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No Biochemical test Medium and Its composition Nutrient sugar broth 1 Carbohydrates Fermentation test

10% aq. Soln of Test sugar: 10. ml Peptone water 90. ml 1% Andrades reagent 1. ml pH 7.0 Glucose ______________Xylose _______________ Maltose _______________ Mannitol _______________ Sucrose _______________ Lactose _______________

Indicator Andrads reagent

Procedure

Interpretation(+)

Interpretation(-) No color change. (Color less media)

Inoculate loopful Acid produced by Incubate at 37. c 24 hrs. fermentation, lowers Observe for Acid & Gas the pH of medium so indicator turns to pink in color. & Gas is collected in Durhams vial as gas bubble

& No gas bubble in Durhams vial

1 % Trptone broth 2 Indole Production test Tryptone NaCL D/w pH 1.00 gm. 0. 50 gm. 100 ml. 7.5

Distribute 5 ml in each tubes And autoclave them.

Glucose phosphate broth 3 Metyl-Red test Glucose K2HPO4 0.5 gm 0.5 gm

Peptone D/w pH

0.5 gm 100 ml 7.0

Distribute 5 ml in each tubes And autoclave them.

Inoculate loopful Ehrlichs Incubate at 37. c 24 hrs. reagent After incubation --Add 5 dropes of Xylene OR --Shake well --Allow the two layers Kovaks separate reagent --Add slowly 1ml Ehrlich reagen in slanting position of tube -- then observe it. Inoculate loopful Metyl red Incubate at 37. c 24 hrs. Indicator After incubation (pH indicator) --Add 2-3 drops of Methyl-red indicator and mix well. -- Note :transfer half volume of broth in other tube for Vi-P test

Development of Pinc/red color is bright fuchin red not developed at color (ring) at the the interphage. the interphase of reagent and the broth with in second

Development of Stable red color in broth indicate M-R positive.

Development of yellow or other color

No Biochemical test Medium and Its composition 4 VogusProskauer test Glucose phosphate broth Glucose K2HPO4 0.5 gm 0.5 gm

Indicator 5 % alcoholic - napthol & 40% KOH.

Peptone D/w pH

0.5 gm 100 ml 7.0


Bromothymol blue (pH indicator) yellow6.0 Blue-----7.6

Distribute 5 ml in each tubes And autoclave them. 5 Citrate utilization test Simmons citrate agar slant Sodium citrate 0.20 gm. MgSO4 0.02 gm. NaCl 0.50 gm. NH4H2PO4 0.10 gm. K2HPO4 0.10 gm. Bromothymol blue 0.008 gm. Agar 3.00 gm. D/w 100. 00 ml pH 6.9 Urea broth KH2PO4 0.91 gm Na2HPO4 0.95 gm. Yeast extrate 0.01 gm. n Phenol red dye Sol 0.001gm. 40 % Urea Sol 5.0 ml. pH 6.8 D/w water 95.0 ml. Note: Add sterilized urea after autoclaving
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Procedure Inoculate loopful Incubate at 37. c 24 hrs. After incubation -Add 0.6 ml -napthol per ml. broth and 0.2 ml KOH soln per ml. broth --Shake very well and allow it good aeration --Read result after 15-20 minutes (Dont read after 1hr.) Streak heavily on surface of agar slant Incubate at 37. c 24 hrs -- then observe it.

Interpretation(+) Development of red color with in15 min. --

Interpretation(-) Development of other color.?

Positive test is NO growth shown by color occure on slant change :From green to deep blue color of slant

Urease activity test

Phenol red dye pH indicator) yellow6.8 Blue-----8.4

Inoculate loopful Incubate at 37. c 24 hrs then observe it.

Red color of medium shows increase in pH due to urease activity.

No color change. (Color less media)

No Biochemical test Medium and Its composition

Indicator

Procedure

Interpretation(+)

Interpretation(-)

2 % Peptone broth 7 Hydrogen sulphide production test Peptone NaCl D/w pH 2.0 gm. 0.5 gm. 100. ml 7.2

Lead acetate. (paper strip)

Inoculate loopful. Place wet lead acetate at the neck of the tube such 2 /3 strip projected below the cotton plug. Incubate at 37. c 24 hrs Inoculate loopful. Place red litmus paper strip the neck of the tube such 2/3 strip projected below the cotton plug. Incubate at 37. c 24 hrs After incubation --Add 0.5 ml. reagent A -- Add 0.5 ml. reagent B Observe red color with in 30 seconds

Strip will appear blackish due to formation of Lead sulfhide

No change in color of strip

Ammonia production test &

Peptone nitrate broth Meat extract 0.3 gm Peptone 0.5 gm Potassium nitrate 0.1 gm D/w 100. ml pH 7.5 Distribute 5 ml in each tubes And autoclave them.

Red litmus paper

Litmus paper turns to red color due to Ammonia production.

No color change of strip

Nitrate reduction test

napthylamine (Reagent A) Sulphanilic acid (Reagent-B)


10% aq. Sol of Ferric chloride
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If ,color is not seen --Add pinch of Zinc dust and put for 2 min. -- if color is not change to red ,the test is positive Immediately appear an intense green color on slant indicates the presence of PAA

If color of broth is changed to red after addition of Zinc dust, It indicates as negative Green color will not appear

Phenylalanine agar 9 Phenylalanine deamination test Phenylalanine Yeast extract Na2HPO4 NaCl Agar D/w pH 0.2 gm 0.3 gm 0.1gm 0.5 gm. 2.0 gm 100. ml 6.9

Streak heavily on surface of agar slant Incubate at 37. c 24 hrs After incubation --Add 5 drops of FeCl3 on surface of slant -- then observe it.

No Biochemical test Medium and Its composition 10 Catalase test Nutrient agar slant Meat extract 0.3 gm NaCl 0.5 gm Peptone 1.0 gm Agar 3.0gm D/w 100.0 ml pH 7.2 Triple sugar iron agar slant Meat extract 0.3 gm Yeast extract 0.3 gm Peptone 1.5 gm Protease peptone 0.5 gm Lactose 1.0 gm Glucose 0.1 gm. Sucrose 1.0. gm Ferrous sulphate 0.02 gm Sodium thiosulphate 0.03 gm Sodium chloride 0.50 gm Agar 3.00 gm D/w 100 ml pH 7. 4 Milk Agar Plate 12 Pigmentation test Nutrient agar Milk 90.0 ml 10.0 ml

Indicator 3% H2O2

Procedure Streak out agar slant Incubate at 37. c 24 hrs After incubation --Add drops wise H2O2 on growth of organisms.

Interpretation(+) Rapid appearance and sustainable production of gas bubbles on the slant.

Interpretation(-) No bubble formation occur

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Triple Sugar Iron test

Phenol red dye (pH indicator) yellow6.8 Blue-----8.4

Streak a loopful of test culture on slant surface and stab the same the same culture in to the butt of the slant - Incubate at 37. c 24 hrs After incubation ,observe for Aci / Gas / H2S production

Lactose fermenting: Slant: Yellow(Acidic) Butt : Yellow(Acidic) Lactose non fermenting Slant: Pink(Alkaline) Butt : Yellow(Acidic) Non fermenting Slant: Pink(Alkaline) Butt : Pink(Alkaline) H2S production: Black color appear Gas production resulting in cracking of butt and slant

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add sterile milk after autoclave to media and pour it

Streak the plate by four flame method Incubate at 37. c 24 hrs After incubation ,observe colony characteristic (pigment of colony).

Pigment will be seen against white back ground

Growth occur but no pigmentation

No Biochemical test Medium and Its composition

Indicator

Procedure

Interpretation(+)

Interpretation(-)

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Gelatin Hydrolysis test

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Motility test

Nutrient gelatin Meat extract 0.3 gm Peptone 1.0 gm Gelatin 15.0 gm D/w 100 gm pH 7.2 Motility agar Meat extract 0.3 gm NaCl 0.5 gm Peptone 1.0 gm Agar 1.0gm D/w 100.0 ml pH 7.2

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CInoculate loopful Incubate at 37. c 24 hrs then observe it.

At low temperature (2-10 c), incubated Medium remains in a liquid state

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Stab the culture

slant

with Dispersive growth will appear

At low temperature (2-10 c), incubated Medium remains in a semi-solid state Only growth occurs

Nutrient broth Meat extract 0.3 gm NaCl* 0.5 gm Peptone 1.0 gm Effect of 100.0 ml 16 Temperature D/w pH * 7.2 * Note: Adjust pH and Salt Effect of salt as per 17 concentration concentration requirements for different tests MacConkeys agar 18 Lactose Peptone 2.0 gm fermentation Lactose 1.0 gm test NaCl 0.5 gm Bile salts 0.5 gm Neutral red 3.0 mg. Crystal violet 1.0 mg D/w 100.0 ml Agar 3.0 gm. pH 7.4 No Biochemical test Medium and Its composition 15 Effect of pH

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Inoculate loopful Turbidity will be Incubate*at 37. c 24 hrs seen due to growth, then observe it. compare to uninoculate broth * Note: Incubate tubes at different temperature to test effect of temperature Streak the plate by four flame method Incubate at 37. c 24 hrs After incubation ,observe colony characteristic

NO turbidity

Neutral red dye

Lactose fermenting Lactose non colonies appear fermenting pink in color colonies do not appear pink in color

Indicator

Procedure

Interpretation(+)

Interpretation(-)

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Starch hydrolysis test

Starch agar plate 10% soluble starch Nutrient agar

Lugols Iodine

5.0 gm. 100.0 ml

Add soluble starch in N. Agar and autoclave it

Streak the plate by four flame method Incubate at 37. c 24 hrs After incubation ,observe zone of utilization of starch --Flood the plate with lugols iodine

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