Week 9 B New: Gram (–) Bacteria Identification Last week’s: Exp 24: IMViC Part A: Indole Test: - Lab Two, pg 166 Part B: Citrate Test Lab Two, pg 170 Exp 25: Hydrogen Sulfide Test & Motility Lab Two, pg 178 Exp 26: Urease Test Lab Two, pg 182 Exp 30: Oxidase Test: Lab Two, pg 200 New: Gram (–) Bacteria Identification Exp 62: ID of Human Staph. and Streptococcus Pathogens Part 3: Blood and MSA Part 5: Coagulase Exp 29: Catalase Test

Ex 24: IMViC Part A: Indole Test Ex 25: Hydrogen Sulfide Test (& motility) SIM – a combination of three tests in one tube. These tests are: Sulfur reduction – ability of the bacterium to reduce a sulfur metabolite in the medium, producing H2S (a colorless gas that generates a black color in the agar medium); sulfates serve as an inorganic source of energy (chemolithotroph); medium contains ferrous (iron) sulfate to detect the production of H2S (which is colorless gas) Indole production – if a red color develops in the Kovac’s reagent at the top of the agar (2nd tube from the left) , the bacterium IS able to convert tryptophane to indole (Kovac’s mixed with indole turns red); if the Kovac's reagent color does not change (remains a golden/yellow - middle tube) then indole is not produced Motility – if there is a distinct “feathering” out pattern that extends from the original stab (tube on right - this is not SIM medium), then the bacterium is motile. Sometimes the black color (if the bacterium can reduce the sulfur-containing substrate) makes it difficult to see the feathering pattern (if it is there).

Experiment 24: Indole Test SIM agar contains tryptophan Tryptophan is an essential amino acid; some organisms possess an enzyme, tryptophanase, that can metabolize it. Indole is a product of tryptophan metabolism. Kovac’s reagent reacts with the indole and produces a cherry red layer on the top of the media [presence of cherry red layer means that the organism can break down tryptophan (indole-positive reaction)] Used to distinguish between microorganisms that can break down the amino acid tryptophan.

Exp 25: Hydrogen Sulfide Test The SIM medium contains peptones (inc cysteine) and sodium thiosulfate. Some microorganisms have an enzyme that metabolizes cysteine (a sulfur-containing amino acid). Microorganisms produce H2S as a product. Ferrous sulfate is an indicator; it combines with the gas and turns black in the medium. Motility – if there is a distinct “feathering” out pattern that extends from the original stab it means that the org. has a flagella. Used to determine which organisms produce H2S gas by reducing sulfur-containing compounds present in the medium.

Ex 24: IMViC Part D: Citrate Test Citrate utilization – this test determines if a bacterium can use citrate as the sole source of carbon. this test determines if a bacterium can use citrate as the sole source of carbon. If the color of the medium has changed from green to BLUE, the bacterium IS able to use citrate as the sole energy and carbon source. If the color remains green, then the bacterium can not use citrate as the sole source of carbon. Usually a bacterium that can not utilize citrate will also not grow. However, there are rare circumstances in which a bacterium might grow a little but not produce the blue color. This sometimes does occur if a large amount of inoculum is placed on the slant.

Ex 24: IMVic Part D: Citrate Utilization Test Citrate is broken down into pyruvic and acetic acids, with CO2. CO2 reacts with Na+ to produce sodium bicarbonate as a byproduct. Sodium bicarb. is basic and turns indicator bromthymol blue. Bottom line: those organisms that ferment citrate will show growth on the slant and blue coloration (citrase +); lack of citrate fermentation will cause no growth and medium will remain green (citrase -). Used to determine which microorganisms can use the molecule citrate as the only carbon source to ferment Those that can break down citrate to produce energy contain the enzyme citrate permease that transports citrate into the cell; citrate is metabolized using the enzyme citrase which also occurs in the Krebs cycle.

Exp. 26: Urease Test Urea hydrolysis – another test for the ability to break down a molecule (urea). In this case it is not hydrolysis of a complex polymer, but simply the splitting of urea into ammonia and CO2. When he inoculated broth changes in color to PINK. This indicates that the bacterium was able to degrade urea (with the enzyme urease.

Exp 26: Urease Test Key identifier for Proteus vulgaris, which has the enzyme urease. Urease is a hydrolytic enzyme that breaks down urea and produces CO2, H2O, and ammonia. Phenol red (pH indicator) turns yellow in acidic conditions and red in basic conditions Organisms that possess urease will turn the broth pink (ammonia is a product and it is basic!) urease + Used to determine if a microorganism can degrade urea.

Exp. 30: Oxidase Test Oxidase enzyme activity - detects activity of cytochrome oxidase (one of the enzymes in the bacterium's electron transport system - assumes the bacterium is an aerobe or facultative anaerobe) - After incubation, add 2-3 drops of p-aminodimethylaniline oxidate to the inoculated area. - Presence or absence of a color CHANGE from pink to maroon and finally to purple occurs in 10-30 seconds after adding the reagent, the bacterium is considered positive for oxidase enzyme activity. If no color change takes place, or the change is a slightly darker pink, the bacterium is considered negative for oxidase activity.

Exp. 30: Oxidase Test Cytochrome oxidase is part of the electron transport chain and transfers electrons from a donor molecule to oxygen. Aerobic bacteria, and some facultative anaerobes, exhibit oxidase activity Pseudomonas is oxidase positive Enterobacteriaceae are oxidase negative Test reagent oxalate changes color in the presence of oxidase. Maroon-black = oxidase + No change in color = oxidase - Used to determine which organisms utilize the enzyme cytochrome oxidase.

Results To ID Gram Negative Bacteria Serratia marcescens (SM)   Notation MICROORGANISM E.coli (EC) P. aeruginosa (PA) P.vulgaris (PV) E. aerogenes (EA) Serratia marcescens (SM) Morphology shape/ arrangement rods TSA (cultural characteristics) color, size, form, margin, elevation, texture white, small, circular, entire, convex , wet white/green, small, circular, entire, convex, mucoid white, small, circular, undulate, wet white, small, circular, entire, convex, wet red centers, small, circular, entire, convex, wet TSI Slant Alk or A TSI Butt Alk or A (G) TSI H2S +/- H2S prod. SIM Indole Prod. +/- tryptophanase ■ SIM H2S SIM Motility +/- motility Citrate Ferm. +/- ferm. Urease Activity +/- urease Methyl Red +/- VP (Voges-Proskauer) Oxidase Activity +/- oxidase Catalase Activity +/- catalase NO3 (nitrate reduction) Coagulase Actvity +/- coagulase + - positive AG - acid with gas MF - mannitol fermentor - - negative ± - variable LF - lactose fermentor Lucia Testa: Bio 156 Enterobacter aerogenes Biohazard: 1 Respiration: aerobic Motility: yes Industrial implications: EA is being evaluated for Hydrogen production. About: EA is part of the normal intestinal flora of humans and have been isolatef from soil, water, vegetable, meats, plants, dairy products, and cosmetics. Human Health: EA is an important nosocomial pathogen responsible for a variety of infections, usually opportunistic.

Results To ID Gram Negative Bacteria Serratia marcescens (SM)   Notation MICROORGANISM E.coli (EC) P. aeruginosa (PA) P.vulgaris (PV) E. aerogenes (EA) Serratia marcescens (SM) Morphology shape/ arrangement rods TSA (cultural characteristics) color, size, form, margin, elevation, texture white, small, circular, entire, convex , wet white/yellow, small, circular, entire, convex, mucoid white, small, circular, undulate, wet white, small, circular, entire, convex, wet red centers, small, circular, entire, convex, wet +/- growth MAC LF or NLF/ color EMB LF or LNF/ color TSI Slant Alk or A TSI Butt Alk or A (G) TSI H2S +/- H2S prod. SIM Indole Prod. +/- tryptophanase ■ SIM H2S SIM Motility +/- motility Citrate Ferm. +/- ferm. Urease Activity +/- urease Oxidase Activity +/- oxidase + - positive AG - acid with gas MF - mannitol fermentor - - negative ± - variable LF - lactose fermentor Lucia Testa: Bio 156

Media for Gram Negative Bacteria MacConkey agar - Contains crystal violet, bile salts, lactose, pH indicator neutral red - Crystal violet inhibits Gram(+) bacteria: selective Lactose fermentor – red colony, non-fermenter – transparent: differential Image: MacConkey's Plates:Gram negative, lactose fermenting E. coli in plate on right (pink) and Gram-negative, non-lactose fermenting Salmonella in plate on left (tan/colorless). EMB agar: selective– The dye Methylene Blue and Eosin prevent growth of gram(+) bacteria -Differentiate- Lactose differentiates lactose-fermenting (acids produced will precipitate dyes) and lactose non-fermenting gram(-) bacteria). Image E coli- Lactose fermenter: bluish black with green luster PA- Lactose-nonfermenter: transparent color EA- Lactose fermenter MacConkey Agar (MAC) Eosin Methylene Blue Agar (EMB)

Results To ID Gram Positive Bacteria Staph. epidermidis (SE)   Notation MICROORGANISM Staph. epidermidis (SE) Staph. aureus (SA) B. subtilis (BS) Strep. salivarius (SS) M. luteus (ML) Morphology shape/ arrangement cocci/ cluster rods cocci/ chain TSA (cultural characteristics) color, size, form, margin, elevation, texture PEA +/- growth ■ MSA MF or NMF/ color MAC EMB Blood Agar (cultural characteristics) Blood Hemolysis У α β Starch Hydrolysis +/- amylase Gelatin Liquefac. +/- gelatinase Oxidase Activity +/- oxidase Catalase Activity +/- catalase NO3 (nitrate reduction) +/- Coagulase Actvity +/- coagulase + - positive AG - acid with gas MF- mannitol fermentor - - negative ± - variable LF- lactose fermentor Lucia Testa: Bio 156 3 major species of of Staph are Staph. Aureus, Staph. Epidermidis, and Staph. Saprophyticus. ( last two generally avirulent) Staph. Aureus (pathogenic) infections are often responsible for abcesses on skin (boils, acne, carbuncles). Staph. Aureus produces a variety of metabolic end products which play roles in its pathogenicity Including coagulase (causes clot formation) Hemolysisns (active against red bllod cells) When there is a poss of staph infection, isolation and id of Staph. Aureus is imp.

Exp 62: ID of Human Staph. And Strep. Pathogens: Blood and MSA Review Mannitol Salt Agar (MSA) Selective & Differential Media a. Blood agar: for fastidious bact. (streptococcus, styphylococcus, etc.) Hemolysin – destroy red blood cell Alpha-hemolysis: incomplete hemolysis – green ring Beta-hemolysis: complete hemolysis – clear zone Gamma-hemolysis: absence of hemolysis b. Mannitol salt agar: Selective - most bacteria cannot grow except staphylococcus due to high salts Differential- Mannitol-fermenting species of Staph will change the phenol-red color. Other Staph will grow without color change. Blood Agar

Exp 65: ID of Human Staph. And Strep. Pathogens: Part 5: Coagulase Test Images: The coagulase test: the upper positive reaction has gelled, while the lower negative reaction is liquid. Coagulase is an enzyme produced by Staphylococcus aureus that converts fibrinogen to fibrin. In the laboratory, it is used to differentiate between various types of Staphylococcus. S. aureus is coagulase-positive (negativity preculdes S aureus). Coagulase is tightly bound to the surface of the bacteria S. aureus and can coat its surface with fibrin upon contact with blood. It has been proposed that fibrin-coated staphylococci resist phagocytosis making the bacteria more virulent. The coagulase test uses rabbit plasma that has been inoculated with a staphylococcal colony. The tube is then incubated at 37 degrees Celsius for 1-1/2 hours. If negative then continue incubation up to 24 hours. If positive (i.e., the suspect colony is S. aureus), the serum will coagulate,[2] resulting in a clot (sometimes the clot is so pronounced that the liquid will completely solidify). If negative, the plasma remains liquid. The negative result may be S. epidermidis but only a more detailed identification test can confirm this. Although manual says that test needs to be read within 4 hours- it is OK to read next week.

Medium: TSA Plate Chemical Components: Hydrogen peroxide Property/Enzyme Testing For: Catalase degrades H2O2 Results: + = bubbling or foaming - = no bubbling Interpretation of Results: During aerobic respiration H2O2 and superoxides are produced. Their accumulation is toxic unless they can be enzymatically degraded. Catalase rapidly degrades H2O2. If catalase is present, free O2 bubbles up when added to the grown culture. (Anaerobes do not synthesize catalase, peroxidase, or superoxidase thus O2 is poisonous to them.)

Exp 63: Bacitracin Test Blood Agar Streptococcus pyogenes - Members af Strep are responsible for greater number of infectious disease than any other organism. Gram pos and nutritionally fastedius requiring enriched media to grow. Alpha and gamma are usually avirulent. Beta hemolytic most associated with pathogenicity. Bacitracin Test Beta-hemolytic Streptococcus are spherical bacteria that produce hemolysins capable of completely lysing (bursting open) red blood cells. When grown on sheep blood agar, colonies of beta-hemolytic Streptococcus are encircled by visible areas of clearing where beta hemolysis has occurred. - Strep pyogenes is the only Beta Himolyitc streptococci sensitive to Bacitracin- used to test for strep throat. Beta-hemolytic streptococci belonging to Group A Streptococcus (Streptococcus pyogenes) causes "strep throat". - The bacitracin susceptibility test is used to distinguish Group A streptococci, which cause ninety percent of acute streptococcal infections in humans, from other streptococci. When grown on blood agar, Group A streptococci are sensitive to (killed by) the antibiotic bacitracin . A sterile disk impregnated with bacitracin is placed on the first sector of an isolation plate before incubation. A zone of inhibition (area with no growth) will be seen around the disk after incubation if the organism is a Group A beta-hemolytic Streptoccus. Other beta-hemolytic streptococci are resistant to (not killed by) bacitracin. Their colonies will thus grow right up to the disk of bacitracin. Streptococcus salivarius is a species of spherical, Gram-positive bacteria which colonize the mouth and upper respiratory tract of humans a few hours after birth, making further exposure to the bacteria harmless. The bacteria is considered an opportunistic pathogen, rarely finding its way into the bloodstream, where it has been implicated in septicemia cases in people with neutropenia. It is usually alpha hemolytic and sensitive to many antibiotics. Streptococcus pyogenes

Exp. 29: Catalase Test Catalase enzyme activity (degradation of hydrogen peroxide into water and oxygen). Catalase is the enzyme that breaks hydrogen peroxide (H2O2) into H2O and O2. Hydrogen peroxide is often used as a topical disinfectant in wounds, and the bubbling that is seen is due to the evolution of O2 gas. H2O2 is a potent oxidizing agent that can wreak havoc in a cell; because of this, any cell that uses O2 or can live in the presence of O2 must have a way to get rid of the peroxide. One of those ways is to make catalase. Most higher organisms produce catalase, but in bacteriology this test is usually used to differentiate staphylococci (Catalase positive) from streptococci (Catalase negative). Catalase is nearly ubiquitous among organisms that can grow in the presence of oxygen (air). ..The major function of catalase within cells is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes - primarily that of the electron transport pathway. After incubation, add 3-4 drops of 3% hydrogen peroxide to the plate and allow it to flow over the entire surface of the slant (to be sure it contacts the bacteria on the slant). Observe for presence or absence of bubbling or foaming. Presence indicates that the hydrogen peroxide is being broken down (by catalase), with the release of oxygen gas (causes the bubbling). The absence of foaming/bubbling indicates that catalase is not present (or is not active for some reason).

Medium: Citrated plasma Chemical Components: plasma Property/Enzyme Testing For: Coagulase forms fibrin Results: + = clot formation - = no coagulation Interpretation of Results: Indicative of Staphylococcus aureus. In host tissues, coagulase forms fibrin meshwork to surround organisms of infected tissues to protect it from nonspecific host resistance such as phagocytosis and anti-staph activity in normal serum.

Lab One, Part 3: Blood and MSA Lab One, Part 5: Coagulase Test Exp 62: ID of Human Staph. and Strep. Pathogens Exp 29: Catalase Test Culture: SE, SA, SS (Streptococcus salivarius) Lab One, Part 3: Blood and MSA Media: 1 Blood plate, 1 MSA plate. Procedure: (refer to pg 428) Divide the bottom of the plates in 3 parts. Streak inoculate each third. Incubate at 37oC for 24 hrs. Lab One, Part 5: Coagulase Test Materials: three 10X75 mm tubes, 1ml pipet Medium: Rabbit Plasma Add 0.5ml rabbit plasma to each tube. Label each tube (1 org/tube). Inoculate with loop. Per table Media: 3 BHI slants Culture: SE, SA, SS Procedure: (refer to pg 196) Streak inoculate with loop. Incubate at 37oC for 24 hrs. Next week: Add 3% H2O2 Media: Mannitol Salt (MSA) Blood Agar Test Reagents: 3% Hydrogen Peroxide Rabbit Plasma Organisms: Staphylococcus aureus (SA) Staphylococcus epidermidis (SE) Streptococcus salivarius (SS) Students will streak the organisms on MSA and Blood, inoculate Rabbit plasma (coagulase test), and inoculate slants for catalase test. EX65 1 plate of each per table: MSA and Blood Divide the plates into three sections with permanent marker and inoculate with the three organisms. Incubate EX30 Three tubes of BHI slants per table: inoculate each tube with one of the organisms This will be used to perform the Catalase test next week. Incubate Coagulase test: EX 65 Three 10x75mm tubes per table Step 1: add 0.5ml (use a 1ml pipet) of rabbit plasma to each of three tubes (one for each organism) Step 2: add a loopful of organism and label tubes then incubate. Step 3: Incubate (Although the book says read within 4 hours, they will be fine until next week) *If there is time practice gram stain (we will put out g+ and G- organisms for practice) Gram staining practice Week 9 B