Media

Basic ordinary

Enriched

Differential &Selective

CHO

Yeast &

Fungi

Anaerobic

Characteristic

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•

• Enrichedغنية فيها اضافات البكتريا محتاجاها علشان تنمو وبالذات البكتريا الممرضة •

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

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

• Selective واحد يعيش والباقي يموت )تختارة( MOفيها مادة تخلي •

• Differential \ يكون االختالف في اللونMO مختلف عن الـ MOفيها مادة تخلي شكل الـ • التاني غالبا

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

Plate Media

Nutrient Agar

Blood Agar

Chocolate Agar

MAC

Liquid Media

Nutrient Broth

Peptone H2O

Cooked Meat

Thioglycolate

Media

Sabouraud’s media

Solid&

Slant

Nutrient Agar Slant

DeepAgar

Gelatin Media

Loffler’s Serum

L-J Media

SimmonCitrate

ChristensenUrea

Blood AgarContain growth factors & other complex organic substances like

blood, serum, etcBetter for Fastidious Pathogenic bacteria

Enriched media

Selenite broth that used for Salmonella

Provides nutrient & environmental conditions that favor the growth of certain organisms but not suitable for others.

Enrichment media

MacConkey’s media

Simmon’s Citrate media

Contains chemicals & dyes that inhibits the growth of certain bacteria

While not interfere with the growth of other bacteria.Addition of bile salt to the media make this media selective to

Pathogenic Enteriococci (Salmonella & Shigella)

Selective media

Differentiate ( ) the bacteria by change in the color of the growing colonies

Differential media

Triple sugar iron (TSI)

Lysin iron agar (LIA)

Sulfide indole motility (SIM)

Used to test the organism for Metabolic activity Or Metabolic products Or Metabolic

requirementsUseful in identification of the type of the bacteria.

Characteristic media

Cultivation of bacteriameat extract & Pepton &

0.5% NaCl, neutral PHlight yellow transparent fluid

Fluid Basic

Ordinary Media

Nutrient

Broth

Peptone

Water

Indole ProductionBase for sugar media

1% Pepton + 0.5% NaCl + water

Nutrient AgarAgar Plate

Isolation of bacteria(Streaking for isolation)

Agar SlantShort Storage

(3-6 weeks)

Deep AgarProlonged Storage

(6 months)

Gelatin Media Gelatin Liquefaction

Proteolytic activity

N.B + 10-15 % Gelatin

N.B + 1-5 – 2 % Agar

Enriched

BloodAgar

Chocolate Agar

Loffler’sserum Lowentsein

Blood Agar

Alpha

Incomplete (partial) & green zone

Streptococcus

Pneumonia

Beta

Complete & Clear zone

Staph. aureus

St. Pyogenes

Gamma

No Change

St. Faecalis

Differentiate M.O according 2 Hemolytic activity

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

Lowenstein Jensen Loffler’s Serum Chocolate

Mycobacterium TB Corynbacterium DiphtheriaH. Inflenza

&Neisseria

Malachite green (Selective& Enriched) Horse serumHeated sheep

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

Differential & Selective

MACSelective Inhibits the growth of

Gm+Ve due to the presence of Crystal violet &

Bile saltsGm –Ve

bacteria grow well

Differential Differentiate ( ) bacteria on the basis of a color

change reaction

MAC contains: Lactose Neutral red

Simmon’s Citrate

Upon citrate utilization the PH of the media will be

increased causing change in color of the media into

blueDue to Bromothymol Blue

Example of Non-Lactose Fermenters Example of Lactose Fermenters

Salmonella & Shigella & Proteus species& Pseudomonas aeruginosa

E. Coli & Klebsiella

Colorless Pink Colony

No fermentation & PH indicator remains Purple Non-Fermenter

Fermentation with the production of acid(Yellow color) but no gas

Fermenter-Acid Producer

Fermentation with the production of acid(Yellow color) and gas (Bubbles in Durham tube)

Fermenter-Acid & Gas Producer

Broth media + Sugars (Glucose & Galactose & Lactose & Mannose & Maltose)

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

+ Durham's tube (Gas indicator)

Fluid Saboura

ud’s

Yeast & Fungi

Sterility Test(Saliva

&Candida )

4% Glucose & 5.6 Acidic

PH

Incubation 25C for 10

days

Thioglycollate

AerobicAnaerobicFacultative

Sterility Test

Na Thioglycollate

Cystine (Reducing

agents)

Small amount of Agar

(Decrease oxygen diffusion )

Methylene green

Methylene blue

Resazorine(O2 indicator)

Incubation 30-35C for 7 days

Cooked meat for Anaerobic ONLYGlutathione

LactoseSucrose Glucose

FAS

Na2S2O3

TSICharacteristic

media Used in

Identification of Enteric organisms

Hydrolysi

s

StarchCasein

Gelatin

Starch HydrolysisTest the ability of the organism to produce:

Exoenzyme Amylase 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 HydrolysisTest the ability of the organisms to produce:

Proteolytic exoenzymes )Proteinase which hydrolyze casein(

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

Hydrolysis of casein Form more soluble & transparent compounds )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:

Exoenzyme Gelatinsae which liquefy gelatin.

Gelatin hydrolysis )Liquefaction( is indicated by:

loss in ability to solidify even after refrigeration

Production

Catalase

Oxidase

HemolysinUrease H2S

Ammonia

Nitrate

Acid Or Gas

Catalase Production

Test the ability of the organism to produce:

Catalase enzyme that degradates H2O2 O2 + H2O + Air bubbles.

H2O2 is added to the bacterial mediaPresence of gas bubbles means that the organism produces

catalase

Oxidase ProductionTest the presence of Cytochrome C in the respiratory chain.

Aerobic organisms with Cytochrome C can oxidize amines to form

colored products.

This Test is specific for Pseudomonas Aeruginosa.

Wet F. Paper with

1% N,N,N',N' Tetra methyl - P-Phenylene-Diamine )TMPD( (Kovac Oxidase

reagent)

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

A purple color is produced

Hemolysin ProductionTest the ability of the organism to produce:

Exoenzyme Hemolysin which has destructive effect on the blood

cellsBlood Agar

Beta

Complete & Clear zone

St. Pyogenes

Alpha

Incomplete (Partial) & green zone

St. Pneumoni

a

Gamma

No Change

St. Faecalis

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 ProductionH2S 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

CHO Media No

Fermentation

No Acid No Gas

Phenol Red = Red

Fermentation

Acid Production /

No Gas

Phenol Red = Yellow

Fermentation

Acid & Gas Production

Phenol Red = Yellow

Bubble in Durham’s

tube

Nitrate ReductionTest the ability of the organism to produce:

Nitrate reductase enzyme 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

IndoleMR VP

Citrate

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

The presence of E. coli The presence of Enterobacter & KlebsiellaIndicate fecal contamination

of food & waterDoes not Indicate fecal contamination because

they are widespread in soil & grass

Indole TestTest 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 Dimethyl Aminobenzaldehyde 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

E. Coli Klebsiella and Enterobacter

Produces acidic products from glucose

PH drop below 4.4

Adding MR indicator Cherry red color (Positive MR test)

Produce neutral products from glucose

(Ethyl alcohol & Acetyl methyl carbinol)

PH rise above 6.2

Adding MR indicator Yellow color

(Negative MR test)

Vogas ProskaurTestTest 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)

E. ColiKlebsiella and Enterobacter

Produces acidic products from glucose

PH drop below 4.4

Adding Barritt's A & B

Slight Yellow or (No Change)

)Negative VP test(

Produce neutral products from glucose

(Ethyl alcohol & Acetyl methyl carbinol)

PH rise above 6.2

Adding Barritt's A & B

Pink color

)Positive VP test(

)Color may take 20-30 mins to develop(

MR & VP tests is done on MR-VP broth media(contains glucose & peptone)

MR & VP tests:• E. Coli is (MR+/VP-)• Klebsiella & Enterobacter aerogenes is (MR-/VP+)

Citrate TestTest 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)