MICROBIOLOGY LABORATORY EXERCISE 7.a --IMViC TEST Biochemical test for identification of enteric organisms: Basis: metabolic action of microorganisms on the culture media Used for the identification of enteric organisms/ gram-negative bacilli One of the earliest sets of test used for the identification of enteric bacilli includes such organisms as Klebsiella, Enterobacter, Citrobacter and Escherichia coli This acronym stands for •I - Indole •M- Methyl red •V - Voges – Proskauer •( i ) is inserted for euphony •C – Citrate INDOLE Indole, a benzyl pyrrole, is one of the metabolic degradation product of amino acid tryptophan Indole positive bacteria produce tryptophanase, an enzyme that is capable of hydrolyzing and diaminating tryptophan, thus producing: - indole - pyruvic acid - ammonia Materials: 2% Peptone broth tube Test organisms Ether indicator: Erlich/Kovac's reagent (para-dimethyl-aminobenzaldehyde) Procedure: -Inoculate 1 loopful of the test organism into the tube of peptone broth. Incubate at 370C for 24-48 hours. -Next laboratory period, add 1 ml. of ether. -Shake well and allow to stand for a few minutes until the ether rises to the surface. -Gently add about, 1cc. of Kovac’s or Erlich’s reagent down the side of the tube so that it forms a ring between the medium and the ether layer. Observation: Positive result •Bright red or purple ring •If indole has been produced by the organism it will, being soluble in ether, it will be concentrated in the ether layer and upon the addition of Erlich’s reagent, a positive result is the production of a purple ring at the junction of the medium and the ether layer. Negative result – Yellow color
- no red or purple ring METHYL RED TEST: All enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes Both the MR and VP tests are used to determine what end products result when the test organism degrades glucose MR test is a quantitative test for acid production, requiring positive organism to produce strong acids (lactic, acetic, formic) from glucose via the mixed acid fermentation pathway End result is based on the final pH reached only those organism that can maintain low ph of about ph 4-4.5 can be called methyl red – positive organisms that are MR (+) are always VP (-) Materials: MR-VP broth medium (contains 10% glucose) Test organisms Methyl red ph indicator Procedure: Inoculate 1 loopful of the test organism into a tube MR-VP medium. Incubate for 24-48 hours at 370C. Next laboratory period, add 5 to 10 drops of methyl red reagent. Mix thoroughly and observe the results. Observation: žPositive result – cherry red/bright red color •ph 4-4.5 •Ex. Salmonella, Escherichia, Citrobacter, Proteus, Morganella and Providencia žNegative result – Yellow color •At neutral pH the growth of the bacteria is not inhibited •The bacteria thus begin to attack the peptone in the broth, causing the pH to rise above 4.5 •At this pH, methyl red indicator produce a yellow color •Ex. Enterobacter and Klebsiella
Negative result •Remains Yellow to Amber; no change in color Ex. E. coli CITRATE TEST: VOGES-PROSKAUER TEST: -is a test for the detection of acetyl-methyl carbinol (acetoin) which is also a degradation product of glucose Materials: MR-VP medium (contains 10% glucose) Test organism Potassium Hydroxide Alpha-napthanol reagent When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a burgundy color/crimson red color (a positive VP test) organisms that are VP (+) are always MR (-) acetyl-methyl carbinol + Potassium Hydroxide ↓ dimethyl-carbinol ↓ guanidine compounds ↓ Crimson red Procedure: -Inoculate MR-VP medium with 1 loopful of the test organism Incubate for 48 hours at 370C. Next laboratory period add 0.6 ml. 5% alphanapthol reagent. -Mix and shake the mixture lightly. Add 0.2 ml (5drops). of 40% potassium hydroxide reagent (KOH). -Mix and shake the mixture lightly. Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10 to 15 minutes. Observation: ž Positive result •Crimson Red color •Presence of Acety methyl carbinol Ex. Enterobacter and Klebsiella ž
ža test depends upon the ability of the organism, to utilize citrate as the sole source of carbon and energy growth ž Materials: •Solid media : Simmon’s Citrate Agar •Liquid media: Kosher’s Media •Test Organism žSimmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6 •uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color (neutral pH) •Growth of bacteria in the media leads to development of a Prussian blue color at more alkaline pH's (around 7.6) (positive citrate) ž Procedure: Inoculate the test organism on the medium by stab streaking. Incubate at 370C for 24 - 48 hours. Observe. Observation: žPositive result •Deep blue/ Prussian blue color •indicating that the test organism has been able to utilize citrate for energy source • Ex. Enterobacter, Klebsiella, Salmonella, Citrobacter Microorgan Indole MR VP Citrate and Providencia ism ž E. coli + + žNegative result •Retains its original color (Green) Enterobacter + + •Ex. Escherichia, Shigella and Morganella Citrobacter
-
+
-
+
ž
Exercise 7.b Lysine-Iron agar test: Intended Use •Lysine Iron Agar is used for the differentiation of enteric organisms based on their ability to decarboxylate or deaminate lysine and to form hydrogen sulfide
Components: Hydrogen Sulfide formation: ž-Organisms which produce hydrogen sulfide gas will exhibit a black precipitate in the butt of the tube ž ž-LIA is not as reliable an indicator of H2S as are KIA and TSI ž ž-Splitting of the agar in the butt indicates gas production. Procedures: Inoculate the organism into the Lysine Iron Agar medium Lysine deamination: •Reaction in the slant • Lysine decarboxylation •Reaction in the butt ž-is an aerobic process which occurs on the slant of the media ž-If the bacterium can deaminate Lysine in the slant, it will produce an unknown orange product ž-This orange product will react with the Bromcresol Purple ž-If the organism has the ability to deaminate lysine, the ammonia produced will react with the ferric ammonium citrate to produce a dark red color on the slant of the tube -Positive deamination: red slant -Negative deamination: purple slant žThe genus that will give a positive red slant are Proteus, Providencia, Citrobacter and Morganella Lysine decaboxylation: ž -is an anaerobic process which occurs in the butt of the media ž -Due to overneutralizing the acid formed from glucose fermentation ž -The bacteria that is inoculated into the butt of this tube can ferment the glucose, producing a low or acidic pH -This induces the bacteria to form the carboxylase enzyme which removes the -COOH from the lysine molecule, creating an amine end product, that is alkaline in pH ž ž -If the organism has the ability to decarboxylate lysine, it produces an amine endproduct which reacts with the pH indicator to give a purple color in the butt of the tube (Positive decarboxylation: purple butt) (Negative decarboxylation: yellow butt) žOrganisms that can decarboxylate lysine : Salmonella, Klebsiella, Enterobacter, Serratia
Stab the butt twice all the way down, then streak the slant using a straight wire needle Incubate at 37OC for 18-24 hours.
Reading LIA reactions: ž SLANT
ž
BUTT
P/P
- /+
purple slant lysine deaminase negative
purple butt lysine decarboxylase positive
P/Y
-/-
purple slant lysine deaminase negative
yellow butt lysine decarboxylase negative
R/Y
+/ -
red slant lysine deaminase positive
yellow butt lysine decarboxylase negative
-you cannot have a (+) slant with a (+) butt! Exercise 7.c Sulfide Indole motility test (SIM): ž-A semi-solid culture medium used to detect: •sulfide formation •indole production •motility ž-Used for the for the identification of Enterobacteriaceae Procedures: - Stab the Sulfide-Indole-Motility (SIM) medium with the organism once with a straight wire needle to the bottom of the tube -Incubate at 37 C for 24 hours. -Read the motility before adding Ehlichs / Kovac’s reagent. Observations:
Test organism A. Hydrogen sulfide production -The sulfide reaction is indicated by blackening of the medium along the line of inoculation or in the bottom of the tube -the reaction is derived from organisms bearing sulfur-containing amino acids -Hydrogen sulfide reacts with ferrous sulfate to form ferrous sulfide which is seen as a black colored substance ž(+) H2S organisms are always motile B. Motility Determination POSITIVE TEST (MOTILE) žmotile organisms migrate from the stab line and diffuse into the medium, causing turbidity žThey may exhibit fuzzy streaks of growth at the stab line NEGATIVE TEST (NON-MOTILE) žbacterial growth accentuated along stab line, surrounding mediums remains clear CONTROL MEDIUM (Uninoculated) žno growth, medium remains clear
Procedures: Inoculate 5 loopful of the test organism into the urea christensen broth. Incubate at 370C for 24 hours. Observe for color change. Observation: -POSITIVE •dark pink color •The pH indicator phenol red changes to a deep pink color in the presence of ammonia turning the medium alkaline •particularly helpful in identifying Proteus from other species due to the rapid action of the enzyme by Proteus compared to other non-lactose fermenting enterics •Also (+) to Klebsiella, Haemophilus and Citrobacter -NEGATIVE •If the medium retains its original color ž ž ž ž
C. Indole Production ž -Add about 13drops of Ehrlichs / Kovac’s reagent to the tube POSITIVE TEST a red or purple ring indicates that indole is produced at the junction of the medium and the reagent -NEGATIVE TEST a yellow color is observed. Exercise 7.d Urease test: ž-Urease, an enzyme produced by some microorganisms, attacks the nitrogen and carbon bond in amide compounds and forms the alkaline end product ammonia ž-Microorganism that possess the enzyme urease have the capability of hydrolyzing urea with the release of ammonia and carbon dioxide Urea + Water Ammonia & Carbon dioxide ž Materials: Urea Christensen broth
Exercise 7.e CYTOCHROME OXIDASE TEST: The cytochromes are iron-containing hemoproteins that act as the last link in the chain of aerobic respiration by transferring electrons (hydrogen) to oxygen, with the formation of water. Cytochrome oxidase or Warburge’s respiratory enzyme – the enzyme which reduces molecular oxygen to water to compete the last link in the chain of aerobic respiration The test is most helpful in screening colonies suspected of being one of the Enterobacteriaceae (all negative) and in identifying colonies suspected of being a pseudomonas specie or a neisseria specie which are all positive. (Pseudomonas, Vibrio, Aeromonas, Neisseria and Comamonas) žThe cytochrome oxidase test utilize certain reagent dyes, such as the “redux dye: tetramethylparaphenylenediamine dihydrochloride”, that substitute for oxygen as artificial electron acceptors. ž ž In the reduced state the dye is colorless, however, in the presence of cytochrome oxidase and atmospheric oxygen, tetramethyl-
paraphenylenediamine dihydrochloride is oxidized, forming indophenol blue color. Materials: Culture plate of organism Cytochrome oxidase paper strip (Pathotec oxidase strip) (tetra-methyl-para-phenylene diamine dihydrochloride) Procedures: -The test is commonly performed by one of the two methods: -The direct plate technique in which 2 to 3 drops reagent are directly poured into the isolated bacteria colonies growing on the plate medium. ž -The indirect paper strip procedure. In which a few drops of the reagent are either added to a filter paper trip or commercial disk or strips impregnated that dried reagent are used. ž -In either method, a loopful of suspected colony is smeared into the reagent zone of the filter paper.
-Do not place anything in the other tube which serves as control Incubate broth tube at 370C. Examine at 24 hours. Result: Positive Coagulase – a clot is formed Negative Coagulase – no clot is formed This usually detects bound coagulase, which is the clumping factor attached to the cells and acts directly on the fibrinogen. ž ž Procedures: Divide the slide into two sections with grease pencil. -Place a small drop of normal saline on each area. -Emulsify one or two colonies of microorganism on blood agar plate on each drop to make a smooth suspension. -Add a drop of undiluted plasma to one of the drop of saline and stir gently with a wire loop. -Do not put anything in the other drop that serves as control. -Observe for clumping of the microorganism microscopically. Positive - clumping / Negative - no clumping
Observations: Bacterial colonies having cytochrome oxidase actively will develop a deep blue color to purple to black at the inoculation site within seconds. Exercise 7.f COAGULASE TEST: ž Some bacteria produce coagulase, an enzyme and as a precursor of thrombin like substance which coagulate plasma. žThere are two kinds of coagulase which could be present in the microorganism – the free coagulase and – the bound coagulase. žThis enzyme is demonstrated in vitro by two methods. •The Test Tube Method •The Slide Method ž-Differentiate pathogenic from non-pathogenic Staphylococcus. Tube Method: The method measures free coagulase. ž
Exercise 7.g CATALASE TEST: ž-Some bacteria produce the enzyme catalase, which is an iron porphyrin protein ž-This enzyme is responsible for the decomposition of hydrogen peroxide to water and oxygen H2O2
CATALASE
H 2 O + O2
ž Procedures: -Emulsify about 1 or 2 colonies of Staphylococcus organism from a plate culture with a drop of saline on a slide. -Add a drop of hydrogen peroxide. -Observe for air bubbles ž Observation: ž žPositive test •presence of air bubbles •Bubbling upon the addition of hydrogen peroxide is indicative of the presence of catalase for this organism. •Note, most aerobic organism make catalase. •Differentiates Staphylococcus from Streptococcus All Staph (+) All Strep (-) ž
Materials: •Citrated or heparinized human or rabbit plasma is diluted with isotonic saline or other suitable diluent •Overnight broth culture of microorganism. ž
Negative test – no air bubbles
Procedures: -Place about 0.5ml of diluted plasma in each of 2 test tubes. -To one tube add 5 drops of an overnight broth culture of the organism.
-Bacteria ferment carbohydrates in different ways depending upon the enzymes they process. -In bacteriologic test systems, acidity therefore is an indication of fermentation and is detected by observing
EXERCISE NO. 7.h Carbohydrate Fermentation
color change of pH indicators as acid products are formed. ž Materials: 1. 2. 3. 4. 5.
Lactose broth 1% (with Durham’s tube) Sucrose broth 1% Mannitol broth 1% Dextrose semi-solid agar 5% Slant agar culture with Coliform, Klebsiella and E. coli
ž ž
Indicator dye is “Bromcresol purple” ž Procedures: 1) Arrange the tubes properly and label each with the corresponding sugar. 2) Inoculate the test organism into this different carbohydrate test tube. 3) Incubate at 370C for 24 hours. 4) Next laboratory period observe the color changes and gas production. Acid production -phenol red turns into yellow color -No acid production-color remains purple EXERCISE NO. 7.i. Triple Sugar Iron Reaction The TSI reaction provides a means for the presumptive identification of gram negative enteric bacteria. The rationale of this test is the fact that many of the gram negative bacteria can be differentiated on the basis of: 1) Acid production from fermentation of carbohydrates 2) Gas formation 3) Production of hydrogen sulfide (H2S) Indicator - Phenol red Materials: Triple Sugar Iron Agar tubes Culture of enteric bacteria ž Procedures: 1) Fish out a colony from plate culture of bacteria and inoculate it by making a stab to the butt portion of the TSI medium. Then pull the needle up to the surface of the agar and make a zigzag streaking on the slant portion. Cover the tube loosely with a cotton plug. 2) Incubate the tubes for 24 hours and observed the reaction produced in each test tube. Record your observation. Yellow-acid
Red-alkaline
-red slant/red butt – at least 2 sugars fermented -red slant/yellow butt – only one sugar is fermented, usually Lactose -yellow slant/yellow butt – no sugar fermented -black discoloration - presence of ferrous sulfide (H2S)
