1. PHT313
Lab. No. 4

2. Classification of Bacteria

3. Most Important Species
Pseudomonas
Aerobic, non-fermentative, motile, oxidase-positive gram-negative bacilli.
Most Important Species
P.aeruginosa
opportunistic pathogen
causes UTI, wound infections and otitis media

4. Identification of pseudomonas species
(I) Microscopical examination
(morphology):
A) Gram’s Stain:
Gram –ve
Non-sporeforming bacilli ,
having single arrangement.

5. B) Examination of Motility:
Using the “Hanging Drop technique”
Pseudomonas is highly motile by means of polar flagella.

7. (II) Cultural characteristics:
It grows on simple media.
It usually produces exopigments.
1) Growth on nutrient agar:
Its growth on nutrient agar
showing greenish discolouration
due to exopigment production.

8. 2) Growth on Cetrimide Agar:
Principle:
Cetrimide agar is a highly selective medium for pseudomonas species due to presences of cetrimide which inhibits the growth of other bacteria.
It contains also MgCl2 & K2So4 to facilitate production of the charactaristic green pigment of pseudomonas.

9. Results:
Only Pseudomonas species can grow on cetrimide agar showing growth of pale colonies with diffusion of green pigmentation.

10. Procedure: Inoculate Cetrimide agar plate with the test
organism by streaking.
Flam & Cool
2. Incubate the plate at 35oC for 24 hrs.

11. 3) Growth on MacConkey’s agar:
Principle:
MacConkey’s agar is a selective and differential medium
selective medium for gram –ve bacteria (bile salt & crystal violet inhibit the growth of gram +ve bacteria).
Test sugar: lactose.
pH indicator: neutral red ( yellow in alkaline, pink in acidic pH).

12. Gram –ve bacteria are classified into:
Lactose fermenter
(Pink colonies)
Lactose non-fermenter
(pale colonies)

13. Inoculate MacConkey’s agar plate with the test organism by streaking.
Procedure:
Inoculate MacConkey’s agar plate with the
test organism by streaking.
Flam & Cool
2. Incubate the plate at 35oC for 24 hrs.

14. Results: Pink colonies Lactose fermenter Pale colonies
Lactose nonfermenter

16. Tetramethyl p-phenylene diamine
(III) Biochemical reactions:
1)Oxidase test:
Principle:
Tetramethyl p-phenylene diamine
(oxidase reagent)
colourless
Cytochrome oxidase enzyme
Indophenol
(Purple colour)

17. Results: +ve Test: Appearance of purple colour within 1-2 min.
No colour
purple colour
-ve test
+ve test

18. Results: +ve Test: Appearance of purple colour within 1-2 min.
No colour
purple colour
-ve test
+ve test

19. 2) Nitrate Test: Principle: Red diazonium salt If no red colour!
zinc dust
Nitrate reductase
Further reduction
Nitrate
nitrite
Nirtogen (N2)
α-naphthyl amine (nit. A)
Sulphanilic acid (nit. B)
Red diazonium salt
If no red colour!
Add zinc dust (reducing agent)

20. Procedure: No red colour Add zinc dust Red colour Nit.A test m.o
Nit. B
Incubate at 35oC for 24 hrs
No red colour
Nitrate broth
Add zinc dust

21. Results: No red colour after addition of zinc dust
Red colour after addition of nit.A & nit.B
Red colour after addition of zinc dust
No red colour after addition of zinc dust
Reduction of Nitrate to nitrite
-ve reduction
Further reduction to Nitrogen

22. 3) Oxidation Fermentation (O/F) Test:
Principle:
O/F medium ( Hugh and Leifson Medium) is a specifically formulated medium (sensitive O/F medium) to detect weak acids produced from saccharolytic Gram’s –ve bacteria.
i.e: Its composition differs from ordinary fermentation medium to be more sensitive to detect the small amount of weak acids produced by Gram’s –ve bacteria.

23. Principle: To be more sensitive this medium contains:
Higher conc. Of sugar to increase amount of acid produced.
Lower amount of peptone to reduce formation of alkaline amines which neutralize weak acids formed.
Lower conc. Of agar making the medium semisold to facilitate diffusion of acid throughout the medium.

24. Procedure:
1 ml liquid paraffin O F

25. O-/F+ O-/F- O+/F+ O+/F- Results: Positive Test: Non Saccharolytic
Fermentative
Oxidative

26. 4) Growth on Triple Sugar Iron (TSI) agar:
Principle:
This medium contains three types of sugars (lactose, sucrose and dextrose).
The conc. of lactose and sucrose is 10 times that of dextrose.
It also contains phenol red indicator.

27. Principle:
butt
slant

28. Results: 1. No Fermentation: Butt: alkaline (red)
Slant: alkaline (red)

29. a) Initial reaction: 2. Dextrose Fermentation: (after 10 – 12 hrs)
acid
Butt: acidic (yellow)
Slant: acidic (yellow)

30. b) Delayed reaction: 2. Dextrose Fermentation: (after 24 hrs) O2
Peptone O2
Small amount of acid
Alkaline amines
Butt: acidic (yellow)
Slant: alkaline (red)

31. 3. Lactose Fermentation:
Peptone O2
Large amount of acid
Alkaline amines
Butt: acidic (yellow)
Slant: acidic (yellow)

32. Principle:
This medium contains also ferrous sulfate as an indicator for H2S production.
H2S + FeSo4
FeS
Black ppt. of ferrous sulfid

33. Procedure:

34. Results: acidic (yellow) acidic (yellow) acidic (yellow) Butt: Slant:
H2S Production:
acidic (yellow)
alkaline (red)
alkaline (red)
-ve
+ve
-ve

36. Practicle Work Gram’s stain (spot) Growth on Cetrimide agar
Growth on MacConkey agar
Oxidase test.
Nitrate test.
O/F test.
TSI test.