7658686 Classification Identification

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Laboratory procedures employed in theidentification of bacteria

1. Isolation of organism in pure culture2. Bacterial colony morphology3. Microscopic morphology and Staining reaction4. Biochemical test 5. Serological procedure

6. Antibiotic sensitivity



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 almost pure bacterial culture

specimen of sputum, stool, materials from the skin andbody orifices usually contains mixture of organism

- Spread plate, streak plate, and pour plate are

techniques used to isolate pure culture



Laboratory Cultivation

y Cultivation is the process of growing microorganisms bytaking bacteria from the infection site by some means of specimen collection and growing them in the artificialenvironment of the laboratory

y

For the in vitro environment of the bacteria, required nutrientsare supplied in a culture medium

y culture - organisms that grow and multiply in or on a culturemedia



Culture Medium- is 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 arenutrient broths and agar plates

- specialized media are sometimes required for microorganism and cellculture growth



Based on Chemical CompositionComplex 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 media contain all the elements that most bacteria needfor growth and are non-selective, so they are used for thegeneral cultivation and maintenance of bacteria kept inlaboratory culture collections

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



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 specialnutrients Blood agar is an enriched medium in which nutritionally rich whole blood

supplements the basic nutrients Chocolat e agar is enriched with heat -treated blood (40-45°C), 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 forG

ram-negative bacteria Mannitol Salt Agar (MSA) which is selective for Gram (+) bacteria and differential for



Alpha Hemolytic Streptococci

Incomplete lysis of RBC¶s

Beta Hemolytic Streptococci

Complete lysis of RBC¶s

Gamma Hemolytic Streptococci

No lysis of RBC¶s



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 to

visibly indicate the defining characteristics of a microorganismEx. 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



Bacterial colony morphology Bacteria grow on solid media as colonies

colony is defined as a visible mass of microorganisms alloriginating from a single mother cell, when inoculated into

appropriate 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



To identify the following colonial characteristics/culture characteristics:

WHOLE SHAPE OF COLONY EDGE /MARGIN OF COLONY



ELEV ATION 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

OPACIT Y OF COLONY:transparent (clear), opaque,translucent (almost clear, but distorted visionlike looking through frosted glassiridescent (changing colors in reflected light)

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

EMULSIFIABILIT Y OF COLONY:Is it easy or difficult to emulsify? Does it form a uniform suspension, a granularsuspension, or does not emulsify at all?



SURFACE OF COLONY:smooth, mucoid/glistening, rough, dull (opposite of glistening),rugose (wrinkled)

Smooth - colonies gives the appearance of homogeneity and uniform

texture without appearing as liquid or as mucoid coloniescharacteristically isolated from fresh wild type organism such asgram- negative enterobacteria Ex. Salmonella, Shigella

Mucoid - colonies exhibits a water-like glistening confluent

appearance commonly seem among organism which fromslime layer or capsule. Ex. Kleb. pneumoniae, S. pneumoniae

Rough colonies are granulated and rough in appearance, usually

produced by mutant strain that lacks surface protein andpolysaccharide of freshly isolated wild-type parent organism



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: shapearrangement staining reaction



I. Bacterial Shape and Arrangementy Bacterial Shape

determined by the configuration of the cell wall detected by brightfield microscopy of stained smear

y Bacterial Arrangement

y is the result of the number of plane division the organism may undergoand how the cell remain attached afterwards

y divides only across their short axis

y 3 conventional forms :

Spherical (cocci) Rod (bacilli) Spirals



Spherical (Cocci)

y Shape:y round like a ball, perfect sphere or globe

y Variations :

1. Ovoid shape - both sides rounded ends are pointed

Ex. Streptococcus2. Lancet -shape - one end is pointed, other end is flat

Ex. Pneumococcus

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

Ex. Neisseria



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. Pairs diplococci

- common with lancet -shaped and coffee-bean

shaped spherical resulting from one plane divisionwith daughter cell separatingEx. Streptococcus pneumoniae

Neisseria gonorrheae



4. Clusters staphylococci- common with spherical resulting from many plane

division with daughter cell in grape-like agglomeration

Ex. Staphylococcus aureus

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



Rods (Bacilli)y

Shapey cell appears longer than wide or cylindrical form

y both sides parallel and ends are convex

y varies in actual form depending on the species

y divides only across their short axisy Variations :

1. Clubbed/drumstick shaped swollen on one end

Ex. Clostridium diphtheriae/C. tetani

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

3. Fusiform both sides parallel, ends pointed



Arrangements:1. Singly occurs as a single rod

2. Chain result from one plane division with daughtercell remain attached to one another

Ex. 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 cigarette arrangement like bundles

Ex. Mycobacterium leprae

6. Serpentine commonly seen with virulent strainof Mycobacterium tuberculosis



I t r i t f r

Coccobacilli- when a rod is short & wide / l- t hese f or is int er ediat e bet ween a s herical

and rodEx. aemophil s, r cella

Vibrio - a entl cur e bact eria (comma-shaped) -

it is an

int

er

mediat

e b

et

ween a rod a

nd a spiralEx. Vibrio cholerae



Spiralsy bacteria with more than one somatic curve

y may be regarded as bacillary forms trusted in the form of a helix

y no characteristic cell arrangement

y most occurs singly

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

y 2 types :

1. Flexible spirals that can contract and relax move by creeping

movement Ex. Spirochetes

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

Ex. Spirillum



SPIRILLUM- whose long axis remains rigid when in

motion

Ex. Campylobacter jejuni

SPIROCHETE

whose long axis bends when in motionGenus 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



II. Bacterial size

y all linear measurements in microbiology are expressed inmetric units

the basic unit of the metric system is the meter m

y centimeter cm (1/100th of a m)

- the largest unit of length used for measuring microorganismy micrometer µm

- visible only with high powered microscope

- unit of measurement most frequently used in microbiology

y

1µm = 1/1000 of a mmy Cocci = 0.4-2µm

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

y Spirals = 1-4µm in length

y nanometer nm - commonly used to measure virus

y Angstrom smallest unit of measurement





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 dye Acid (anionic) Dyes - chromophore is the negative ion dye

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



y Preparing smears for staining

1. Smear preparation

- depends on the physical state; if in liquid state spread thesmear out

- Bacteria on slide

2. Air Dry

- preserve the morphology of the bacteria

- allow the smear to adhere to the slide

3. Bacteria are HE AT FIX ED 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



Positive Staining Negative staining

Appearance of

organisms

Colored by dye Clear and colorless

Background Not stained

(generally white)

Stained

(dark gray or black)

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



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



ram-staining

y Hans Christian Gram (1884), a Danish doctor, accidentally

stumbled on a method which still forms the basis for theidentification of bacteria; which divided almost all bacteria intotwo large groups

y The reagents needed:

y Crystal Violet (Primary Stain)

y Iodine Solution (Mordant)

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

- Increase the affinity of a stain to the specimen

y Decolorizer (ethanol is a good choice, mixture of acetone alcohol)y Safranin (Counterstain)

y Counterstain gives contrasting color to the primary stain



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.



PRINCIPLE:

y Gram reaction is based on the structure of the bacterial cell wall

y ram-positive bacteria

y the peptidoglycan appears to act as a permeability barrier preventingloss of crystal violet -iodine-complex

y When gram-positive bacteria are treated with alcohol, the alcoholcauses coagulation and dehydrateion of the thick layer of peptidoglycan resulting in shrinkage of pores preventing C VI-complexfrom escaping and the bacteria remain deep purple

y Reaction to Gram staining is also believed to be asso. With proteincomplex Magnesium ribonucleate which is absent in Gram (-) org.

y ram Negative bacteria

y peptidoglycan is very thin in gram (-) bacteria and has larger pores

y Alcohol readily penetrates the lipid-rich outer layer of the cell wall andextracts enough lipid thus increasing the porosity further

y alcohol more readily removes the deep purple C VI-complex fromgram (-) bacteria thus becomes decolorized

y The outer membrane is then permeabilized by the decolorizer, andthe pink safranin counterstain is trapped by the peptidoglycan layer



Divides bacteria into 2 groups

y Gram (+) : violet

y

Gram (-) : red

Dictome of Gram Staining

y All COCCI are Gram Positive except Neisseria group,Moraxella (Branhamella) catarrhalis and Veilonella

y All BACILLI are Gram Negative except the acid fast

organisms (Mycobacterium, Nocardia) , Sporeformers(Bacillus, Clostridum) and Corynebacterium species

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

Gram Negative



Acid Fast Stainingy

Acid-fast stain is a useful differential staining procedure that specifically stains all members of the genera mycobacteria

y 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

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

y 2 methods

y Ziehl-Neelsen method

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

y Cold Kinyoun technique

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



y Divides bacteria into 2 groups

y Acid - Fast organism: red

y Non Acid Fast organism: blue

y The reagents needed

1. Primary stain: Carbol fuchsin

2. Decolorizer: Acid Alcohol

3. Counterstain: Methylene Blue



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 Bunsen

burner 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



Positive Staining Negative staining

Capsule

Flagella

Endospore

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

capsule, flagella, inclusion granule, endospore and etc.



Biochemical Test

y various species of organism exhibits characteristicpattern of substrate utilization, metabolic product formation and sugar fermentation

y Enzyme based test based on its reaction with a substratey Catalase, oxidase, indole, urease

y Reactions in glucose fermentation broth

y Reactions in lactose fermenation broth

y Starch hydrolysis of test strains

y Nitrate Broth reactions

y 60% of common pathogens can be identified bymetabolic test



Serological procedurey

Antigen and antibody determinationy Serological Tests

y Use group specific antiserum isolated from the plasma of animals that have been sensitized to the organism

y The antiserum contains antibody proteins that react withantigens on the unknown organism.

y Procedures: agglutination, precipitation test, hemagglutinationinhibition, complement fixation, ELISA, RIA, Western blot assay

y Advantages:

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

culture

y Can be used to identify organisms that cant be grown onmedium



Antibiotic sensitivityy

antibiotic sensitivity is a term used to describe the susceptibilityof bacteria to antibiotics

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

y Methods of testing:

y Broth dilution

y The lower the dilution, the greater the antibiotic content

y Agar dilution

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

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

y



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

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

y Bacteria are not able to grow around antibiotics

to which they are sensitive

y If the organism is killed or inhibited by theconcentration of the antibiotic, there will beNO growth in the immediate area around the disc:

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

y Many charts have a corresponding column that also gives the MIC(minimal inhibitory concentration) for that drug



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7658686 Classification Identification

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