - infectious elementary bodies and intracellular reticulate bodies. The document outlines their structures, metabolisms, growth and multiplication processes, pathogenic mechanisms, epidemiologies, diagnoses and treatments.
This document summarizes various Rickettsiaceae genera including Rickettsia, Orientia, Ehrlichia, Coxiella, and Bartonella. It describes their characteristics such as being small, gram-negative, obligate intracellular parasites transmitted by arthropod vectors. Specific diseases caused by each genus are outlined, such as typhus fever caused by Rickettsia transmitted by lice and fleas, scrub typhus caused by Orientia transmitted by chiggers, and Q fever caused by Coxiella transmitted between animals and humans. Laboratory diagnosis and treatment options are also briefly discussed.
This document discusses Mycoplasma, a genus of bacteria that lacks a cell wall. It notes that Mycoplasma pneumoniae and Ureaplasma urealyticum are important human pathogens that can cause respiratory and genital diseases. M. pneumoniae specifically can cause upper respiratory infections, tracheobronchitis, and atypical pneumonia. Laboratory diagnosis of Mycoplasma involves culture, microscopy, and serological tests. Treatment involves tetracycline or erythromycin.
This document discusses Rocky Mountain spotted fever, a potentially fatal disease caused by the bacterium Rickettsia rickettsii that is transmitted through tick bites. It presents a case study of an 8-year old girl from Tennessee who was initially diagnosed with gastroenteritis but later tested positive for R. rickettsii after presenting with fever, rash, and other symptoms. Key details include that the disease causes vasculitis and thrombocytopenia, symptoms typically emerge 2-14 days after a tick bite, and doxycycline is the recommended treatment to prevent severe outcomes or death from infection.
Rickettsiae and Chlamydiae are obligate intracellular bacteria that can cause disease in humans and animals. Rickettsiae include genera that are transmitted by arthropods like ticks and fleas, and parasites of gut cells or white blood cells. Chlamydiae have a unique life cycle alternating between infectious elementary bodies and intracellular reticulate bodies, and can cause respiratory diseases as well as ocular and urogenital infections in humans. Both are treated with tetracycline or erythromycin, and control relies on prevention of transmission from arthropod vectors or infected animals.
The document describes a study that aimed to predict non-coding RNAs (ncRNAs) in clinically important Mycoplasma species through comparative genomic analysis. The approach involved extracting intergenic regions from Mycoplasma genomes, searching for sequence similarity across related organisms, parsing alignments using certain cutoffs, and using the parsed alignments as input for the QRNA program to predict putative ncRNAs. Several ncRNAs were predicted for each organism and some showed similarity to known bacterial and eukaryotic ncRNAs.
Rickettsiae are small, obligate intracellular bacteria that can cause diseases in humans. They are transmitted through arthropod vectors like ticks and lice. Rickettsiae are divided into three groups based on the diseases they cause: spotted fever, typhus, and scrub typhus. Upon entering the body through a bite or broken skin, they infect endothelial cells. Some escape the phagosome and enter the cell cytoplasm, while others are released when the cell lyses. Doxycycline is the treatment of choice and preventing exposure to ticks can reduce risk of infection. Laboratory diagnosis involves serologic testing to detect antibodies in paired serum samples.
This document summarizes rickettsial diseases, which are caused by obligate intracellular parasites called Rickettsia. Rickettsia species are transmitted to humans via the bites of infected arthropods like fleas, lice, ticks and mites. Some key Rickettsia species and the diseases they cause include R. prowazekii which causes epidemic typhus, R. typhi which causes endemic typhus, and R. rickettsii which causes Rocky Mountain spotted fever. Symptoms include fever, headache, rash and potentially serious complications if left untreated. Diagnosis involves isolation of the bacteria or serological tests to detect antibodies. Treatment is with doxycycline or tetracycline.
1119 East 5th Street, Royal Oak3, Forsale2Julie Thayer
This listing describes a 1925 Craftsman style bungalow located in Royal Oak, Michigan. It has been updated with features like a new kitchen, bathrooms, floors, windows, and more. The home has 3 bedrooms, 2.5 bathrooms, and 1800 square feet of living space. It is located in a quiet neighborhood within walking distance of downtown Royal Oak and its amenities. The home is being listed for $410,000.
The document discusses Clearleap Sentinel Monitor, a software tool that provides live stream monitoring, stream integrity monitoring, real-time session performance monitoring, and a visual trending toolset to analyze KPI performance, MOS scoring, blockiness, audio compliance, and other quality metrics for streaming media.
The passage of a federal spending bill contains protections for state-legal medical marijuana programs that prohibit the DOJ from using funds to interfere. This represents a historic step away from the federal war on drugs towards respecting state marijuana laws. However, the summary also highlights the tragic story of an 8-year-old girl in New York who died waiting for emergency access to medical marijuana despite the state's new law, prompting pleas to the governor to take action to help suffering families.
AOS Studley is the largest global corporate real estate advisor focused solely on representing tenants. It has over 950 employees across 30 countries. The document provides details on AOS Studley's expertise in tenant representation, workplace consulting, project management, and strategic consulting. It also includes international case studies and references for clients such as Carrefour, Microsoft, and SwissLife.
Here are the answers with simple present or present continuous verbs:
1) She has a bath every evening.
2) My husband is always tasting the food while I'm cooking! It's very annoying.
3) Luke is seeing the doctor now.
4) He is having a party next weekend.
5) This coffee doesn't taste right.
6) We are going to see John and Susie next month.
7) What do you think about the war ?
8) She has a headache.
9) It is cold today.
10) They don't have a car.
11) I can't see anything, I can't work the telescope?
12) The wa
What do you believe are the greatest challenges facing the sector or industry you would like to specialize in at IE? What role do you hope to be able to play in this sector or industry in the medium term?
The document provides a vision statement for the Phoenix Tears Foundation, a 501(c)3 non-profit organization. It outlines plans to expand the foundation's capacity and financial resources through the release of a line of trusted CBD products. It then describes proposed projects including a multi-level care facility in Colorado Springs, Colorado and a medical clinic in Las Vegas, Nevada that would provide both Eastern and Western cancer treatment modalities and incorporate a Phoenix Tears Day Spa and Boutique. The goal is for these projects to be funded by sales of Phoenix Tears branded CBD products and attract angel investors.
Corfu is a Greek island in the Ionian Sea that has the shape of a sickle. The document provides a map of Corfu town with landmarks like the old fortress, Asian Art Museum, Archaeological Museum, and Palace. It asks questions about directions between locations, where to find ferry tickets and mail letters, and where cricket players train.
The document provides examples of questions and statements using the Greek verbs for "can" and "can't" (εγώ= I can, εσύ = you can, etc.) in both the affirmative and negative form. It includes examples asking about abilities, permissions, and requests for help using these verbs. The document then has exercises for rearranging words into questions and statements.
This document provides an overview of mycobacteriology and techniques used for tuberculosis screening and diagnosis. It discusses the PPD skin test and IGRAs for TB screening, as well as acid-fast staining and culture techniques used to identify mycobacteria in clinical specimens. Key points covered include the characteristics of the mycobacteria genus, the Runyon classification system for grouping nontuberculous mycobacteria, and the multi-step process for decontaminating, staining, and culturing specimens to diagnose mycobacterial infections.
Explain why a mycoplasma PCR kit might give a negative result when u.pdfflashfashioncasualwe
Explain why a mycoplasma PCR kit might give a negative result when used to test for
mycoplasma in a cell line containing mycoplasma
Solution
Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures.
This organism can modify many aspects of cell physiology, rendering experiments that are
conducted with contaminated cells worthless. Because of their small size, Mycoplasma can pass
through filters used to prevent bacterial and fungal contamination and potentially spread to all
the cultures in a laboratory. It is essential that all new cell cultures entering a laboratory and all
cell banks are tested for presence of Mycoplasma. It is recommended that two techniques be
used, selected from a PCR-based methos, indirect staining and an agar and broth culture. These
three tests for detecting Mycoplasma take one day to 3-4 weeks and such tests should be an
obligatory component of quality control in every tissue culture laboratory.
Mycoplasma refers to any of over 200 species of tiny bacteria that invade cell cultures and may
even persist unrecognized for some time. They can come from sources as mundane as other cells
introduced to lab via media, sera or lab personnel. So, it is important to test your cell lines for
mycoplasma regularly. Consequences to your cells of mycoplasma infestation include changes in
growth rates, gene expression, morphology, metabolism and viability.
Even though most lab contamination can be blamed on as few as eight main culprit species, these
differ enough that no one test can find them all. And the little blighters are hardy – most
antibiotics used in cell culture are ineffective against mycoplasmas. For example, mycoplasmas
lack a cell wall, which is the key to penicillin’s success. Flawless cell culture technique is one of
the best preventive tools and testing to ascertain which cultures are clean and which cultures are
infected – so you can take quick and aggressive action before infestation spreads.
The M.hyorhinis strain is picked up by the PCR method of detection. This is the quickest but
also the least sensitive, mycoplasma assay. In this method, you perform PCR on samples of your
suspected cell cultures, using primers specific for mycoplasma DNA, usually mycoplasma’s 16S
rRNA genes. Running the PCR product on a gel shows presence of mycoplasma DNA by bands
of distinct sizes.
One major limitation of PCR is that prior information about target sequence is necessary in order
to generate the primers that will allow its selective amplification. This means that, typically, PCR
users must know precise sequences upstream of target region on each of the two single-stranded
templates in order to ensure that DNA polymerase properly binds to primer-template hybrids and
subsequently generates entire target region during DNA synthesis. Like all enzymes, DNA
polymerases are also prone to error, which in turn causes mutations in PCR fragments that are
generated. Specificity of PCR fragments can .
Mycobacterium are rod-shaped bacteria that resist decolorization when stained. The genus Mycobacterium contains over 71 species that can infect humans and animals. M. tuberculosis is an intracellular pathogen that causes tuberculosis in humans. It has a complex cell wall containing lipids and prevents the phagosome from fusing with lysosomes, allowing it to survive and replicate inside macrophages. Diagnosis involves microscopy, culture, nucleic acid tests, and the tuberculin skin test. Treatment of tuberculosis in Indonesia follows national guidelines and standards of care.
This document provides an overview of mycobacteriology, including Mycobacterium tuberculosis and other mycobacteria. It discusses the PPD skin test and newer interferon gamma release assays for detecting TB infection. It describes the Runyon classification system for grouping mycobacteria based on pigment production. Key aspects of processing specimens, staining, culturing, and identifying mycobacteria are summarized, along with the most common mycobacteria that can cause disease. The document emphasizes the importance of safety protocols when working with mycobacteria in the laboratory.
This document summarizes information about mycobacteriology and tuberculosis. It discusses the PPD skin test and IGRAs for detecting TB infection. It describes acid-fast staining and characteristics of the mycobacteria genus. The document outlines the specimen processing, culture, and identification of mycobacteria, including M. tuberculosis. It also summarizes tuberculosis disease characteristics and methods for direct detection of M. tuberculosis from respiratory specimens.
This document provides an overview of mycobacteriology. It discusses acid-fast bacilli (AFB) staining and taxonomy. Laboratory safety level 3 is required for working with mycobacteria. Diagnostic testing includes the tuberculin skin test, interferon-gamma release assays, and molecular detection of TB from respiratory specimens. Culture media like Middlebrook and Lowenstein-Jensen are used to grow mycobacteria. Identification has transitioned to methods like MALDI-TOF mass spectrometry from older biochemical techniques. Common pathogens include Mycobacterium tuberculosis and non-tuberculous mycobacteria.
1) M. tuberculosis is the bacterium that causes tuberculosis. It is an acid-fast, rod-shaped bacterium that grows very slowly and infects alveolar macrophages in the lungs.
2) Diagnosis of tuberculosis involves acid-fast staining of sputum samples under a microscope to identify the bacteria, tuberculin skin tests, and culture tests that can take weeks for colonies to form.
3) Treatment requires a lengthy, multi-drug cocktail including isoniazid, rifampin, pyrazinamide, and ethambutol to kill both actively dividing bacteria and prevent drug resistance from developing.
This document provides information about mycobacteria, including their classification, identification, laboratory safety protocols, and diagnostic testing. It discusses the taxonomy and characteristics of mycobacteria species, with a focus on Mycobacterium tuberculosis. Methods covered include acid-fast staining, automated detection systems, PCR, and interferon gamma release assays for diagnosis of tuberculosis.
MC3 - Week 3 Microbial Growth and Control.pptMCFototana1
This document discusses microbial growth and methods for controlling microbial growth. It covers the following key points:
1) Microbial growth refers to the increase in number of microbial cells through processes like binary fission rather than an increase in cell size. Growth is influenced by available nutrients, temperature, pH, oxygen levels and other environmental factors.
2) Methods for controlling microbial growth include physical agents like heat, radiation, filtration and desiccation as well as chemical disinfectants, antiseptics and antibiotics. These work by killing microbes or inhibiting their growth.
3) Proper control of microbial growth is important for preventing disease transmission and food spoilage. A variety of chemical and physical methods can be
Bananas are highly perishable tropical fruits that are easily contaminated by microbes. Common microbes that cause banana spoilage include Fusarium, Mucor, Collectotrichum, and Rhizopus fungi, as well as Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella, and E. coli bacteria. Microbes are introduced through physical damage during handling and storage or contamination from handlers. Proper handling, refrigeration, drying, packaging, and discarding of spoiled bananas can help prevent microbial growth and spoilage.
Streptomycin is an antibiotic derived from Streptomyces griseus that is effective against both gram-positive and gram-negative bacteria, including those that cause tuberculosis. It is produced through fermentation of S. griseus in a nutrient-rich medium, followed by purification using techniques like filtration, precipitation, and chromatography. Streptomycin works by binding to the bacterial ribosome and inhibiting protein synthesis. It has applications as an antibiotic in treating diseases and as a pesticide in agriculture.
This document discusses microbial strain improvement techniques. It describes how raising gene dose can increase yields for products involving one or few genes, benefiting biomass or primary metabolite production. Mutation techniques like spontaneous and induced mutation are explored, noting their mutation rates and limitations. Selection methods for auxotroph mutants are covered, including replica plating, penicillin selection, and enrichment procedures to isolate mutants unable to grow in minimal medium. Overall, the document provides an overview of genetic engineering methods for enhancing microbial production of metabolites.
This document provides an overview of diagnostic microbiology. It discusses the goals of clinical microbiology laboratories in testing specimens to identify microorganisms causing illness and providing antimicrobial susceptibility results. It also describes various laboratory procedures used, including microscopy, culture-based techniques, immunological and molecular assays. Specimen collection, processing, staining methods, and interpretation of culture results are discussed in detail.
The document discusses diagnostic microbiology and the role of the clinical microbiology laboratory. The key responsibilities of the laboratory include testing specimens to identify microorganisms causing illness, providing antimicrobial susceptibility results, and advising physicians. Important techniques used in diagnosis include microscopy, culture, antigen detection methods like ELISA, and molecular methods like PCR. Proper specimen collection, transport, and processing are essential for accurate diagnostic testing.
This document discusses industrial enzymes and their production through microbial sources. It describes that enzymes can be produced from plants, animals, and microorganisms, but microbes are preferred for large-scale production due to their ability to be genetically manipulated and grown at low costs. The key steps in microbial production include identifying a suitable source microbe, inoculum preparation through screening and isolation, cultivation through solid-state or submerged fermentation, enzyme extraction from cells or culture, and purification using techniques like chromatography, electrophoresis, or adsorbent gels.
This document discusses various methods for identifying bacteria, including traditional phenotypic methods, immunochemical methods, and genotypic molecular methods. Phenotypic methods involve examining bacterial morphology, staining characteristics, growth requirements, and biochemical reactions. Immunochemical methods like immunofluorescence and ELISA use antigen-antibody reactions for identification. Molecular identification methods analyze bacterial DNA sequences. Correct specimen collection, handling, and transport are essential for accurate identification. Identification determines clinical significance and appropriate treatment.
Cancer is characterized by uncontrolled cell proliferation. Many factors can cause cancer, including external factors like chemicals and radiation, and internal factors like hormones and genetic mutations. While there are 92 approved anticancer drugs, effective therapies are still lacking for many types of cancer. New drugs are needed that are more selective for cancer cells to reduce side effects from long-term treatment. In vitro screening methods are used to identify potential drug candidates, including assays to test cell viability, proliferation, and morphology. Promising candidates then advance to in vivo testing using animal models of cancer like chemically-induced tumors in mice. The goal is to find drugs that can effectively treat cancer while avoiding side effects.
Project 1 Assignment Presentation Ppt by Tekalign.pptxTekalignKebede1
This document presents information on rapid and alternative techniques for microbiological examination of foods. It discusses rapid techniques such as immunological-based methods like ELISA and lateral flow assays, nucleic acid-based methods like PCR and multiplex PCR, and direct epifluorescent filter technique. Alternative techniques described include dye-reduction tests, electrical methods, and ATP determination. The presentation provides details on the principles, applications, advantages and limitations of these various rapid and alternative microbiological examination methods.
This document provides an overview of mycobacteriology and the identification and diagnosis of mycobacteria species. It discusses the characteristics of acid-fast bacilli, current identification methods including genetic sequencing and mass spectrometry, mycobacteria taxonomy and disease-causing species. It also summarizes specimen collection and processing, culture methods, acid-fast staining, and molecular detection of tuberculosis.
Lonza Bioscience provides a variety of life science research products and services including cell culture media, reagents, assays, primary cells, stem cells, and cell therapy services. They have expertise in areas such as cell culture, cell biology, bioassays, electrophoresis, and rapid microbial detection. Lonza Bioscience manufactures most of their offerings and maintains high quality standards in compliance with cGMP. They aim to support customers through technical services and reliable customer service.
The 4th quarter 2005 sales plan for Wheaton Science Products outlines goals of increasing knowledge of accounts and customers through working with top sales reps and trade shows. Specific objectives include closing existing opportunities in the 4th quarter, improving use of CRM systems, and mastering the quoting process. Key revenue goals are to increase total revenue 7% through securing large orders from Pfizer and BMS, pursuing potential at AdVantx, and increasing sales in the Cambridge, MA and Longwood Medical Area locations with major accounts.
Alcan Inc. produces pharmaceutical and scientific packaging and laboratory equipment through its Wheaton Science Products division. Wheaton offers a broad range of glass, plastic, and metal containers, caps and closures, liquid handling products, cell culture products, freezers, staining ware, vials, bottles, chromatography products, and barcoding and critical cleaning services to pharmaceutical, biotech, and laboratory customers globally. Wheaton is recognized for its brand, quality products, and customer service.
The document discusses bottled water processing and filtration solutions from Merck Millipore. It focuses on ensuring water quality and safety by removing contaminants and microorganisms through various filtration steps. Merck Millipore offers a range of filtration products tailored for clarification, prefiltration, tank protection, final filtration, and process control to produce a clean and safe bottled water product.
This document summarizes the products and services of AllCells, a leading provider of human primary cells. It offers over 800 healthy and diseased primary cell products that are used in areas like drug discovery, stem cell research, and regenerative medicine. AllCells has a supply chain and tissue collection clinic that enables efficient procurement of these cells. It provides a variety of normal and diseased primary cells and tissues, as well as custom processing and isolation services. AllCells aims to advance scientific research through its high quality and biologically relevant primary cell products.
Ernie Desmarais - Reichert Business Plan 09.18.15Ernie Desmarais
This document provides an executive summary and business plan for Reichert's AI business unit. The plan outlines Reichert's surface plasmon resonance (SPR) products, which are label-free biomolecular interaction systems. The target market is pharmaceutical, biotech, and universities. The plan details Reichert's goals of driving profitability, raising customer awareness, delivering high quality products and services, and growing the business. It analyzes competitors including GE Biacore, and identifies strengths in experience and opportunities in becoming an established player in the 4-channel SPR market.
The document provides information on Reichert Life Science and their surface plasmon resonance (SPR) systems. It discusses Reichert's 15 years of SPR expertise and over 200 publications using their SPR instruments. It also highlights the key features and advantages of Reichert SPR systems including their robust fluidics, lower costs, increased sample flexibility, and commitment to customer service and support. Several application examples are described demonstrating Reichert SPR's use in areas like protein-protein interactions, enzyme kinetics, lipid-peptide binding, carbohydrate-receptor interactions, and cell-protein binding.
2. slide 2
What Contaminations Can You See Under
A Microscope?
Bacteria
small black specks
pH change/ cloudy media
mistaken for cell debris
definite movement
Fungus
Filamentous Strands
web-like mesh
3. slide 3
What Contaminations Can You See Under
A Microscope?
Yeast
oval/round in shape
smaller than cells
appear as bright beads
reflect light
form branched chains
Mycoplasma
INVISIBLE!
5. slide 5
Mycoplasma
Smallest, simplest prokaryotes
Size ranges from 0.2 to 0.8 µΜ
Many species cannot be removed by
filtration
Cannot be visualized, even at high
concentrations
Lacks Rigid Cell Wall
Not affected by traditional antibiotics
used in cell culture
Limited Biosynthetic capabilities
Utilize nutrients from “host”
6. slide 6
Adverse Effects of Mycoplasma
Contamination
DNA fragmentation associated with mycoplasmal nucleases
Induction of Apoptosis
Alters Gene Expression in contaminated cells
Affects Cell Metabolism
Changes in Signal Transduction
7. slide 7
Mycoplasma - Effects
Inhibition of cell growth
Inhibition of cell metabolism
Disruption of nucleic acid synthesis
Chromosomal aberrations
Changes in cell membrane antigenicity
Alteration of DNA transfection
efficiency
Increased sensitivity
to inducers of apoptosis
DNA fragmentation due to
Mycoplasmal nucleases
NOT apoptosis
CELL DEATH
Production of viruses
compromised
8. slide 8
Most Common Species
Mycoplasma orale (human)
20-40%
Mycoplasma hyorhinis (swine)
10-40%
Mycoplasma arginini (bovine)
20-30%
Mycoplasma fermentans (human)
10-20%
Mycoplasma hominis (human)
10-20%
Acholeplasma laidlawii (bovine)
5-20%
10. slide 10
Sources
Cross contamination from infected cultures
Laboratory Personnel
Culture Reagents (i.e. bovine serum)
Original isolate tissue (<1%)
11. slide 11
Continuous Monitoring
Monitoring of U937 cells for the presence of mycoplasma
0
0.2
0.4
0.6
0.8
1
1.2
1.4
week1 week 2 week3 week 4 week 5 week 6
MycoAlert®ratio
12. slide 12
Summary
Mycoplasma are a real problem in cell culture
Changes in gene expression
Changes in cell functioncytotoxicity
Mycoplasma contamination is often invisible
Infection from Laboratory personnel and reagents requires
routine monitoring
Mycoplasma testing of new cells is essential
14. slide 14
MycoAlert® for Mycoplasma detection
The assay detects the activity of two enzymes found in
mycoplasma and other mollicutes
Enzymes are associated with energy generation pathways that
result in ATP synthesis
The enzymes are found in all 6 of the main mycoplasma cell
culture contaminants and the majority of mollicute species
Being an enzyme assay, MycoAlert® only detects live
mycoplasma
The enzymes are not found in eukaryotic cells
17. slide 17
Assay Sensitivity
HepG2 cells were infected with mycoplasma at the concentration listed, the cells
were then cultured for 4 hours before being tested with a range of techniques
A. Laidlawii Culture Fluorescence PCR MycoAlert®
0 Negative Negative Negative Negative
20 Positive Positive Negative Positive
200 Positive Positive Positive Positive
2000 Positive Positive Positive Positive
20000 Positive Positive Positive Positive
200000 Positive Positive Negative Positive
0.1
10.0
1.0
100.0
CFU/ml
Mycoalert®ratio
0 20 200 2000 20000 200000
Samples provided, enumerated and tested by the European Collection of Cell Cultures (ECACC) with the exception of the MycoAlert® testing which was performed by Lonza
18. slide 18
Effect of Common Media Components
10
0.1
1
RPMI
Penicillin/
Streptomycin
10% DMSO
Trypsin/
EDTA
Serum 20%
Non Essential
Amino Acids
Glutamate
Sodium
Pyruvate
EMEM
Iscove’s
Negative
Mycoplasma positive
M. faucium added to create the positive samples
MycoAlert®Ratio
20. slide 20
Best Practices
Receive cells
Quarantine & test for mycoplasma
NEGATIVE
Put cells in normal
culture
Prepare cryo
samples
Monitor frequently
Use in experimental
protocols
POSITIVE
Destroy cells
Inform source
Quarantine cells
Treat with MycoZap™
Inform source
Monitor daily for
mycoplasma
Keep in quarantine until
infection eradicated for
4 weeks
21. slide 21
Best Practices
We strongly recommend to discard contaminated cultures
It is not known if all cell functions return upon elimination
In cases where fresh stocks cannot be obtained one should
consider treatment of the cells…
22. slide 22
Treatments
Standard Antibiotics- most prominent
Time-consuming (prolonged periods of >6 weeks needed)
Usually the success rates are relatively low…
Low antibiotic concentration
Heat-instability of the antibiotic
Early termination of the treatment
Formation of resistant mycoplasma
Recontamination possible
MycoZap™ Reagent
Fast (clear contamination in little as 4 days)
Highly reliable and definite elimination of mycoplasma
23. slide 23
MycoZap™ Reagent
for Mycoplasma Elimination
Minimal toxic effect on host cells
Optimized reagent mix- combination of antibiotic and antimetabolic
agents
Reagent 1 is a detergent
Integrates into the mycoplasma cell wall and
disintegrates the mycoplasma particle
Pretreatment will reduce the mycoplasma titer drastically
Reagent 2 is an antibiotic
Mycoplasma particles which survived this treatment by
hiding in cell clusters or cell crinkles will be destroyed
subsequently by the antibiotic
25. slide 25
MycoZap™ Summary
Easy to use
Simply add Reagent to your culture
Universal
One protocol to eliminate all mollicutes
Complete
All required reagents are in one kit, one protocol
Effective clean up
Combination of antibiotic and antimetabolic agents- disrupts
mycoplasma
Minimum toxic effect on host cells
Allows you to rescue priceless or high value cell line from
destruction and enables lab to continue using cell line and add
value to their investment
Hello everyone
My name is ……. and I am the Life Science specialist for the Cell Discovery Division of Lonza Bioscience.
I would like to talk to you today about mycoplasma. This is something every cell biologist has heard about
There seem to be three camps of people when you start to discuss mycoplasma, those who are aware and test regularly – we salute you! Those who are aware , know they ought to test but somehow never quite get round to it and those who definitely know that their lab is clean even though they have never tested and therefore can ignore any issues.
Hopefully this webinar will demystify the whole mycoplasma issue, raise awareness of the issues and offer convenient and really easy solutions to ensure that the fear – whilst we can’t guarantee to make it disappear will definitely become more manageable.
Mycoplasma –Background (What is it, what effects it can have), Detection techniques, Our Solution for detection. Best Practices for Clean up.
Some questions to ask before doing presentation or demo
What would be the consequences of an infected cell line to you and your lab?
How would the loss of a cell line adversely affect your lab and your research?
What routine testing can you keep in house to ensure you are mycoplasma free?
Bacterial infections of media ( especially media containing no antibiotics) can be quite common. The infection is normally quite rapid and can be seen by a pH change in the media as well as the media becoming cloudy. When observed under light microscopy the bacteria can be seen as small black specks which when closely observed are under motion. Observation is necessary for low lying infections as bacteria can often be mistaken as cell debris. Cell debris will only undergo Brownian motion ( a slight trembling type motion) whereas bacteria will show a defined ( sometimes quite rapid) activity.
Fungus is one of the easier contaminations to spot when established as it is usually floating on the surface of the culture media. When observed down the microscope it appears in the early stages of infection it appears as filamentous
strands associated with the cells but can be quite difficult to spot. As the infection progresses this web of strands gets larger and more intermeshed.
Yeast infections usually produce oval/round shaped organisms which are quite easy to spot as they reflect the light well so appear quite bright under light microscopy. They are characterised by their tendency to appear as groups of “buds” in the early stages. If left to progress they form branched chains rather like beads. They are distinctly smaller than cells.
Mycoplasma
There is no picture in this section as mycoplasmal infections cannot be seen under light microscopy like bacteria, fungus and yeast. They do not produce any black moving specks or bright beads. They do not even affect the pH of the media or its clarity. The most disturbing aspect of a mycoplasma infection is that it is virtually invisible.
Usually the first indication that cells are infected with mycoplasma is the niggling doubt that the cells are not “acting quite right”. This may be noted by a difference in the population doubling rate or the response to a normal drug dosing regime. Unfortunately by the time these effects are seen the culture is invariably heavily infected and the best course of action is to discard it immediately before other precious stocks are also infected.
This electron micrograph shows mycoplasma attached to the cell. From this picture it becomes more apparent why these infections are so detrimental to the cell. The mycoplasma are almost like parasites requiring the cell to work much harder just to stay alive. They deplete essential nutrients from the media making life in the flask a struggle.
Mycoplasmas are from the family Mollicutes, which includes Acholeplasma, Ureaplasma and other species. However the term Mycoplasma is most often used as a ‘cover-all’. More than 180 species have been identified of which 20 distinct Mycoplasma and Acholeplasma species from human, bovine and swine have been isolated from cell culture.
There are 6 species that account for 95% of all mycoplasma infections, these are M.orale, M.arginii, M.fermentans, M.salivarum, M.hyorhinis and A.laidlawii.
Mycoplasmas are widespread in nature as parasites of humans, mammals, reptiles, insects and plants. They are the smallest and simplest prokaryotes, they lack a rigid cell wall and are surrounded by a single plasma membrane. The lack of cell wall means that they are resistant to a number of commonly used antibiotics and are therefore difficult to eradicate.
They are dependant on their hosts for many nutrients as their biosynthetic capabilities are limited.
(Mycoplasma photos are of M. Hyorhinis, M. Orale and M. Pnemoniae)
Mycoplasma have long been recognised as common contaminants of cell lines in continuous culture. Unlike the turbid growth that is commonly associated with bacterial and fungal contamination, the presence of mycoplasma can go undetected for months.
As the mycoplasma compete with the cell lines for the nutrients in culture media, one of the first signs is a slow down in cell proliferation and slight changes in cellular responses and gene expression. They can cleave DNA to give the appearance of apoptosis, and also induce gene expression – for example IL-2 and interferons.
Their metabolism can also cause aberrant results with tetrazolium assays, and so could mask any cytotoxic effects of compounds and cause shifts in IC50 values. This is the result of the ability of certain mycoplasma species to reduce the tetrazolium salts and cause an increase in coloured product.
Mycoplasma contamination can alter experimental outcomes significantly and can have serious consequences as virtually every cellular process can be altered by the infection
Mycoplasmas grow slowly and colonies may take up to 3 weeks to develop and are typically very small.
There are currently different Mycoplasma detection methods:
Culture:
Advantage: Properly done this offers the greatest sense of security
Disadvantage: Costly and can take up to a month for results.
Indirect tests measuring biochemical markers and other
characteristics associated with mycoplasmas
DNA Flourochrome staining
Advantages: These are typically faster than direct culture methods. Can detect the non-cultivable mycoplasmal strains that direct culture may miss.
Disadvantages: They do require a higher level of contamination 10 to the 4 organisms/ml for detection. Sometimes hard to read
PCR
Advantages: Rapid, 6 to 8 hours, and inexpensive. requires small amount of sample 200-400 ul.
Disadvantages: Subject to False Positives
When the presence of Mycoplasma is suspected, it is usually confirmed by one of these three methods: DNA staining (Hoechst or DAPI), culture or PCR.
PCR requires both skill and time, and is currently not accepted by the FDA. Hoechst or DAPI DNA staining is subjective, especially with low contamination, requiring expertise, also time to prepare samples. Culture of mycoplasma is definitive but difficult, so is usually sent off to a specialist lab. Both PCR and Hoecsht staining will pick up mycoplasmal DNA from non-viable organisms, and therefore gives false positives. Also not all PCR primers will detect all the relevant contaminating species, for example the Takara kit does not pick up Acholeplasma, and A.laidlawii is a common contaminant.
A comparison of detection technologies. A HepG2 cell line was infected with mycoplasma at a range of CFUs per ml. The cells were cultured for 4 hours post infected as per ECACC SOPs and then tested by culture, fluorescence, PCR and MycoAlert. These results do not show absolute detection limits due to the culture period employed by ECACC which will have allowed time for some degree of mycoplasma multiplication. ECACC have developed their own PCR system as they have continued problems with all commercial kits as is illustrated by the data they supplied above.
The effect of common media components that may be found in samples tested with MycoAlert were tested to see if they interfered with the assay. All sample were made up in RPMI media, positive samples had the addition of M. faucium mycoplasma. The results show that none of the components used interfered significantly with the assay, positives remained positive and negatives remained negative.
The use of DMSO showed a reduction in the positive ratio and a more negative negative ratio. DMSO is known to inhibit luciferase to a degree at high concentrations. It should be noted that the concentration of DMSO found in cell culture would normally be no greater than 1%, only concentrated samples for freezing in liquid Nitrogen would contain as high as 10% and we do not recommend testing such samples without expansion into media.
Trypsin/EDTA showed a reduced positive ratio, EDTA at high concentrations inhibits luciferase.
20% Serum showed a reduced positive ratio, serum darkens the sample and quenches the light emitted by the reaction to a degree.