Non-Fermentative Gram-Negative Rods Pseudomonas spp

Classification of Bacteria

Gram-negatitive Bacilli Oxidase Test Oxidase positive Oxidase Negative O/F O/F O+/F+ O+/F+ O+/F- Pseudomonadaceae Vibrionaceae Enterobacteriaceae

Characters of Pseudomonas Gram-negative bacilli belonging to Pseudomonadaceae Motile by means of a single polar flagellum. Non spore forming Capsulated "Polysaccharide capsule" Aerobic Breakdown glucose by oxidation i.e. Oxidative Oxidase and catalase positive It has very simple nutritional requirements i.e. non fastidious The most important pathogenic organism is Ps. aeruginosa Optimum temperature is 37 C, and it is able to grow at 42 C It is resistant to high concentrations of salts, dyes, weak antiseptics, and many antibiotics Common inhabitants of soil, water, GIT

Ps. aeruginosa is opportunistic pathogen and associated with a variety of infections including: Urinary tract infections Wound and burn with blue green pus Respiratory system infections (Pneumonia) Eye infection and may lead to blindness Ear infection (external ear or otitis media) Meningitis A variety of systemic infections Ps. aeruginosa produce two types of soluble pigments: Pyoverdin or fluorscein: It is yellow-green pigment and fluorescent Pyocyanin: It is a blue-green pigment and non-fluorescent

Identification of Ps. aeruginosa Laboratory diagnosis Specimen: Urine, pus, sputum, CSF, blood, skin swap according to the type of infection Microscopical Examination Gram Stain: Gram-negative rods Motility Test: Hanging Drop Techniques Semisolid agar medium Motile

Cultural Characteristics On Nutrient agar: Colonies are surrounded by bluish green coloration On selective media "Cetermide" Pigments are more obvious On Blood agar -hemolytic colonies On MacConkey agar Pale yellow colonies i.e. non lactose fermenters Ps. aeruginosa able to grow at 42 C for 3 days

Cultural Characteristics Ps. aeruginosa on cetrimide agar Gram Stain of Pseudomonas Ps. aeruginosa on Nutrient agar

Biochemical Reactions Oxidase positive Breakdown glucose oxdatively Nitrate Reductase negative Gelatinase positive Utilize Citrate

Oxidase Test: Principal Alternative substrate for Cytochrome Oxidize the reagent from colorless to purple color Oxidase Reagent Indophenol Cytochrome Oxidase Tetramethyl-P-Pheneylenediamine Colorless Purple color Pseudomonas Vibrio Play role in aerobic respiration

Use platinum loop (not used nichrome) or wood stick Results Method: hold a piece of the oxidase test paper with forceps and touch onto an area of heavy growth Use platinum loop (not used nichrome) or wood stick Results Color change to purple within: 10 seconds = positive 10 - 60 seconds = delayed positive >60 seconds = negative Negative Positive

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

O/F Test: Principal O/F medium differs from carbohydrate fermentation medium to be more sensitive to detect the small amount of weak acids produced by M.O. O/F medium is more sensitive due to: Higher % of glucose to increase amount of acid produced Lower % of peptone to reduce formation of alkaline amines which neutralize weak acids formed Lower % of agar making the medium semisolid to facilitate diffusion of acid throughout the medium

O/F Test: Procedure Each organism is inoculated into two tubes of glucose O/F medium Inoculation is carried out as a stab to within 1 cm of the bottom of the tube One of which is covered with mineral oil to exclude oxygen Incubate at 37°C for 24 hours.

Non-Saccharolytic O-/F Open & covered remain green O/F Test: Results There are three types of reactions possible Reaction 1 Reaction 2 Reaction 3 Non-Saccharolytic O-/F Alcaligenes faecalis Open & covered remain green Oxidative O+/F- Pseudomonas Open turns yellow Fermentative O+/F+ Enterobacteriaceae Both turn yellow

Gelatin Liquifaction Test: Principle Certain bacteria are capable of producing a proteolytic exoenzyme called gelatinase Gelatinase hydrolyze the protein (solid) to amino acids (liquid) At temperature below 25°C, gelatin will remain a gel, but if the temperature rises about 25°C, the gelatin will be liquid. Gelatin hydrolysis has been correlated with pathogenicity of some microorganisms Pathogenic bacteria may breakdown tissue & spread to adjacent tissues Pseudomonas Gelatinase Incubation at 37/overnight Gelatinase hydrolyze the protein to aminoacids Nutrient gelatin Amino acids Liquid at > 25 C Nutrient gelatin Protein/Polypeptides Solid

Gelatinase Test: Procedure Stab M.O. If tube remains solid No change -ve E. coli Incubate at 37 C overnight If tube liquefied at > 25 C +ve Nutrient gelatin Ps. aeruginosa

Gelatin Liquifaction Test Method Stab a nutrient gelatin tube with inoculums of the tested organism Inoculated nutrient gelatin tube is incubated at 37°C for 24 h Result If a tube of gelatin liquefy indicates positive test (Ps. aeruginosa) If a tube of gelatin remains solid indicates negative test (E. coli) Positive test Negative test

Nitrate Reductase Test Principle To determine the ability of an organism to reduce nitrate to nitrites or free nitrogen gas Method Inoculate a nitrate broth with tested M.O. incubate for 24 hrs at 37°C. After incubation, add 1 ml of sulphanilic acid and 1 ml of -naphtylamine to nitrate broth tube Result The production of a red color occurs in the presence of nitrite indicates the ability of the organism to reduce nitrate to nitrite. To broths showing a negative reaction add a few particles of zinc. The appearance of a red color indicates that nitrate is still present and hence has not been reduced by the organism. If the solution does not change color the organism has reduced the nitrate through nitrite to nitrogen gas.

Nitrate Reductase Test: Principal Further reduction Nitrate reductase Nitrate (NO3) Nitrite (NO2) Nitrogen gas N2 Sulfanilic acid α-naphthylamine Red diazonium salt If no red color! Add zinc dust (reducing agent)

Nitrate Reductase Test: Procedure Red color M.O. 1m Sulfanilic acid Incubate at 37oC for 24 hrs 1m -naphthylamine Nitrate broth Add zinc dust No red color

Nitrate Reductase Test: Results Red color after addition of sulfanilic acid & -naphtylamine No red color after addition of zinc dust Red color after addition of zinc dust Reduction of Nitrate to nitrite -ve reduction Nitrate unreduced Nitrate reduced into nitrite and further reduction to Nitrogen

Practical Work Gram stain Growth on Cetrimide agar Oxidase test O/F test Nitrate reductase test Gelatinase test Citrate Utilization Test (See under Enterobacteriacea)