1. IDENTIFICATION OF BACTERIA

2. BIOCHEMICAL REACTION
The most widely used tests in microbiology laboratories.

3. BIOCHEMICAL TESTS Sugar fermentation Iodole production Methyl red test
Voges-Proskauer test
Citrate utilization
Urease test
Catalase production
Oxidase reaction
Triple sugar iron medium

4. OTHER TESTS Litmus milk Nitrate reduction Production of ammonia
Hydrogen sulphide production
Methylene blue reduction
Egg yolk reaction
Growth in presence of KCN

5. SUGAR FERMENTATION Tested in sugar media
Acid production is shown by change in color of the medium to pink/red
Gas produced get collected in Durham’s tube

6. SUGAR FERMENTATION

7. UREASE Urea-is a diamide of carbonic acid
All amides are easily hydrolysed with the release of ammonia and CO2 – by the organisms that can hydrolyse urea.
The amonia reacts in solution to form ammonium carbonate,resulting in alkalinization of the medium
Step 1:Urea+water→(in the presence of urease) ammonia + carbondioxide
Step 2: phenopthaline(colourless PH<8.1) →in alkaline media due to ammonia become pink red(PH >8.1)

8. Media and reagents Stuart’s urea broth: Yeast extract
Monopotassium phosphphate
Disodium phosphate
Urea
Phenol red
Distilled water
PH-6.8

9. Media and reagents Christensen’s urea agar: Peptone Glucose
Sodium chloride
Monopotassium phosphate
Urea
Phenol red
Agar
Distilled water
PH – 6.8

10. POSITIVE CONTROL: proteus species, klebsiella
Negative control: escherichia coli
Procedure: the broth medium is inoculated with a loopful of a pure culture of the test organism , the surface of the agar slant is streaked with the test organism and inoculated at 35deg C for 18 to 24 hrs

11. Less active species may take 3 or more days.
RESULT : organism that hydrolyse urea rapidly may produce pisitive reaction within 1-2 hrs
Less active species may take 3 or more days.
In stuarts broth:red colour through out the media indicates alkalinazitation and urea hydrolysis
In christensen’s media:
1. rapid urea spliters- (proteus species) produce red colour throughout the media.
2. slow urea splitters(klebsiella species) red colour initially in slant only ,gradually converting the entire tube.
3. no urea hydrolysis – media remain original yello colour.

12. UREASE TEST EXAMPLES POSITIVE:Proteus Klebsiella H.pylori
NEGATIVE: E.Coli
Salmonella

13. COMPOSITE MEDIA(TSI)
COMPOSITE MEDIA-different properties of the bacterium can be studied and interpreted using a single medium.
TSI(Triple sugar iron agar):
a)tests whether the bacterium utilizes glucose only , or lactose and sucrose also either by oxidative or fermentative method.
b)gas formation present or not
C)H2S production present or not.

14. Triple sugar iron agar
Medium
Beef extract – 3g
yeast extract – 3g
peptone 20g
glucose 1g
lactose 10g
sucrose 10g
ferric chloride ,Nacl – 5g
sodium thiosulphate 0.3g
agar- 12g
phenol red, 0.2 %solution 12ml
distilled water -1 liter
heat to dissolve the solids add the indicator solution mix and tube sterile at 121 degree Celsius for 15 min and cool to form slop with deep 3 cm butts.

15. TSI – triple sugar iron agar:
Kligler iron agar:
Standard test for testing H2S production
It is a multi-test medium
Disadvantage chemical interaction, acid production from fermentable sucrose may inhibit blackening of the iron indicator.

16. Method :
Streak a heavy inoculum over the surface of the slope and stab into the butt, incubate aerobically at 37 degree Celsius for 24 hrs.

17. TSI MEDIA-Consist of a butt and slant
INTERPRETATION:
After inoculation,if slant remains red and butt becomes yellow it indicates that all the three sugars are fermented.(eg)E.coli
Bubbles in the butt indicates gas production.(eg)Klebsiella spp
Blackening of the medium indicates H2S production.(eg)Proteus spp

18. TSI MEDIA

19. Oxidative fermentative (OF) test: Principle, procedure and results
The oxidative-fermentative (OF) test was developed by Hugh and Leifson in They developed OF media to differentiate between oxidative bacteria (that produces acid from carbohydrates under aerobic condition only) and fermentative bacteria (that produces acid both under aerobic and anaerobic conditions).

20. Saccharolytic microorganisms degrade glucose either fermentatively or oxidatively.
The end products of fermentation are relatively strong mixed acids that can be detected in a conventional fermentation test medium. However, the acids formed in oxidative degradation of glucose are extremely weak and less, and the more sensitive oxidation fermentation medium of Hugh and Leifson’s OF medium is required for the detection.  The medium was made by increasing the amount of glucose above that found in medium used to detect fermentation and by decreasing the amount of peptone.

21. The OF medium of Hugh and Leifson differs carbohydrate fermentation media as follows:
The concentration of agar is decreased to 2% from 3%, making it semisolid in consistency (This assists in the determination the motility of the organism).
The concentration of peptone is decreased from 11% to 2%. (decreasing the amount of alkaline product produced by the metabolism of peptone; thus reducing the neutralizing effect of these products).
Carbohydrate concentration is increased by 0.5% to 1.0% (The increased concentration of glucose in the medium enhances the production of these weak acids to a level that can be detected by bromthymol blue indicator.)

22. Principle:
The oxidative-fermentative test determines if certain gram-negative rods metabolize glucose by fermentation or aerobic respiration (oxidatively). During the anaerobic process of fermentation, pyruvate is converted to a variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during fermentation will turn the bromthymol blue indicator in OF media from green to yellow in the presence or absence of oxygen .
Certain nonfermenting gram-negative bacteria metabolize glucose using aerobic respiration and therefore only produce a small amount of weak acids during  glycolysis and Krebs cycle. The decrease amount of peptone and increase amount of glucose facilitates the detection of weak acids thus produced.
 Dipotassium phosphate buffer is added to further promote acid detection.

23. Uses: OF Test is used to determine if gram-negative bacteria metabolize carbohydrates oxidatively, by fermentation, or are nonsacchrolytic (have no ability to use the carbohydrate in the media).
Media: Hugh and Leifson’s OF basal medium; the constituents are as follows:
Sodium chloride : 5.0 g
Di-potassium phosphate : 0.3 g
Peptone : 2.0 g
Bromthymol blue:0.03 g
Agar: 3.0 g
Glucose : 10 g
Water : 1000 ml
The pH should be adjusted to 7.1 prior to autoclaving. After the medium is autoclaved at 121°C for 15 minutes, a filter sterilized solution of 10% solution of carbohydrate is aseptically added to the medium to a final concentration of 1%.

24. Procedure
Inoculate two tubes of OF test medium with the test organism using a straight wire by stabbing “half way to the bottom” of the tube.
Cover one tube of each pair with 1 cm layer of sterile mineral oil or liquid paraffin (it creates anaerobic condition in the tube by preventing diffusion of oxygen), leaving the other tube open to the air.
Incubate both tubes at 35oC for 48 hours (Slow growing bacteria may take 3 to 4 days before results can be observed)
Interpretation
Acid production is detected in the medium by the appearance of a yellow color. In the case of oxidative organisms; color production may be first noted near the surface of the medium.

26. Oxidative Fermentative Test Following are the reaction patterns:
Open (Aerobic) Tube
Covered (Anaerobic) Tube
Metabolism
Acid (Yellow)
Alkaline (Green)
Oxidative
Fermentative
Non saccharolytic (glucose not metabolised)

27. Fermentative result: Acid production on both (open and covered) tubes
Fermentative result:  Acid production on both (open and covered) tubes. The acid produced changes the pH indicator, bromthymol blue, from green to yellow. e.g. Escherichia coli 
Oxidative result: Acid production in the open tube (aerobic) and not the oil-covered tube (anaerobic) indicates an oxidative result. Nonfermenting bacteria that metabolize glucose via oxidative metabolism give an oxidative result. e.g. Pseudomonas aeruginosa 
Non saccharolytic (Negative OF result): Nonsacchrolytic bacteria give a negative OF result. The negative result is indicated by no color change in the oil-covered tube and in some cases an increase in pH (pH 7.6) changing the bromthymol blue from green to blue in the top of the open tube. The increase in pH is due to amine production by bacteria that break down the peptone (protein) in the medium. e.g. Alcaligenes faecalis.
Quality Control
Glucose Fermenter: Escherichia coli
Glucose oxidizer: Pseudomonas aeruginosa
Nonsaccharolytic: Moraxella species