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CHO
Differential &
&
Selective
Fungi
Enriched
Basic ordinary
Yeast
Anaerobic
Media
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
‫‪• 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 ‪ MO‬واحد يعيش والباقي يموت (تختارة)‬
‫‪• Differential‬‬
‫فيها مادة تخلي شكل الـ ‪ MO‬مختلف عن الـ ‪ MO‬التاني غالبا ً يكون االختالف في اللون‬
‫‪Characteristic‬‬
‫•‬
‫•‬
‫•‬
‫ميديا بستخدمها في التعرف على البكتريا عن طريق الميتابوليزم بتاعها ( ‪)Sugar fermentation‬‬
Nutrient
Agar
MAC
Plate
Media
Chocolate
Agar
Blood Agar
Nutrient
Broth
Sabouraud’s
media
Peptone H2O
Liquid
Media
Thioglycolate
Media
Cooked Meat
Nutrient
Agar
Slant
Christensen
Deep
Agar
Urea
Solid
&
Simmon
Gelatin
Slant
Citrate
Media
L-J
Loffler’s
Media
Serum
Enriched
media
Enrichment
media
Selective
media
Contain growth factors & other complex organic substances like
blood, serum, etc
Better for Fastidious Pathogenic bacteria
Provides nutrient & environmental conditions that favor the
growth of certain organisms but not suitable for others.
Characteristic
media
Selenite broth
that used for
Salmonella
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)
Differential
media
Blood Agar
MacConkey’s
media
Simmon’s Citrate
media
Differentiate ( ) the bacteria by change in the color of the growing
colonies
Used to test the organism for 
Triple sugar iron
(TSI)
Metabolic activity Or Metabolic products Or Metabolic
requirements
Lysin iron agar
(LIA)
Useful in identification of the type of the bacteria.
Sulfide indole
motility (SIM)
Nutrient
Broth
Cultivation of bacteria
meat extract & Pepton &
0.5% NaCl, neutral PH
light yellow transparent fluid
Fluid Basic
Ordinary Media
Peptone
Water
Indole Production
Base for sugar media
1% Pepton + 0.5% NaCl + water
Agar Plate
N.B + 1-5 – 2 % Agar
Nutrient Agar
Semisolid Basic
Ordinary Media
N.B + 10-15 % Gelatin
Agar Slant
Deep Agar
Gelatin Media
Gelatin
Liquefaction
Isolation of bacteria
(Streaking for isolation)
Short Storage
(3-6 weeks)
Prolonged Storage
(6 months)
Proteolytic activity
Enriched
Blood
Chocolate
Loffler’s
Agar
Agar
serum
Lowentsein
Blood Agar
Alpha
Beta
Gamma
Incomplete (partial) & green zone
Complete & Clear zone
No Change
Streptococcus
Staph. aureus
Pneumonia
St. Pyogenes
Differentiate M.O according 2 Hemolytic activity
N. Agar  100 C 55 C  5-10 % Sheep or Ox Blood
St. Faecalis
Chocolate
Loffler’s Serum
Lowenstein Jensen
H. Inflenza
&
Neisseria
Corynbacterium Diphtheria
Mycobacterium TB
Heated sheep
blood
Horse serum
Malachite green (Selective& Enriched)
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
MAC
Simmon’s Citrate
Selective
Differential
Inhibits the growth of Gm+Ve
due to the presence of Crystal
violet & Bile salts
Differentiate ( ) bacteria on the
basis of a color change reaction
Gm –Ve bacteria grow well
 Lactose  Neutral red
MAC contains:
Upon citrate utilization the PH
of the media will be increased
causing change in color of the
media into  blue
Due to Bromothymol Blue
Example of Lactose Fermenters
Example of Non-Lactose Fermenters
E. Coli & Klebsiella
Salmonella & Shigella & Proteus species
& Pseudomonas aeruginosa
Pink Colony
Colorless
Broth media + Sugars
(Glucose & Galactose & Lactose & Mannose & Maltose)
+
Phenol Red (Yellow in Acidic PH & Purple/Red in Basic PH)
+ Durham's tube (Gas indicator)
Non-Fermenter
Fermenter-Acid Producer
Fermenter-Acid & Gas
Producer
No fermentation & PH indicator remains Purple
Fermentation with the production of acid
(Yellow color) but no gas
Fermentation with the production of acid
(Yellow color) and gas (Bubbles in Durham tube)
Yeast & Fungi
Incubation 25C
for 10 days
Fluid
Sabouraud’s
4% Glucose &
5.6 Acidic PH
Sterility Test
(Saliva &Candida)
Aerobic
Anaerobic
Incubation 30-35C
for 7 days
Facultative
Sterility Test
Thioglycollate
Methylene green
Na Thioglycollate
Methylene blue
Cystine
Resazorine
(Reducing agents)
(O2 indicator)
Small amount of
Agar
(Decrease oxygen
diffusion)
Cooked meat for Anaerobic ONLY
Glutathione
Lactose
Sucrose
Glucose
FAS
Na2S2O3
TSI
Characteristic
media
Used in
Identification of
Enteric organisms
Casein
Starch
Gelatin
Hydrolysis
Starch Hydrolysis
Test 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 Hydrolysis
Test 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
Urease
H2S
Hemolysin
Ammonia
Oxidase
Catalase
Nitrate
Production
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 media
Presence of gas bubbles means that the organism produces catalase
Oxidase Production
Test 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 Production
Test the ability of the organism to produce:
Exoenzyme Hemolysin which has destructive effect on the blood cells
Blood Agar
Beta
Alpha
Gamma
Complete & Clear zone
Incomplete (Partial) & green
zone
No Change
St. Pyogenes
St. Pneumonia
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 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
CHO Media
No Fermentation
Fermentation
Fermentation
No Acid No Gas
Acid Production / No Gas
Acid & Gas Production
Phenol Red = Red
Phenol Red = Yellow
Phenol Red = Yellow
Bubble in Durham’s tube
Nitrate Reduction
Test 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
MR
VP
Indole
Citrate
IMViC
IMViC tests used for  Identification & Differentiation of Enterobacteriaceae
(Klebsiella & Enterobacter & E. Coli) ( All are Lactose Fermenters)
The presence of Enterobacter & Klebsiella
The presence of E. coli
Does not Indicate fecal contamination because 
they are widespread in soil & grass
Indicate fecal contamination
of food & water
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 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
Klebsiella and Enterobacter
E. Coli
Produce neutral products from glucose
(Ethyl alcohol & Acetyl methyl carbinol)
 PH rise above 6.2
Adding MR indicator  Yellow color
(Negative MR test)
Produces acidic products from glucose
 PH drop below 4.4
Adding MR indicator  Cherry red color
(Positive MR test)
Vogas ProskaurTest
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)
Klebsiella and Enterobacter
E. Coli
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)
Produces acidic products from glucose
 PH drop below 4.4
Adding Barritt's A & B 
Slight Yellow or (No Change)
(Negative VP test)
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 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)