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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.)