Medical

Novobiocin Susceptibility Test   for differentiating of  Staphylococcus spp . which included a novobiocin disk test. Principle  Susceptibility to novobiocin is determined by placing a novobiocin-impregnated paper disk on a agar plate seeded with the organism under investigation. As the organism multiplies during incubation to produce a lawn of confluent growth, cells are exposed to the antibiotic diffusing into the agar from the paper disk. If the bacteria are susceptible to novobiocin, there will be a formation of visible zone of inhibition around the disk, representing an area where the antibiotic concentration has prevented bacterial growth. No zone of inhibition around the disk represents that organism is resistant to the anitibiotic.   Novobiocin Disc: Disk is prepared by impregnating 5ug of novobiocin onto high quality 6mm diameter filter paper disks (commercially available). Method 1.  ...

ONPG  The ability of bacteria to ferment lactose depends on two enzymes, beta-galacto permease and beta-galactosidase.  beta-galacto permease  allows lactose to enter the bacterial cell wall, where it is then broken down into glucose and galactose by beta-galactosidase. The glucose and galactose can then be metabolized by the bacteria. However, some organisms lack  beta-galacto permease  and appear as late or non-lactose-fermenters. Principle O-nitrophenyl-beta-D-galactopyranoside (ONPG) is an artificial substrate structurally similar to lactose with the exception that glucose is substituted with an o-nitrophenyl group. Unlike lactose, the substrate O-nitrophenyl-beta-D-galactopyranoside (ONPG) is capable of penetrating the bacterial cell without the presence of  beta-galacto permease . Media: ONPG broth:  Na2HPO4 (9.46 g), phenylalanine (4 g), ONPG (2 g), KH2PO4 (0.907 g), per 1000 mL, pH 8.0 ONPG disk:  ...

The Triple Sugar Iron (TSI) Test  To determine the ability of an organism to ferment glucose, lactose, and sucrose, and their ability to produce hydrogen sulfide. Principle The Triple Sugar Iron (TSI) test is a microbiological test named for its ability to test a microorganism’s ability to ferment sugars and to produce hydrogen sulfide. An agar slant of a special medium with multiple sugars constituting a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate is used for carrying out the test.  . Due to the building of acid during fermentation, the pH falls. The acid base indicator Phenol red. is incorporated for detecting carbohydrate fermentation that is indicated by the change in color of the carbohydrate medium from orange red to yellow in the pres...

Blood Agar (BAP) Blood Agar (BA) are  enriched medium  used to culture those bacteria or microbes that do not grow easily. Such bacteria are called “fastidious” as they demand a special, enriched nutritional environment as compared to the routine bacteria.  Composition of Nutrient Agar 0.5% Peptone 0.3% beef extract/yeast extract 1.5% agar 0.5% NaCl Distilled water  Blood Agar is made from Nutrient Agar more: 5% Sheep Blood pH should be from 7.2 to 7.6 (7.4) Preparation of Blood Agar 1.     Suspend 28 g of nutrient agar powder in 1 litre of distilled water. 2.     Heat this mixture while stirring to fully dissolve all components. 3.     Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes. 4.     Once the nutrient agar has been autoclaved, allow it to cool but not solidify. 5.   ...

Mueller Hinton Agar (MHA)       The major use of Mueller Hinton Agar is for antimicrobial susceptibility testing. Composition of MHA Ingredients   Beef Extract                                  2.00 gm Acid Hydrolysate of Casein         17.50 gm Starch                                           1.50 gm Agar                                             17.00 gm Distilled Water                             1000 ml Final pH 7.3 ± 0.1 at 25ºC Why Mueller Hinton agar is used for antibiotic susceptibility tes...