CLINICAL BACTERIOLOGY LABORATORY IDENTIFICATION AND DIFFERENTIATION OF THE FAMILY ENTEROBACTERIACEAE OUTLINE • Introduction o Biochemical Tests • Different Biochemical Test o Acetate Utilization Test o Amino Acid Decarboxylase Test o Casein Hydrolysis Test o Citrate Utilization Test o Gelatin Hydrolysis Test o Hydrogen Sulfide Test o Indole Test o Kliger’s Iron Agar Test o Lipase Test o Lysine Iron Agar Slants Test o Litmus Milk Test o Methyl Red Test o Motility Test o MUG Test o ONPG Test o Phenol Red Fermentation Test o Phenylalanine Agar Test o Pyruvate Broth Test o Sulphur Reduction Test o Triple Sugar Iron o Urease Test o Voges-Proskauer Test INTRODUCTION BIOCHEMICAL TESTS • Biochemical tests are among the most important methods for microbial identification. Routine biochemical tests include tests for carbohydrate fermentation (Figure 2.18(A)), methyl red (Figure 2.18(B)), citric acid utilization (Figure 2.18(C)), and hydrogen sulfide production (Figure 2.18(D)). • (A) Carbohydrate fermentation test. (B) Methyl red test. (C) Citric acid utilization test. (D) Hydrogen sulfide production test. (E) Gram-positive anaerobic cocci series II (Streptococcus series) (Streptococcus mutans). (F) Gram-negative anaerobic nonspore bacillus series II (black pigment produced) (Porphyromonas gingivalis). • Microbial biochemistry tests shorten the time required to identifymicrobes , reduce costs, and ensure or enhance the accuracy of identification of an unknown sample. It is the fastest developing trend in microbial identification. In recent years, the rapid commercial test kits foranaerbic bacteria have become available. DIFFERENT BIOCHEMICAL TESTS ACETATE UTILIZATION TEST • Acetate utilization test is employed in the qualitative procedures to differentiate species of Shigella from Escherichia coli and non-fermentative gram negative bacteria. • Objective o To differentiate organisms based on ability to use acetate as the sole source of carbon. • Uses o Generally used to differentiate Shigella spp. from Escherichia coli. PRINCIPLE • This test is used to differentiate an organism capable of using acetate as the sole source of carbon. Organisms capable of using sodium acetate grow on the medium, resulting in an alkaline pH, turning the indicator from green to blue. • Organic acids have been used widely as an aid in the differentiation of Enterobacteriaceae, usually in formulae that contained organic nitrogen sources. Most bacteria, however, can use citrate and acetate in the presence of organic nitrogen. The differentiation of groups is based on the ability or failure of the test culture to utilize acetate in a medium devoid of trace organic nitrogen but sodium acetate as the sole source of carbon. Most Shigella and Proteus species are unable to utilize acetate and therefore fail to grow. Majority of Escherichia coli and closely related organisms grow well within 24-48. • Acetate utilization is indicated by formation of blue colour, which is due to the utilization of sodium acetate and subsequent formation of an alkaline reaction detected by the presence of bromothymol blue indicator. PROCEDURE 1. Emulsify a portion of the colony from a pure, 18-24 hour culture in 1.0ml of sterile physiological saline (0.85%). 2. With a straight inoculating needle, inoculate acetate slant lightly from an 18- to 24-hour culture. Do not inoculate from a broth culture but rather from a plate. 3. Incubate at 35°-37°C for up to 7 days. EXPECTED RESULTS • Positive : Medium becomes alkalinized (blue) as a result of the growth and use of acetate. • Negative : No growth or growth with no indicator change to blue.
AMINO ACID DECARBOXYLASE TEST • Amino acids are metabolized variably by gram negative aerobic and facultatively anaerobic bacteria as well as gram positive cocci. These amino acids are decarboxylated, hydrolysed or deaminated depending on the organism and the amino acid in question. In decarboxylation, the enzymes break the bond holding the carboxylic (-COOH) group to the rest of the amino acid. There are three decarboxylase enzymes that is routinely tested for – arginine decarboxylase, ornithine decarboxylase, and lysine decarboxylase. The production of lysine, arginine, ornithine decarboxylase by various members of Enterobacteriaceae offers an important parameter to other biochemical tests for differentiating bacteria within closely related groups. • Objective o To differentiate decarboxylase producing Enterobacteriaceae from other gram negative rods. PRINCIPLE • Decarboxylases are a group of substrate specific enzymes that are capable of reacting with the carboxyl (COOH) portion of amino acids, forming alkaline-reacting amines and byproduct carbon dioxide. Some microorganisms possess such an enzyme which allows their detection. The test thus measures the enzymatic ability (decarboxylase) of an organism to decarboxylate (or hydrolyze) an amino acid to form an amine. Decarboxylation, or hydrolysis, of the amino acid results in an alkaline pH. The increased pH of the medium is detected by color change of the pH indicators bromcresol purple and cresol red present which results in a color change from orange to purple. PROCEDURE • Glucose-Nonfermenting Organisms 1. Prepare a suspension (≥McFarland No. 5 turbidity standard) in brain-heart infusion broth from an overnight culture (18 to 24 hours old) growing on 5% sheep blood agar. 2. Inoculate each of the three decarboxylase broths (arginine, lysine, and ornithine) and the control broth (no amino acid) with 4 drops of broth. 3. Add a 4-mm layer of sterile mineral oil to each tube. 4. Incubate the cultures at 35°-37°C in ambient air. 5. Examine the tubes at 24, 48, 72, and 96 hours. • Glucose-Fermenting Organisms 1. Inoculate tubes with 1 drop of an 18- to 24-hour brain- heart infusion broth culture. 2. Add a 4-mm layer of sterile mineral oil to each tube. 3. Incubate cultures for 4 days at 35°-37°C in ambient air. 4. Examine the tubes at 24, 48, 72, and 96 hours. EXPECTED RESULTS • Positive : Alkaline (purple) color change compared with the control tube • Negative : No color change or acid (yellow) color in test and control tube. Growth in the control tube. USES • Arginine decarboxylase test aids in differentiating enteric bacteria with closely related physiological characteristics. • Lysine decarboxylase test assist in the identification of Salmonellae (+ve) and Shigellae (-ve). CASEIN HYDROLYSIS TEST • Casein, the major milk protein, is a macromolecule composed of amino acid subunits linked together by peptide bonds (CO —NH). It makes around 85% of the protein found in milk as well as the white color of milk. Casein is way too large to enter the cell membrane. Before their assimilation into the cell, proteins must undergo step-by-step degradation into peptones, polypeptides, dipeptides, and ultimately into their building blocks, amino acids. These are mediated by extracellular enzymes called proteases. The function of these proteases is to cleave the peptide bond CO –NH by introducing water into the molecule. The reaction then liberates the amino acids which are low-molecular in weight which can be transported through the cell membrane for use in the synthesis of structural and functional cellular proteins. • Objectives o To determine the ability of the organism to degrade the casein protein. o To differentiate the organism on the basis of production of exoenzyme proteinase (caseinase) PRINCIPLE • Some microorganisms have the ability to degrade the casein protein by producing proteolytic exoenzyme, called proteinase (caseinase). For demonstration of such an activity, in the lab, milk agar is used. The medium is composed of nutrient agar supplemented with milk that contains the protein substrate casein. Similar to other proteins, milk protein is a colloidal suspension that gives medium its color and opacity because it deflects light rays rather than transmitting them. • Following inoculation and incubation of the agar plate cultures, organisms secreting proteases will exhibit a zone of proteolysis, which is demonstrated by a clear area surrounding the bacterial growth. This loss of opacity is the result of a hydrolytic reaction yielding soluble, non-colloidal amino acids, and it represents a positive reaction. • In absence of protease activity, the medium surrounding growth of organism remains opaque, w/c is a negative reaction. PROCEDURE 1. Inoculate organism on plate either a straight line or a zig-zag. 2. Incubate the plate at 25°C or 37°C. 3. Examine milk agar plate cultures for the presence or absence of a clear area, or zone of proteolysis, surrounding the growth of each of the bacterial test organisms. RESULT INTERPRETATION • Positive Test : Clearing is observed around and/or beneath colony growth (hydrolysis). • Negative Test : No clearing is observed around and/or beneath the inoculum.
USES • Helpful in identifying bacteria that grow in milk • Differentiating among Enterobacteriaceae, Bacillaceae, and several other families. • For the differentiation of aerobic actinomycetes based on casein proteolysis. • Identification of organisms that can hydrolyse casein, such as Streptomyces, Pseudomonas , and Actinomadura CITRATE UTILIZATION TEST • This test is among a suite of IMViC Tests (Indole, Methyl-Red, Vogues-Proskauer, and Citrate) that are used to differentiate among the Gram-Negative bacilli in the family Enterobacteriaceae. PRINCIPLE • Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. The medium contains citrate as the sole carbon source and inorganic ammonium salts (NH4H2PO4) as the sole source of nitrogen. • Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of converting citrate to pyruvate. Pyruvate can then enter the organism’s metabolic cycle for the production of energy. Growth is indicative of utilization of citrate, an intermediate metabolite in the Krebs cycle. • When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to blue above pH 7.6. • Christensen developed an alternative citrate test medium that does not require the organism to use citrate as a sole carbon source. Christensen’s medium contains both peptone and cysteine. Thus citrate-negative bacteria can also grow on this medium. A positive reaction shows that the organism can use citrate but not necessarily as the sole carbon source. PROCEDURE 1. Streak the slant back and forth with a light inoculum picked from the center of a well-isolated colony. 2. Incubate aerobically at 35 to 37C for up to 4-7 days. 3. Observe a color change from green to blue along the slant. RESULT INTERPRETATION • Positive Reaction : Growth with color change from green to intense blue along the slant. o Examples : Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter, Serratia, Providencia, etc. • Negative Reaction : No growth and No color change; Slant remains green. o Examples : Escherichia, Shigella, Morganella, Yersinia etc. QUALITY CONTROL • Citrate Positive : Klebsiella pneumoniae ATCC 13883 (growth; blue color) • Citrate Negative : Escherichia coli ATCC 25922 (no growth or trace of growth) GELATIN HYDROLYSIS TEST • Gelatin is a protein derived from the connective tissues of vertebrates, that is, collagen. It is produced when collagen is boiled in water. Gelatin hydrolysis detects the presence of gelatinases. Gelatinases are proteases secreted extracellularly by some bacteria which hydrolyze or digest gelatin. The production of gelatinases is used as a presumptive test for the identification of various organisms, including Staphylococcus sp., Enterobacteriaceae, and some gram-positive bacilli. PRINCIPLE • This test is used to determine the ability of an organism to produce extracellular proteolytic enzymes (gelatinases) that liquefy gelatin, a component of vertebrate connective tissue. • The reaction occurs in two sequential steps: in first reaction gelatinases hydrolyze gelatin into polypeptides and then polypeptides are further converted into amino acids. The amino acid is taken up by the cell and used for metabolic purposes. • The presence of gelatinases is detected using a nutrient gelatin medium. When an organism produces gelatinase, the enzyme liquefies the growth medium by hydrolyzing gelatin present in the medium. PROCEDURE • There are several methods for determining gelatinase production, all of which make use of gelatin as the substrate. The standard and most commonly employed method is the nutrient gelatin stab method. 1. Inoculate the gelatin deep with 4 to 5 drops of a 24-hour broth culture. 2. Incubate at 35°-37°C in ambient air for up to 14 days.
• Note: Incubate the medium at 25°C if the organism grows better at 25°C than at 35°C. 3. Alternatively, inoculate the gelatin deep from a 24-hour-old colony by stabbing 4-5 times, 0.5 inch into the medium. 4. Remove the gelatin tube daily from the incubator and place at 4°C to check for liquefaction. • Note: Do not invert or tip the tube, because sometimes the only discernible liquefaction occurs at the top of the deep where inoculation occurred. 5. Refrigerate an un-inoculated control along with the inoculated tube. Liquefaction is determined only after the control has hardened (gelled). • Nutrient gelatin plate method 1. Stab-inoculate a heavy inoculum of an 18- to 24-hour- old test bacteria onto culture plates prefilled with nutrient gelatin (23 g/liter nutrient agar, 8 g/liter gelatin). 2. Incubate inoculated nutrient gelatin plates at 35oC for 24 hours. o Note: In some cases, plates are flooded with saturated ammonium sulfate to precipitate unhydrolyzed gelatin, making the clear zones easier to see. Results are often observed within 5 to 10 minutes after flooding with saturated ammonium sulfate. EXPECTED RESULTS • Positive : Partial or total liquefaction of the inoculated tube (the control tube must be completely solidified) at 4°C within 14 days. On plates, gelatin hydrolysis is indicated by clear zones around gelatinase-positive colonies. • Negative : Complete solidification of the tube at 4°C. On plates, no clear zones around colonies are observed. Gelatin hydrolysis. A, Positive; note liquefaction at top of tube. B, Uninoculated tube . Gelatin hydrolysis. A, Positive B, Negative USES • This test is used to determine the ability of an organism that produce gelatinases. • This test is helpful in identifying and differentiating species of Serratia, Proteus, Bacillus, Clostridium, Pseudomonas and Flavobacterium. • This test differentiates pathogenic Staphylococcus aureus which is gelatinase-positive from non-pathogenic epidermidis which is gelatinase negative. • This test can be used to differentiate Serratia and Proteus species which are gelatin positive from other members of Enterobacteriaceae family. • Bacillus anthracis, B. cereus and several other members of the genus are gelatinase-positive, as are Clostridium tetani and perfringens. HYDROGEN SULFIDE TEST • Some microorganisms have an ability to reduce sulfur (Sulphur) containing compounds to hydrogen sulfide during metabolism which is commonly employed as a test measure for their identification in laboratories. Numerous methods are used to detect H2S production by micro-organisms which vary with the source of sulfur and the metal salts used to indicate H2S formation. SIM is more sensitive in the detection of H2S than either TSI or KIA, because of its semisolid nature, its lack of interfering carbohydrates, and the use of peptonized iron as an indicator. However, Lead acetate paper is 10 times more sensitive than other media. • Objectives o To determine whether the microbe reduces sulfur-containing compounds to sulfides to produce hydrogen sulfide gas. PRINCIPLE • An iron compound and a sulfur compound are included in the test medium to test for the production of hydrogen sulfide gas. Hydrogen sulfide is produced if the sulfur compound is reduced by the bacterial strain. This test thus determines whether the microbe reduces sulfur-containing compounds to sulfides during the process of metabolism. H2S is produced by certain bacteria through reduction of sulphur containing amino acids like cystine, methionine or through the reduction of inorganic sulphur compounds such as thiosulfates, sulfates or sulfites during protein degradation or when anaerobic respiration shuttles the electrons to sulfur instead of to oxygen. In either case H2S is produced (hydrogen sulfide gas) which reacts with the iron compound to form the black precipitate of ferric sulfide. The black color acts as an indicator for the presence of hydrogen sulfide. The detection of hydrogen sulphide (H2S) gas produced by an organism. is used mainly to assist in the identification of that particular organism. PROCEDURE • In Sulphite indole motility (SIM) medium 1. Inoculate the organism into labeled tube by means of stab inoculation. 2. Incubate the inoculated tubes at 37°C for 24-48 hours. 3. Observe for the formation of black precipitate on the medium.
• In Kligler iron agar (KIA) and Triple Sugar Iron Agar (TSIA) 1. Inoculate the test organism into KIA and incubate it at appropriate temperature over night. 2. Observe for blackening of the medium. • Lead acetate paper test 1. Inoculate a tube or bottle of sterile peptone water or nutrient broth with the test organism. 2. Insert a lead acetate paper strip in the neck of the bottle or tube above the medium, and stopper well. 3. Incubate the inoculated medium at 35-37oC, and examine daily for a blackening of the lower part of strip. RESULTS • Positive result : Blackening on the medium • Negative result : No blackening on the medium USES • It is used mainly to assist in the identification of members of family Enterobacteriaceae and occasionally to differentiate other bacteria such as Bacteroidessps and Brucella sps. • The test aids in the identification and differentiation of members of Enterobacteriaceae (enterics) from other Gram- bacilli. • It is especially helpful in identifying Salmonella, Francisella, and Proteus species. INDOLE TEST • This test demonstrate the ability of certain bacteria to decompose the amino acid tryptophane to indole, which accumulates in the medium. Indole production test is important in the identification of Enterobacteria. Most strains of E. coli, P. vulgaris, P. rettgeri, M. morgani and Providencia species break down the amino acid tryptophan with the release of indole. This is performed by a chain of a number of different intracellular enzymes, a system generally referred to as “tryptophanase.” It is used as part of the IMViC procedures,a tests designed to distinguish among members of the family Enterobacteriaceae. • A variation on this test using Ehrlich’s reagent (using ethyl alcohol in place of isoamyl alcohol, developed by Paul Ehrlich) is used when performing the test on non-fermenters and anaerobes. PRINCIPLE • Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that express tryptophanase enzyme. Indole is generated by reductive deamination from tryptophan via the intermediate molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine (-NH2) group of the tryptophan molecule is removed. Final products of the reaction are indole, pyruvic acid, ammonium (NH4+) and energy. Pyridoxal phosphate is required as a coenzyme. • When indole is combined with Kovac’s Reagent (which contains hydrochloric acid and p-dimethylaminobenzaldehyde in amyl alcohol) the solution turns from yellow to cherry red. Because amyl alcohol is not water soluble, the red coloration will form in an oily layer at the top of the broth. • In the spot test, indole combines, in the filter paper matrix, at an acid pH with p-Dimethylaminocinnamaldehyde (DMACA) to produce a blue to blue-green compound. Indole Spot Reagent has been reported to be useful in detecting indole production by members of the family Enterobacteriaceae and certain anaerobic species. PROCEDURE 1. Take a sterilized test tubes containing 4 ml of tryptophan broth. 2. Inoculate the tube aseptically by taking the growth from 18 to 24 hrs culture. 3. Incubate the tube at 37°C for 24-28 hours. 4. Add 0.5 ml of Kovac’s reagent to the broth culture. 5. Observe for the presence or absence of ring. RESULT INTERPRETATION • Positive : For mation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the medium within seconds of adding the reagent. o Examples : Aeromonas hydrophila, Aeromonas punctata, Bacillus alvei,Edwardsiella sp., Escherichia coli, Flavobacterium sp., Haemophilus influenzae, Klebsiella oxytoca, Proteus sp. (not P. mirabilis and P. penneri), Plesiomonas shigelloides,Pasteurella multocida, Pasteurella pneumotropica, Enterococcus faecalis, and Vibrio sp. • Negative : No color change even after the addition of appropriate reagent. o Examples : Actinobacillus spp., Aeromonas salmonicida, Alcaligenes sp., most Bacillus sp., Bordetella sp., Enterobacter sp., Lactobacillus spp., most Haemophilus sp., most Klebsiella sp., Neisseria sp., Pasteurella haemolytica, Pasteurella ureae, Proteus mirabilis, P. penneri, Pseudomonas sp.,Salmonella sp., Serratia sp., Yersinia sp. • Indole Spot Reagent Result o Positive reaction : The development of a blue colour within 3 minutes. o Negative reaction : The development of a pink colour within 3 minutes.
SPOT INDOLE TEST • Method 1. Place several drops of 1% p- dimethylaminocinnamaldehyde reagent on a piece of filter paper until saturation. 2. With an inoculating loop or wooden applicator stick, pick a portion of an 18-24 hour isolated colony from a non-selective media and rub it onto the reagent saturated area of the filter paper. 3. Observe for colour development within 1 to 3 minutes. • Expected Results o Positive : A positive reaction is denoted by the appearance of a blue to blue-green color change on the bacterial smear within 2-3 minutes. o Negative : Negative reactions remain colorless or light pink. o Note: Positive reaction is Red-violet in the case of Providencia alcalifaciens. USES • To differentiate Proteus mirabilis (indole negative) from all other Proteus species (indole positive). • To differentiate Klebssiella pneumoniae (indole negative) from Klebsiella oxytoca (indole positive). • To differentiate Citrobacter freundii (indole negative) from Citrobacter koseri (indole positive). QUALITY CONTROL • Positive Control : Escherichia coli NCTC 10418 • Negative Control : Proteus mirabilis NCTC 10975 KLIGLER'S IRON AGAR TEST • The Kligler’s Iron Agar test employs a medium for the identification of Enterobacteriaceae, based on double sugar fermentation and hydrogen sulphide production. In 1918, Kligler described a medium for detection of H2S and differentiation of Salmonella spp. Bailey and Lacey further modified the medium by substituting phenol red indicator for Andrade indicator. This medium became known as KIA. It is recommended for determination of H2S production by enteric gram-negative bacilli and for detection of H2S produced by some strains of Pseudomonas. • Objective o To differentiate organisms by demonstrating hydrogen sulfide production and fermentation of dextrose and lactose. PRINCIPLE • Kligler Iron Agar, in addition to peptone, HM peptone B and yeast extract, contains lactose and glucose (dextrose), which enables the differentiation of species of enteric bacilli. Phenol red is the pH indicator, which exhibits a colour change in response to acid produced during the fermentation of sugars. • Fermentation of dextrose results in production of acid, which turns the indicator from red to yellow. Since there is little sugar i.e. dextrose, acid production is very limited and therefore a reoxidation of the indicator is produced on the surface of the medium, and the indicator remains red. However, when lactose is fermented, the large amount of acid produced, avoids reoxidation and therefore the entire medium turns yellow. • The combination of ferrous sulphate and sodium thiosulphate enables detection of hydrogen sulfide production, which is evidenced by a black color either throughout the butt, or in a ring formation near top of butt. • Lactose non-fermenters (e.g., Salmonella and Shigella ) initially produce a yellow slant due to acid produced by the fermentation of the small amount of glucose (dextrose). When glucose (dextrose) supply is exhausted in the aerobic environment of the slant, the reaction reverts to alkaline (red slant) due to oxidation of the acids produced. The reversion does not occur in the anaerobic environment of the butt, which therefore remains acidic (yellow butt). • Lactose fermenters produce yellow slants and butts because of lactose fermentation. The high amount of acids thus produced helps to maintain an acidic pH under aerobic conditions. Tubes showing original colour of the medium indicates the fermentation of neither glucose (dextrose) nor lactose. • Gas production (aerogenic reaction) is detected as individual bubbles or by splitting or displacement of the agar by the formation of cracks in the butt of the medium. PROCEDURE 1. With an inoculating needle, pick the center of well-isolated colonies obtained from solid culture media. The medium is recommended for the identification of colonies picked off from plating media such as MacConkey Agar, Bismuth Sulphite Agar, or Desoxycholate Citrate Agar, etc. 2. Stab the center of the medium into the deep of the tube to within 3-5mm from the bottom. 3. Withdraw the inoculating needle and streak the surface of the slant. 4. Loosen closure on the tube before incubating. 5. Incubate aerobically at 35ºC. for 18-48 hours. 6. Read tubes for acid production of the slant/butt, gas, and hydrogen sulfide reactions. EXPECTED RESULTS • Carbohydrate Fermentation : o Positive Test for Slant Reaction – Yellow (acid) o Negative Test for Slant Reaction – Red (alkaline) o Positive Test for Butt Reaction – Yellow (acid) o Negative Test for Butt Reaction – Red (alkaline) • KIA Color Reactions : o Red slant/ yellow butt – dextrose (+), lactose (-) o Yellow slant/ yellow butt – dextrose (+), lactose (+) o Red slant/ red butt – dextrose (-), lactose (-)
• Hydrogen Sulfide Production : o Positive Test – Black color throughout medium, a black ring at the juncture of the slant and butt, or a black precipitate in the butt o Negative Test – No black color development • Gas Production : o Positive Test – Bubbles in the medium, cracking and displacement of the medium, or separation of the medium from the side and bottom of the tube o Negative Test – No bubbles and no separation or displacement of the medium. USES • The test is recommended for the differential identification of gram-negative enteric bacilli from clinical and non clinical samples on the basis of the fermentation of dextrose, lactose and H2S production. • It is used as a differentiation medium for typhoid, dysentery and allied bacilli. • It differentiates Salmonella Typhi from other Salmonellae and also Salmonella Paratyphi A from Salmonella Scottmuelleri and Salmonella Enteritidis. • Kligler Iron Agar test differentiates lactose fermenters from the nonfermenters. LIPASE TEST • Lipid is the general term used to describe all types of fats. Fats are formed by ester linkage between three molecules of fatty acids and one molecule of glycerol. Simple fats are known as triglycerides or triacylglycerols. Triglycerides are composed of glycerol and three long chain fatty acids. Enzymes that break simple fat into its component fatty acid and glycerol are known as lipase. Some microorganisms possess the enzyme lipase. Thus they can be classified based on their ability to produce and secrete lipases. A variety of simple fats can be used for this determination, depending on the type of organism being tested. • Objectives o To determine the ability of microorganisms to produce the enzyme lipase. o To identify the bacteria on the basis of its lipase activity PRINCIPLE • Egg Yolk Agar is a differential and enriched medium used in the isolation and presumptive differentiation of different species based on their lipase activity among others. The non-selective medium supplemented with a suspension of egg yolk, supplies lecithin and free fats, substrates needed to detect lecithinase and lipase production and proteolytic activity. Microorganisms that possess the enzyme lipase hydrolyze the free fats present in the medium to form glycerol and free fatty acids. Consequently, the release of insoluble free fatty acids results in the formation of an iridescent sheen (oil on water) that can be seen when the plate is held at an angle to a light source. Since lipase is not very diffusible, it produces a reaction only on the surface of the agar in the immediate vicinity of the colony. PROCEDURE 1. Take a loopful of test organism and streak it as a straight line on the plate. 2. Incubate anaerobically in a gas pak jar immediately after streaking and transfer into the incubator maintained at 35-37oC for 24-48 hours for anaerobes and for aerobes incubate the plate at 35-37oC for 24-48 hours. 3. Examine the plate for the formation of an iridescent sheen. RESULTS • Positive test : A positive lipase test is noted by the appearance of an iridescent sheen (oil on water) immediately around colonies that can be seen when the plate is held at an angle to a light source. • Negative test : A negative lipase test is indicated by the absence of an iridescent sheen. • Clostridium sporogenes. Lipase positive; iridescent sheen on agar surface when plate is held at an angle to the light source. USES • The lipase test is used to detect and enumerate lipolytic bacteria, especially in high-fat dairy products. • A variety of other lipid substrates, including corn oil, olive oil, and soybean oil, are used to detect differential characteristics among members of Enterobacteriaceae, Clostridium, Staphylococcus, and Neisseria. • Several fungal species also demonstrate lipolytic ability. LYSINE IRON AGAR (LIA) SLANTS TEST • Lysine iron agar (LIA) slants tests organisms for the ability to deaminate lysine or decarboxylate lysine. Lysine deamination is an aerobic process which occurs on the slant of the media. Lysine decarboxylation is an anaerobic process which occurs in the butt of the media. PRINCIPLE • Lysine iron agar contains lysine, peptones, a small amount of glucose, ferric ammonium citrate, and sodium thiosulfate. The medium has an aerobic slant and an anaerobic butt. When glucose is fermented, the butt of the medium becomes acidic (yellow). If the organism produces lysine decarboxylase, cadaverine is formed. Cadaverine neutralizes the organic acids formed by glucose fermentation, and the butt of the medium reverts to the alkaline state (purple). If the decarboxylase is not produced, the butt remains acidic (yellow). If oxidative deamination of lysine occurs, a compound is formed that, in the presence of ferric ammonium citrate and a coenzyme, flavin mononucleotide, forms a burgundy color on the slant. If deamination does not occur, the LIA slant remains purple. Bromocresol purple, the pH indicator, is yellow at or below pH 5.2 and purple at or above pH 6.8.
PROCEDURE 1. The medium is tubed, sterilised and slanted so that a short slant and deep butt are formed. 2. With a straight inoculating needle, inoculate LIA by twice stabbing through the center of the medium to the bottom of the tube and then streaking the slant. 3. Cap the tube tightly and incubate at 35°-37°C in ambient air for 18 to 24 hours. 4. Examine at 18 – 24 and 40 – 48 hours for growth and color changes in tube butt and slant, and for blackening at the apex of slant. EXPECTED RESULTS • Lysine iron agar. A, Alkaline slant/alkaline butt (K/K). B, Alkaline slant/alkaline butt, H2S positive (K/K H2S+). C, Alkaline slant/acid butt (K/A). D, Red slant/acid butt (R/A). E, Uninoculated tube. • Lysine Decarboxylation (detected in butt): o Positive Test : Purple slant/purple butt (alkaline), the butt reaction may be masked by H2S production o Negative Test : Purple slant/yellow butt (acid), fermentation of glucose only • Lysine Deamina tion (detected on slant): o Positive Test : Red slant o Negative Test : Slant remains purple • H2S Production : o Positive Test : Black precipitate o Negative Test : No black color development o Gas production : demonstrated by the presence of bubbles or cracks in the medium USES • This test is used to differentiate gram-negative bacilli based on decarboxylation or deamination of lysine and the formation of hydrogen sulfide (H2S). • It employs a sensitive medium for the detection of lactose-fermenting and non lactose-fermenting salmonellae. • Lysine Iron Agar is specified in standard methods for Salmonella testing. LITMUS MILK TEST • Milk is an excellent medium for the growth of microorganisms because it contains the milk protein casein, the sugar lactose, vitamins, minerals and water. Litmus milk is a milk-based medium used to distinguish between different species of bacteria. The lactose (milk sugar), litmus (pH indicator), and casein (milk protein) contained within the medium can all be metabolized by different types of bacteria. The test differentiates microorganisms based on various metabolic reactions in litmus milk, including reduction, fermentation, clot formation, digestion, and the formation of gas. • Objectives o To determine an organism’s ability to metabolize litmus milk. o To differentiate among microorganisms that enzymatically transforms different milk substrates into varied metabolic end products. PRINCIPLE • The major milk substrates capable of transformation are the milk sugar lactose and the milk proteins casein, lactalbumin, and lactoglobulin. To distinguish among the metabolic changes produced in milk, a pH indicator, the oxidation reduction indicator litmus, is incorporated into the medium. Litmus milk then forms an excellent differential medium in which microorganisms can metabolize milk substrates depending on their enzymatic complement. A variety of different biochemical changes result. • Fermentation of lactose is demonstrated when the litmus turns pink as a result of acid production. If sufficient acid is produced, casein in the milk is coagulated, solidifying the milk. With some organisms, the curd shrinks and whey is formed at the surface. Some bacteria hydrolyze casein, causing the milk to become straw colored and resemble turbid serum. Additionally, some organisms reduce litmus, in which case the medium becomes colorless in the bottom of the tube. PROCEDURE 1. Inoculate with 4 drops of a 24-hour broth culture. 2. Incubate at 35°-37°C in ambient air. 3. Observe daily for seven days for alkaline reaction (litmus turns blue), indicator reduction, acid clot, acid reaction (litmus turns pink), rennet clot, and peptonization. 4. Record all changes. EXPECTED RESULTS • Multiple changes can occur over the observation period. • Positive test o Acid pH : pink to red color o Alkaline pH (K): purplish- blue color o Reduction : white o Acid curd : hard curd with clear supernatant (whey) o Digestion : Dissolution of clot with clear, grayish, watery fluid and a shrunken, insoluble pink clot o Rennet curd : soft curd followed by peptonization (alkaline pH, supernatant brown) o Gas production : bubbles in coagulated milk • Negative test : o Color and consistency remain same.
USES • The litmus milk test differentiates members of the Enterobacteriacaeae from other gram-negative bacilli based on the enterics’ ability to reduce litmus. • It is commonly used to differentiate members within the genus Clostridium. • It mainly aids in the identification and differentiation of Enterococcus, and Lactic acid bacteria. The media may also be used to grow lactic acid bacteria. METHYL RED (MR) TEST • The methyl red (MR) test detects the production of sufficient acid during the fermentation of glucose and the maintenance of conditions such that the pH of an old culture is sustained below a value of about 4.5, as shown by a change in the colour of the methyl red indicator which is added at the end of the period of incubation. • Clark and Lubs developed MR-VP Broth which allowed both the MR and VP tests to be performed from the same inoculated medium by aliquoting portions to different tubes. PRINCIPLE • Some bacteria have the ability to utilize glucose and convert it to a stable acid like lactic acid, acetic acid or formic acid as the end product. • These bacteria initially metabolise glucose to pyruvic acid, which is further metabolized through the ‘mixed acid pathway to produce the stable acid. The type of acid produced differs from species to species and depends on the specific enzymatic pathways present in the bacteria. The acid so produced decreases the pH to 4.5 or below, which is indicated by a change in the colour of methyl red from yellow to red. • In the methyl red test (MR test), the test bacteria is grown in a broth medium containing glucose. If the bacteria has the ability to utilise glucose with production of a stable acid, the colour of the methyl red changes from yellow to red, when added into the broth culture. • The mixed acid pathway gives 4 mol of acidic products (mainly lactic and acetic acid), 1 mol of neutral fermentation product (ethanol), 1 mol of CO2, and 1 mol of H2 per mol of glucose fermented. The large quantity of acids produced causes a significant decrease in the pH of culture medium. PROCEDURE 1. Prior to inoculation, allow medium to equilibrate to room temp. 2. Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium. 3. Incubate aerobically at 37 degrees C. for 24 hours. 4. Following 24 hours of incubation, aliquot 1ml of the broth to a clean test tube. 5. Reincubate the remaining broth for an additional 24 hours. 6. Add 2 to 3 drops of methyl red indicator to aliquot. 7. Observe for red color immediately. RESULT INTERPRETATION • Positive Reaction : A distinct red color (A) o Examples : E. coli, Yersinia sps, etc. • Negative Reaction : A yellow color (B) o Examples : Enterobacter aerogenes, Klebsiella pneumoniae, etc. • A weak positive is red-orange. If an orange color is seen, incubate the remainder of the broth for up to 4 days and repeat the test after further incubation. In this case it may also be helpful to set up a duplicate broth at 25C. USES • Originally the paired MR-VP tests were used to distinguish between members of the family Enterobacteriaceae, but now they are used to characterize other groups of bacteria including Actinobacteria. QUALITY CONTROL • Klebsiella pneumoniae ATCC 13883 —MR negative (yellow) • Escherichia coli ATCC 25922 —MR positive (red) MOTILITY TEST • Motility is the ability of an organism to move by itself by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility. Motile bacteria move using flagella, thread like locomotor appendages extending outward from the plasma membrane and cell wall either single flagellum or multiple flagella. Motility has long been recognized as an important taxonomic tool and biological characteristic of microorganisms. The presence of flagella occurs primarily in bacilli but there are a few flagellated cocci, thus motility is a very important means of identification in the family Enterobacteriaceae. From the early days in the field of microbiology, the ability of bacteria to move has been used as a means of differentiation & classification of organisms. • Objective o To determine the motility of bacterium. o To differentiate between motile & non-motile bacteria. PRINCIPLE • Motility by bacterium is mostly demonstrated in a semi solid agar medium. In semi-solid agar media, motile bacteria ‘swarm’ and give a diffuse spreading growth that is easily recognized by the naked eye.The medium mainly used for this purpose is SIM medium (Sulphide Indole Motility medium) which is a combination differential medium that tests three different parameters, Sulfur Reduction, Indole Production and Motility. This media has a very soft consistency that allows motile bacteria to migrate readily through them causing cloudiness. The inoculum is stabbed into the center of a semisolid agar deep. Bacterial motility is evident by a diffuse zone of growth extending out from the line of inoculation. Some organisms grow throughout the entire medium, whereas others show small areas or nodules that grow out from the line of inoculation. The non-motile bacteria will only grow in the soft agar tube and only the area where they are inoculated.
PROCEDURE 1. Touch a straight needle to a colony of a young (18- to 24- hour) culture growing on agar medium. 2. Stab once to a depth of only 1/3 to ½ inch in the middle of the tube. Be sure to keep the needle in the same line it entered as it is removed from the medium. 3. Incubate at 35°-37°C and examine daily for up to 7 days. 4. Observe for a diffuse zone of growth flaring out from the line of inoculation. EXPECTED RESULTS • Positive : Diffuse, hazy growths that spread throughout the medium rendering it slightly opaque. • Negative : Growth that is confined to the stab-line, with sharply defined margins and leaving the surrounding medium clearly transparent. USES • It is used for the differentiation of microorganisms on the basis of motility in a laboratory setting. • Performed to assign taxonomic classification to organisms. • Motility tests are important in characterization of pathogens. • The tests are often employed in identification protocols in the family Enterobacteriaceae • Motility test is also used for the species differentiation of gram positive cocci, Enterococci. Enterococcus faecium and E. faecalis are non-motile, whereas E. gallinarum and E. casseliflavus/E. flavescens generally are motile. MUG TEST • It has been reported that the enzyme β-glucuronidase is present in most strains of E. coli (97%).Organisms other than E. coli (e.g., Salmonella, Shigella, Staphylococcus, Streptococcus, etc.) also possess the enzyme β-glucuronidase. Hence, the detection of the β-glucuronidase enzyme is commonly employed in laboratories to identify and differentiate such organisms. • The substrate, 4-methylumbelliferyl- β-D-glucuronide (MUG), is both sensitive and selective for detection of β-glucuronidase activity. Hence, MUG test in conjunction with oxidase, indole, and lactose fermentation can be performed to effectively identify E. coli & related organisms. • Objective o To identify glucuronidase activity in organisms by fluorescence, under a long-wave (365nm) UV light source. PRINCIPLE • E. coli and other Enterobacteriaceae produce the enzyme β-d-glucuronidase, which hydrolyzes β-d-glucopyranosid-uronic derivatives to aglycons and d-glucuronic acid. The substrate 4-methylumbelliferyl- β-d-glucuronide is impregnated into disk and is hydrolyzed by enzyme to yield 4-methylumbelliferyl moiety, which fluoresces blue under long wavelength ultraviolet light. Hence, if test organism produces enzyme glucuronidase it will break down the substrate which will ultimately result in a fluorescence indicating a positive test. Incase of absence of desired enzyme activity, the substrate is not broken down resulting in no fluorescence on test, which is a negative test. PROCEDURE 1. Wet the disk with 1 drop of water. Do no saturate. 2. Using a wooden applicator stick, rub a portion of a colony from an 18- to 24-hour-old pure culture onto the disk. 3. Incubate at 35°-37°C in a closed container for up to 2 hours. 4. Observe disk using a 366-nm ultraviolet light. EXPECTED RESULTS • Positive : Electric blue fluorescence • Negative : Lack of fluorescence MUG test. A, Positive. B, Negative USES • The test is used to presumptively identify various genera of Enterobacteriaceae. • To characterize verotoxin-producing Escherichia coli. (verotoxinproducing strains of E. coli do not produce MUG, and a negative test result may indicate the presence of a clinically important strain.) • It aids in the detection of Escherichia colifrom water and food samples. ONPG TEST • The ability of bacteria to ferment lactose depends on two enzymes, permease and beta-galactosidase. Permease allows lactose to enter the bacterial cell wall, where it is then broken down into glucose and galactose by beta-galactosidase. Glucose and galactose can then be metabolized by the bacteria. Some organisms lack permease and appear as late or non-lactose-fermenters. • The ONPG test is considered to be a very sensitive test for lactose-fermentation. O-nitrophenyl-beta-D-galactopyranoside (ONPG), an artificial substrate, is incorporated into this test and acts as the substrate for the beta-galactosidase to ascertain the particular enzyme activity which subsequently aids in the identification and differentiation of different organisms. • Objective o To determine the ability of an organism to produce β-galactosidase enzyme. PRINCIPLE • O-nitrophenyl-beta-D-galactopyranoside (ONPG) is an artificial substrate structurally similar to lactose with the exception that glucose is substituted with an o-nitrophenyl group. Unlike lactose, the substrate O-nitrophenyl-beta-D-galactopyranoside (ONPG) is capable of penetrating the bacterial cell without the presence of permease. • In the broth method of testing, the organism is taken from a medium containing a high concentration of lactose and is inoculated into the ONPG Broth. If the organism possesses beta-galactosidase, the enzyme will split the beta-galactoside bond, releasing o-nitrophenol which is a yellow-colored compound. This indicates a positive test.
• In the disk method, the organism to be tested is taken from a medium containing a high concentration of lactose. A dense suspension (turbidity equivalent to a McFarland 3) is prepared. An ONPG disk is added to 0.5ml of the suspension. If the organism possesses beta-galactosidase, the enzyme will split the beta-galactoside bond, creating a yellow color change in the suspension. Organisms with strong beta-galactosidase activity can produce a positive reaction a few minutes after inoculation of the ONPG medium; other organisms may take up to 24 hours. PROCEDURE • For ONPG disk method 1. Place an ONPG disk into a sterile tube and add 0.2 mL saline. 2. Heavily inoculate the tube with a loopful of the test isolate. 3. Incubate at 35-37°C for up to 4 hours. 4. Examine for color change of the disk. • For broth method 1. Bring test medium to room temperature. 2. Inoculate the test medium with heavy inoculum from a pure 18-24 hour culture. 3. Incubate aerobically, with loose caps, at 35- 37ºC. 4. Examine for a yellow color development at 1 hour. 5. If no color change seen after an hour of incubation, continue incubation for up to 24 hours. EXPECTED RESULTS • Positive : Development of a yellow colouration (presence of β-galactosidase) o Note: The fluid and disc will turn any shade of yellow if positive for galactosidase enzyme. • Negative : No colour development (absence of enzyme) OPNG test. A, Positive. B, Negative. USES • The test is used in differentiating members of the Enterobacteriaceae and other microorganisms based on beta-D-galactosidase activity. • The test distinguishes late lactose fermenters from non – lactose fermenters of Enterobacteriaceae. PHENOL RED FERMENTATION TEST • Fermentation media are used to differentiate organisms based on their ability to ferment carbohydrates incorporated into the basal medium. • Phenol Red Broth Medium with various added carbohydrates serves as a differential medium by aiding in differentiation of various species and genera by their ability to ferment the specific carbohydrate, with the production of acid or acid and gas. • The carbohydrate source can vary based on test requirements. The common broth media used are: o Phenol Red Glucose Broth o Phenol Red Lactose Broth o Phenol Red Maltose Broth o Phenol Red Mannitol Broth o Phenol Red Sucrose Broth • Objective o To determine the fermentation reactions of pure cultures of microorganisms. PRINCIPLE • Carbohydrate fermentation is the process microorganisms use to produce energy. Most microorganisms convert glucose to pyruvate during glycolysis; however, some organisms use alternate pathways. A fermentation medium consists of a basal medium containing a single carbohydrate (glucose, lactose, sucrose, mannitol etc.) for fermentation. However, the medium may contain various color indicators. In addition to a color indicator to detect the production of acid from fermentation, a Durham tube is placed in each tube to capture gas produced by metabolism. The carbohydrate fermentation patterns shown by different organisms are useful in differentiating among bacterial groups or species. • Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate (glucose, lactose, or sucrose). Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow. If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia. When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an air bubble will be trapped inside the Durham tube. If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed. PROCEDURE 1. Aseptically inoculate each test tube with the test microorganism using an inoculating needle or loop. Alternatively, inoculate each test tube with 1-2 drops of an 18- to 24-hour brain-heart infusion broth culture of the desired organism. 2. Incubate tubes at 35-37°C for 18-24 hours. 3. Check for color changes or formation of gas. RESULT INTERPRETATION • Acid production : o Positive : After incubation the liquid in the tube turns yellow (indicated by the change in the color of the phenol red indicator). It indicates that there is drop in the pH because of the production of the acid by the fermentation of the carbohydrate (sugar) present in the media. o Negative : The tube containing medium will remain red, indicating the bacteria cannot ferment that particular carbohydrate source present in the media.
• Gas Production o Positive : A bubble (small or big depending up amount of gas produced) seen in inverted Durham tube. o Negative : No bubble in the inverted Durham tube i.e. bacteria does not produce gas from the fermentation of that particular carbohydrate present in the media i.e. anaerogenic organism. USES • It is recommended to determine the fermentation reaction of carbohydrates for the differentiation of microorganisms. • It is useful in identifying Gram negative bacilli, especially Enterobacteriaceae. PHENYLALANINE AGAR TEST • Biochemical tests are the tests used for the identification of bacteria species based on the differences in the biochemical activities of different bacteria. Bacterial physiology differs from one type of organism to another. The differences in carbohydrate metabolism, protein metabolism, fat metabolism, production of certain enzymes, ability to utilize a particular compound etc. help them to be identified. • On such test is the the phenylalanine agar test which tests for the ability of some specific species to convert amino acid phenylalanine to phenylpyruvic acid; an important reaction in the differentiation of Enterobacteriaceae. Based on this criterion, the test is carried out for the differentiation of Proteus and Providencia group from other members of Enterobacteriaceae which lack such an activity. • Objective o To determine the ability of an organism to oxidatively deaminate phenylalanine to phenylpyruvic acid. PRINCIPLE • Phenylalanine agar, also known as phenylalanine deaminase medium, contains nutrients and DL-phenylalanine. The phenylalanine serves as the substrate for enzymes, which are able to deaminate it to form phenylpyruvic acid. Yeast extract in the medium supports the growth of the organisms. Sodium chloride maintains osmotic equilibrium. • Microorganisms that produce phenylalanine deaminase remove the amine (NH2) from phenylalanine. The reaction results in the production of ammonia (NH3) and phenylpyruvic acid. The phenylpyruvic acid is detected by adding a few drops of 10% ferric chloride which acts as a chelating agent ; a green colored complex is formed between these two compounds indicating a positive test. If the medium remains a straw color, the organism is negative for phenylalanine deaminase production. METHOD 1. Using a loopful of inoculum from an 18-24 hour pure culture, streak the slant surface using a fishtail motion or inoculate phenylalanine slant with 1 drop of a 24-hour brain-heart infusion broth. 2. Incubate inoculated slant aerobically at 35ºC. for 18-24 hrs. 3. Following incubation, apply 4-5 drops of a 10% Ferric Chloride solution directly to the slant. 4. Gently agitate the tube and observe for the development of a green color within 1-5 minutes. EXPECTED RESULTS • Positive : Green color develops on slant after ferric chloride is added within 1-5 minutes after applying ferric chloride reagent. • Negative : Absence of a green color reaction. Negative results will take on a yellow color due to the color of the ferric chloride. Phenylalanine deaminase. A, Positive. B, Negative. USES • It is recommended for use in the differentiation of gram-negative enteric bacilli based on the ability of the microorganisms to produce phenylpyruvic acid by oxidative deamination. The genera Morganella, Proteus, and Providencia can be differentiated from other members of the Enterobacteriaceae family. PYRUVATE BROTH TEST • Biochemical tests are the tests used for the identification of bacterial species based on the differences in the biochemical activities of different bacteria. Bacterial physiology differs from one type of organism to another. • The ability of bacteria to form organic compounds by metabolizing certain carbohydrates and related compounds is a widely used method for the identification of microorganisms. • One such test is the pyruvate broth test which tests for the ability of some specific species to utilize substrate pyruvate. • Objective o To determine the ability of an organism to utilize pyruvate to produce acidic end products. PRINCIPLE • A source of pyruvate is added to the culture broth to determine whether the microorganism is able to use pyruvate, resulting in the formation of metabolic acids. Acid is produced as a metabolic waste when pyruvate broth is inoculated with bacteria that are capable of metabolizing pyruvate.
• Acid production causes a decrease in pH which results in a color shift in the medium. Bromothymol blue is the acid-base indicator in the media. It is greenish-blue at an alkaline pH, and shifts to yellow when acid is produced during fermentation of the pyruvate. After incubation, yellow media is indicative of a positive fermentation reaction. PROCEDURE 1. Lightly inoculate the pyruvate broth with an 18- to 24-hour culture of the organism from 5% sheep blood agar. 2. Incubate at 35°-37°C in ambient air for 24 to 48 hours. 3. Examine for a yellow reaction. EXPECTED RESULTS • Positive : A positive pyruvate utilization result is indicated by a color change of the broth from greenish-blue to yellow. • Negative : A negative pyruvate utilization result is indicated by no color change and the resultant broth remaining a greenish-blue color. USES • The test aids in the differentiation between Enterococcus faecalis (positive) and Enterococcus faecium (negative). • It can be used as a part of an identication tests for other organisms capable of utilizing pyruvate. SULPHUR REDUCTION TEST • SIM medium (Sulphide Indole Motility medium) which is a combination differential medium that tests three different parameters, Sulfur Reduction, Indole Production and Motility. • As the name suggests, it is commonly used to test a microbe for the ability to produce the gas hydrogen sulfide (H2S). The “S” in SIM stands for sulfur. • Objective o To test for the ability of an organism to reduce sulphur o To differentiate gram-negative enteric bacilli on the basis of sulfide production PRINCIPLE • Organisms which produce the enzyme thiosulfate reductase can reduce sulfur to hydrogen sulfide gas. This happens when the strain either degrades the amino acid cysteine during protein degradation, or when anaerobic respiration shuttles the electrons to sulfur instead of to oxygen. In the SIM tubes, the medium contains casein and animal proteins as amino acid sources, sodium thiosulfate as a source of sulfur and ferrous ammonium sulfate as the H2S indicator. Cysteine is a sulfur containing amino acid present in the SIM medium. • The enzymes cysteine desulfurase and thiosulfate reductase catalyze hydrolysis reactions that produce H2S. This gas combines with the ferrous ammonium sulfate forming an insoluble, black ferrous sulfide precipitate. The black color thus acts as an indicator for the presence of hydrogen sulfide. • In the laboratory, a fresh culture of the organism is inoculated with a single stab using straight needle through the center of the medium. Following incubation, the tube is observed for H2S production (blackening of the medium). PROCEDURE 1. Touch a straight needle to a colony of a young (18- to 24- hour) culture growing on agar medium. 2. Stab once to a depth of only 1/3 to ½ inch in middle of the tube. 3. Incubate at 35°-37°C and examine daily for up to 7 days. 4. Observe for blackening of the medium on line of inoculation. EXPECTED RESULTS • Positive : Darkening of the medium (a black precipitate) or blackening of the line of inoculation indicates the presence of bacteria producing hydrogen sulfide. • Negative : A negative H2S test is denoted by the absence of blackening. USES • It is used to differentiate sulfur reducing members of the genera Salmonella, Shigella and Proteus from the negative Moranella morganii and Providencia rettgeri. • The production of hydrogen sulphide is a useful diagnostic test in the identification of enteric bacteria. TRIPLE SUGAR IRON • Most bacteria have the ability to ferment carbohydrates, particularly sugars. Among them, each bacteria can ferment only some of the sugars, while it cannot ferment the others. Thus, the sugars, which a bacteria can ferment and the sugars, which it cannot is the characteristic of the bacteria and thus an important criterion for its identification. • 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. All of these ingredients when mixed together and allowed solidification at an angle result in an agar test tube at a slanted angle. The slanted shape of this medium provides an array of surfaces that are either exposed to oxygen-containing air in varying degrees (an aerobic environment) or not exposed to air (an anaerobic environment) under which fermentation patterns of organisms are determined. • Objective o 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 agar test employing Triple Sugar Iron Agar is designed to differentiate among organisms based on the differences in carbohydrate fermentation patterns and hydrogen sulfide production. Carbohydrate fermentation is indicated by the production of gas and a change in the colour of the pH indicator from red to yellow. • To facilitate the observation of carbohydrate utilization patterns, TSI Agar contains three fermentative sugars, lactose and sucrose in 1% concentrations and glucose in 0.1% concentration. 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 products are built and the pH rises. This is indicated by the change in colour of the medium from orange red to deep red. Sodium thiosulfate and ferrous ammonium sulfate present in the medium detects the production of hydrogen sulfide and is indicated by the black color in butt of the tube. • To facilitate the detection of organisms that only ferment glucose, the glucose concentration is one-tenth the concentration of lactose or sucrose. The meagre amount of acid production in the slant of the tube during glucose fermentation oxidizes rapidly, causing the medium to remain orange red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the butt of the tube since it is under lower oxygen tension. • After depletion of the limited glucose, organisms able to do so will begin to utilize the lactose or sucrose. To enhance the alkaline condition of the slant, free exchange of air must be permitted by closing the tube cap loosely. PROCEDURE 1. With a straight inoculation needle, touch the top of a well- isolated colony. 2. Inoculate TSI by first stabbing through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant. 3. Leave the cap on loosely and incubate the tube at 35°- 37°C in ambient air for 18 to 24 hours. 4. Examine the reaction of medium. EXPECTED RESULTS • An alkaline/acid (red slant/yellow butt) reaction : It is indicative of dextrose fermentation only. • An acid/acid (yellow slant/yellow butt) reaction : It indicates fermentation of dextrose, lactose and/or sucrose. • An alkaline/alkaline (red slant, red butt) reaction : Absence of carbohydrate fermentation results. • Blackening of the medium : Occurs in the presence of H2 • Gas production : Bubbles or cracks in the agar indicate the production of gas (formation of CO2and H2) Triple sugar iron agar. A, Acid slant/acid butt with gas, no H2S (A/A). B, Alkaline slant/acid butt, no gas, H2S-positive (K/A H2S+). C, Alkaline slant/alkaline butt, no gas, no H2S (K/K). D, Uninoculated tube . USES • The test is used primarily to differentiate members of the Enterobacteriaceae family from other gram-negative rods. • It is also used in the differentiation among Enterobacteriaceae on the basis of their sugar fermentation patterns. UREASE TEST • Urea Agar was developed by Christensen in 1946 for the differentiation of enteric bacilli. The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme urease. PRINCIPLE • Urea is the product of decarboxylation of amino acids. • Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24 hours. • Weakly positive organisms may take several days, and negative organisms produce no color change or yellow as a result of acid production. RAPID UREASE TEST (RUT) • The rapid urease test (RUT) is a popular diagnostic test for diagnosis of Helicobacter pylori. It is a rapid, cheap and simple test that detects the presence of urease in or on the gastric mucosa. It is also known as the CLO test (Campylobacter-like organism test). This test uses a procedure called gastric endoscopy and biopsy to collect stomach lining cells. • • The test is performed at the time of gastroscopy. A biopsy of mucosa is taken from the antrum of the stomach, and is placed into a medium containing urea and an indicator such as phenol red. The urease produced by H. pylori hydrolyzes urea to ammonia, which raises the pH of the medium, and changes the color of the specimen from yellow (NEGATIVE) to red (POSITIVE). • The test can also be used to provide an informal assessment of the accuracy of the histopathology result and discrepancies should prompt a review of the histopathology and discussions with the pathologist.
UREA BREATH TEST • Urea breath test is a common non-invasive test to detect Helicobacter pylori also based on urease activity. This is highly sensitive and specific test. • Patient ingests radioactively labeled Urea (either radioactive carbon-14 or non-radioactive carbon-13). If infection is present, the urease produced by Helicobacter pylori hydrolyzes the urea to form ammonia and labeled bicarbonate that is exhaled as CO2. The labeled CO2 is detected either by a scintillation counter (Carbon-14) and a isotope ratio mass spectrometry or by mass correlation spectrometry (Carbon-13). USES • This test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease. • This test can be used as part of the identiﬁcation of several genera and species of Enterobacteriaceae, including Proteus, Klebsiella, and some Yersinia and Citrobacter species, as well as some Corynebacterium species. • It is also useful to identify Cryptococcus spp., Brucella, Helicobacter pylori, and many other bacteria that produce the urease enzyme. • Directly, this test is performed on gastric biopsy samples to detect the presence of H. pylori. PROCEDURE 1. Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate slant with 1 to 2 drops from an overnight brain-heart infusion broth culture. 2. Leave the cap on loosely and incubate the tube at 35°- 37°C in ambient air for 48 hours to 7 days. 3. Examine for development of pink color for as long as 7 days. RESULT INTERPRETATION • Positive Reaction : Development of an intense magenta to bright pink color in 15 min to 24 h. o Examples : Proteus spp, Cryptococcus spp, Corynebacterium spp, Helicobacter pylori, Yersinia spp, Brucella spp, etc. • Negative Reaction : No color change. o Examples : Escherichia, Shigella, Salmonella, etc. QUALITY CONTROL • Positive : Proteus vulgaris (ATCC13315) • Weak positive : Klebsiella pneumoniae (ATCC13883) • Negative : Escherichia coli (ATCC25922) VOGES-PROSKAUER (VP) TEST • Voges and Proskauer, in 1898, first observed the production of a red color after the addition of potassium hydroxide to cultures grown on specific media. Harden later revealed that the development of the red color was a result of acetyl-methyl carbinol production. In 1936 Barrit made the test more sensitive by adding alpha-naphthol to the medium before adding potassium hydroxide. PRINCIPLE • The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethyl carbinol from glucose fermentation. If present, acetylmethyl carbinol is converted to diacetyl in the presence of ∝ - naphthol, strong alkali (40% KOH), and atmospheric oxygen. The ∝ -naphthol was not part of the original procedure but was found to act as a color intensiﬁer by Barritt and must be added ﬁrst. The diacetyl and quanidine-containing compounds found in the peptones of the broth then condense to form a pinkish red polymer. • 2 pyruvate = acetoin + 2CO2 • acetoin + NADH + H+ = 2,3-butanediol + NAD+ PROCEDURE 1. Prior to inoculation, allow medium to equilibrate to room temp. 2. Using organisms taken from an 18-24 hour pure culture, lightly inoculate the medium. 3. Incubate aerobically at 37 degrees C. for 24 hours. 4. Following 24 hours of incubation, aliquot 2 ml of the broth to a clean test tube. 5. Re-incubate the remaining broth for an additional 24 hours. 6. Add 6 drops of 5% alpha-naphthol, and mix well to aerate. 7. Add 2 drops of 40% potassium hydroxide, and mix well to aerate. 8. Observe for a pink-red color at the surface within 30 min. Shake the tube vigorously during the 30-min period. RESULT INTERPRETATION • Positive Reaction : A pink-red color at the surface o Examples : Viridans group streptococci (except Streptococcus vestibularis), Listeria, Enterobacter, Klebsiella, Serratia marcescens, Hafnia alvei, Vibrio eltor, Vibrio alginolyticus, etc. • Negative Reaction : A lack of a pink-red color o Examples : Streptococcus mitis, Citrobacter sp., Shigella, Yersinia, Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio parahaemolyticus etc. • A copper color should be considered negative. A rust color is a weak positive reaction. QUALITY CONTROL • VP positive : Enterobacter aerogenes (ATCC13048) • VP negative : Escherichia coli (ATCC25922)