Principles of biochemical tests commonly used in the identification of gram-negative bacteria

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1 Dr. Khoramrooz 1 In the name of God Department Of Microbiology Yasouj University of Medical Science Principles of biochemical tests commonly used in the identification of gram-negative bacteria By: Dr. S. S. Khoramrooz, Ph.D. Department of Microbiology, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran

2 Introduction Historically, the identification of bacteria based on: Colony morphology, Gram staining, Biochemical testing. Based on the phenotype of microorganisms This method required a lot of time and lots of space in incubators. Over time, biochemical tests were miniaturized and multitest systems were developed Resulting in time and space savings. More recently, automated systems have been developed Dr. Khoramrooz 2

3 Clinical microbiology entered the next generation of test systems for the identification of bacteria: Molecular biology assays. Based on nucleic acid sequences, are highly sensitive, specific, and rapid, thereby providing accurate results in a few hours or less. More accurate than examining the phenotype. In this lecture discuss the principles of biochemical tests commonly used in the identification of gram-negative bacteria. Dr. Khoramrooz 3

4 Characters of Enterobacteriaceae All Enterobacteriaceae Gram-negative rods Ferment glucose with acid production Reduce nitrates into nitrites Oxidase negative Facultative anaerobic Motile except Shigella and Klebsiella Non-capsulated except Klebsiella Non-fastidious Grow on bile containing media (MacConkey agar) Dr. Khoramrooz 4

5 Classification of Enterobacteriaceae Enterobacteriaceae Lactose fermenters E. coli, Citrobacter, Klebsiella, Enterobacter Non-lactose fermenter Salmonell, Shigella Proteus, Yersinia There are several selective and differential media used to isolate distinguishes between LF & LNF The most important media are: MacConkey agar Eosin Methylene Blue (EMB) agar Salmonella Shigella (SS) agar In addition to Kiligler Iron agar (KIA) Dr. Khoramrooz 5

6 Tests To Know Case Study Tests Indole Methyl Red/Voges Proskauer Citrate H 2 S production in SIM Urea hydrolysis Motility Lactose fermentation Glucose fermentation & gas production Decarboxylation of amino acis Fermentation of sugars Reaction on selective media Dr. Khoramrooz 6

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8 Selection of Primary Isolation Media Used to recover the significant species of bacteria from specimens that may harbor a mixture of microorganisms. Microbiologists must know the composition of each formula and the purpose and relative concentration of each chemical or compound that is included. Not sufficient to know that bile salts are included in selective media In SS agar contains about 5 times the concentration of bile salts compared with MacConkey agar and is more inhibitory to E.coli and more selective for the recovery of Salmonella species from stool cultures. Dr. Khoramrooz 8

9 For the recovery of the Enterobacteriaceae from clinical, three general types of media are available: 1) nonselective media for primary isolation (e.g., blood agar); 2) Selective or differential agars (e.g., MacConkey and Hektoen enteric agars); 3) enrichment broths. Dr. Khoramrooz 9

10 Selective Isolation Media MacConkey and EMB agars are only moderately inhibitory and are designed primarily to prevent growth of gram positive bacteria from mixed cultures. Dr. Khoramrooz 10

11 Growth of Enterobacteriaceae on MacConkey agar Colorless colonies Pink colonies Uninoculated plate Lactose non feremters Lactose feremters Salmonella, Shigella, E. coli, Citrobacter Proteus Klebsiella, Enterobacter Dr. Khoramrooz 11

12 MacConkey Agars Intended Use MacConkey agars are slightly selective and differential plating media mainly used for the detection and isolation of gram-negative organisms from clinical, dairy, food, water, pharmaceutical, cosmetic, and other industrial sources. MacConkey Agar is used for isolating and differentiating lactose-fermenting from lactose-nonfermenting gramnegative enteric bacilli. MacConkey Agar Base is used with added carbohydrate in differentiating coliforms based on fermentation reactions. Dr. S. S. Khoramrooz 12

13 MacConkey Agar without Crystal Violet is used for isolating and differentiating enteric microorganisms while permitting growth of staphylococci and enterococci. The medium can be used also to separate Mycobacterium fortuitum and M. chelonae from other rapidly growing mycobacteria. MacConkey Agar without Crystal Violet or Salt and MacConkey Agar without Salt are used for isolating and differentiating gram-negative bacilli while suppressing the swarming of most Proteus species. Dr. S. S. Khoramrooz 13

14 Principles of the Procedure Lactose is a fermentable carbohydrate. When lactose is fermented, a local ph drop around the colony cause a color change in the ph indicator (neutral red) and bile precipitation. Dr. S. S. Khoramrooz 14

15 Bile salts, bile salts no. 3, oxgall and crystal violet are selective agents that inhibit growth of gram-positive organisms. Magnesium sulfate is a source of divalent cations. Agar is the solidifying agent. Dr. S. S. Khoramrooz 15

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17 Expected Results Lactose-fermenting organisms grow as pink to brick-red colonies with or without a zone of precipitated bile. Lactose-nonfermenting organisms grow as colorless or clear colonies. Swarming by Proteus spp. is reduced on MacConkey agars without salt. Dr. S. S. Khoramrooz 17

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19 Limitations of the Procedure 1. Although MacConkey media are selective primarily for gram-negative enteric bacilli, biochemical and, if indicated, serological testing using pure cultures are recommended for complete identification. 2. Incubation of MacConkey Agar plates under increased CO2 has been reported to reduce the growth and recovery of a number of strains of gram-negative bacilli. Dr. S. S. Khoramrooz 19

20 Intended Use Eosin Methylene Blue Agar, Levine is a slightly selective and differential plating medium for the isolation of gramnegative enteric bacteria. Principles of the Procedure The eosin Y and methylene blue dyes in Levine EMB Agar render the medium slightly selective in that they inhibit gram- positive bacteria to a limited degree. Dr. S. S. Khoramrooz 20

21 These dyes also play a role in differentiating between lactose fermenters and lactose nonfermenters due to the presence or absence of dye uptake in the bacterial colonies. Coliforms, as lactose-fermenting organisms, are visualized as blue-black colonies, whereas colonies of Salmonella and Shigella, as lactose nonfermenters, appear colorless, transparent or amber. Some gram-positive bacteria, such as fecal streptococci, staphylococci and yeasts, will grow on this medium and usually form pinpoint colonies. Dr. S. S. Khoramrooz 21

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23 Expected Results Typical colonial morphology on Eosin Methylene Blue Agar, Levine is as follows: Dr. S. S. Khoramrooz 23

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25 Highly Selective Isolation Media Used Primarily for Gastrointestinal Specimens Media are made highly selective by the addition of a variety of inhibitors to their formulas, generally in higher concentrations than in MacConkey and EMB agars. These media are used primarily to inhibit the growth of E. coli and other "coli forms," but they allow Salmonella and Shigella species to grow out from stool specimens The most commonly used are Salmonella-Shigella (SS) agar, xylose lysine deoxycholate (XLD) agar, and Hektoen enteric (HE) agar. Dr. Khoramrooz 25

26 Intended Use XL (Xylose Lysine) Agar Base is used for the isolation and differentiation of enteric pathogens and, when supplemented with appropriate additives, as a base for selective enteric media. XLD Agar is the complete Xylose Lysine Desoxycholate Agar, a moderately selective medium recommended for isolation and differentiation of enteric pathogens, especially Shigella species. Dr. S. S. Khoramrooz 26

27 Principles of the Procedure Xylose is incorporated into the medium because it is fermented by practically all enterics except for the shigellae. This property enables the differentiation of Shigella species. Lysine is included to enable the Salmonella group to be differentiated from the nonpathogens. Without lysine, salmonellae rapidly would ferment the xylose and be indistinguishable from nonpathogenic species. Dr. S. S. Khoramrooz 27

28 After the salmonellae exhaust the supply of xylose, the lysine is attacked via the enzyme lysine decarboxylase, with reversion to an alkaline ph, which mimics the Shigella reaction. To prevent similar reversion by lysine-positive coliforms, lactose and sucrose (saccharose) are added to produce acid in excess. Degradation of xylose, lactose and sucrose generates acid products, which in the presence of the ph indicator phenol red, causes a color change in the medium from red to yellow. Dr. S. S. Khoramrooz 28

29 To add to the differentiating ability of the formulation, an H2S indicator system, consisting of sodium thiosulfate and ferric ammonium citrate, is included for the visualization of the hydrogen sulfide produced, resulting in the formation of colonies with black centers. The nonpathogenic H2S producers do not decarboxylate lysine; therefore, the acid reaction produced by them prevents the blackening of the colonies. Dr. S. S. Khoramrooz 29

30 XLD Agar is both a selective and differential medium. It utilizes sodium desoxycholate as the selective agent and, therefore, it is inhibitory to grampositive microorganisms. Dr. S. S. Khoramrooz 30

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33 Expected Results Degradation of xylose, lactose and sucrose generates acid products, causing a color change in the medium from red to yellow. Hydrogen sulfide production under alkaline conditions causes colonies to develop black centers. This reaction is inhibited by the acid conditions that accompany carbohydrate fermentation. Lysine decarboxylation in the absence of lactose and sucrose fermentation causes reversion to an alkaline condition and the color of the medium changes back to red. Typical colonial morphology and reactions on XLD Agar are as follows: Dr. S. S. Khoramrooz 33

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37 Limitations of the Procedure 1. Red, false-positive colonies may occur with some Proteus and Pseudomonas species. 2. Incubation in excess of 48 hours may lead to falsepositive results. 3. S. Paratyphi A, S. Choleraesuis, S. pullorum and S. gallinarum may form red colonies without black centers, thus resembling Shigella species. 4. Some Proteus strains will give black-centered colonies on XLD Agar. Dr. S. S. Khoramrooz 37

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42 Intended Use Hektoen Enteric (HE) Agar is a moderately selective medium used in qualitative procedures for the isolation and cultivation of gram-negative enteric microorganisms, especially Shigella, from a variety of clinical and nonclinical specimens. Dr. S. S. Khoramrooz 45

43 Principles of the Procedure The selective nature of Hektoen Enteric Agar is due to the incorporation of bile salts in the formulation. These substances inhibit gram-positive organisms but also can be toxic for some gram-negative strains. Dr. S. S. Khoramrooz 46

44 This medium contains three carbohydrates, lactose, sucrose (saccharose) and salicin, for optimal differentiation of enteric pathogens The lactose concentration is higher than in many other media used for enterics in order to aid in the visualization of enteric pathogens and minimize the problem of delayed lactose fermentation. Ferric ammonium citrate and sodium thiosulfate in the medium enable the detection of hydrogen sulfide production. The indicator system, consisting of acid fuchsin and bromthymol blue, has a lower toxicity than that of many other enteric media, resulting in improved recovery of enteric pathogens. Dr. S. S. Khoramrooz 47

45 Procedure A nonselective medium should also be streaked to increase the chance of recovery when the population of gram-negative organisms is low and to provide an indication of other organisms present in the specimen. Incubate plates, protected from light, at 35 ± 2 C for hours. Dr. S. S. Khoramrooz 48

46 DO NOT AUTOCLAVE. Dr. S. S. Khoramrooz 49

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49 Salmonella mixed with normal fecal flora. Dr. S. S. Khoramrooz 52

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51 Hektoen Enteric (HE) Agar: Composition Peptone 1.2% Yeast extract 0.3% Bile salts 0.9% Lactose 1.2% Sucrose 1.2% Salicin 0.2% Sodium chloride 0.5% Ferric ammonium citrate Acid fuchsin Thymol blue Agar 1.4% ph = 7.6 Dr. Khoramrooz 54

52 HE Agar: Growth of Enteric Pathogens and Commensals High bile salt concentration inhibits growth of grampositive and gram-negative intestinal commensals, and thereby selects for pathogenic Salmonella (bileresistant growth) present in fecal specimens. Salmonella species as non-lactose and non-sucrose fermenters that produce H2S form colorless colonies with black centers. Shigella species (non-lactose and non-sucrose fermenters, no H2S production) form colorless colonies. Lactose and sucrose fermenters (E. coli, K. pneumoniae) form orange to yellow colonies due to acid Dr. Khoramrooz production. 55

53 Intended Use Bismuth Sulfite Agar is a highly selective medium used for isolating Salmonella spp., particularly Salmonella Typhi, from food and clinical specimens. Dr. S. S. Khoramrooz 56

54 Principles of the Procedure Dextrose is an energy source. Bismuth sulfite indicator and brilliant green are complementary in inhibiting gram-positive bacteria and members of the coliform group, while allowing Salmonella to grow luxuriantly. Ferrous sulfate is included for detection of H2S production. When H2S is present, the iron in the formula is precipitated, giving positive cultures the characteristic brown to black color with metallic sheen. Dr. S. S. Khoramrooz 57

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56 For isolation of Salmonella spp. from clinical specimens, inoculate fecal specimens and rectal swabs onto a small area of one quadrant of the Bismuth Sulfite Agar plate and streak for isolation. This will permit the development of discrete colonies. Incubate plates at 35 C. Examine at 24 hours and again at 48 hours for colonies resembling Salmonella spp. Dr. S. S. Khoramrooz 59

57 Expected results The typical discrete S. Typhi surface colony is black and surrounded by a black or brownish-black zone which may be several times the size of the colony. By reflected light, preferably daylight, this zone exhibits a distinctly characteristic metallic sheen. Plates heavily seeded with S. Typhi may not show this reaction except near the margin of the mass inoculation. Dr. S. S. Khoramrooz 60

58 In these heavy growth areas, this organism frequently appears as small light green colonies. This fact emphasizes the importance of inoculating plates so that some areas are sparsely populated with discrete S. Typhi colonies. Other strains of Salmonella produce black to green colonies with little or no darkening of the surrounding medium. Dr. S. S. Khoramrooz 61

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60 Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. DO NOT AUTOCLAVE. Use the medium the same day it is prepared. Dr. S. S. Khoramrooz 63

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62 Intended Use Brilliant Green Agar is a highly selective medium for the isolation of Salmonella other than S. Typhi from feces and other materials. Principles of the Procedure Brilliant green dye inhibits gram-positive bacteria and a majority of gram-negative bacilli. Phenol red serves as a ph indicator and yields a yellow color as a result of acid production in the fermentation of the lactose and/or sucrose in the medium. Dr. S. S. Khoramrooz 65

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64 Procedure A less selective medium and a nonselective medium should also be streaked to increase the chance of recovery when the population of gram-negative organisms is low and to provide an indication of other organisms present in the specimen. Incubate plates, protected from light, at 35 ± 2 C for hours. If negative after 24 hours, reincubate an additional 24 hours. Dr. S. S. Khoramrooz 67

65 Salmonella on BPLS Agar. The colonies are red because the bacterium does not ferment lactose or sucrose. Escherichia coli on BPLS Agar. The colonies are yellow due to the low ph which is caused by the production of acid during fermentation of lactose and/or sucrose. Dr. S. S. Khoramrooz 68

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67 Differential Identification Characteristics Dr. Khoramrooz 70

68 Carbohydrate utilization Lactose degradation; Differentiate those (lactose fermenter LF]) and those that are nonlactose fermenters (NLFs). Examples of sugars used to differentiate bacteria include lactose, maltose, rhamnose, sucrose, raffinose, and arabinose Bacteria can utilize carbohydrates: oxidation (aerobically), fermentatively (anaerobically),or both. Bacteriat hat can grow either aerobically or anaerobically are called facultative anaerobes. Some bacteria are asacchrolytic. They do not use any carbohydrate; instead they use other organic molecules for energy and carbon sources. Dr. Khoramrooz 71

69 Oxidation/Fermentation (O/F) Test Principle : To determine the ability of bacteria to breakdown glucose oxidative or fermentative O/F medium ( Hugh and Leifson Medium) is formulated to detect weak acids produced from saccharolytic M.O. O/F medium contains Sugar (glucose 1%) Low percentage of Agar and Peptone ph indicator (Bromothymol blue) Alkaline Blue Neutral Green Acidic Yellow Dr. Khoramrooz 72

70 Intended Use OF (Oxidation Fermentation) media are used for the determination of oxidative and fermentative metabolism of carbohydrates by gram-negative rods on the basis of acid reaction in either the open or closed system. Summary and Explanation OF Medium was developed by Hugh and Leifson who described the taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by gram-negative bacteria. Dr. S. S. Khoramrooz 73

71 They showed that when an organism is inoculated into two tubes of OF Basal Medium containing a carbohydrate and the medium in one of the tubes is covered with melted petrolatum prior to incubation, the patterns of metabolism are of differential significance. Oxidative organisms only produce an acid reaction in the open tube with little or no growth and no acid formation in the covered tube. Fermentative organisms will produce an acid reaction in both types of tubes. Changes in the covered agar are considered to be due to true fermentation, while changes in the open tubes are due to oxidative utilization of the carbohydrate present. If the carbohydrate is not utilized by either method, there is no acid production in either tube. Dr. S. S. Khoramrooz 74

72 Principles of the Procedure Dextrose is the most important carbohydrate for use in OF Basal Medium; however, certain organisms may metabolize other carbohydrates even if they are unable to utilize dextrose. Prepared tubed media containing arabinose, dextrose, dulcitol, fructose, galactose, lactose, maltose, mannose, raffinose, rhamnose, salicin, sorbitol, sucrose and xylose are provided. Dr. S. S. Khoramrooz 75

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74 Procedure Inoculate a pair of OF tubes of each carbohydrate used with each organism being tested. The tubes should be stabbed to approximately 1/4 inch from the bottom using an inoculating needle and a light inoculum. Overlay one tube of each pair with sterile mineral oil. Incubate tubes at 35 ± 2 C in an aerobic atmosphere for 48 hours. Do not discard as negative until after 4 days of incubation. Dr. S. S. Khoramrooz 77

75 Expected Results Record results as acid (A) or alkaline/no change ( ). Also record whether or not the organism is motile as evidenced by the appearance of growth away from the line of inoculation. Typical reaction patterns are as follows. Dr. S. S. Khoramrooz 78

76 Limitations of the Procedure 1. The acid reaction produced by oxidative organisms is apparent at the surface and gradually spreads throughout the medium. If the oxidation is weak or slow, however, an initial alkaline reaction at the surface of the open tube may persist for several days and eventually convert to an acid reaction. 2. If an organism is unable to grow on OF Basal Medium, Cowan recommends adding either 2% serum or 0.1% yeast extract to each carbohydrate tube. Dr. S. S. Khoramrooz 79

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79 Triple Sugar lron Agar Kligler Iron Agar Lactose Fermentation Glucose fermentation Gas Production (H2 & CO2 ) H2S Production Dr. Khoramrooz 83

80 TSI and KIA glucose lactose Ferrous sulfate ph indicator: phenol red Proteins Dr. Khoramrooz 84

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82 Intended Use Triple Sugar Iron Agar (TSI Agar) is used for the differentiation of gram-negative enteric bacilli based on carbohydrate fermentation and the production of hydrogen sulfide. Principles of the Procedure TSI Agar contains three sugars (dextrose, lactose and sucrose), Phenol red for detecting carbohydrate fermentation Dr. S. S. Khoramrooz 86

83 Ferrous ammonium sulfate for detection of hydrogen sulfide production (indicated by blackening in the butt of the tube). Carbohydrate fermentation is indicated by the production of gas and a change in the color of the ph indicator from red to yellow. To facilitate the detection of organisms that only ferment dextrose, the dextrose concentration is one-tenth the concentration of lactose or sucrose. The small amount of acid produced in the slant of the tube during dextrose fermentation oxidizes rapidly, causing the medium to remain red or revert to an alkaline ph. In contrast, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower oxygen tension. Dr. S. S. Khoramrooz 87

84 After depletion of the limited dextrose, 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. If the tube is tightly closed, an acid reaction (caused solely by dextrose fermentation) will also involve the slant. Dr. S. S. Khoramrooz 88

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86 Procedure To inoculate, carefully touch only the center of an isolated colony on an enteric plated medium with a cool, sterile needle, stab into the medium in the butt of the tube, and then streak back and forth along the surface of the slant. Several colonies from each primary plate should be studied separately, since mixed infections may occur. Dr. S. S. Khoramrooz 90

87 Incubate with caps loosened at 35 C and examine after hours for carbohydrate fermentation, gas production and hydrogen sulfide production. Any combination of these reactions may be observed. Do not incubate longer than 24 hours because the acid reaction in the slant of lactose and sucrose fermenters may revert to an alkaline reaction. Dr. S. S. Khoramrooz 91

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89 Expected Results Carbohydrate fermentation is indicated by a yellow coloration of the medium. If the medium in the butt of the tube becomes yellow (acidic), but the medium in the slant becomes red (alkaline), the organism being tested only ferments dextrose (glucose). A yellow (acidic) color in the slant and butt indicates that the organism being tested ferments dextrose, lactose and/or sucrose. A red (alkaline) color in the slant and butt indicates that the organism being tested is a nonfermenter. Dr. S. S. Khoramrooz 93

90 Hydrogen sulfide production results in a black precipitate in the butt of the tube. Gas production is indicated by splitting and cracking of the medium. Dr. S. S. Khoramrooz 94

91 1. Hydrogen sulfide production may be evident on Kligler Iron Agar but negative on Triple Sugar Iron Agar. Studies by Bulmash and Fulton showed that the utilization of sucrose could suppress the enzymatic mechanisms responsible for H2S production. Padron and Dockstader8 found that not all H2S-positive Salmonella are positive on TSI. 2. Sucrose is added to TSI to eliminate some sucrose-fermenting lactose-nonfermenters such as Proteus and Citrobacter spp. 3. Further biochemical tests and serological typing must be performed for definite identification and confirmation of organisms. Dr. S. S. Khoramrooz 95

92 4. Do not use an inoculating loop to inoculate a tube of Triple Sugar Iron Agar. While stabbing the butt, mechanical splitting of the medium occurs, causing a false positive result for gas production. 5. A pure culture is essential when inoculating Triple Sugar Iron Agar. If inoculated with a mixed culture, irregular observations may occur. 6. Tubes should be incubated with caps loosened. This allows a free exchange of air, which is necessary to enhance the alkaline condition on the slant. Dr. S. S. Khoramrooz 96

93 Red/Red Alkaline /Alkaline K/K Lactose -/Glucose Yellow/Yellow Acid/Acid A/A Lactose +/Glucose + Red/Yellow Alkaline/Acid K/A Lactose -/Glucose + Gas - H2S - Red/Yellow Alkaline/Acid K/A Lactose -/Glucose + Gas + H2S - Red/Yellow Alkaline/Acid K/A Lactose -/Glucose + Gas - H2S + Red/Black Alkaline/Acid K/A Lactose -/Glucose + Gas + H2S + Dr. Khoramrooz 97

94 Result Reaction on KIA Example Butt color Slant color H 2 S Result Red Red Negative Alk/Alk/- (No action on sugars) Non fermenter e.g. Pseudomonas Yellow Red Negative A/Alk/- (Glucose fermented without H 2 S) LNF e.g. Shigella Yellow Red Positive black in butt A/Alk/+ (Glucose fermented with H 2 S) LNF e.g. Salmonella & Proteus Yellow Yellow Negative A/A/- (three sugars are fermented) Dr. Khoramrooz LF e.g. E. coli, Klebsiella, Enterobacter 98

95 Ortho- Nitrophenyl - β D- galactopyranosidetest Organisms that are delayed lactose-fermenters appear as nonfermenting colonies. The o-nitrophenyl β D- galactopyranoside (ONPG) and the p- nitrophenyl-b-d-galactopyranoside (PNPG) tests determine whether the organism is a delayed lactose fermenter (one that lacks the enzyme β-galactoside permease but possesses β- galactosidase) or a true nonlactose fermenter (NLF). The test can be performed by making a heavy suspension of bacteria in sterile saline and adding commercially prepared ONPG disks or tablets. The suspension is incubated at 37' C, and positive results can generally be seen within 6 hours. Dr. Khoramrooz 99

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97 IMViC Test Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests: The following four tests comprise a series of important determinations that are collectively called the IMViC series of reactions The IMViC series of reactions allows for the differentiation of the various members of Enterobacteriaceae. Dr. Khoramrooz 101

98 Principle IMViC: Indole test Certain microorganisms can metabolize tryptophan by tryptophanase The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia The presence of indole is detected by addition of Kovac's reagent. Tryptophanase Tryptophane amino acids Indole + Pyurvic acid + NH3 Kovac s Reagent Red color Dr. Khoramrooz in upper organic layer` 102

99 IMViC: Indole test Result: A bright pink color in the top layer indicates the presence of indole The absence of color means that indole was not produced i.e. indole is negative Negative test e.g. Klebsiella Positive test e.g. E. coli Special Features: Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-). Dr. Khoramrooz 103

100 Intended Use SIM Medium is used to differentiate enteric bacilli on the basis of sulfide production,indole formation and motility. Dr. S. S. Khoramrooz 104

101 Summary and Explanation Hydrogen sulfide production, indole formation and motility are distinguishing characteristics which aid in the identification of the Enterobacteriaceae, especially Salmonella and Shigella. SIM Medium, therefore,is useful in the process of identification of enteric pathogens. Dr. S. S. Khoramrooz 105

102 Principles of the Procedure Sodium thiosulfate and ferrous ammonium sulfate are indicators of hydrogen sulfide production. The ferrous ammonium sulfate reacts with H2S gas to produce ferrous sulfide,a black precipitate. The casein peptone is rich in tryptophan,which is attacked by certain microorganisms resulting in the production of indole. The indole is detected by the addition of chemical reagents following the incubation period. Dr. S. S. Khoramrooz 106

103 Motility detection is possible due to the semisolid nature of the medium. Growth radiating out from the central stab line indicates that the test organism is motile. Dr. S. S. Khoramrooz 107

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105 Procedure Loosen caps, boil and cool before use. Using growth from a pure culture, stab an inoculating needle two-thirds of the distance to the bottom in the center of the tube. Incubate tubes with loosened caps for18-24 hours at 35±2 C in anaerobic atmosphere. Dr. S. S. Khoramrooz 109

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107 IMViC test Methyl Red-Voges Proskauer (MR-VP) Tests Principle Glucose Acidic pathway Or Neutral pathway Mixed acids ph less than 4.4 Methyl Red indicator Red color MR positive E. coli Acety methyl carbinol (ACETOIN) VP positive Klebsiella Dr. Khoramrooz solution A solution B Pink color 111

108 Butylene Glycol Pathway of Glucose Fermentation In the butylene glycol pathway pyruvic acid to acetoin and butylene glycol. Acetoin and butylene glycol are detected by oxidation to diacteyl at an alkaline ph. Addition of -naphthol which forms a red-colored complex with diacetyl. Important biochemical property used for the identification of Klebsiella, Enterobacter, and Serratia. Dr. Khoramrooz 112

109 IMViC test: MRVP test Method Inoculate the tested organism into MRVP broth Incubate the tubes at 37 C for 24 hours For methyl red: Add 6-8 drops of methyl red reagent. For Voges-Proskauer: Add 12 drops of solution A ( -naphthol), mix, 4 drops of Solution B (40% KOH), mix Dr. Khoramrooz 113

110 Results IMViC test: MR/VP test Methyl Red test Voges-Proskauer test Pink: Positive VP (Klebsiella) Red: Positive MR (E. coli) No pink: Negative VP (E. coli) Yellow or orange: Negative MR (Klebsiella) Dr. Khoramrooz 114

111 Intended Use MR-VP Medium and MR-VP Broth (Methyl Red- Voges Proskauer Medium/Broth, also known as Buffered Peptone- Glucose Broth) are used for the differentiation of bacteria by means of the methyl red and Voges-Proskauer reactions. Dr. S. S. Khoramrooz 115

112 Principles of the Procedure Methyl red-positive organisms produce high levels of acid during fermentation of dextrose, overcome the phosphate buffer system and produce a red color upon the addition of the methyl red ph indicator. In the Voges-Proskauer test, the red color produced by the addition of potassium hydroxide to cultures of certain microbial species is due to the ability of the organisms to produce a neutral end product, acetoin (acetylmethylcarbinol), from the fermentation of dextrose. The acetoin is oxidized in the presence of oxygen and alkali to produce a red color. This is a positive Voges-Proskauer reaction. Dr. S. S. Khoramrooz 116

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114 Procedure Using a light inoculum, inoculate tubes of MR-VP media with 18- to 24-hour pure cultures. Incubate tubes aerobically at 35 2 C for a minimum of 48 hours but preferably for 5 days. Prepare the methyl red indicator: 0.1 g of methyl red in 300 ml of 95% ethyl alcohol. Add sufficient purified water to make 500 ml. After the appropriate incubation period, aseptically remove aliquots (1 ml for the VP test) of the medium and conduct the following tests: Dr. S. S. Khoramrooz 118

115 1. Methyl Red Test: Add 5 drops of methyl red indicator to an aliquot of the broth. Interpret the color result immediately. 2. Voges-Proskauer Test: Empty the contents (15 drops) from the reagent A dropper 5 drops from the reagent B dropper into 1 ml of broth culture. Shake well after the addition of each reagent to aerate the sample. Dr. S. S. Khoramrooz 119

116 Expected Results 1. Methyl Red Test a. Positive red color at surface of the medium. b. Negative yellow color at surface of the medium. 2. Voges-Proskauer Test A positive reaction is indicated by the development of a distinct red color which occurs within 5 minutes. Certain species within Enterobacteriaceae genera may react differently or give variable results. Consult appropriate texts for reactions of specific species. Dr. S. S. Khoramrooz 120

117 Limitations of the Procedure 1. Results of the MR and VP tests need to be used in conjunction with other biochemical tests to differentiate genus and species within the Enterobacteriaceae. 2. A precipitate may form in the potassium hydroxide reagent solution. This precipitate has not been shown to reduce the effectiveness of the reagent. Dr. S. S. Khoramrooz 121

118 3. Most members of the family Enterobacteriaceae give either a positive MR test or a positive VP test. However, certain organisms such as Hafnia alvei and Proteus mirabilis may give a positive result for both tests. 4. Incubation time for the Methyl Red test cannot be shortened by increasing the dextrose concentration in the medium or by heavily inoculating the broth. 5. Incubate MR-negative tests for more than 48 hours and test again. Dr. S. S. Khoramrooz 122

119 6. Read the VP test at 48 hours. Increased incubation may produce acid conditions in the broth that will interfere with reading the results. 7. VP reagents must be added in the order and the amounts specified or a weak-positive or false-negative reaction may occur. A weak-positive reaction may be masked by a copper-like color which may form due to the reaction of KOH and α-naphthol. 8. Read the VP test within 1 hour of adding the reagents. The KOH and α-naphthol may react to form a copper-like color, causing a potential false-positive interpretation. 9. Due to the possible presence of acetoin, diacetyl or related substances in certain raw materials, the use of media low in these substances (such as MR-VP media) is recommended for this test. Dr. S. S. Khoramrooz 123

120 Dr. S. S. Khoramrooz 124

121 Citrate Utilization Test Principle: Citrate Pyruvate CO2 + Na + H2O Na 2 CO 3 Simmone s Citrate media Contains Citrate as a sole of C source Alkaline, ph Positive test Blue colour Bromothymol blue Positive test: Klebsiella, Enterobacter, Citrobacter Dr. Khoramrooz Negative test: E. coli 125

122 Citrate Utilization Test Method Streak a Simmon's Citrate agar slant with the organism Incubate at 37 C for 24 hours. Dr. Khoramrooz 126

123 Result Examine for growth (+) Citrate Utilization Test Growth on the medium is accompanied by a rise in ph to change the medium from its initial green color to deep blue Positive Klebsiella, Enterobacter Dr. Khoramrooz Negative E. coli 127

124 Intended Use Simmons Citrate Agar is used for the differentiation of gram negative bacteria on the basis of citrate utilization. Principles of the Procedure Organisms able to utilize ammonium dihydrogen phosphate and sodium citrate as the sole sources of nitrogen and carbon, respectively, will grow on this medium and produce an alkaline reaction as evidenced by a change in the color of the bromthymol blue indicator from green (neutral) to blue (alkaline). Dr. S. S. Khoramrooz 128

125

126 Procedure Inoculate slants with growth from a pure culture using a light inoculum. Incubate all tubes for 4 days at 35 ± 2 C in an aerobic atmosphere. Expected Results A positive reaction is indicated by growth with an intense blue color in the slant. A negative reaction is evidenced by no growth to trace growth with no change in color (medium remains dark green). Dr. S. S. Khoramrooz 130

127 Dr. S. S. Khoramrooz 131

128 Dr. S. S. Khoramrooz 132

129 Dr. S. S. Khoramrooz 133

130 Amino Acid Decarboxylation Enterobacteriaceae contain decarboxylases with substrate specificity for amino acids, and are detected using Moeller decarboxylase broth overlayed with mineral oil for anaerobiosis. Moeller broth contains: glucose for fermentation, peptone and beef extract, amino acid, pyridoxal, ph indicator bromcresol purple. Dr. Khoramrooz 134

131 Amino Acid Decarboxylation If an Enterobacteriaceae contains amino acid decarboxylase, amines produced by decarboxylase action cause an alkaline ph, and bromcresol purple turns purple. Lysine, ornithine, and arginine are utilized. A base broth without amino acid is included in which glucose fermentation acidifies the broth, turning the bromcresol purple yellow. Dr. Khoramrooz 135

132 Amino Acid Decarboxylation 1 Lysine Cadaverine Ornithine Putrescine Arginine Citrulline Ornithine Putrescine 1 Conversion of arginine to citrulline is a dihydrolase reaction Dr. Khoramrooz 136

133 Amino Acid Decarboxylation Decarboxylation patterns are essential for the genus identification of Klebsiella, Enterobacter, Escherichia, and Salmonella. Decarboxylation patterns are also essential for the species identification of Enterobacter aerogenes, Enterobacter cloacae, Proteus mirabilis, and Shigella sonnei. Dr. Khoramrooz 137

134 Amino Acid Decarboxylation Lys Orn Arg Klebsiella + Enterobacter +/ + +/ Escherichia + +/ /+ Salmonella Dr. Khoramrooz 138

135 Amino Acid Decarboxylation Lys Orn Arg E. aerogenes + + E. cloacae + + P. mirabilis + P. vulgaris Shigella D + Shigella A-C Dr. Khoramrooz 139

136 Phenylalanine Deaminase Reaction Enterobacteriaceae utilize amino acids in a variety of ways including deamination. Phenylalanine is an amino acid that forms the keto acid phenylpyruvic acid when deaminated. Phenylpyruvic acid is detected by addition of ferric chloride that forms an intensely dark olive-green colored complex when binding to phenylpyruvic acid. The deamination of phenylalanine is an important biochemical property of Proteus, Morganella, and Providencia. Dr. Khoramrooz 140

137 Intended Use Decarboxylase media are used in the biochemical differentiation of gram-negative enteric bacilli based on the production: Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Decarboxylase Medium Base, with added arginine, lysine or ornithine is used for the same purpose. Lysine Decarboxylase Broth is used for differentiating microorganisms based on lysine decarboxylation. Dr. S. S. Khoramrooz 141

138 Summary and Explanation Moeller introduced the decarboxylase media for detecting the production of lysine and ornithine decarboxylase and arginine dihydrolase. These media are a useful adjunct to other biochemical tests for the speciation and identification of the Enterobacteriaceae and other gram-negative bacilli. The production of OD is particularly useful for differentiating Klebsiella and Enterobacter species. Klebsiella species are non-motile and, except for K. ornithinolytica, do not produce ornithine decarboxylase, while most Enterobacter species are motile and, except for E. agglomerans, usually produce this enzyme. Dr. S. S. Khoramrooz 142

139 Principles of the Procedure Pyridoxal is an enzyme co-factor for the amino acid decarboxylase. Dextrose is a fermentable carbohydrate. Bromcresol purple and cresol red are ph indicators. The amino acids lysine, ornithine or arginine are added to the basal medium at a concentration of 10.0 g/l to detect the production of the enzyme specific for these substrates. Dr. S. S. Khoramrooz 143

140 When the medium is inoculated with a bacterium that is able to ferment dextrose, acids are produced that lower the ph of the medium and change the color of the indicator from purple to yellow. The acidic condition also stimulates decarboxylase activity. If the organism produces the appropriate enzyme, the amino acid in the medium is degraded, yielding a corresponding amine. Dr. S. S. Khoramrooz 144

141 Dr. S. S. Khoramrooz 145

142 Decarboxylation of lysine yields cadaverine. while decarboxylation of ornithine yields putrescine. Arginine is first hydrolyzed to form ornithine, which is then decarboxylated to form putrescine. The production of these amines elevates the ph of the medium, changing the color of the indicator from yellow to purple or violet. If the organism does not produce the appropriate enzyme, the medium remains acidic (yellow). Dr. S. S. Khoramrooz 146

143 Each isolate to be tested must also be inoculated into a tube of the basal medium that does not contain the amino acid. If this tube becomes alkaline, the test is invalid. To obtain the appropriate reactions, the inoculated tubes must be protected from air with a layer of sterile mineral oil. Exposure to air may cause alkalinization at the surface of the medium, which could cause a decarboxylasenegative organism to appear positive. Dr. S. S. Khoramrooz 147

144 Dr. S. S. Khoramrooz 148

145 Expected Results Compare the color of tubes of media containing the specific amino acids with the color of control tubes of basal media (without amino acid) that have been inoculated with the same isolate. If inoculated control tubes show an alkaline reaction, the test is invalid; i.e., Improperly performed or the test organisms Degrade the peptone sufficiently to produce an alkaline reaction in the absence of a specific amino acid. The medium becomes purple to violet if the reaction is positive (alkaline). A yellow color indicates a negative test; i.e., the organism does not produce the appropriate enzyme. Dr. S. S. Khoramrooz 149

146 Dr. S. S. Khoramrooz 150

147 The lysine iron agar (LIA) test is a tubed agar slant. It contains the amino acid lysine, glucose, ferric ammonium citrate, and sodium thiosulfate. The ph indicator is bromcresol purple. LIA is used primarily to determine whether the bacteria decarboxylate or deaminate lysine. H2S production is also detected in this medium. LIA is inoculated in the same manner as a TSI agar slant. LIA is most useful in conjunction with TSI in screening stool specimens for the presence of enteric pathogens, differentiating Salmonella spp. (lysine-positive) from Citrobacter spp. (lysinenegative). Dr. S. S. Khoramrooz 151

148 Decarboxylation occurs anaerobically only; the presence of a dark purple butt is positive for lysine decarboxylation. The production of H2S can mask the purple color in the butt of the tube. Because H2S production in LIA occurs only in an alkaline environment, a black precipitate indicating H2S is also a positive result for decarboxylation. Dr. S. S. Khoramrooz 152

149 LIA is also useful in differentiating Proteus, Morganella, and Providencia spp. from most other members of Enterobacteriaceae This group of enterics deaminates (attacks the NH2 group instead of the carboxyl group) amino acids. In the LIA slant, deamination of lysine turns the original light purple color slant to a plum or reddish purple color; the butt turns yellow because of glucose fermentation. Dr. S. S. Khoramrooz 153

150 Dr. S. S. Khoramrooz 154

151 IPViC Reactions for Initial Grouping of the Enterobacteriaceae Indole Phenylalanine deaminase Voges-Proskauer Citrate Dr. Khoramrooz 155

152 Principle Urease Test Urea agar contains urea and phenol red Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3 Ammonia combines with water to produce ammonium hydroxide, a strong base which ph of the medium. in the ph causes phenol red r to turn a deep pink. This is indicative of a positive reaction for urease Urea Urease CO2 + NH3 H2O NH4 OH in ph Method Streak a urea agar tube with the organism incubate at 37 C for 24 h Dr. Khoramrooz Phenol Red Pink Positive test 156

153 Result Urease Test If color of medium turns from yellow to pink indicates positive test. Proteus give positive reaction after 4 h while Kelebsiella and Enterobacter gave positive results after 24 h Dr. Khoramrooz Positive test Negative test 157

154 Intended Use Urea Agar and Urease Test Broth are used for the differentiation of organisms, especially the Enterobacteriaceae, on the basis of urease production. Dr. S. S. Khoramrooz 158

155 Principles of the Procedure The urea medium of Rustigian and Stuart is particularly suited for the differentiation of Proteus species from other gram negative enteric bacilli capable of utilizing urea. Unable to do so in Urease Test Broth because of limited nutrients and the high buffering capacity of the medium. To provide a medium with greater utility, Urea Agar was devised by Christensen with peptone and dextrose included and reduced buffer content to promote more rapid growth of many of the Enterobacteriaceae and permit a reduction in incubation time. Dr. S. S. Khoramrooz 159

156 The complete Urea Agar contains 15.0 g/l of agar in addition to the ingredients in the base medium. When organisms utilize urea, ammonia is formed during incubation which makes the reaction of these media alkaline, producing a red-pink color. Consequently, urease production may be detected by the change in the phenol red indicator. Dr. S. S. Khoramrooz 160

157 Urease medium

158 Dr. S. S. Khoramrooz 162

159 Dehydrated Product BBL Urea Agar Base 1. Dissolve 29 g of the powder in 100 ml of purified water. Mix thoroughly. Sterilize by filtration. 2. Suspend 15 g of agar in 900 ml of purified water. 3. Autoclave at 121 C for 15 minutes. 4. Cool to 50 C and add 100 ml of the sterile Urea Aga Base. 5. Mix thoroughly and dispense aseptically in sterile tubes. 6. Cool tubed medium in a slanted position so that deep butts are formed. 7. Do not remelt the complete medium. 8. Test samples of the finished product for performance using stable, typical control cultures. Dr. S. S. Khoramrooz 163

160 Procedure Using a heavy inoculum (2 loopfuls) of growth from an 18- to 24-hour pure culture (TSI Agar or other suitable medium), inoculate the broth or agar (streaking back and forth over the entire slant surface). Dr. S. S. Khoramrooz 164

161 Do not stab the butt since it serves as a color control. For broth, shake tubes gently to suspend the bacteria. Incubate tubes with loosened caps at 35 2 C in an incubator or water bath. Observe reactions after 2, 4, 6, 18, 24 and 48 hours. For agar, continue to check every day for a total of 6 days; even longer incubation periods may be necessary. Dr. S. S. Khoramrooz 165

162 The production of urease is indicated by an intense pink-red (red-violet) color on the slant or throughout the broth. The color may penetrate into the agar (butt); the extent of the color indicates the rate of urea hydrolysis. Dr. S. S. Khoramrooz 166

163 A negative reaction is no color change. The agar medium remains pale yellow to buff; the broth remains yellowish orange. Dr. S. S. Khoramrooz 167

164 Urea Agar Base 1. The alkaline reaction produced in this medium after prolonged incubation may not be caused by urease activity. False positive reactions may occur due to the utilization of peptones (especially in slant agar by Pseudomonas aeruginosa, for example) or other proteins which raise the ph due to protein hydrolysis and the release of excessive amino acid residues. To eliminate possible protein hydrolysis, perform a control test with the same test medium without urea. 2. Do not heat or reheat the medium because urea decomposes very easily. Dr. S. S. Khoramrooz 168

165 3. Urea Agar detects rapid urease activity of only the urease positive Proteus species. For results to be valid for the detection of Proteus, the results must be read within the first 2-6 hours after incubation. Urease-positive Enterobacter, Citrobacter or Klebsiella, in contrast, hydrolyze urea much more slowly, showing only slight penetration of the alkaline reaction into the butt of the medium in 6 hours and requiring 3-5 days to change the reaction of the entire butt. Dr. S. S. Khoramrooz 169

166 Urea Broth 1. To rule out false positives due to protein hydrolysis (as opposed to urea hydrolysis) that may occur in the medium after prolonged incubation, perform a control test with the same test medium without urea. 2. Do not heat or reheat the medium because urea decomposes very easily. Dr. S. S. Khoramrooz 170

167 3. The high buffering system in this medium masks urease activity in organisms that are delayed positive. This medium is therefore recommended for the detection of urease activity in all Proteus spp., Providencia rettgeri and ureasepositive Providencia stuartii. M. morganii slowly hydrolyzes urea and may require approximately a 36 hour incubation for a strong urease-positive reaction to occur. If in doubt as to a result, compare with an uninoculated tube or incubate for an additional 24 hours. 4. Variations in the size of the inoculum can affect the time required to reach positive (alkaline, ph 8.1) results. Dr. S. S. Khoramrooz 171

168 Motility From left to right: + + Dr. Khoramrooz 172

169 SIM Sulfide, Indole, Motility Dr. Khoramrooz 173

170 Initial Grouping of the Enterobacteriaceae (VP=Voges Proskauer, PDA=Phenylalanine Deaminase) GENERA VP PDA Klebsiella POSITIVE NEGATIVE Enterobacter POSITIVE NEGATIVE Serratia POSITIVE NEGATIVE Hafnia POSITIVE NEGATIVE Pantoea POSITIVE NEGATIVE Dr. Khoramrooz 175

171 Initial Grouping of the Enterobacteriaceae GENERA VP PDA Proteus 1 NEGATIVE POSITIVE Morganella NEGATIVE POSITIVE Providencia NEGATIVE POSITIVE 1 Proteus mirabilis: Dr. 50% Khoramrooz of strains VP positive 176

172 Initial Grouping of the Enterobacteriaceae GENERA VP PDA Escherichia NEGATIVE NEGATIVE Shigella NEGATIVE NEGATIVE Edwardsiella NEGATIVE NEGATIVE Salmonella NEGATIVE NEGATIVE Citrobacter NEGATIVE NEGATIVE Yersinia NEGATIVE NEGATIVE Dr. Khoramrooz 177

173 Initial Grouping of the Enterobacteriaceae 1 GENERA INDOLE CITRATE Escherichia POSITIVE NEGATIVE Shigella POSITIVE 2 Yersinia POSITIVE 3 Edwardsiella POSTIVE NEGATIVE NEGATIVE NEGATIVE 1 VP negative, PDA negative 2 Shigella groups A, B, and C variably positive for indole production (25-50%), group D Shigella negative. 3 Dr. Khoramrooz Yersinia enterocolitica 50% positive 178

174 Initial Grouping of the Enterobacteriaceae 1 GENERA INDOLE CITRATE Salmonella NEGATIVE POSITIVE 2 Citrobacter NEGATIVE POSITIVE 1 VP negative, PDA negative 2 Salmonella serotype Paratyphi A and Typhi negative Dr. Khoramrooz 179

175 Key Characteristics of the Enterobacteriaceae E coli Shi A- C Shi D Ed Sal Cit TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR A/A Ak/ A A Yer A/A / / + / + Ak/ A + + Ak/ A Ak/ +/ A A/A Ak/ (1) RT=room temperature +/ Dr. Khoramrooz +/ +/ / + +/ RT (1) + 180

176 Kle A/A pne Kle A/A oxy En A/A aer En A/A cloa Serr A/A (1) Haf Ak/ A Pan A/A Alk/ A Key Characteristics of the Enterobacteriaceae TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR / /+ +/ /+ +/ /+ /+ (1) Produces DNase, lipase, and gelatinase Dr. Khoramrooz 181

177 Key Characteristics of the Enterobacteriaceae Prot mir a Prot vulg TSI ON GAS H2S VP IND CIT PDA UR MO LYS OR AR Ak/ A Mor Ak/ Pro v + + +/ +/ + + +s + A/A +/ + + / s A Ak/ A s = swarming motility Dr. Khoramrooz 182

178 Biochemical Characteristics of Escherichia coli and Shiglla E. coli E. coli O157:H7 Shigella TSI A/Ag A/Ag Alk/A Lactose + + ONPG + + /+ 1 Sorbitol + +/ Indole + + +/ Methyl red VP Citrate Lysine + + Motility Shigella sonnei (group D) ONPG + Dr. Khoramrooz 183

179 Biochemical Characteristics of Salmonella Most Serotypes Typhi Paratyphi A TSI Alk/A Alk/A Alk/A H2S (TSI) + + (weak) Citrate + Lysine + + Ornithine + + Dulcitol + + Rhamnose + + Indole Methyl red VP Dr. Khoramrooz 184

180 Dr. Khoramrooz 185

181 The End Dr. Khoramrooz 186

182 t Dr. Khoramrooz 187

183 Dr. Khoramrooz 188

184 Intended Use Malonate Broth is used for differentiating Enterobacter from Escherichia based on malonate utilization. Principles of the Procedure Malonate Broth contains ammonium sulfate, which is the sole source of nitrogen in the medium; Sodium malonate is the sole source of carbon. Increased alkalinity resulting from malonate utilization causes the indicator, bromthymol blue, to change color from green to blue. Dr. S. S. Khoramrooz 189

185 Dr. S. S. Khoramrooz 190

186 Procedure 1. Inoculate tubes with a loopful of test organism. 2. Incubate at 35 2 C for hours. 3. Examine tubes for a change in the color of the medium from green to blue. Expected Results Malonate utilization is indicated by a change in the color of the medium from green to blue: Positive: Blue Negative: Green Dr. S. S. Khoramrooz 191

187 Dr. S. S. Khoramrooz 192

188 Intended Use SS Agar and Salmonella Shigella Agar are moderately selective and differential media for the isolation of pathogenic enteric bacilli, especially those belonging to the genus Salmonella. This formulation is not recommended for the primary isolation of Shigella. Dr. S. S. Khoramrooz 193

189 Principles of the Procedure SS Agar and Salmonella Shigella Agar are designated as moderately selective media based upon the degree of inhibition of gram-positive microorganisms that they inhibit due to their content of bile salts, brilliant green and citrates. Differentiation of enteric organisms is achieved by the incorporation of lactose in the medium. Organisms that ferment lactose produce acid which, in the presence of the neutral red indicator, results in the formation of red colonies. Lactose nonfermenters form colorless colonies. Dr. S. S. Khoramrooz 194

190 The latter group contains the majority of the intestinal pathogens, including Salmonella and Shigella. The sodium thiosulfate and ferric citrate enable the detection of hydrogen sulfide production as evidenced by colonies with black centers. Dr. S. S. Khoramrooz 195

191 Procedure A nonselective medium should also be streaked to increase the chance of recovery when the population of gram-negative organisms is low and to provide an indication of other organisms present in the specimen. Incubate plates, protected from light, at 35 ± 2 C for hours. If negative after 24 hours, reincubate an additional 24 hours. Dr. S. S. Khoramrooz 196

192 Dr. S. S. Khoramrooz 197

193 Expected Results Typical colonial morphology on Salmonella Shigella Agar is as follows: Dr. S. S. Khoramrooz 198

194 Dr. S. S. Khoramrooz 199

195 Limitation of the Procedure Due to the relatively high level of selectivity, some Shigella strains may not grow on SS Agar and Salmonella Shigella Agar and, therefore, these media are not recommended for the primary isolation of Shigella. Media recommended for the isolation of Shigella are Hektoen Enteric and XLD agars. Dr. S. S. Khoramrooz 200

196 Intended Use Selenite Broth (Selenite-F Broth) is used as an enrichment medium for the isolation of Salmonella from feces, urine, water, foods and other materials of sanitary importance. Principles of the Procedure The peptone provides essential nitrogenous and carbon compounds. The lactose in the medium serves to maintain a uniform ph. Dr. S. S. Khoramrooz 201

197 When selenite is reduced by the growth of bacteria, alkali is produced, and such increase in ph would lessen the toxicity of the selenite and result in overgrowth of extraneous bacteria. The acid produced by lactose fermentation serves to maintain a neutral or slightly decreased ph. The function of the phosphate is two-fold; it serves to maintain a stable ph and lessens the toxicity of the selenite, thus increasing the capacity of the medium. Sodium selenite inhibits many species of grampositive and gram-negative bacteria including enterococci and coliforms. Dr. S. S. Khoramrooz 202

198 Dr. S. S. Khoramrooz 203

199 Dr. S. S. Khoramrooz 204