microbial bio_aaser
DESCRIPTION
evaluation tests for microorganisms in labTRANSCRIPT
BIOCHEMICAL EVALUATIONSfor Microorganisms
Aaser Mohamed AbdelazimAssistant lecturer of Biochemistry
Zagazig Vet. Medicine [email protected]
Decarboxylase test1Principles: 1.Some M.Os have the ability to produce Decarboxylase E which induce decarboxylation of some amino acids as Ornithine and Lysine.2.Media changed from acidic to alkaline and can detected by any PH indicator like bromo cresol purple which changed to purple in alkaline media
++
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Ornithine lysine Ornithine lysine
Arginine Arginine
Dihydroxylation
Decarboxylation
Decabxylase test
Results
Amino acid
Amines + co2
Decarboxylase
Carbohydrate Fermentation Tests (Phenol Red Broth)2
Purpose: Used to diagnose the fermenting bacteria especially gram – ve enteric bacteria
1. Ability the M.Os to ferment simple sugar(glucose, lactose, succrose) with production of acid and gas
2. We put durhum`s tube to collect gas 3. PH indicator or (phenol red) which changed to yellow in acidic media
and to pinkish red in alkaline media.4. Peptone: marker for no utilization of sugar and production of alkaline
media
principles
Media: one sugar + peptone+ Phenol red
Results of carbohydrates fermentation Results of carbohydrates fermentation
Utilize the sugarUtilize the sugar Do not utilize the sugarDo not utilize the sugar Do not utilize the sugar and utilize peptone Do not utilize the sugar and utilize peptone
Neutral No change in
indicator color
Acid Acid + GasMedia changed
alkaline
Pink Pink yellowyellow
Diagnosis Diagnosis
M.Os Glucose Lactose Sucrose
(2) S . Aureus Acid Acid Acid
(3) P. Vulgaris Acid + Gas -ve Acid +gas
(4) P. Aeruginosa
-ve -ve -ve
(5) E. Coli Acid + Gas Acid + gas -ve
Glucose
S. Areus: acid all P. aeruginosa: not all E. coli: except sucroseP. vulgaris: execpt lactose
S. Areus: acid all P. aeruginosa: not all E. coli: except sucroseP. vulgaris: execpt lactose
Lactose Sucrose
Starch Hydrolysis Test3Purpose :Diagnose bacteria which produce exoenzyme amylase and ability to hydrolyze starch
Starch Starch Amylase
Glucose Glucose
Large molecules can not enter to bacterial cells
Small can be utilized by cells
Media = starch + Iodine + agar plate Starch agar plate
Results
Starch agar before adding Iodine
Starch Agar after adding iodine
Iodine
Diagnosis Clear zone of inhibition indicates +ve results
M.Os: B. subtilis
Citrate Utilization Test4Purpose: To test the ability of M.Os to produce Citrase and utilization of citrate as a source of carbon(photoorganotropics and chemoorganotropics).
One of IMViC test (Indole, Methyl red, Voges-Proskauer, and Citrate) used to diagnose enterobacteraece
Media: Simmon`s citrate agarMedia: Simmon`s citrate agar
Sodium citrate Sodium citrate Ammonium Ammonium
Nitrogen source Carbon source
Indicator: bromothymol blueIndicator: bromothymol blue
Blue Blue Green
neutral Alkaline
Yellow or green Yellow or green
Results of citrate utilization test Results of citrate utilization test
Utilize citrate Utilize citrate Not utilize citrate Not utilize citrate
Ammonia remain
Alkaline media
Blue
Citrate remain
Acidic media
Citrate utilization test
Diagnosis
1. Enterobacter coloace: +ve2. Klebsiella. Pneumonae: +ve 3. E. coli: -ve
Gelatin Liquefaction TestGelatin Liquefaction Test5Purpose :Test the ability of some M.Os to produce exoenzyme Gelatinase that liquefy gelatin into polypeptide and then to many amino acids
Gelatin (liquid)
Inoculation of M.O
incubated in refrigeratorTo allow the
gelatin to melt
Results
Then let in room temp. for more 1 hour
Solid Liquid
+ve -ve
Diagnosis Diagnosis p. Aeruginosa E. Coli
Control tube should be done to
Coagulase TestsCoagulase Tests6Purpose: 1.Test the ability of some Staph. Species to produce coagulase E. 2.Bacteria secrete Coagulase to protect it self by forming clotting in surround the tissues and protect itself from phagocytosis or antibodies.3.The test used to diagnose pathogenic staph. Aureus that are catlase possitive. To induce clotting to plasma.
Methods Methods
Identify free coagulase
Identify the cell bounded
coagulase
Slide method Tube method
Clumping Clotting Rabbit plasma used
to diagnose Staph. aureus
Phenylalanine Deaminase7Purpose: Test the ability of some enterobacteraece to produce exoenzyme phenylaninedeaminase (Morganella, Providencia, Proteus).
Phenylalanine Phenyle pyruvate + ammonia Deaminase
Detected by adding ferric chloride as oxidizing agent
Green Green
10% ferric chloride
-ve no change in
color
+ve Green color
Test should be read rapidly as
green color fades
Precipitation ReactionsPrecipitation Reactions81. Serological test examine the reaction between the homologus
antibodies with their antigens. 2. When the reaction occur a visible line of PPT will appeared3. The line due to the formation of lattic
Antibody have 2 binding site for
antigen
Antigen has multiple site for
antibody binding
Copmlex lattic formation: Due to reaction of antigen with more than one antibody
Several wells were installed in the gel and in the central well put 6 Abs and on the peripheral wells add antigens specific reaction will form line of ppt.
Several wells were installed in the gel and in the central well put 6 Abs and on the peripheral wells add antigens specific reaction will form line of ppt.
9 Triple sugar Iron agar
Purpose :To differentiate between fermenting bacteria and that reduce hydrogen sulphide H2S
Media: TSI media
Peptone+ glucose+ lactose+sucrose+thiosulphate
Indicator: phenol red
Yellow Red
PH above 6.8 (Alkaline ) PH less 6.8 (Acidic)
A B C D E F G H
Results of triple sugar iron agar Results of triple sugar iron agar
Ferment sugar
One or two or three sugar
With gas
No ferment
Without gas
Black color Red color
Reduce sulphide
Yellow
10 Casease test
Purpose: To identify bacteria produce casease
Media Milk agar contain casein white color
Casein Polypeptides
Results: Only bacillus subtilis only gives + results
Clear zone around colony
casease
Litmus milk—casein precipitation
11 Urease test
Purpose Identify bacteria produce Urease Diagnose genius proteus from enteric bacteria
Media :Urea , phenol red indicator
Urea Ammonia CO2 +
Red pinkish color indicate Proteus vulgaris
Urease
12 Methyl red and Voges- proskauer test (MR/VP)
Purpose Identify bacteria that produce stable acids when ferment glucose and produce mixed acid fermentation
Media :Glucose, peptone,phosphate buffer
Indicator Methyl red (MR)Methyl red (MR)
Red
Yellow Orange
+ - Need farther inoculation
I – Methyl red test
II – Voges proskauer test (VP)
Purpose :Identify bacteria produce acetion during 2,3 butandiol fermentation Diagnose enterobacteria
Some bacteria don’t produce strong acid to change the indicator
Add Baritt`s reagent A(alphanaphthol ) and Baritt`s reagent B (potassium hydroxide )
After 48 hours
Red color
Indicates + results Indicates + results
13Lipase test
Purpose :Identify Bacteria produce Lipase
Triglycerides Glycerol + Fatty acids
Media :Agar , tributyrin
Spirit blue lipase testMethylene blue tributyrin lipase
14 DNase test
Purpose Bacteria produce DNase
Media :Agar, emulsion of DNA, peptides , methyle green dye(MG).
Reaction : DNA with MG gives green blue color
Results : clearing around the M.Os
- Serratia marcescens - Streptococcus pyogens
+E. coli
-
1515 Nitrate reduction test
purpose: Bacteria produce Nitrate reductase
Nitrate (NO3) Nitrite (NO2) or nitrogen (N2)
Nitrite (NO2) + sulfanilic acid + α- naphathylamine
NO2
Red color
No color
No reduction Reduction to ferther N2
Add zinc dust to distinguish between negative and positive reactions
Pink color
Means nitrate presents and reduced
by Zinc
No color
Nitrate reduced to ferther N2
Results Results
-Pseudomonas aeruginosa -Corynebacterium xerosis E. coli
+ -Red No change
Need ferther invistigationBy add ZINC
Red No color
Corynebacterium xerosis
Pseudomonas aeruginosa +
Reduce NO3 to N2
1616 Oxidase test
Purpose :•Oxidase postive (pseudomonacae) from oxidase negative (enterbacterace) . It is a hallmark test for the Neiserria•Cytochrome oxidase respiratory chain
Cytochrome oxidase transports electrons to final acceptor O2
Phynelenediamine a redox dye act as artificial an electron donor
Test : when dye added to the media when E is persent it changed to purple color Results of oxidase test
1717 SIM medium (sulfur reduction test, Indole production, Motility
I- Sulfur reduction test
Media: cysteine, sodium thiosulfate, peptonized iron(ferrous sulphate)
Results: H2S reacts with Iron gives Black colorResults: H2S reacts with Iron gives Black color
II- Indole production
Bacteria produce tryptophanase
Tryptophane Ammonia + Pyruvate + Indole Indole
Kovac`s reagent HCl +
Dimethylaminobenzaldehyde+Amyl alcohol
Red layer Add
III- Motility
Stab inoculation to indicates motility
Stab inoculation
Radiating growth indicates motility
Results
Staph. Typhimurium Staph aureus
H2S production and motile Negative
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Aaser Mohamed Abdelazim Assistant lecturer of Medical Biochemistry and Molecular Biology
Zagazig Vet. [email protected]
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