1. Non-Fermentative Gram-Negative Rods
Pseudomonas spp

2. Classification of Bacteria

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

4. 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

5. 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

6. 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

7. 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

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

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

10. 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

11. 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
seconds = delayed positive
>60 seconds = negative
Negative
Positive

12. 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

13. 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

14. 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.

15. 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

16. 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

17. 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

18. 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

19. 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.

20. 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)

21. 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

22. 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

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