Antibiotic sensitivity test

Many bacteria have developed the ability to become resistant to antibiotics. These bacteria are now a major threat in our hospitals. Antibiotic resistant bacteria include Methicillin Resistant Staphylococcus aureus (MRSA)

Materials Required   Petriplate containing microbial culture(For example, Escherichia coli)  Inoculation loop Bunsen burner Saline solution McFarland solution MHA plate Cotton swab Antibiotic disks(Streptomycin (S), Ciprofloxacin (CIP), Chloramphenicol (C), Doxycycline (D), Penicillin G (P), Gentamycin (G) Tooth pick Incubator Ruler  

Select a pure culture plate of one of the organisms to be tested.   Select a pure culture plate of one of the organisms to be tested. Aseptically emulsify a colony from the plate in the sterile saline solution. Mix it thoroughly to ensure that no solid material from the colony is visible in the saline solution. Repeat until the turbidity of the saline solution visually match that of the standard turbidity. Take a sterile swab and dip it into the broth culture of organism. Gently squeeze the swab against the inside of the tube in order to remove excess fluid in the swab. Take a sterile Mueller-Hinton agar (MHA) plate or a nutrient agar (NA) plate. Use the swab with the test organism to streak a MHA plate or a NA plate for a lawn of growth. After the streaking is complete, allow the plate to dry for 5 minutes. Antibiotic discs can be placed on the surface of the agar using sterilized forceps. Gently press the discs onto the surface of the agar using flame sterilized forceps or inoculation loop. Carefully invert the inoculated plates and incubate for 24 hours at 37° C. After incubation, use a metric ruler to measure the diameter of the zone of inhibition for each antibiotic used. Compare the measurement obtained from the individual antibiotics with the standard table to determine the sensitivity zone. Compare the measurement obtained from the individual antibiotics to the standard table to determine whether the tested bacterial species is sensitive or resistant to the tested antibiotic.  

* Müller-Hinton agar is a microbiological growth medium that is commonly used for antibiotic sensetivity testing. It is also used to isolate and maintain Neisseria and Moraxella species. It typically contains: 1-2.0g beef extract 2-17.5g casein hydrolysate 3-1.5g starch 4-17.0g agar dissolved in 1 liter of distilled water. 5- pH adjusted to neutral at 25 °C .

It has a few properties that make it excellent for antibiotic use It has a few properties that make it excellent for antibiotic use.1- First of all, it is a non-selective, non-differential medium. This means that almost all organisms plated on here will grow. 2-Additionally, it contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the antibiotics. 3-it is a loose agar. This allows for better diffusion of the antibiotics than most other plates,a better diffusion leads to a truer zone of inhibition.