Media Basic ordinary Enriched Differential & Selective CHO Yeast & Fungi Anaerobic Characterist ic All media must be sterile & the basic conditions for autoclaving Temperature = 121 o C Pressure = 15 PSI Time = 20 minutes
Jan 06, 2016
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)