All media must be sterile & the basic conditions for autoclaving
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All media must be sterile & the basic conditions for autoclaving   Temperature = 121 o C  Pressure = 15 PSI  Time = 20 minutes. Ordinary أكتر ميديا بتستخدم في نمو البكتريا Enriched غنية فيها اضافات البكتريا محتاجاها علشان تنمو وبالذات البكتريا الممرضة Enrichment

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All media must be sterile & the basic conditions for autoclaving 

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All media must be sterile & the basic conditions for autoclaving 

 Temperature = 121oC  Pressure = 15 PSI  Time = 20 minutes


  • Ordinary

  • أكتر ميديا بتستخدم في نمو البكتريا

  • Enriched

  • غنية فيها اضافات البكتريا محتاجاها علشان تنمو وبالذات البكتريا الممرضة

  • Enrichment

  • فيها حاجات تزود العدد بتاع البكتريا اللي موجودة بكمية قليلة علشان أزود عددها عن الـ N. Flora

  • Selenite broth  allow growth of Salmonella & prevent E. Coli

  • Selective

  • فيها مادة تخلي MO واحد يعيش والباقي يموت (تختارة)

  • Differential

  • فيها مادة تخلي شكل الـ MO مختلف عن الـ MO التاني غالباً يكون الاختلاف في اللون

  • Characteristic

  • ميديا بستخدمها في التعرف على البكتريا عن طريق الميتابوليزم بتاعها (Sugar fermentation )


  • Cultivation of bacteria

  • meat extract & Pepton &

  • 0.5% NaCl, neutral PH

  • light yellow transparent fluid

  • Indole Production

  • Base for sugar media

  • 1% Pepton + 0.5% NaCl + water


N.B + 1-5 – 2 % Agar

N.B + 10-15 % Gelatin


Differentiate M.O according 2 Hemolytic activity

N. Agar  100 C 55 C  5-10 % Sheep or Ox Blood


Simmon Citrate Agar

  • Upon citrate utilization the PH of the media will be increased causing change in color of the media into  blue

  • Due to Bromothymol Blue

  • Ability of MO to use citrate as carbon source for energy.

  • Degrade citrate producing CO2 which react with Na & water forming Na carbonate (alkaline product) which change color or BTB from green into deep prussian blue


Broth media + Sugars

(Glucose & Galactose & Lactose & Mannose & Maltose)

+

Phenol Red (Yellow in Acidic PH & Purple/Red in Basic PH)

+ Durham's tube (Gas indicator)


Cooked meat for Anaerobic ONLY

Glutathione


Characteristic media

Used in

Identification of Enteric organisms


  • Starch Hydrolysis

  • Test the ability of the organism to produce:

  • ExoenzymeAmylase which breaks down the Starch (Complex CHO of large molecule  Cannot pass through the cytoplasmic membrane) into Monosaccharide (MS = Simple can be used by the organism)

 Inoculate the Organism in Starch agar + add I2

 Amylase producing organism is surrounded by a clear zone(MS)

while the remaining of the media will stain with the violet color


  • Casein Hydrolysis

  • Test the ability of the organisms to produce:

  • Proteolyticexoenzymes (Proteinase which hydrolyze casein)

  • Casein  Main protein of milk Responsible for the white color of milk.

  • Hydrolysis of casein  Form more soluble & transparentcompounds (peptides &aa)

  • Upon growing the organism on casein media the area surrounding the proteinase producing organism will appear transparent.

  • Casein hydrolysis is called Peptonization or Proteolysis.


  • Gelatin Hydrolysis (Liquefaction)

  • Test the ability of the organism to produce:

  • ExoenzymeGelatinsae which liquefy gelatin.

  • Gelatin hydrolysis (Liquefaction) is indicated by:

  • loss in ability to solidify even after refrigeration


  • Catalase Production

  • Test the ability of the organism to produce:

  • Catalase enzyme that degradatesH2O2 O2 + H2O + Air bubbles.

  • H2O2 is added to the bacterial media

  • Presence of gas bubblesmeans that the organism produces catalase


  • Oxidase Production

  • Test the presence of Cytochrome Cin the respiratory chain.

  • Aerobic organisms with Cytochrome Ccan oxidize amines to form coloredproducts.

  • This Test is specific for Pseudomonas Aeruginosa.

  • Wet F. Paper with

  • 1% N,N,N',N' Tetra methyl - P-Phenylene-Diamine (TMPD) (KovacOxidase reagent)

  • allow to dry & Pick bacterial colony with sterile toothpick  add to F. Paper

  • A purple color is produced


  • Hemolysin Production

  • Test the ability of the organism to produce:

  • ExoenzymeHemolysin which has destructive effect on the blood cells


  • Urease Production

  • Test the ability of the organism to produce:

  • Urease enzyme which splits urea in urea media to form Ammonia + CO2

  • Accumulation of Ammonia will produce alkaline PH  Turns the color of indicator (phenol red) into Pink


  • H2S Production

  • H2S from Organic S or Inorganic S

  • Hydrogen Sulfide is detected by iron salt.

  • The presence of black precipitate is indication of H2S production.

  • Inoculate media peptone iron agar or TSI (Na2S2O3)

  • Black color will indicate H2S production


  • Sugar (CHO) Fermentation

  • Test the ability of the organism to produce:

  • Acid or Acid & Gas upon sugar fermentation


  • Nitrate Reduction

  • Test the ability of the organism to produce:

  • Nitrate reductaseenzyme which can reduce nitrate into nitrite

 Inoculate organism into nitrate broth

 Incubate at 37 C for 48 hrs

 Add 1 ml of coupling reagent

(sulfanilic acid & 1 ml of dimethyl alpha naphthyl amine reagent)

If the organism produce nitrate reductase the nitrate in the media will be reduced into nitrite & Color become red precipitate


  • Ammonia Production

  • Test the ability of the organism to:

  • Degradate the organic nitrogen in the protein into ammonia.

 Inoculate organism in 4% peptone water

 Incubate at 37 c for 2, 4, 7 days

 Add Nessler’s reagent.

Appearance of Yellow-Orange or brown color indicates +Ve test


IMViC tests used for  Identification & Differentiation of Enterobacteriaceae (Klebsiella & Enterobacter & E. Coli) ( All are Lactose Fermenters)


  • Indole Test

  • Test the ability of organism to break down tryptophan into indole.

  • Incubate tryptophan (Peptone) broth media with the tested organism.

  • The Presence of indole can be detected by Kovac’s reagent

  • (Para DimethylAminobenzaldehyde in amyl alcohol)

Kovac's reagent (yellow color) reacts with indole & produce

(red color) on the surface of the test tube.


MR Test


VP Test


  • Methyl Red Test

  • Test the ability of organism to ferment the glucose & produce acids which will change the color of M.R (PH indicator) into red color


  • VogasProskaurTest

  • Test the ability of organism to ferment the glucose & produce neutral products which will change the color of indicator into Pink color

  • The reagents used for the VP test are

  • Barritt's A (Alpha-Napthol) & Barritt's B (Potassium-Hydroxide)


  • MR & VP tests is done on MR-VP broth media

  • (contains glucose & peptone)

  • MR & VP tests:

    • E. Coli is (MR+/VP-)

    • Klebsiella & Enterobacteraerogenes is (MR-/VP+)


  • Citrate Test

  • Test the ability of organism to utilize citrate as its only source of carbon.

  • Simmon’s Citrate media used in this test

  • Bacteria can break citrate into organic acids & CO2 CO2 form a basic compound (Na2CO3)

Adding Bromothymol Blue Detects the presence of Na2CO3 by turning into blue (+Ve test)


Eosin-Methylene blue medium

  • Lactose / Esoin & MB

  • Permit differentiation between enteric lactose fermenters and non-fermenters

  • Alos in identification of E. coli


  • Lactose fermenter: purple black

  • Non- Lactose fermenter: colorless

  • E.coli: metallic green sheen


Motility Test

  • The medium contain triphenyltetrazolium which is reduced into red color by the stabbed bacterial growth.

  • Motile bacteria appear as diffused growth (with red color)

  • Non-Motile bacteria appear as single line of growth (the original stabbed line with pin color)


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