Description: Hygromycin B, an aminoglycosidic antibiotic produced by Streptomyces hygroscopicus, is used for the selection and maintenance of prokaryotic and eukaryotic cells transfected with the hygromycin resistance gene, hph. Hygromycin B kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis. The resistance gene codes for a kinase that inactivates Hygromycin B through phosphorylation. Cloning of the resistance gene and fusion with eukaryotic promoters has resulted in the development of vectors that permit selection for resistance to Hygromycin B in both prokaryotic and eukaryotic cells.

Use: The working concentration for the purpose of selection varies with cell type, media, growth conditions and cell metabolic rate. Recommended concentration for the selection of resistant cells is 25 – 1000 μg/ml. Commonly used concentrations for selection are 200 μg/ml for mammalian cells, 20-200 μg/ml for plant cells & bacteria cells and 200-1000 μg/ml for fungi. Your optimum concentration should be tested experimentally.

QUESTION 1: What is the stability of Hygromycin B solution?

ANSWER: Hygromycin B solution is stable for at least two years at +4°C and about one month at +37°C.

QUESTION 2: What colour is Hygromycin B solution?

ANSWER: The colour can vary from light yellow to a dark brown or caramel colour. The concentrated Hygromycin solution tends to have a darker appearance.

QUESTION 3: What is the working concentration for selection?

ANSWER: The working concentration for the purpose of selection varies with cell type, media, growth conditions and cell metabolic rate. Recommended concentration for the selection of resistant cells is 25-1000 μg/ml. Commonly used concentrations for selection are 200 μg/ml for mammalian cells, 20-200 μg/ml for plant cells & bacteria cells and 200-1000 μg/ml for fungi. Your optimum concentration should be tested experimentally.

QUESTION 4: How can non-transfected cells escape antibiotic selection?

ANSWER: Cells can escape selection if the antibiotic is used at too low concentration or if the cell density on the plate is too high. Additionally, cells rapidly proliferating are killed faster than these, only slowly proliferating. Control cells should die within 5-7 days after addition of the antibiotic allowing colonies of resistant cells to form by 10–14 days.

antibiotic, gene selection, hygromycin b, media, aminoglycoside

QUESTION 5: How do I determine the Toxic Concentration?

ANSWER: Hygromycin B is added to the culture medium at a concentration that varies with the cell type transfected. A titration experiment for each cell type may therefore be performed to determine the amount of Hygromycin B needed to kill non-transfected cells. The working concentration for mammalian cell selection is normally between 50 μg/ml and 1mg/ml, Plant cells: 20-200 μg/ml, Bacteria: 20-200 μg/ml and Fungi: 200 μg-1mg/ml. Your appropriate concentration should be tested experimentally.

QUESTION 6: How do I perform a Dose Response curve?

ANSWER: To determine the minimum concentration of antibiotic required to kill your non-transfected host cell line.
A.) Test arranges of concentrations (5-6) to ensure that you determine the minimum concentration necessary for your cell line.
B.) Seed cells at approximately 20-25% confluency on the appropriate number of plates for each time plate and allow cells to adhere overnight. For cells that require higher densities for viability, increase the number of cells seeded.
C.) The next day, substitute culture medium with medium containing varying concentration of the antibiotic.
D.) Replenish the selective medium every 3-4 days.
E.) Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents the growth of nontransfected cells. Select the concentration that kills the majority of the cells in the desired number of days, usually 7-10 days.

QUESTION 7: How do I maintain Hygromycin resistant phenotype of transfected cell lines?

ANSWER: To maintain Hygromycin resistant phenotype of transfected cell lines and for the elimination of revertants cells may be regularly cultured in culture medium containing Hygromycin B at the same concentration used for the initial selection.

QUESTION 8: Replacement of Media?

ANSWER: Replacement of culture media containing Hygromycin B is needed only if nutritional components are consumed by the cells cultured. Acidification of the culture medium is a normally a sign of consumption. Utilizing phenol red or media containing phenol red will aid in the detection of acidification. In this case the media will turn yellow.

QUESTION 9: Is Hygromycin B sensitive to acids?

ANSWER: It is sensitive to high concentrations of acids; however, a brief exposure to dilute acids does not affect its stability.

QUESTION 10: Can we increase the sensitivity of our cells to Hygromycin B

ANSWER: The sensitivity to Hygromycin B can be increased by increasing the pH of the medium. Sensitivity appears to be greater at lower salt concentrations.

QUESTION 11: What is the enzyme that inactivates Hygromycin B?

ANSWER: Hygromycin phosphotransferase (hpt) inactivates the antibiotic Hygromycin B through phosphorylation. The hygromycin phosphotransferase gene (denoted hpt, hph or aphIV) codes for hygromycin phosphotransferase, and is utilized as selectable marker gene for both plant and animal systems.

QUESTION 12: What is the Mode of Action:

ANSWER: Binds to the 30S ribosomal subunit and affects the fidelity of translation.

QUESTION 13: How can I roughly assess hygromycin purity?

ANSWER: The colour of a hygromycin solution is a good indicator: the clearer it is, the purer. Therefore, caramels to brown colored solutions are of inferior grade. The colour difference may not be very dramatic with a freshly made low concentration hygromycin solution.

QUESTION 14: What is the spectrum of inhibition?

ANSWER: An aminoglycoside antibiotic that inhibits protein synthesis in bacteria, fungi and higher eukaryotes. Spectrum also stated as Gram (+), Gram (-) bacilli aerobes and facultative anaerobes, only.

QUESTION 15: Is Hygromycin B hazardous?

ANSWER: Yes. It is considered toxic/hazardous material. Care should be taken to avoid contact with skin & eyes. Please consult & review Material safety data sheet (M.S.D.S.) prior to handling.