Medical

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 presence of acids.  In case of oxidative decarboxylation of peptone, alkaline produ

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.     When the agar has cooled to 45-50 °C, Add 5% (vol/vol) sterile defibrinated blood that has b

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 testing? 1.     It is a non-selective, non-differential medium. This means that almost all organisms plated on here will grow. 2.     It contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the  antibiotics . It also mediates the rate of diffusion of the antibiotics through the agar. 3.     It is a loose agar. This allows for better diffusion of the antibiotics than most other plates. A bette

   Gram stain reagents These stains can be ordered as a complete kit from VWR (#l5204-004) or can be reconstituted as follows:   Crystal violet crystal violet (90% dye content)                             20.0 g ethanol (95%)                                                          200 mL ammonium oxalate                                                  8.0 g dH 2 0                                                                        800 mL Mix   first   two   ingredients   and   let   sit   overnight   or   until   dye   goes into solution.      Add remaining ingredients and filter before use. Gram's iodine iodine crystals                                                         1.0 g potassium iodide                                                     2.0 g dH 2 0                                                                        300 mL                      Decolorizer acetone                                            

   Cytochrome oxidase   – this test determines the presence of cytochrome oxidase enzymes.        Oxidase reagent Tetramethyl-P-Phenylenediamine Dihydrochloride         1.0g d-H 2 O                                                                               100 mL The use of an iron-containing metal inoculation loop can lead to a false- positive reaction. Use only plastic or platinum loops for this test. 1.       Add an inoculum of pure 18 -24 hour old bacterial culture to the test strip impregnated with reagent. RESULTS:             Positive: purple color within 5-10 seconds (reactions that occur after 10 seconds are negative).( Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella, Pasteurella,   Moraxella, Helicobacter pylori,   Legionella pneumophila, etc.)       Negative: no purple color.(  Family  Enterobacteriaceae ) Quality Control    Positive: Pseudomonas aerugenosa   Negative: Escherichia coli

     Catalase  test – this test determines bacterial production of catalase enzymes.   1.       Place a drop of hydrogen peroxide (3% H 2 O 2 - reagent grade) on a microscope slide or in the concave surface of a hanging drop slide. 2.       With a sterile loop, collect a sample of 18-24 hour old pure bacterial culture.   3.       Place the loop in the hydrogen peroxide.   4.       If the test is positive, there will be immediate bubbling or foaming, and liberation of O 2 gas. 5.       Record results. Positive :   If the mixture produces bubbles or froth, the organism is said to be 'catalase-positive (   Staphylococcus  and  Micrococcus are catalase-positive. Other catalase-positive organisms  include  Listeria ,  Corynebacterium diphtheriae ,  Burkholderia cepacia ,  Nocardia , the family  Enterobacteriaceae  ( Citrobacter, E.coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia ),  Pseudom onas ,