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Mycoplasma Contamination | Cell Culture Mikoplazme kontaminacije | kultura stanica

We include here for cell culture labs, information on mycoplasma contaminations and the latest mycoplasma forum discussions for all your contamination questions and discussions. Mi uključuju ovdje za stanične kulture učionice, informacije na mikoplazme contaminations i najnovije rasprave mikoplazme forum za sve vaše kontaminacije pitanja i rasprave.

Mycoplasma Background Mikoplazme Pozadina

Mycoplasmas are a genus of bacteria that lack a cell wall. Mycoplasmas su rod bakterija koji nedostaje stanica zida. Due to a lack of a cell wall, they are resistant to many antibiotics including penicillin or other beta-lactam antibiotics that target cell wall synthesis. Zbog nedostatka ćeliju zid, oni su otporni na mnoge antibiotike, uključujući penicilin ili drugi beta-laktamski antibiotici da Target Cell zid sinteza.

Mycoplasma are either parasitic or saprophytic. Mikoplazme su ili parazitski ili saprophytic. Several Mycoplasma are disease-causing in humans, including M. pneumoniae, which is an important cause of atypical pneumonia and other respiratory disorders, and M. genitalium, which is believed to be involved in pelvic inflammatory diseases. Nekoliko mikoplazme su-uzrok bolesti u ljudi, uključujući i M. pneumoniae, koja je važan uzročnik atipične pneumonije i drugih respiratornih poremećaja, i M. genitalium, koji je vjerovao da budu uključeni u prsni upalne bolesti. They may cause or contribute to some cancers. Oni svibanj uzrokovati ili doprinijeti nekih vrsta rakova.

Genus Mycoplasma Opis mikoplazme

The genus Mycoplasma is one of several genera within the class Mollicutes. U opis mikoplazme je jedan od nekoliko rodova unutar klase Mollicutes. Mollicutes are bacteria which have small genomes, lack a cell wall and have a low GC-content (18-40 %). Mollicutes su bakterije koje su mali genocid, nedostatak jednog zida stanica i imaju nizak GC-sadržaj (18-40%).

There are over 100 recognized species of the genus Mycoplasma. Postoji preko 100 priznatih vrsta na opis mikoplazme. Their genome size ranges from 0.58 1.38 megabase-pairs. Njihova genom veličine raspona od 0,58 1,38 megabit-parova. Mollicutes are parasites or commensals of humans, animals (including insects), and plants; the genus Mycoplasma is by definition restricted to vertebrate hosts. Mollicutes su parazita ili commensals ljudi, životinje (uključujući i kukci), i biljke, opis mikoplazme je po definiciji ograničene na kralježnjaci Vojskama. Cholesterol is required for the growth of species of the genus Mycoplasma as well as certain other genera of mollicutes. Kolesterol je potreban za rast vrsta, opis mikoplazme, kao i nekih drugih rodova od mollicutes. Their optimum growth temperature is often the temperature of their host if warmbodied (eg 37 degrees Celsius in humans) or ambient temperature if the host is unable to regulate its own internal temperature. Njihova optimalna temperatura rasta je često temperaturu njihova vojska i ako warmbodied (npr. 37 stupnjeva Celzij u čovječji) ili ako je temperatura ambijenta u vojsci se ne može regulirati svoje vlastite unutarnje temperature. Analysis of 16S ribosomal RNA sequences as well as gene content strongly suggest that the mollicutes, including the mycoplasmas, are closely related to either the Lactobacillus or the Clostridium branch of the phylogenetic tree (Firmicutes sensu stricto). Analiza 16S ribosomal RNA sekvence gena, kao i sadržaj snažno sugeriraju da mollicutes, uključujući mycoplasmas, usko su vezani uz bilo Lactobacillus ili Clostridium Branch of the phylogenetic tree (Firmicutes sensu stricto).

Cell Lines and Mycoplasmas Stanične linije i Mycoplasmas

In cell cultures, mycoplasmas may induce cellular changes, including chromosome aborations, changes in metabolism and cell growth. U kulture stanica, stanični mycoplasmas svibanj prouzrokovati promjene, uključujući kromosom aborations, promjene u metabolizmu i rast stanica. Severe mycoplasma infections may destroy a cell line or render expensive or precious cell lines useless. Olujno mikoplazme infekcije svibanj uništiti jedan cell line ili skuplji ili pružiti dragocjenu stanične linije beskorisno.

Cell Culture Contamination with Mycoplasma Kultura stanica kontaminacije s mikoplazme

Mycoplasmas are often found in research laboratories as contaminants in cell culture. Mycoplasmas se često naći u istraživačkim laboratorijima kao kontaminanti u stanične kulture. Mycoplasmal cell culture contamination occurs due to contamination from individuals or contaminated cell culture medium ingredients. Mycoplasmal kulture stanica kontaminacija nastaje zbog kontaminacije od pojedinaca ili kontaminirane stanične kulture srednje sastojaka.

Mycoplasma Detection Methods Mikoplazme Detection Metode

Mycoplasma cells are usually smaller than 1 µm and therefore quite difficult to detect with conventional microscopy methods. Mikoplazme stanice su obično manji od 1 μ m i stoga vrlo teško detektirati s konvencionalne metode mikroskopije. Detection techniques include: Detekcija tehnike uključuju:

• PCR
• Real-time PCR Real-time PCR
• Plating on sensitive Agar Plating na osjetljive Agar
• DNA stains including DAPI or or Hoescht Stain DNA mrlje uključujući DAPI ili ili Hoescht mrlja

Hoechst Stain Mycoplasma Contamination Detection Protocol Hoechst mrlja mikoplazme kontaminacije Detection Protocol

A Hoechst (Sigma, catalog #:H6024) staining method for Mycoplasma: A Hoechst (Sigma, katalog #: H6024) staining method za mikoplazme:

1. For Adherent cells, place a sterile cover glass in petri dish. Za prijanja stanica, mjesto sterilnom cover stakla u Petri dish. Add 2-3 drops of cell suspension in the middle of coverglass (depending on the growth rate of your cells). Dodajte 2-3 kapi cell suspension u sredini coverglass (ovisno o stopi rasta vašeg stanica).
2. Then carefully add 2-3 ml of medium without disturbing the cells. Tada pažljivo dodati 2-3 ml u mediju bez uznemiravanje ćelije. Grow the cells until they are 50 -70% confluent. Širi se stanice dok su 50 -70% sliva.

Fixing Cells: Učvršćivanje Cells:

1. Prepare cell Fixative which is a mix fo 3:1 Methanol : glacial acetic acid (M:AA). Pripremite stanica Fixative koji je mix za 3:1 metanol: glacial octena kiselina (M: AA). Make sure you prepare this fresh before use. Pobrinite se za to da pripremite ovu svježu prije upotrebe.
2. Discard medium from growing cells. Odbaci iz srednje uzgoj stanica. Add 2 ml of the Methanol : glacial acetic acid M:AA solution dropwise onto the side of the plate (not directly onto the cells). Dodaj od 2 ml metanol: glacial octena kiselina M: AA rješenje dropwise na strani na tanjur (ne izravno na stanice). Swirl the plate and discard solution. Vrtložni tanjuru i odbaciti rješenjem.
3. Add 2 ml of M:AA, swirl and leave on for 2-3 minutes at room temperature. Dodati 2 ml, M: AA, brazda na vodi i ostaviti na za 2-3 minuta na sobnoj temperaturi.
4. Discard M: AA solution and rinse with water Odbaci M: AA rješenje i isprati s vodom
5. Leave to air dry (you can keep the plate until you are ready to stain) Hoecht stain (cat# 33258 from Sigma) stock concentration = 0.1 mg/ml in water You can filter sterilize at this stage using a 0.2uM filter Wrap container in aluminium foil Store at 4 oC Working solution Prepare fresh each time as necessary Make 1:1000 dilution (0.1 µg/ml) of stock in water Ostavite da se sušiti zrakom (možete čuvati u tanjur dok ne budete spremni za mrlja) Hoecht mrlja (# 33258 cat iz Sigma) stock concentration = 0,1 mg / ml vode u Možete filtrirati sterilize u ovoj fazi pomoću 0.2uM filter Wrap u kontejner aluminijska folija Trgovina na 4 oC Radno rješenje Pripremite svježe svaki put kao što je potrebno Make 1:1000 dilution (0,1 μ g / ml) zaliha vode u

Staining Cells: Staining Cells:

1. Add enough Hoechst stain to cover the entire cover slip (use 1-2ml for a small Petri dish). Dodaj dovoljno Hoechst mrlja da pokriju cijeli cover slip (1-2ml koristiti za male Petrijeva zdjelica).
2. Leave the stain on for 45 minutes - 1 hour (in the dark). Ostavite mrlja na za 45 minuta - 1 sat (u mraku).
3. Rinse well with water. Dobro isprati s vodom.
4. Mount on a microscope slide (mountant mix: 22 mM Citric Acid; 55.6 mM disodium phosphate; 50% glycerol, pH mix to 5.5) place a drop or two on cover slip (cells up) and place cover slip (cells down) on slide. Montiranje na microscope slide (mountant mix: 22 mm, limunska kiselina, 55,6 mm disodium fosfat; 50% glicerola, pH 5,5 do mix) mjesto pad ili dva na cover slip (stanica gore) i mjesto cover slip (ćelije dolje) na slajd .
5. Visualise under fluorescence microscope Visualise fluorescencija pod mikroskop

Note: store Hoechst stain at 4oC at least, or aliquot into the freezer for long-term storage. Napomena: spremišta Hoechst mrlja na 4oC barem, ili aliquot u zamrzivač za dugoročno skladištenje.