General Microbiology Laboratory Biochemical Tests H 2 S

General Microbiology Laboratory Biochemical Tests

H 2 S Production v In this lab we have chosen two bacteria that are H 2 S producers and motile, and one bacterium that is nonmotile and does not produce H 2 S. v Salmonella is a facultatively anaerobic gram-negative rod that occurs in humans, warm- and cold-blooded animals, food, and the environment. It is H 2 S positive and motile. v Proteus is a gram-negative facultatively anaerobic rod that occurs in the intestines of both humans and a wide variety of animals and polluted waters. It is motile and produces H 2 S. v Klebsiella ( inflammation of the lungs) is a facultatively anaerobic gram-negative rod that occurs in human feces, clinical specimens, soil, water, fruits, and vegetables. It is nonmotile and does not produce H 2 S. Mohammed laqqan

H 2 S Production in SIM v The most convenient medium for testing for indole and/or hydrogen sulfide production is SIM medium (SIM is an acronym for sulfide, indole, and motility). the SIM medium contains peptones (Cysteine) and sodium thiosulfate as substrates, and ferrous ammonium sulfate, Fe(NH 4)SO 4, as the H 2 S indicator. v Hydrogen sulfide is produced when amino acids containing sulfur (Cysteine ) are metabolized by microorganisms. v Once H 2 S is produced, it combines with the ferrous ammonium sulfate, forming an insoluble, black ferrous sulfide precipitate that can be seen along the line of the stab inoculation. If the organism is also motile, the entire tube may turn black. This black line or tube indicates a positive H 2 S reaction; absence of a black precipitate indicates a negative reaction. v SIM agar may also be used to detect the presence or absence of motility in bacteria as well as indole production. Mohammed laqqan

Hydrogen Sulfide Production This is a tube semisolid agar that can also be used to demonstrate bacterial motility. It is inoculated by stabbing the wire loop (or preferably a straight wire inoculating needle) straight down the middle of the agar to about onefourth the depth of the medium and withdrawing the wire along the same path. Mohammed laqqan

H 2 S Production in SIM v How to Perform Test: Stab SIM media with inoculating needle. v Property it tests for: This test is used to help differentiate species of the family Enterobacteriaceae. This test is used to determine the ability to reduce sulfur into H 2 S. v Media and Reagents Used: SIM media contains the sulfur containing amino acid, cysteine, sodium thiosulfate, & peptonized iron or ferrous sulfate. v Reading Results: H 2 S will react with the iron or ferrous sulfate and produce a black precipitate. A positive result has a black or brown precipitate present and a negative result has no black precipitate. v Any blackening of the medium is considered a positive test for H 2 S. Mohammed laqqan

Triple sugar Iron Agar (TSIA) v Triple sugar iron agar tests for 3 things---sugar fermentation (glucose/lactose/sucrose), CO 2, and H 2 S. It is a good medium for some bacteria, although you can run all of these tests in other ways, such as running individual sugar discs, SIM for H 2 S, and phenol red glucose for CO 2. Gases are easy to spot. The H 2 S is identified the same way as in SIM, but be sure that the black is Inside of the medium. v Carbon dioxide is identified by cracks and bubbles inside of the medium, sometimes a few bubbles and sometimes enough to push the slant up to the top. v There are 3 possibilities of sugar reactions and different reactions in different areas (butt vs. slant) of the medium, so the physiology behind it is pretty complex. The outcome of sugar use is always acid, so the p. H indicator phenol red will turn yellow--reported as A. No use of the sugar or alkaline by-products (which is NO sugar use) from the other non-sugar nutrients in the medium will cause the indicator to stay the same color red/orange or maybe even change it to a red---reported as a K. Mohammed laqqan

v The reactions in TSIA are reported as slant (A or K), butt (A or K), a circle around the butt for CO 2, and + for H 2 S. For example K/A +H 2 S = red slant, yellow butt, with both CO 2, and H 2 S. v The fermentation of the sugars causes the anaerobic butt to turn yellow and stay yellow. However, if only glucose is used, even though the slant turns yellow only after a few hours it will revert to red because the protein in the medium is broken down to alkaline products when the small amount of glucose is used up. If lactose and/or sucrose are used, the large amount of fermentation products neutralizes the basic products and the slant stays yellow. v Therefore, • A/A = glucose and lactose and/or sucrose are used • K/A = glucose alone • K/K = no sugars used v There is no way to get a A/K reaction when using a G-rod on this medium. IF YOU do, it means that § 1) you did not inoculate correctly with a stab and streak § 2) you inoculated something other than a G- rod. Mohammed laqqan

Principle v The triple sugar iron (TSI) agar test is generally used for the identification of enteric bacteria (Enterobacteriaceae). v It is also used to distinguish the Enterobacteriaceae from other gram-negative intestinal bacilli by their ability to catabolized glucose, lactose, or sucrose, and to liberate sulfides from ferrous ammonium sulfate or sodium thiosulfate. v TSI agar slants contain a 1% concentration of lactose and sucrose, and a 0. 1% glucose concentration. The p. H indicator, phenol red, is also incorporated into the medium to detect acid production from carbohydrate fermentation Mohammed laqqan

SOME COMMON SUGAR REACTIONS IN TSIA v NOTE: This is a stab and streak inoculation. THIS MEDIUM IS RUN ON GRAM - RODS ONLY! A/A E. coli, Yersinia, Aeromonas, Vibrio K/A Salmonella, Edwardsiella, Shigella K/K nonfermenters such as Pseudomonas and others A/K erroneously inoculated a Gram + or some other weird thing Mohammed laqqan

Mohammed laqqan

v How to Perform Test: : v 1 -Inoculate the medium using an inoculating NEEDLE. Stab the inoculum down through the butt, then pull the needle out and streak up the slant ( Do NOT take another inoculum to do the slant). 2 -Incubate at 30 or 37 degrees C. v Property it tests for: § Identify the various sugar reactions in TSIA. § Identify the presence of carbon dioxide and hydrogen sulfide gases. v § § § Media and Reagents Used: TSIA per unknown Reading Results: A/A = yellow throughout K/A = red slant, yellow butt K/K = red or red/orange throughout carbon dioxide = bubbles or breaks in medium black precipitate = hydrogen sulfide Mohammed laqqan

From left to right: Uninoculated control Red slant and red butt, no black color= no fermentation of glucose, sucrose or lactose. No Hydrogen sulfide produced Red slant and black butt= no lactose or sucrose fermentation, H 2 S has been produced Red slant with yellow butt= no lactose or sucrose fermentation, lactose is fermented, no H 2 S has been produced Yellow slant, yellow butt and black coloration= Lactose, sucrose and glucose fermented, and H 2 S has been produced Yellow slant, yellow butt and lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented, H 2 S has not been produced but gas has been produced Yellow slant, yellow butt and no lifting and/or cracking of media, no black coloration= Lactose, sucrose and glucose fermented, H 2 S has not been produced nor has gas been produced Mohammed laqqan

Limitation of the procedure (1) If screw-cap tubes are used, leave the caps loose after inoculating the tubes to prevent excessive disruption of the agar should large amounts of gas be produced during incubation. (2) Record the butt as acid production if the black color of Fe. S masks the color in the butt. (3) Hydrogen sulfide production may be evident on Kligler Iron Agar (4) Do not use an inoculating loop to inoculate a tube of Triple Sugar Iron Agar. While stabbing the butt, mechanical splitting of the medium occurs, causing a false positive result for gas production Mohammed laqqan

End of lecture Mohammed laqqan