Basic microbiology laboratory tests.pptx

Laboratory examinations

1. LAB 02 Lecture # 01 Biochemical Identification Tests

2. Topics: 1 ) Catalase Test 2) Simmons Citrate Test 3) Triple Sugar Iron Test

3. Catalase Test PRINCIPLE The catalase test is used to detect the presence of the enzyme catalase in bacteria. Catalase is an enzyme that converts hydrogen peroxide into water and oxygen. The bacteria that contain this enzyme are usually aerobic (need oxygen) or facultative anaerobes (can live with or without oxygen ). Catalase producing bacteria will produce O 2 when mixed with H 2 O 2. 2H 2 O 2 -------------> 2H 2 O + O 2 (Bubbles) Methods: i ) Slide Test Method ii) Tube Test Method

4. PURPOSE Identification for gram-positive & gram-negative organisms. It is a primary test used in the differentiation of staphylococci (catalase + ve ) and streptococci (catalase – ve ). Also valuable in differentiating aerobic and obligate anaerobic bacteria. PROCEDURE Place a small amount of a bacterial colony on a clean glass slide. Add one to two drops of 3% hydrogen peroxide . Slide catalase test results: Hydrogen peroxide was added directly to the culture on a microscope slide. A positive reaction produced by Staphylococcus aureus is indicated by bubbling; a negative reaction produced by Streptococcus pyogenes is indicated by lack of bubbling.

5. RESULTS Positive: Rapid bubble formation with evolution of O 2 Negative: No bubble formation

6. Simmons Citrate Test PRINCIPLE Ability of bacteria that utilize citrate as the carbon source is identified through citrate utilization test. The medium contains citrate as a carbon and energy source whereas salt of ammonia is present in the medium as nitrogen source. When bacteria that produces enzyme citrate permease catalyze citrate, pyruvate is produced that enter the Krebs cycle for generation of energy. Metabolism of citrate result in the degradation of salts of ammonia resulting in production of ammonia thus pH of medium gets increased and indicator bromothymol blue changes color from forest green to blue at pH greater than 7.6

7. PROCEDURE Prepare the Simmon’s citrate agar slant in test tubes. Simmons citrate media was prepared in distilled water, this media along with other apparatus required was autoclaved. With the help of pipette 5 ml media was dispensed in the labelled test tubes that were allowed to solidify for formation of slants . Inoculate the freshly streaked 18-24 hrs test organisms and control organisms , was streaked on the slants in zig zag manner without rupturing media, in separate test tubes with the aid of inoculating loop. Autoclaved plugs were fixed on the open end of test tubes, and inoculated test tubes were kept in the incubator for 24-48 hrs. at 37 degrees centigrade . Observe the color change in the medium

8. RESULTS After 24 hrs of incubation results were observed change in color of slants from green to blue indicated that bacteria utilized citrate as carbon source and if the color remains same that is indication of organisms to be negative for enzyme citrate permease . Positive: Klebsiella pneumoniae . Negative: E.coli

9. Triple Sugar Iron Test (TSI) PRINCIPLE Purpose: This test is performed to know the pattern of carbohydrase fermentation and hydrogen sulphide production in organisms. T riple sugar iron is differential media with sucrose and lactose 1%, and glucose 0.1 percent thus it is used for differentiating bacteria on the basis of 1 ) carbohydrates either glucose, lactose, or sucrose they ferment and also 2) hydrogen sulphide gas they produce or not. Indicator used is phenol red in the media. When carbohydrates are fermented the pH is decreased that result in change in color from orange red to yellow In case of peptone alkalization color is changed from orange red to deep red. Moreover , production of black insoluble ppt indicates that gas has been produced. This test is used to differentiate members of Enterobacteriaceae from other gram-negative rods.

10. PROCEDURE TSI media was prepared in distilled water in conical flask, and along with other required apparatus such as test tubes, inoculating loop, blue tip box, cotton plugs were autoclaved at 121 degrees centigrade for twenty mints . In a laminar flow hood media was dispensed in the test tubes with the help of pipette using blue tips. Test tubes were allowed to stand for few mints. in a tilted manner for the formation of sharp slants. Isolated colony from freshly prepared streaked culture was picked and first direct stabbing in the butt of the agar was done and without taking the needle of the loop out of the test tube then zig zag streaking was done on the slant in very gentle way and test tubes were incubated . Observe the test tubes after 24 hours of incubation.

11. RESULTS The color and composition of the media in the tubes will be changed due to gas ( Hydrogen Sulphide ) production and also producing of cracks in the media inside the tubes . Cracks produced due to Hydrogen Sulphide gas inside the media . Yellow color of the media in the tubes determined by the fermentation of glucose or sucrose. Different bacterial species change color of butt and slop differently; Sometime butt and slop both are yellow Sometime butt and slop both are black. Sometime butt is black and slop is yellow. Sometime butt is yellow and slop is black.