Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli Author: Dr. M.M. Henton and Dr. J.A. Picard Licensed under a Creative Commons Attribution license. TABLE OF CONTENTS INTRODUCTION...3 Table 9.1: Identifying characteristics of several genera of nonfermentative bacilli...3 Table 9.2: Reactions of some Gramnegative, glucose nonfermenting bacteria...4 Bordetella and Alcaligenes spp....4 Table 9.3: Differential characteristics of the genus Bordetella and other morphologically and physiologically similar genera...6 Table 9.4: Differential characteristics for Gramnegative, nonfermentative bacteria with phenotypic characteristics similar to those of Bordetella and Alcaligenes species...6 Table 9.5: Differential characteristics of B. bronchiseptica, B. avium, Alcaligenes spp., and CDC groups IVc and Oligella. All are motile (except B. parapertussis), oxidase (except B. avium and B. parapertussis) and catalasepositive, and grow on MacConkey agar)...7 Riemerella Anatipestifer...7 Moraxella spp....7 Table 9.6: Summary of the disease and sites of isolation of some the Moraxella species...8 Table 9.7: Differentiation of the Moraxella/Brahamnella species...8 Pseudomonas spp. and other related genera...9 Table 9.8: Pathogenicity and the normal habitat of Pseudomonas/Burkholderia species of veterinary importance...9 1 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli Table 9.9: Main characteristics of the pathogenic Pseudomonas/Burkholderia species... 10 Table 9.10: Identification of common members of the Pseudomonaceae (all are motile)... 12 Shewenella... 13 Table 9.11: Identification of Shewenella species (all are Gramnegative curved or straight rods, catalase and oxidase positive, motile and grow in 3% NaCl. Negative for arginine dehydrolase, lysine or ornithine decarboxylase and indole production)... 14 Flavobacterium and related bacteria... 14 APPENDIX 1... 15 2 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli INTRODUCTION Many Gramnegative bacteria fall into this group and include: Alcaligenes spp., Acinetobacter spp., Bordetella spp, Branhamella spp., Flavobacterium spp., Francisella tularensis, Moraxella spp., Neisseria spp., Pseudomonas spp., and Burkholderia spp. Very few isolates are clinically significant, with most being isolated as normal commensal flora or as sample contaminants. Bacteria that are potentially pathogenic include Bordetella bronchiseptica, Pseudomonas aeruginosa, Burkholderia mallei, Burkholderia pseudomallei and Moraxella bovis. These bacteria are particularly difficult to identify as they show few biochemical reactions, and commercial tests such as the API 20 NE are recommended. Although dealt with in a separate chapter, note that Brucella spp. are similar to this group. Bacteria of veterinary significance will be discussed under their respective headings. Tables 9.1, 9.2 and 9.3 list the characteristics of some of these bacteria. As they are a diverse and difficult group to identify, an identification key is included to assist in grouping them. Table 9.1: Identifying characteristics of several genera of nonfermentative bacilli Genus Metabolism Motility Oxidase Acinetobacter Alcaligenes Bordetella Flavobacterium Moraxella Oxidative or nonsaccharolytic Oxidative Nonsaccharolytic Oxidative Oxidative (some strains are slow fermenters) Oxidative or nonsaccharolytic Nonmotile Motile by means of peritrichous flagella Motile by peritrichous flagella Variable. Those species motile aro peritrichous flagella are B. bronchiseptica, B. avium, and B. hinzii) Nonmotile Nonmotile Negative Positive Positive Most are positive except B. parapertussis is negative and B. avium is variable. Positive Positive Growth on MacConkey agar Good growth except for some strains of A. lwoffii Good growth Good growth B. parapertussis and B. bronchiseptica Poor or negative Scant or negative Additional characteristics Special growth factors are not required. Acid production from glucose is weak (A. baumannii) or lacking (A. lwoffii). Cells appear coccoid in Gramstained preparations. Most strains are penicillin resistant. Obligate aerobe. Glucose may be oxidized slowly (5 days); xylose is oxidized rapidly (24 hours) Strict aerobe, although some strains utilize nitrate instead of oxygen as the final electron acceptor. B. bronchiseptica rapidly splits urea (within 4 hours) Supplemental nitrogen and B complex vitamins required for growth of many strains. Yellow pigment often produced. No denitrification of nitrates. Growth optimal at 30 C. All species are resistant to polymyxin B. Most species are weak indole positive (F. odoratum is indole negative). Most strains are fastidious in growth requirements, some requiring serum supplement. Strict aerobes. May appear as coccobacilli on Gram s stain. Highly susceptible to penicillin. 3 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli Table 9.2: Reactions of some Gramnegative, glucose nonfermenting bacteria Acid from glucose Haemolysis Growth on MacConkey agar Motility Catalase Oxidase Nitrate reduction Urease Indole Gelatinase Penicillin susceptibility Pigment Alcaligenes faecalis R A. xylosidans v R A. denitrificans v v A. piechaudii R Acinetobacter calcoaceticus v R A. lwoffii v Branhamella catarrhalis () S Moraxella caviae β (w) S M. cuniculi S M. bovis β () () S M. ovis (β) () S Neisseria canis S Yellow N. flavescens S Yellow N. sicca O v S v (Yellow) N. lactamica O S Yellow N. denitrificans O S v(yellow) N. mucosa O S v(yellow) N. weaveri vβ v S N. elongata v (S) Flavobacterium meningosepticum O (α) R v(yellow) F. indologenes O vα v v () () R Yellow F. odoratum (α) R v(yellow) F. multovorum O (α) R Yellow Weeksella zoohelcum v(α) S CDC group EF4 (O) v * Yellow/tan O =oxidative, = unreactive, (O)= most strains oxidative, = positive reaction, () = most strains positive, () = most strains negative, v = variable, S = susceptible, R = resistant, * = data unavailable, β = betahaemolysis, α = alphahaemolysis, () = most strains. BORDETELLA AND ALCALIGENES SPP. These are small, motile, nonfermentative, Gramnegative rods that are catalase and oxidase positive. Bordetella bronchiseptica is the cause of respiratory disease in mammals and B. avium the cause of turkey coryza and respiratory disease in poultry. Alcaligenes species are saprophytes present in the intestinal tracts of vertebrates. They may be opportunistic invaders and are difficult to distinguish from Bordetella. Bordetella parapertussis has been reported in sheep in Europe, and B. hinzii is not known to be pathogenic. 4 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli Specimens Specimens for bacterial isolation include nasal swabs, tracheal washings and pneumonic lungs. If nasal swabs are to be taken from animals where the nasal orifice is small, flexible swabs designed for human infants should be used. Direct microscopy As Bordetella is a small Gramnegative coccobacillus, smears made directly from specimens are not very useful. Isolation They can all be isolated on blood agar. A selective medium described by Smith and Baskerville (See Appendix 1) can be used for contaminated lung samples. Bordetella bronchiseptica and B. avium forms blue, convex, smooth colonies 1 2 mm in diameter after 48 72 hours of incubation at 37 C. Escherichia coli and Klebsiella species form yellow colonies and Alcaligenes and Pseudomonas species green colonies. For isolates from dogs and rabbits, the medium should be prepared without gentamicin. After 24 hours of incubation on sheep and horse blood agar, B. bronchiseptica forms small, convex, smooth colonies that may be betahaemolytic. The colonies of B.avium are similar, but nonhaemolytic. Phase modulation occurs in both species and is thought to be due to loss of a capsulelike structure on subculture. Phase I colonies are convex and shiny, phase II are larger circular and convex with a smooth surface and phase III are large, flat, and granular with irregular edges. The colonies on MacConkey agar are small, pale with a pinkish hue and amber discolouration of the underlying medium. Bordetella colonies tend to be small after 24 hours, enlarging greatly at 48 hours, whereas Pseudomonas and Alcaligenes colonies are large from the start. Biochemical identification Differential characteristics for these genera are in Tables 9.3, 9.4 and 9.5. All will grow on MacConkey agar, are catalase and oxidasepositive and produce an alkaline slant with an alkaline or no reaction in the butt of a TSI slant. Bordetella bronchiseptica, B. avium and Alcaligenes species are motile. The API rapid NFT will identify Bordetella bronchiseptica and Alcaligenes. Bordetella avium is not listed, but will assimilate adipate but not caprate, while A. faecalis assimilates caprate but not adipate. The API20E system will also identify Bordetella species. However, the CDC group may also be confused with Bordetella and Alcaligenes. Haemagglutination test Bordetella bronchiseptica possesses a haemagglutinin that will haemagglutinate washed sheep red blood cells. A young 24hour culture should be used, as older cultures tend to lose their haemagglutinating 5 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli ability. Two colonies of a suspected B. bronchiseptica culture are suspended in a drop of physiological saline on a slide. An equal volume of a 3% suspension of washed sheep red cells is added and mixed. To check for autoagglutination, controls should include a suspension of colonies without red blood cells and a suspension of red blood cells alone. B. bronchiseptica will autoagglutinate the red cells within 12 minutes. Serology Tube agglutination, microagglutination and ELISA procedures have been developed for B. avium and B. bronchiseptica. Detection of dermonecrotic toxin of Bordetella bronchiseptica Bordetella bronchiseptica produces an intracellular, heatlabile toxin that is lethal when injected intraperitoneally into mice, and produces necrosis when inoculated intradermally into guinea pigs. Table 9.3: Differential characteristics of the genus Bordetella and other morphologically and physiologically similar genera Characteristics Bordetella Alcaligenes Brucella Haemophilus Riemerella Strictly parasitic Saprophytic Localise on respiratory cilia Strictly aerobic () () Growth requirements: Thiamine Nicotinamide X and/or V factor Ferments carbohydrates Nitrate reduction d d Litmus milk/ alkaline d Oxidation of amino acids Tetrazolium reduction d Table 9.4: Differential characteristics for Gramnegative, nonfermentative bacteria with phenotypic characteristics similar to those of Bordetella and Alcaligenes species Bacterial Glucose Growth on Rods/ Type of Catalase Oxidase Motility genera oxidation MacConkey cocci flagella Bordetella R peritrichous Alcaligenes d R peritrichous Pseudomonas/ Burkholderia d d * R polar Flavobacterium d d R Moraxella () d C Eikenella R Acinetobacter d d C * Burkholderia mallei is nonmotile 6 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli Table 9.5: Differential characteristics of B. bronchiseptica, B. avium, Alcaligenes spp., and CDC groups IVc and Oligella. All are motile (except B. parapertussis), oxidase (except B. avium and B. parapertussis) and catalasepositive, and grow on MacConkey agar) Bacteria genera Urease Nitrate Oxidation of reduction Glucose Malonate B. bronchiseptica B. avium B. parapertussis B. hinzii A. faecalis A. xylosoxydans A. denitrificans d A. piechaudii CDC group IVc2 d O. ureolytica Simmon s citrate RIEMERELLA ANATIPESTIFER Riemerella anatipestifer (previously designated Pasteurella anatipestifer) causes septicaemia and respiratory disease in poultry, especially ducks. It is catalase and oxidasepositive, and does not grow on MacConkey agar. It prefers microaerophilic conditions. It is variable for urease, negative for nitrate and is usually gelatine positive. Acid production from sugars is normally negative, except glucose and maltose that are positive. Its biochemical reactions are listed in Table 9.3. MORAXELLA SPP. Moraxella are short, plump Gramnegative rods (1 1,5 x 1,52,5 µm) found characteristically in pairs. Some strains approach a completely coccoid shape. They are strict aerobes, oxidative, oxidase and catalasepositive, nonmotile and do not attack carbohydrates. Although they grow on nonenriched media, their growth is enhanced by the addition of blood or serum. The optimal temperature for growth is 33 35 C. Most Psychrobacter phenylpyruvica strains will grow on MacConkey agar, but M. bovis and M. lacunata will not. A summary of the differential characteristics is included in Table 9.7. LABORATORY DIAGNOSIS Moraxella bovis Specimens 7 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli A swab of the lachrymal secretions is taken from the deep canthus of the eye. Ideally BTA plates should be inoculated immediately after collection. If this is not possible, each swab is placed in about 1 2 ml of sterile distilled water, to prevent desiccation, and the specimens taken to the laboratory within two hours after collection. Table 9.6: Summary of the disease and sites of isolation of some the Moraxella species Species Host Natural habitat Disease M. bovis Cattle Conjunctiva or nasopharynx of Infectious bovine keratoconjunctivitis Horses cattle older than 2 years Conjunctivitis M. lacunata Many animal species Opportunistic pathogen: septicaemia, Mucous membranes of abortions Humans animals Conjunctivitis M. ovis Sheep Mucous membranes Not pathogenic Psychrobacter phenylpyruvica Sheep & cattle Pigs Goats Urogenital tract Urogenital tract Intestinal tract Pathogenicity for animals is unknown Table 9.7: Differentiation of the Moraxella/Brahamnella species Characteristic M. bovis B. ovis M. lacunata P. phenylpyruvica Other Moraxella (not Betahaemolysis (blood) equine strains) Growth on MacConkey agar () d Oxidase Catalase () Nitrate reduction () () d Urease DNAse d Peptonisation of milk Gelatinase or Loeffler serum slope () () Direct microscopy Gramstained smears show Gramnegative short, plump diplobacilli. A fluorescent antibody technique will demonstrate and identify M. bovis if sufficient bacterial cells are present. Isolation Inoculate BTA and incubate at 35 C for 48 72 hours. The inoculation of the MacConkey plate is useful to gauge the degree of contamination by other Gramnegative bacteria, as M. bovis does not grow on this agar. Colony identification 8 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli After 48 hours of incubation, small greyishwhite colonies, surrounded by a narrow zone of betahaemolysis are seen. They look similar to streptococci. New isolates are often pilated and erode the agar, sinking into it. Colonial growth autoagglutinates when suspended in saline. Some colonies become nonhaemolytic. Equine strains of M. bovis are nonhaemolytic. So is M. lacunata and P. phenylpyruvica. Some strains of P. phenylpyruvica will grow on MacConkey agar. Moraxella bovis colonies enlarge markedly after 3 days of growth. Biochemical reactions Nonfermentative, nonmotile, indolenegative and all are sensitive to penicillin. M. bovis will slowly pit a Loeffler serum slope and will grow on 5% saline medium. Litmus or Crossley milk inoculated with M. bovis becomes alkaline (blue) = peptonisation, with three zones: a blue upperlayer, a soft blue curd in the centre and the bottom white. This helps distinguish it from other related species. Moraxella species have been differentiated by the analysis of cellular fatty acid. Animal inoculation The inoculation of virulent, haemolytic and pilated strains of M. bovis intraperitoneally into guineapigs or mice results in a fatal infection. PSEUDOMONAS SPP. AND OTHER RELATED GENERA A meaningful number of taxonomic changes have recently been made. This group includes Pseudomonas, Burkholderia, Stenotrophomas, Shewenella and others. Pseudomonas spp. are medium sized (0,5 1,0 x 1,5 5μm), Gramnegative rods. They are strict aerobes, oxidative, catalase and oxidasepositive, and motile by one or several polar flagella. Burkholderia mallei is the only one that is nonmotile. Many produce soluble pigments and most will grow on MacConkey agar. Diseases caused by Pseudomonas species in animals are shown in Table 9.8 and main differential characteristics are in Tables 9.9 and 9.10. B. mallei 9 P age Table 9.8: Pathogenicity and the normal habitat of Pseudomonas/Burkholderia species of veterinary importance Agent Natural habitat Disease in animals Notes nasopharynx of carrier horses B. pseudomallei soil & water P. aeruginosa (Bacillus of green pus) soil & water Glanders or farcy in horses Acute septicaemic disease in man. Melioidosis or pseudoglanders in most mammals. Septicemic disease, affects also the joints and lymph nodes Opportunistic pathogen: Cattle: mastitis, endometritis, abscesses, enteritis & arthritis. Sheep & goats: mastitis, pneumonia, green wool. Horses: metritis, respiratory infections & Zoonosis. Must use a biosafety cabinet when handling this bacterium. Zoonosis. Must use a biosafety cabinet when handling this bacterium. Polymicrobial resistance common

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli S. maltophilia, B. cepacia, P. putida, P. fluorescens, P. stutzeri, Shewanella soil & water mastitis. Dogs & cats: otitis externa, cystitis, endocarditis, dermatitis, wound infections & conjunctivitis. Reptiles: necrotic stomatitis & other necrotic lesions. Rare opportunistic pathogens. P. fluorescens causes food spoilage and lesions in reptiles & fish Sample contaminants Table 9.9: Main characteristics of the pathogenic Pseudomonas/Burkholderia species Characteristic P. aeruginosa B. pseudomallei B. mallei B. cepacia Pigment produced but colonies become orange but colonies are Yellow to cream yellow to brown Odour fruity grapelike Putrid, becoming earthy Sweet Growth on MacConkey Growth at 5 C Growth at 42 C d Oxidation of: Glucose Lactose Arginine dehydrolase () Reduction of nitrate to nitrite Reduction of nitrate to N2 gas v d d Motility LABORATORY DIAGNOSIS Specimens Swabs or tissue samples, taken from various sites with lesions, for culture and antimicrobial sensitivity test. Direct microscopy Seen as a medium sized Gramnegative rod in exudate smears. Not characteristic. A fluorescent antibody test can be used to detect B. mallei and B. pseudomallei Isolation Pseudomonas is nonfastidious and will grow on most bacteriological media. The growth of B. mallei is enhanced by 1% glycerol. A selective medium for B. mallei can be made by adding 1 000 units of polymyxin B, 1250 units of bacitracin and 0,25mg actidione to 100 ml of trypticase soy broth. Incubate aerobically at 37 C for 2448 hours. Some of the saprophytic Pseudomonas species such as P. 10 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli fluorescens will grow poorly if not at all at 37 C, and they need to be incubated at room temperature. Pseudomonas will grow in a candle jar, but prefer normal atmosphere. Colony morphology P. aeruginosa. Large greyishblue spreading colonies (3 4mm) on BTA. It has a characteristic grapelike odour due to aminoacetophenone. Most strains will produce betahaemolysis on BTA. The bacterium is nonlactose fermenting and produces various shades of blue to green pigments (pyocyanin) on MacConkey agar. Red colonies are seen on brilliant green and XLD agars, no H2S is produced. B. pseudomallei. Colony growth varies from smooth to mucoid to rough with a dull wrinkled corrugated surface. Aged colonies develop a yellowtinge. The growth has a characteristic musty odour. Partial and later complete haemolysis is seen on sheep BTA. It is lactosepositive on MacConkey agar (B. mallei is negative) but doesn t grow on deoxylate or SalmonellaShigella agar. 11 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli Table 9.10: Identification of common members of the Pseudomonaceae (all are motile) Oxidase Pyoverdin Glucose Maltose Lactose Mannitol Arginine Lysine Nitrite Nitrate Urea ONPG DNase Aesculin Polymixin Chryseomonas luteola v v v S RNA GROUP III Conamonas acidovoras* v C. terrigena NA S C. testosteroni NA S Flavimonas oryzihabitans v S RNA GROUP I Fluorescent group Pseudomonas V V V S aeruginosa* P. fluorescens V V V S P. putida V v S Stutzeri group P. stutzeri V V S P. mendocina V S CDC group Vb3 V S Alcaligenes group P. alcaligenes V V s P. pseudoalcaligenes V V S P. species group I V S RNA GROUP II Pseudomallei group Burkholderia pseudomallei v R B. mallei B. cepacia W V V V v R B. gladioli V R B. picketti v v v v v R RNA GROUP IV Diminuta group P. dimuta V V P. vesicularis V V V S P. paucimobilis S Shewenella putrifaciens V S RNA GROUP IV Xanthomonas maltophilia v S * Acetamide positive nonmotile 12 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli B. mallei. Growth is slower than that of P. aeruginosa, with smaller colonies at 2448 hours (1 2mm in diameter). Initially the colonies are smooth and white to cream, as they age they become yellow. B. mallei cannot grow on MacConkey agar. The opportunist, B. cepacia may be confused with both B. mallei and B. pseudomallei. Immunological tests Melioidosis: Complementfixation and indirect haemagglutination tests can be used to detect antibodies to B. pseudomallei. Diagnosis is however, based upon isolation and identification of the bacterium. Glanders: Complementfixation, indirect haemagglutination and counter immunoelectrophoresis tests are used in the diagnosis of glanders. False positive reactions may be obtained in areas that are endemic for melioidosis, due to cross reactivity. The mallein test that demonstrates hypersensitivity to B. mallei infection is used to detect carrier horses. Mallein is a glycoprotein extracted from the bacterium. Either subcutaneous injection or instillation into the conjunctival sac will result in a localised swelling in positive animals. Animal inoculation The Shwartzman phenomenon is seen in male guineapigs inoculated intraperitoneally with infective material containing either B. pseudomallei or B. mallei. A localised peritonitis and purulent inflammation of the testes develop in 2 3 days. Antimicrobial sensitivity tests These need to be carried out with most members of this group, but especially P. aeruginosa as multiple drug resistance occurs. Resistance is less common to the aminoglycoside group. SHEWENELLA Members of this genus are marine bacteria. Only S. putrefaciens and S. algae are important as opportunists in animals. Both are positive for H2S production on TSI agar, which is diagnostic. Shewenella algae can grow at 42 C and is haemolytic after 2 days, and S. putrefaciens cannot grow at 42 C and is not known to be haemolytic. The identification of Shewenella species is in Table 9.11. 13 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli Table 9.11: Identification of Shewenella species (all are Gramnegative curved or straight rods, catalase and oxidase positive, motile and grow in 3% NaCl. Negative for arginine dehydrolase, lysine or ornithine decarboxylase and indole production) Characteristic S. algae S. putrefaciens S. amazonensis S. baltica S. benthica S. hanedai S. frigidimarina S. gelidimarina S. oneidensis S. pealeana S. woodyi Luminescence Optimal growth temp 2535 2535 2535 415 #25 2022 1517 2535 2530 25 4 C in 24h D 35 C () 40 C Nitrate () Gelatinase () Lipase Haemolysis () H2S Growth in 0% NaCl Growth in 6% NaCl d Utilization of DGalactose () () DFructose Sucrose D Maltose D D Lactose D Succinate D D Fumarate () D () Citrate Flavobacterium and related bacteria These bacteria are common specimen contaminants that rarely cause disease. The natural habitats of these bacteria are soil, plants, foodstuffs and water sources. Their taxonomy has not yet been fully resolved. They are usually penicillin and polymixin B resistant and most species produce yellow pigments. They are divided into different groups: Group A: Group B: Group C: Group D: Saccharolytic, indole positive. Includes F. meningosepticum, F. group IIb and F. breve. CDC groups IIe, IIh and IIi are similar, but produce no pigment. Asaccharolytic, indole negative. Includes F. odoratum. Saccharolytic, indole negative. Includes F. thalpophilum, F. mizutaii, Sphingobacterium multivorum and S. spiritivorum. Asacchyrolytic, indole positive. Weeksella. Since species that are isolated in a clinical setting, i.e. F. meningosepticum, F. group IIb and F. odoratum, can be resistant to a number of antibiotics i.e. betalactams, tetracyclines, amphenicols and aminoglycosides, it is essential that antimicrobial susceptibility tests be done. As there are usually 14 P age

Chapter 9: Miscellaneous Gramnegative, nonfermentative bacilli errors in disk diffusion tests for this group MIC testing is recommended. These bacteria are often susceptible to the macrolides, potentiated sulphonamides and fluoroquinolones. APPENDIX 1 SmithBaskerville Medium for Bordetella bronchiseptica (Smith and Baskerville 1979) Bacto Peptone Sodium chloride Agar Distilled water 20g 5g 15g 857ml The basal medium is autoclaved at 121 C for 15 minutes and then cooled to 55 C. The following supplementary solutions are mixed together and added to the cooled agar medium. Antimicrobial supplement: Gentamicin Penicillin Furaltadone 0,5ug/ml 20ug/ml 29ug/ml Carbohydrate supplement: Glucose (10%) Lactose (10%) 100ml 100ml Bromothymol blue solution (filter sterilised) 2% Stock solution Bromothymol blue 0,1 N NaOH Distilled water Dilute to 0,2% solution 1g 25ml 475ml 40ml 15 P age