CMLT, 2nd Year Microbiology By- Govind Sah 24 Jan, 2012

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IDENTIFICATION METHOD
The most important task of a bacteriology is to identify the pathogens from the clinical sample so that appropriate treatment can be instituted.

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contd
There are several methods to identified the different type of bacteria.
1. 2. 3. 4. 5. 6.

Isolation in pure form Staining reaction Morphology of bacterial colony Cultural characteristics Metabolism Biochemical properties
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1. Isolation in pure form

Studies on the biochemical, antigenic and other characters

of bacteria can be done only if the organism available in the pure form. Technique: a. Plating on solid culture media- clinical sample is streaked onto a solid medium (like: MacConkey agar, nutrient agar or blood agar) in such a way so as to ensure isolated discrete colonies. b. Use of selective growth condition-most important example of this is the growth of anaerobic bacteria which will not take place in an environment having oxygen.
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2.Staining reaction
a.

The age of the culture is important. In older cultures , staining characteristics either vary or are not brought out well. Simple stains bring out the best morphology. Differential and special stains are necessary to bring out characteristics like: gram negative and gram positive bacteria, Acid fast and non acid fast , spirochetes, capsule and Flagella, etc.

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a. Gram stain
a.

Gram stain divides the bacteria into Gram positive & Gram negative.

The basic procedure goes like this: i. Take a heat fixed bacterial smear. ii. Flood the smear with CRYSTAL VIOLET for 1 minute, then wash with water. [PRIMARY STAIN] iii. Flood the smear with IODINE for 1 minute, then wash with water. iv. Flood the smear with ETHANOL-ACETONE, quickly, then wash with water. [DECOLORI v. Flood the smear with SAFRANIN for 1 minute, then wash with water. [COUNTERSTAIN] vi. Blot the smear, air dry and observe.
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contd
Examine under microscope

i. Gram positive bacteria- violet ii. Gram negative bacteria- pink

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Shape of Bacteria
Bacteria display three basic shapes:

round- cocci, (from the Greek kokkos - a berry), ii. rod shaped bacilli (from the Latin bacillus - a stick or rod), iii. spiral (quelled).
i.

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i. Coccus

Staphylococcus species

Streptococcus species
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ii. Bacillus

Clostridium spp.

Listeria spp.

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Ziehl-Neelsen Staining
Acid-Fast bacilli- red

Mycobacterium tuberculosis
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c. India ink (capsule stain)

The capsule stain employs an acidic stain and a basic

stain to detect capsule production. Capsules are formed by organisms such as Klebsiella pneumoniae . Most capsules are composed of polysaccharides, but some are composed of polypeptides.

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Staining procedure
Place a loopful of India ink on the side of a clean slide A small portion of the solid culture is suspended

in saline on the slide near the ink and then emulsified in the drop of ink, or else, mix a loopful of liquid culture of specimens like CSF with the ink. Place a clean cover slip over the preparation avoiding air bubbles. Press down, or blot gently with a filter paper strip to get a thin, even film Examine under dry objectives followed by oil immersion
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Examine under microscope

Klebsiella pneomonae
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 Spirochetes brownish black

Spirochetes
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Treponema pallidum

Leptospira

3. Morphology of the bacterial colony

Shape: circular, irregular, radiate or rhizoid. ii. Size: diameter in mm iii. Elevation: flat, raised, low convex, dome shaped iv. Margin: Entire, wavy, lobate, filiform v. Surface: smooth, wavy, rough, granular, papillate, glistening etc.
i.

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Shape of the colony

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Elevation of the colony

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Margins of the colony

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4. Cultural characteristics
These provide additional information for the identification of a bacterium.

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A.
i. ii.

On solid medium the following characters are observed

Shape: circular, irregular, radiate or rhizoid. Size: The size of the colony can be a useful characteristic for identification. The diameter of a representative colony may be measured. Elevation: Margin: Entire, wavy, lobate, filiform Surface: smooth, wavy, rough, granular, papillate, glistening etc. Size in mm Texture : dry, moist, mucoid, brittle, viscous, butyrous (buttery). Color : colorless, pink, black, red, bluish-green.
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iii. iv. v. vi. vii.

viii.

Mac Conkey Agar

Enterobacter cloacae on MacConkey Agar: Govind Sah growth with pink colonies

Eschericia coli on MacConkey Agar: growth, with pink colonies

contd.

Staphylococcus aureus
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Streptococcus pyogens

contd

Bacillus subtilis
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Proteus spp.

B. IN A FLUID MEDIUM FOLLOWING CHARACTERS ARE OBSERVED

Degree of growth- Absence, scanty, moderate, abundant etc. ii. Present of turbidity and its nature. iii. Presence of deposit and its character. iv. Nature of surface growth. v. Ease and disintegration and odor.
i.
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5.METABOLISM
To classify the differentiate species following aspects are studied i. Requirement of oxygen ii. The need of co2 iii. Capacity to form pigments iv. Power of hemolysis

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Laboratory Objectives

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Tests To Know
Case Study Tests
Indole Methyl Red/Voges Proskauer Citrate H2S production in SIM Motility Lactose fermentation Sucrose fermentation Glucose fermentation & gas production Triple Sugar Iron Agar (TSI) test

Staphylococcus identification tests

MSA

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Indole Test
Principle:
Indole test is performed to determine the ability of the organism to split tryptophan molecule into Indole. Indole is one of the metabolic degradation product of the amino acid tryptophan Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of Indole, Pyruvic acid and ammonia.

Property it tests for:

This test is performed to help differentiate species of the family

Enterobacteriaceae.

Media and Reagents Used:

Tryptone broth contains tryptophan. Kovacs reagentcontains hydrochloric acid, dimethylaminobenzaldehyde,

and amyl alcoholyellow in color.

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Indole test
Procedure: -Inoculate Tryptone broth with the test organism and incubate for 18 to 24 hrs at 37c -Add 15 drops of Kovacs reagent down the inner wall of the tube Interpretation: -Development of bright red color at the interface of the reagent and the broth within seconds after adding the reagent is indicative of the presence of Indole and is a positive test
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Indole Positive: E.coli Proteus vulgaris Indole Negative: Salmonella spp. Klebsiella spp. Enterobacter aerogens

Methyl Red/Voges-Proskauer (MR/VP)

Properties these test for: Both tests are used to differentiate

species of the family Enterobacteriaceae. Media and Reagents Used: Glucose Broth Methyl Red indicator for MR test Voges Proskauer reagents- A: 5% Alpha-Naphthol & ethanol, B: Potassium Hydroxide; (3:1 ratio) & Deionized Water. Principle of MR test: To test the ability of the organism to produce and maintain stable acid end products from glucose fermentation and to overcome the buffering capacity of the system This is a qualitative test for acid production.
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MR test (contd)
Procedure:

Inoculate the MR/VP broth with a pure culture of the test organism and incubate at 35 for 48 to 72 hrs. Add 5 drops of MR reagent to the broth

Result interpretation:
- Positive result is red (indicating pH below 6) - Negative result is yellow (indicating no acid production)
MR Positive: E. coli MR Negative: Enterobacter aerogenes Enterobacter cloacae Klebsiella spp. Left: negative/Right: positive
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Voges Proskauer Test (acetoin production)

Principle:
To determine the ability of the organisms to produce neutral end product acetyl methyl carbinol (acetoin) from glucose fermentation

Procedure:
Innoculate pure culture of the test organism into MR/VP broth and incubate for 24 hrs at 37c Aliquot 1 ml of the broth to a sterile test tube and add 0.6ml of VP(A) followed by 0.2ml of VP: left + and right VP(B) Shake the tube gently to expose the medium to Positive atmospheric oxygen and allow the tube to Klebseilla pneumoniae remain undisturbed for 10 to 15 mins

Intrepretation:

Enterobacter

Positive : Pinkish red color at the surface of the Negative medium E.coli Negative : Yellow color at the surface of the medium
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Citrate Utilization test:

This test is one of several technique used to assist in the identification of

enterobacteria. The test is based on the ability of an organism to use citrate as its only sole source of carbon and ammonia as its only source of nitrogen. Principle:
The test organism is cultured in a medium which contains sodium citrate, an ammonium salt and the indicator bromothymol blue. Growth in the medium is shown by turbidity and a change in colour of the indicator from light green to blue, due to alkaline reaction following citrate utilization.

Procedure:
Inoculum is streaked over the slant of Simmons citrate agar in a tube and incubated for 24-48 hrs.

Result interpretation:
Growth on the slant and change in colour to blue of the medium indicates positive result. Positive: Klebsiella pneumoniae Govind Sah Negative: E. coli

Left- Positive: Right- Negative

Oxidation-Fermentation (OF) test (Hugh & Leifson)

Principle: Oxidation-Fermentation test is used to determine the oxidative or fermentative metabolism of a carbohydrate or its non utilization. Fermentation is a anaerobic process and bacterial fermenters of carbohydrates are usually facultative anaerobes. Oxidation is a aerobic process and bacterial oxidisers are usually strict aerobes Procedures: -The method described, sometimes referred to as the Hugh and Leifson test employs a semi-solid medium in tubes containing the carbohydrate under test (usually glucose) and a pH indicator -Two tubes are inoculated and one is immediately sealed with paraffin oil to produce anaerobic conditions
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Result interpretation: Oxidising organisms, eg Pseudomonas species, produce an acid reaction in the open tube only Fermenting organisms, eg Enterobacteriaceae, produce an acid reaction throughout the medium in both tubes Organisms that cannot break down the carbohydrate aerobically or anaerobically, eg., Alcaligenes faecalis, produce an alkaline reaction in the open tube and no change in the covered tube
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Motility Test
Property it tests for: This test is done to help differentiate

species of bacteria that are motile from non-motile. Media and Reagents Used: Motility media contains tryptose, sodium chloride, agar, and a color indicator. How to Perform Test: Stab motility media with inoculating needle. Reading Results: If bacteria is motile, there will be growth going out away from the stab line, and test is positive. If bacteria is not motile, there will only be growth along the stab line. A colored indicator can be used to make the results easier to see.
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Motility

From left to right:

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Lactose Fermentation
Property it tests for: This tests for the bacterias ability to ferment

lactose. Media and Reagents Used: Lactose broth contains beef extract, gelatin peptone, and lactose. A phenol red indicator is added to indicate acid production from fermentation. How to Perform Test: Inoculate lactose broth with inoculating loop. Results A positive result is yellow after indicator is added (indicating lactose fermentation) A negative result will have no color change or will be reddish.

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Sucrose Fermentation
Property it tests for: This test is done to help differentiate species of the

family Enterobacteriaceae. This tests the bacterias ability to ferment sucrose and production of acid end-product Media and Reagents Used: Sucrose broth contains beef extract, gelatin peptone, and sucrose. Phenol red indicator is added to indicate an acid end-product. How to Perform Test: Inoculate sucrose broth with inoculating loop. Results A positive result is yellow after indicator is added (indicating sucrose fermentation) A negative result has no color change or is reddish.

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Glucose Fermentation & Gas Production

Property it tests for: This test is done to help differentiate species of
the family Enterobacteriaceae. This tests for the bacterias ability to ferment glucose and produce gas and/or an acid end-product.. Media and Reagents Used: Glucose broth contains beef extract, gelatin peptone, and glucose. A phenol red indicator is added to indicate an acid end-product. A Durham tube is added to indicate gas production. How to Perform Test: Inoculate broth with inoculating loop. Results A positive result for acid is yellow after indicator is added (indicating glucose fermentation) A positive result for gas is a bubble in the Durham tube. A completely negative result has no color change or reddish color and no bubble.
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Sugar Fermentation Tests

Tube 1: Negative acid /Negative gas Tube 2A: Must incubate longer (ambiguous result) Tube 2B: Positive acid /Negative gas Tube 3A: Positive acid/ Positive gas
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Triple Sugar Iron Agar (TSI) test

Principle:
TSI agar is used to determine whether a gram negative rod utilizes glucose and lactose or sucrose fermentatively and forms hydrogen sulphide (H2S). TSI contains 10 parts lactose: 10 parts sucrose: 1 part glucose and peptone. Phenol red and ferrous sulphate serves as indicators of acidification and H2S formation, respectively. The formation of CO2 and H2 is indicated by the presence of bubbles or cracks in the agar or by separation of the agar from the sides or bottom of the tube. The production of H2S requires an acidic environment and is indicated by blackening of the butt of the medium in the tube.

Method:
1. 2. 3.

With a straight inoculating wire, touch the top of a well isolated colony. Inoculate TSI by first stabbing through center of the medium to the bottom of the tube and then streaking the surface of the agar slant. Leave the cap on loosely and incubate the tube for 18-24 hours at 35oC in an incubator.

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Triple Sugar Iron Agar (TSI) test (contd)

Result interpretation:
1.
a b c d

2. 3.

4.

Alkaline slant/no change in the butt (K/NC) = Glucose, lactose and sucrose non-utilizer (alkaline slant/alkaline butt) [figure: 1(d)] Alkaline slant/acid butt (K/A) = Glucose fermentation only. [figure: 1(b)] Acid slant/acid butt (A/A), with gas production = Glucose, sucrose, and/or lactose fermenter. [figure: 1(a)] Alkaline slant/acid butt (K/A), H2S production = Glucose fermentation only. [figure: 1(c)] Quality control: A/A, with gas: E. coli K/A, H2S: Salmonella typhi K/NC: Pseudomonas aeruginosa
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Figure (1): TSI results

Mannitol Salt Agar (MSA)

Property it tests for: This tests for the bacterias ability
to tolerate 7% salt concentration and ferment mannitol. The media is selective because it selects for salt tolerant bacteria.

Media and Reagents: MSA media contains nutrient

agar, mannitol, 7% sodium chloride and phenol red indicator.

How to Perform Test: Inoculate an MSA plate using

streak plate method and incubate 24-48 hours.

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MSA Results
Reading Results:
If the organism is tolerant to salt it will grow.
If the organism is not tolerant to salt it will not grow. If the salt tolerant organism can ferment mannitol then there will be

yellow zones around the colonies. If the salt tolerant organism cannot ferment mannitol then the media will remain pink.

Growth with noGovind Sah fermentation. mannitol

Growth with + mannitol fermentation.

Test for enzymes

Catalase test

Oxidase test
Urease test Coagulase test

Nitrate reduction

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Catalase test
Principle: This test demonstrates the presence of enzyme catalase in the organism. The enzyme catalase mediates the breakdown of hydrogen peroxide (H2O2) into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide (30% for slide test), and the rapid effervescence of O2 bubbles occurs. The lack of catalase is indicated by a lack of bubble production.lase
2H2o2
catalase

2H2o + o2 (gas bubbles)

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Catalase test.
Catalase is present in most cytochrome containing

aerobic and facultative anaerobic bacteria (except streptococcus spp). Hydrogen peroxide forms as one of the oxidative end product of aerobic carbohydrate metabolism. If this is allowed to acculmulate in the bacterial cells it becomes lethal to the bacteria Catalase thus helps in converting H2o2 to H2o and o2 Optimal pH for catalase action is 7.
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Catalase test.
Reagents: 3% hydrogen peroxide stored in dark brown bottle under refrigeration 18 to 24 hrs culture of the organism to be tested Control organisms used: Positive control - Staphylococcus aureus Negative control Streptococcus spp.

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Catalase test.
Methods:

1. Slide method

2. Tube method
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3. Direct plate method

Catalase test.
Precautions Avoid colonies from blood agar 18 to 24 hrs colony only should be used Reagent to be fairly fresh as it is very unstable and easily breaks down on exposure to light Reagent to be stored in brown or amber colored bottle in refrigerator at 4c

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Catalase test.
Positive Micrococcus Staphylococcus Bacillus Listeria monocytogenes Enterobacteriacae Gonococcus & Meningococcus Vibriocholerae Pseudo/Aero/Plesiomonas
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Negative Streptococcus Clostridium

Oxidase Test
Principle Oxidase test is used to determine the presence of bacterial cytochrome oxidase enzyme using the oxidization of the substrate tetramethyl-pphenylenediamine dihydrochloride to indophenol a dark purple colored end product. A positive test (presence of oxidase) indicated by the development of a dark purple colour. No colour development indicates a negative test and the absence of the enzyme.

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Oxidase Test.
Cytochromes are iron containg hemoprotiens and in aerobic respiration they transfer electrons(H) to oxygen to form water. The reagent acts as an artificial electron acceptor substituting the oxygen. In the reduced stage dye is colorless , but in the presence of enzyme cytochrome oxidase dye is oxidised to indophenol blue

Methods

1. Moist filter paper method

2. Direct plate method

Quality controls Positive control- Pseudomonas spp Negative control E. coli

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Oxidase Test..
Positive
Pseudomonas spp. Aeromonas spp. Vibrio spp. Alcaligenes spp. Neisseria spp. Haemophilus sps

Negative
Enterobacteriaceae Acenitobacter spp.

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Oxidase Test..
Precautions Use glass rod or wooden stick Result should be read within 10 secs Do not perform the test from colonies growing in medium having glucose as its fermentation will inhibit the oxidase enzyme activity Reagent should be freshly prepared To be stored in dark bottle Do not use pigmented colonies or colonies fromMacconkey agar
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Urea Hydrolysis (Urease test)

Property it tests for: This test is done to determine a bacterias

ability to hydrolyze urea to make ammonia using the enzyme urease. Media and Reagents Used: Stuarts Urea broth (pH 6.8) contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator. Principle To determine the ability of the organism to split urea forming 2 molecules of ammonia by the action of the enzyme Urease with resulting alkalinity How to Perform Test: Inoculate Urea broth with inoculating loop.

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Reading Results:
Urea broth is a yellow-orange color.

The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made.
Figure in the right shows negative (left) and

Positive (right) results.

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Coagulase test
Principle:
This test is used to differentiate Staphylococcus aureus (positive) from coagulase negative Staphylococci. S. aureus produces two forms of coagulase: bound and free. Bound coagulase or clumping factor, is bound to the bacterial cell wall and reacts directly with fibrinogen. When a bacterial suspension is mixed with plasma, this enzyme causes alteration in fibrinogen of the plasma to precipitate on the staphylococcal cells, causing the cells to clump. Free coagulase is produced extra-cellularly by the bacteria that causes the formation of a clot when S. aureus colonies are incubated with plasma.
-

Method:
A. B. -

Slide test: (for bound coagulase) Place a drop of coagulase plasma on a clean, dry glass slide. Place a drop of distilled water or saline next to the drop of plasma as a control. With a loop or wooden stick, emulsify a portion of the isolated colony being tested in each drop. Mix well and rock the slide gently for 5 to 10 seconds. Tube test: (for free coagulase) Emulsify several colonies in 0.5 ml of rabbit plasma (with EDTA) to give a milky suspension. Incubate tubes at 35oC in ambient air for 4 hrs. Check for clot formation. If negative at 4 hrs, incubate at room temperature overnight and check again for clot formation. Sah Govind

Coagulase Results
Reading Results:
A. Slide test: - Positive: Macroscopic clumping in 10 seconds

or less in coagulated plasma drop and no clumping in saline or water drop. - Negative: No clumping in either drop. - Note: All negative slide tests must be confirmed using the tube test. B. Tube test: - Positive: Clot of any size (a) - Negative: No clot (b)

a
Coagulase Positive : Staphylococcus aureus Coagulase negative: Staphylococcus epidermidis
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Nitrate reduction test

Principle: This test is used to determine the ability of the organism to reduce nitrate to nitrites or fee nitrogen gas. The reduction of nitrate to nitrite is detected by adding sulphanilic acid and alpha-naphthylamine. The sulphanilic acid and nitrite reacts to form a diazonium salt which then reacts with alphanaphthylamine to produce a red, water soluble azo-dye. Purpose: Aid in the species deferentiation of i) Haemophilus duceryi(-) and Haemophilus vaginalis(-) from other Haemophillus spp. ii) Neisseria mucosa(+) from other Neisseria spp Aid in the identification of Enterobacteriaceae(+)
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Nitrate reduction test.

Procedure:
In order to determine if a bacteria can reduce nitrate, the

test organism is inoculated into nitrate broth [an undefined medium that contains large amounts of nitrate (KNO3)]. After incubation, alpha-naphthylamine and sulfanilic acid are added . These two compounds react with nitrite and turn red in color, indicating a positive nitrate reduction test If there is no color change at this step, nitrite is absent. If the nitrate is unreduced and still in its original form, this would be a negative nitrate reduction result. However, it is possible that the nitrate was reduced to nitrite but has been further reduced to ammonia or nitrogen gas (which evolved out). This would be recorded as a positive nitrate reduction result
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Nitrate reduction.
To distinguish between these two reactions, zinc dust

must be added. Zinc reduces nitrate to nitrite. If the test organism did not reduce the nitrate to nitrite, the zinc will change the nitrate to nitrite. The tube will turn red because alpha-naphthylamine and sulfanilic acid are already present in the tube Thus a red color after the zinc is added indicates the negative nitrate reduction test.

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Nitrate reduction test.

Negative Negative Posiitive Positive Nitrate reduced to NH3 or N2 gas, nitrite absent

Nitrate not reduced

Nitrate reduced to nitrite Govind Sah

Nitrate not reduced

Nitrate reduction test.

Addition of Zn dust

or

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Gram positive flowchart

present

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Gram negative flowchart

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Key identification characteristics for Enterobacteriaceae

GENUS/SPECIES Escherichia coli Shiegella Shiegella sonnei Salmonella Klebsiella Pneumo. Enterobacter Serratia Proteus morganella Yersinia Fermentation of G L S (+) (+) (+) (+) (-) (-) (+) (+) (-) (+) (-) (-) (+) (+) (-) (+) (-) (+) (+) (+) (-) (+) (-) (-) (+) (-) (-) (+) (-) (-) Gas M (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-) (-) (+) (+) (-) (+) (-/+) (+) (-) MR (+) (+) (+) (+) (-) (+) (-/+) (+) (+) (+) VP (-) (-) (-) (-) (+) (-) (+) (-) (-) (-) Indole Citrate Urease (+) (-/+) (-) (-) (-) (+) (-) (+) (+) (-/+) (-) (-) (-) (+) (+) (-) (+) (-/+) (-) (-) (-) (-) (-) (-) (+) (+) (-) (+) (+) (-/+) H2S (-) (-) (-) (+) (-) (+) (-) (+) (+) (-)

G: Glucose, L:Lactose, S:Sucrose, M: Manitol,

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MR: Methyl Red, VP: Voges Proskauer

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