Classification and Indentification of Bacteria

7/27/2019 Classification and Indentification of Bacteria

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CLASSIFICATION AND

IDENTIFICATION OF

BACTERIA

School of Environmental Studies


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Isolation (culture)Agar plate

plate/colonies

Liquid media

Identification & taxonomy

Family

GenusSpecies

Type

Strain

After cultureBiochemical (physiological) tests

Genetic tests

Sequencing,

Polymerase chain reaction (PCR)

DNA-DNA homology

Chemical (e.g. fatty acid profiling)

Immunological

Direct detection (i.e. without

culture)

PCR

Antigen detection

Staining (e.g. Gram stain)

Serology (antibody detection)

Key Terms


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Classification

Strain: one single isolate or line

Type: sub-set of species

Species: related strains

Genus: related species

Family: related genera


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Laboratory procedures employed in the

identification of bacteria

1.Isolation of organism in pure culture2.Bacterial colony morphology

3.Microscopic morphology and Staining reaction4.Biochemical test5.Serological procedure6.Antibiotic sensitivity


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Isolation of organism in Pure Culture

Pure culture (axenic culture) Population of cells arising from a single cell

- the approach used for the isolation of organism dependsupon the source of clinical specimen

Blood, spinal fluid and closed abscesses may yield almostpure bacterial culture

specimen of sputum, stool, materials from the skin and body

orifices, waste water etc., usually contains mixture of organism

- Spread plate, streak plate, and pour plate are

techniques used to isolate pure culture


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Laboratory Cultivation

Cultivation is the process of growing microorganisms bytaking bacteria from the infection site by some means ofspecimen collection and growing them in the artificial

environment of the laboratory For the in vitro environment of the bacteria, required

nutrients are supplied in a culture medium

culture- organisms that grow and multiply in or on a

culture media


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Culture Medium- A liquid or gel designed to support the growth of microorganisms- 2 major types of growth media:

- those used for cell culture, which use specific cell types derivedfrom plants or animals

- microbiological culture, which are used for growingmicroorganisms such as bacteria or yeast

-The most common growth media for microorganisms are

nutrient broths and agar plates-specialized media are sometimes required for microorganism and

cell culture growth


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Based on Chemical Composition

Complex Media- Contain some ingredients of unknown composition and/or conc.- is a medium that contains:

carbon source such as glucose for bacterial growth water

various salts needed for bacterial growth a source of amino acids and nitrogen (e.g., beef, yeast extract)

- Nutrient mediacontain all the elements that most bacteria needfor growth and are non-selective, so they are used for thegeneral cultivation and maintenance of bacteria kept in

laboratory culture collections

Defined or Synthetic Media-All components and their concentrations are known


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Functional Types of MediaSupportive or general purpose media- Support the growth of many microorganisms- E.g., Tryptic soy agar

Enriched media- General purpose media supplemented by blood or other special nutrients Blood agar is an enriched medium in which nutritionally rich whole blood

supplements the basic nutrients Chocolate agaris enriched with heat-treated blood (40-45C), which turns brown

and gives the medium the color for which it is named

Selective media- Favor the growth of only selected microorganisms and inhibit growth of others eosin-methylene blue agar (EMB) that contains methylene blue

toxic to Gram (+) bacteria, allowing only the growth of Gram (-) bacteria blood agar (used in strep tests), which contains beef heart blood that becomes

transparent in the presence of hemolytic Streptococcus MacConkey agar for Gram-negative bacteria Mannitol Salt Agar (MSA) which is selective for Gram (+) bacteria and differential for

mannitol


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Differential media

Distinguish between different groups of microorganisms basedon their biochemical characteristics growing in the presenceof specific nutrients or indicators (such as neutral red, phenolred, eosin y, or methylene blue) added to the medium tovisibly indicate the defining characteristics of a microorganism

Ex.Blood agar differentiates hemolytic versus non-hemolytic bacteria MacConkey agar - lactose fermenters versus non-fermenters Eosin methylene blue (EMB), which is differential for lactose and sucrose

fermentation

Mannitol Salt Agar (MSA), which is differential for mannitol fermentation


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EMB Plate

E. coli is seen on the left and E. aerogenes on the right


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Bacterial colony morphology

Bacteria grow on solid media as colonies colony is defined as a visible mass of microorganisms all

originating from a single mother cell, when inoculated intoappropriate medium containing 2% agar and incubated18-24 hours in a favorable atmosphere

therefore a colony constitutes a clone of bacteria all genetically alike Ideally, the colony is the progeny of one, or at most, a few bacteria A colony will usually contain millions of bacterial cells

Colony morphology can sometimes be useful in bacterial identification Colonies are described as to such properties as size, shape, texture,elevation, pigmentation, effect on growth medium


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To identify the following colonial characteristics/culture characteristics:

WHOLE SHAPE OF COLONY EDGE/MARGIN OF COLONY


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ELEVATION OF COLONY(turn the place on end to determine height)

CHROMOGENESIS (pigmentation)- Some bacterial species form an array of pigments: white, red, purple, etc.

Some pigments are contained within the cell (i.e., probably not water soluble) Some pigments readily diffuse throughout the medium (i.e, water soluble)

Some pigments fluoresce in UV light

OPACITY OF COLONY:transparent (clear), opaque,translucent (almost clear, but distorted visionlike looking through frosted glass

iridescent (changing colors in reflected light)CONSISTENCY:

butyrous (buttery), viscid (sticks to loop, hard to get off)brittle/friable (dry, breaks apart)

EMULSIFIABILITY OF COLONY:

Is it easy or difficult to emulsify? Does it form a uniform suspension, a granularsuspension, or does not emulsify at all?


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Microscopic morphology

Provide presumptive identification of an organism

Bacterial Morphology Bacterial cell is a fundamental unit of any living organism All its functions are genetically controlled and performed by that

particular cell structure whether it be physiologic orbiochemical

Bacteria and other microorganism are usually transparent, whichmakes the study of the morphologic detail difficult when they

are examined in the natural state

Routinely used to determine: shapearrangementstaining reaction


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I. Bacterial Shape and Arrangement Bacterial Shape

determined by the configuration of the cell wall

detected by brightfield microscopy of stained smear

Bacterial Arrangement

is the result of the number of plane division the organism mayundergo and how the cell remain attached afterwards

divides only across their short axis

3 conventional forms :

Spherical (cocci) Rod (bacilli) Spirals


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Spherical (Cocci) Shape:

round like a ball, perfect sphere or globe

Variations :

1. Ovoid shape - both sides rounded ends arepointed Ex. Streptococcus

2. Lancet-shape - one end is pointed, other end isflat Ex. Pneumococcus

3. Coffee-bean shape - flat on one side, oppositeside convex or appear as letter D form

Ex. Neisseria


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Arrangements:

1. Singly occurs as a single spherical cell

2. Chain streptococci- common among ovoid-form resulting from oneplane division with daughter cells remained attached

to one another to form a chainEx. Streptococcus pyogenes

3. Pairsdiplococci- common with lancet-shaped and coffee-bean

shaped spherical resulting from one plane divisionwith daughter cell separating

Ex. Streptococcus pneumoniae

Neisseria gonorrheae


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4. Clustersstaphylococci- common with spherical resulting from many plane

division with daughter cell in grape-like agglomerationEx. Staphylococcus aureus

5. Tetrads (Packets of 4)- result from 2 plane division with daughter cell

separating from one another to form group of 4 cellsEx. Micrococcus tetragenous

6. Sarcinae (Packets of 8)- results from many plane division producing cubicalpackets of 8 cellsEx. Sarcina lutea


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Rods (Bacilli) Shape

cell appears longer than wide or cylindrical form

both sides parallel and ends are convex

varies in actual form depending on the species

divides only across their short axis

Variations :

1. Clubbed/drumstick shaped swollen on oneend

Ex. Clostridium diphtheriae/C. tetani

2. Corset-shape both sides swollen, ends flat orconcave Ex. Bacillus anthracis

3. Fusiform both sides parallel, ends pointed


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Arrangements:1. Singlyoccurs as a single rod2. Chain result from one plane division with daughter

cell remain attached to one anotherEx. Bacillus anthracis

3. Palisade arrangement like fence due to slippingmovement of daughter cells (side-by-side)

Common among clubbed shaped rodsEx. Mycobacterium tuberculosis

4. Chinese-letter common with clubbed-shaped rodsresulting from a snapping post divisionmovement of the daughter cells (V shape)

Ex. Corynebacterium diptheriae5. Packets of cigarettearrangement like bundles

Ex. Mycobacterium leprae

6. Serpentinecommonly seen with virulent strainof Mycobacterium tuberculosis


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Intermediate forms

Coccobacilli- when a rod is short & wide/plump- these form is intermediate between a spherical

and rodEx. Haemophilus, Brucella

Vibrio- a gently curve bacteria (comma-shaped)

- it is an intermediate between a rod and a spiralEx. Vibrio cholerae


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Spirals

bacteria with more than one somatic curve may be regarded as bacillary forms trusted in the form of a helix

no characteristic cell arrangement

most occurs singly

different specie vary in size, length, rigidity and amplitude of theircoils

2 types :

1. Flexible spirals that can contract and relax & move by creepingmovement

Ex. Spirochetes

2. Rigid spirals that cannot contract and relax & move byrotation or corkscrew-like motion

Ex. Spirillum


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SPIRILLUM- whose long axis remains rigid when in

motion

Ex. Campylobacter jejuni

SPIROCHETE whose long axis bends when in motion

Genus Treponema char. tightly coil w/ cork screw appearanceEx. Trepanema pallidum

Genus Leptospira less tightly coiled w/ sharp hook-like bends

Ex. Leptospira interrogansGenus Borrelia

much less tightly coiled w/c has theappearance of extremely long undulatingbacillary poresEx. Borrelia recurrentis


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II. Bacterial size

all linear measurements in microbiology are expressed in

metric units

the basic unit of the metric system is the meter m

centimeter cm (1/100th of a m)

- the largest unit of length used for measuring microorganism

micrometer m

- visible only with high powered microscope

- unit of measurement most frequently used in microbiology

1m = 1/1000 of a mm

Cocci = 0.4-2m

Bacilli = 0.2-4m in width by o.5-20m in length

Spirals = 1-4m in length

nanometer nm - commonly used to measure virus

Angstrom smallest unit of measurement


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III. Bacterial Staining Reaction

Staining procedure that applies colored chemicals called dyes tospecimen in order to facilitate identification

Stains - salts composed of a positive and negative ion, one of whichis colored (chromophore color bearing ion), which imparts

a color to cell or cell parts by becoming affixed to themthrough a chemical reaction

Basic (cationic) Dyes - chromophore is the positive ion dyeAcid (anionic) Dyes - chromophore is the negative ion dye

Bacteria are slightly negative, so are attracted to the positive chromophoreof the BASIC DYE


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Preparing smears for staining

1. Smear preparation- depends on the physical state; if in liquid state spread the

smear out- Bacteria on slide

2. Air Dry

- preserve the morphology of the bacteria

- allow the smear to adhere to the slide3. Bacteria are HEAT FIXED to the slide

Heat Fixation

- simultaneously kills the specimen and secures it to the slide

- preserve various cellular component in a natural state withminimal distortion

4. Stain is applied

Staining coloring the microorganisms with a dye


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Positive Staining Negative staining

Appearance of

organisms

Colored by dye Clear and colorless

Background Not stained Stained

Types of Staining:

1. Simple Staining- employs one dye

- most common: methylene blue, crystal violet,carbol fuchsin,safranin

- sufficient to determine size, shape & arrangement- most cells will stain the same color with the dye used


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2. Differential Staining

- employs the use of more than one dye added in several stepsand stained structures are differentiated by color as well asshape

- it is based on the relative affinity of different bacterial cells forthe stains used

- enables microbiologist to differentiate one group from anothera) Gram staining - differentiate gram (+) from gram (-) bacteriab) Acidfast staining - differentiate acidfast from non-acidfast

bacteria


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Gram-staining

Hans Christian Gram (1884), a Danish doctor, accidentallystumbled on a method which still forms the basis for theidentification of bacteria; which divided almost allbacteria into two large groups

The reagents needed:

Crystal Violet (Primary Stain)

Iodine Solution (Mordant)

Mordant - intensifies the stain or coats a structure to make itthicker and easier to see after it is stained

- Increase the affinity of a stain to the specimen

Decolorizer (ethanol is a good choice, mixture of acetone &alcohol)

Safranin (Counterstain)

Counterstain gives contrasting color to the primary stain

Gram Staining


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STEP 2: Flood the entire slide with crystal violet (primary

stain) for 1min. Then rinse with the water.

STEP 3: flood the slide with the iodine solution (mordant)

for 1min. Then rinse with water for 5 seconds. The bacteria

become deeply stained and appear deep purple in color due

to crystal violet-iodine-complex formation

Step 4: addition of the decolorizer, 95% ethanol.

Rinse with water.

Gram (+) cells : purple dye is retained

Gram (-): purple dye is readily removed and appears colorless

STEP 5: Flood the slide with the counterstain, safranin

Again, rinse with water.

Gram (+) cells will incorporate little or no counterstain and will

remain purple in appearance

Gram (-) bacteria take on a pink/red color

Gram Staining

STEP 1: Make a smear. Mounted and heat fixed.


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Divides bacteria into 2 groups

Gram (+) : violet

Gram (-) : red

Dictome of Gram Staining All COCCI are Gram Positive except Neisseria group, Moraxella

(Branhamella) catarrhalis and Veilonella

All BACILLI are Gram Negative except the acid fast organisms(Mycobacterium, Nocardia) , Sporeformers (Bacillus,Clostridum) and Corynebacterium species

Spirals are difficult to stain but when stained, they are Gram

Negative


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Gram nagative bacilli


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Gram positive cocci


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Acid Fast Staining Acid-fast stain is a useful differential staining procedure that

specifically stains all members of the genera mycobacteria The walls of certain bacteria contain long chain fatty acids

(mycolic acid) lending the property of resistance to decolorizationof basic dyes by acid alcohol; thus called acid fast

The high lipid and wax content of the mycobacterial cell walls isthought to be the reason for such impermeability

2 methods

Ziehl-Neelsen method

The procedure utilizes heat and phenol (carbolic acid) to help thepenetration of the dye, carbol fuchsin, to the inside ofmycobacterial cells, which are impermeable to basic dyes in routinestains like in Gram staining

Cold Kinyoun technique

Instead of heat, this technique uses increasing the concentration ofphenol or the inclusion of a detergent in the stain


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Divides bacteria into 2 groups

Acid - Fast organism: red

Non Acid Fast organism: blue

The reagents needed1. Primary stain: Carbol fuchsin

2. Decolorizer: Acid Alcohol

3. Counterstain: Methylene Blue

Acid Fast Staining (Ziehl Neelsen method)


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Acid - Fast Staining (Ziehl-Neelsen method)

STEP 2: Flood the entire slide with Carbol Fuchsin.

STEP 3: Using a Bunsen burner, heat the slides slowly until

they are steaming. Acid fast organisms have a very

hydrophobic surface which resist entry of dyes. Heat is used to

enhance penetration and retention of dye

Maintain steaming for 5 minutes by using low or intermittent

heat (i.e. by occasionally passing the flame from the Bunsenburner over the slides) Then rinse the slide with water.

STEP 4: Flood the slide with 3% acid-alcohol and allow to

decolorize for 5 minutes. Throughout the 5 minutes, continue to

flood the slides with 3% acid-alcohol until the slides are clear of

stain visible to the naked eye. Rinse the slide thoroughly withwater and then drain any excess from the slides.

STEP 5: Flood with the counterstain, Methylene Blue Keep

the counterstain on the slides for 1 minute. Rinse with water.

STEP 1: Make a smear. Mounted and heat fixed

3 S i l St i i


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Positive Staining Negative stainingCapsule

Flagella

Endospore

3. Special Staining- used to color and isolate specific structure of a microorganism like

capsule, flagella, inclusion granule, endospore and etc.


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Biochemical Test

various species of organism exhibits characteristic pattern ofsubstrate utilization, metabolic product formation and sugarfermentation

Enzyme based test based on its reaction with a substrate

Catalase, oxidase, indole, urease

Reactions in glucose fermentation broth

Reactions in lactose fermenation broth

Starch hydrolysis of test strains

Nitrate Broth reactions

60% of common pathogens can be identified by metabolic test


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Biochemical tests for identification of enteric

organisms

Basis: metabolic action of microorganisms

on the culture media

Used for the identification of enteric organisms/

gram negative bacilli


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One of the earliest sets of test used for the identificationof enteric bacilli

includes such organisms as Klebsiella, Enterobacter,

Citrobacter and Escherichia coli

This acronym stands for

I - Indole

M- Methyl red

V - Voges Proskauer

( i ) is inserted for euphony

C - Citrate


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Indole Test

Indole, a benzyl pyrrole, is one of the metabolic degradationproduct of amino acid tryptophan

Indole positive bacteria produce tryptophanase, an enzyme

that is capable of hydrolyzing and diaminating tryptophan,

thus producing: - indole- pyruvic acid

- ammonia

Materials:

2% Peptone broth tubeTest organisms

Ether

indicator: Erlich/Kovac's reagent

(para-dimethyl-aminobenzaldehyde)

I d l M di


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Indole Medium

Tryptone broth, contains extra tryptophan

Trp (tryptophan) can be utilized as a sole carbon andenergy source by some bacteria that produce an enzymetryptophanase

Tryptophan is significant because can be directlyincorporated into proteins, or can be broken down by

organisms with tryptophanase(tryptophanase)

Tryptophan Indole + pyruvic acid + NH3

(C-source) (N-source)

The presence of bi-product Indole can be tested withKorvacs reagent

Korvacs reagent reacts with Indole and turns solutionred


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Procedure:

Inoculate 1 loopful of the test organism into the tube of

peptone broth.

Incubate at 370C for 24-48 hours.

add 1 ml. of ether.

Shake well and allow to stand for a few minutes until the ether

rises to the surface.

Gently add about, 1cc. of Kovacs or Erlichs reagent down the

side of the tube so that it forms a ring between the medium

and the ether layer.


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Positive result

Bright red or purple ring

If indole has been produced

by the organism it will, beingsoluble in ether, it will be

concentrated in the ether layer

and upon the addition of Erlichs reagent, a positiveresult is the production of a purple ring at the

junction of the medium and the ether layer

Negative resultYellow color

- no red or purple ring

M th l d d V P k


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Methyl red and Voges-Proskauer

(MR/VP) Both tests are performed in the same medium (in same

tube!!)

This medium is used to detect both mixed acidfermentation and 2,3 butanediol fermenters

Strains ofE. coliare mixed acid fermenters; theydegrade carbohydrates into acidic end products such as:lactic acid, acetic acid, succinic acid, and formic acid

The Methyl-Red tests for acidic products resulting fromfermentation

These acidic products will drop the pH of the medium topH 4.5 or below

Methyl red pH indicator will turn red if mixed acidfermentation has occurred


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MR/VP (continued)

The Enterobacter-Klebsiella groups produceethanol and 2,3 butanediol The Voges-Proskauer test determines the presence

of acetyl-methyl-carbinol (acetoin), which is a

precursor to 2,3 butanediol The presence of 2,3 butanediol cannot directly be

determined

MRVP broth has several components:

Peptone Glucose

Buffer

MR/VP i f t ti t t f


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MR/VP is a fermentation test for

glucose Usually, the results are opposite for MR and VP

EX:Fermentation (acidic end products) MR+ (VP-)

Glucose Fermentation (alcoholic end products) VP+ (MR-)

No fermentation (no acid or alcohol products) (MR- & VP-)

MR (+) bacteria can produce lactose, succinate, formate,and acetate

VP (+) bacteria can produce acetoin, acetyl-methylcarbinol, ETOH


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Methyle red test

All enterics oxidize glucose for energy; however the end productsvary depending on bacterial enzymes

Both the MR and VP tests are used to determine what endproducts result when the test organism degrades glucose

MR test is a quantitative test for acid production, requiringpositive organism to produce strong acids (lactic, acetic, formic)from glucose via the mixed acid fermentation pathway

End result is based on the final pH reached only those organism that can maintain low ph of about

ph 4-4.5 can be called methyl red positive

organisms that are MR (+) are always VP (-)


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Materials:

MR-VP broth medium (contains 10% glucose)

Test organisms

Methyl red ph indicator


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Procedure:

Inoculate 1 loopful of the test organisminto a tube MR-VP medium.

Incubate for 24-48 hours at 370

C.

Next laboratory period, add 5 to 10 dropsof methyl red reagent.

Mix thoroughly and observe the results.


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Positive result cherry red/bright red color

ph 4-4.5

Ex. Salmonella, Escherichia,

Citrobacter, Proteus, Morganellaand Providencia

Negative result Yellow color

At neutral pH the growth of the bacteria is not inhibited

The bacteria thus begin to attack the peptone in the broth,causing the pH to rise above 4.5

At this pH, methyl red indicator produce a yellow color

Ex. Enterobacterand Klebsiella


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Voges Proskauer test

is a test for the detection of acetyl-methyl carbinol

(acetoin) which is also a degradation product of glucose

Materials:

MR-VP medium (contains 10% glucose)

Test organism

Potassium Hydroxide

Alpha-napthanol reagent

When these reagents are added to a broth in whichacetyl methyl carbinol is present, they turn a burgundy

color/crimson red color (a positive VP test)

organisms that are VP (+) are always MR (-)


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Procedure

Inoculate MR-VP medium with 1 loopful of the test organism

Incubate for 48 hours at 370C.

Add 0.6 ml. 5% alpha-napthol reagent. Mix and shake themixture lightly.

Add 0.2 ml (5drops). of 40% potassium hydroxide reagent(KOH).

Mix and shake the mixture lightly.

Shake the tube gently to expose the medium to atmosphericoxygen and allow the tube to remain undisturbed for 10 to

15 minutes.


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Positive resultCrimson Red color

Presence of Acety methyl carbinol

Ex. Enterobacterand Klebsiella

Negative resultRemains Yellow to Amber; no change in color

Ex. E. coli


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Citrate Test

Simmons Citrate agar is used to determine anorganisms ability to use citrate as a sole carbonsource

Simons citrate agar is a defined medium in

which sodium citrate is the sole carbon source,and ammonium is the sole nitrogen source

Bromothymol blue (BTB) is included as a pHindicator

The medium is initially at pH 6.9, at which BTB isgreen; at a pH greater than 7.6 BTB turns adeep blue


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Citrate Test (continued)

The pH change is induced by CO2, which isgiven off as a by-product of citrate utilization.When it reacts with Na and H2O in the agar itraises the pH above 7.6

The organism must contain the enzyme citraseto degrade citrate

EX:

(citrase)

Citrate CO2 + Na HCO + H2O

(blue color change)


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Procedure:

Inoculate the test organism on the medium by stab streaking.

Incubate at 370C for 24 - 48 hours.

Observe.


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Positive result

Deep blue/ Prussian blue color

indicating that the test organism

has been able to utilize citrate for energy source Ex. Enterobacter, Klebsiella, Salmonella, Citrobacter

and Providencia

Negative result

Retains its original color (Green)

Ex. Escherichia, Shigella and Morganella


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Motility Test

The two tubes onthe left contain anonmotile bacterialspecies. Noticethe clearly visible

line of growth(streak line).The two tubes onthe right contain amotile bacterial

species. Noticethe cloudy mediaand the lessdistinct line ofgrowth.

Molecular differentiation


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Molecular differentiation

Genomics

Gene characterizationSequencing

PCR

Hybridization

% guanine + cytosine


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Serological procedure Antigen and antibody determination

Serological Tests

Use group specific antiserum isolated from the plasma ofanimals that have been sensitized to the organism

The antiserum contains antibody proteins that react with antigens

on the unknown organism. Procedures: agglutination, precipitation test, hemagglutination

inhibition, complement fixation, ELISA, Western blot assay

Advantages:

Highly specific Does not usually require the organism to be isolated into pure

culture

Can be used to identify organisms that cant be grown on medium


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Antibiotic sensitivity antibiotic sensitivity is a term used to describe the susceptibility

of bacteria to antibiotics

Antibiotic susceptibility testing (AST) is usually carried out todetermine which antibiotic will be most successful in treating abacterial infection in vivo

Methods of testing: Broth dilution

The lower the dilution, the greater the antibiotic content

Agar dilution

Disk diffusion the Kirby-Bauer test for antibiotic susceptibility, called the disc

diffusion test, is a standard that has been used for years


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The bacterium is swabbed on the agarand the antibiotic discs are placed on top

The antibiotic diffuses from the disc intothe agar in decreasing amounts the furtherit is away from the disc

Bacteria are not able to grow around antibioticsto which they are sensitive

If the organism is killed or inhibited by theconcentration of the antibiotic, there will be

NO growth in the immediate area around the disc:called the zone of inhibition

The zone sizes are looked up on a standardized chart togive a result of sensititive, resistant, or intermediate

Many charts have a corresponding column that also gives the
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Indicator organism


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coliform

Family Enterobacteriaceae.

Lactose fermenting

Intestinal tract of animals

Free living in the environment

Opportunistic pathogens


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Methods

Multiple-Tube Fermentation

Membrane Filter Technique

Chromogenic Substrate Test


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Membrane Filter Technique


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The plate in the upper left shows typical dark blue fecal coliform colonies

on a membrane filter after incubation on M-FC agar. The plate in the lower

center (purple) is M-FC agar before use, and the plate in the upper right

(blue) is a control plate streaked with an E.c o li culture.

Rapid, quantitative analyses of coliforms


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and E.coli, based on COLILERT cultivation

(IDEXX, U.S.A.)

Documents

Classification and Indentification of Bacteria