Lecture 2 Classification & Identification

8/8/2019 Lecture 2 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 reaction

Gram Stain Acid fast Stain

4. Biochemical characteristics5. Serological procedure6. Antibiotic sensitivity



Isolation of organism in Pure CulturePure culture (axenic culture)

y Population of cells arising from a single cell

y the approach used for the isolation of organism depends

upon the source of clinical specimen

y pure bacterial culture : Blood, spinal fluid and closed abscessesmay yield almost

y mixture of organism : specimen of sputum, stool, materialsfrom the skin and body orifices usually contains

y The specimen is streaked onto solid agar-containing medium so asto separate the bacterial population into individual cells which growas individual colonies



y Techniques to obtain pure culture:

1. streak plate method

y This is the best and fastest of several ways to obtain a PC

y A small amount of desired microbe is obtained with a sterileloop, and a single colony is grown on a separate

quadrant of the agar plate

y

Advantage: recolonize one particular type of bacteria up tothree times on one agar plate

2. pour plate method

y separating one species of bacteria from another by diluting

one loopful of organism into three liquefied nutrient agar plates, with the hopes that one of the platespoured will provide an ideal sample for isolation

y Advantage: requires far less skill than the first method



Laboratory Cultivation

y Cultivation is the process of growing microorganisms bytaking bacteria from the infectionsite by some means of specimencollection and growing them in theartificial environment of thelaboratory

y For the in vitro environment of the bacteria, required nutrients

are supplied in a culture mediumy culture - organisms that grow and multiply in or on a culture

media



Culture Medium

y is a general term referring to the substanceupon which a microorganism is grown

y Ideally designed to mimic the environment in which theorganism naturally grows

y The most common growth media for microorganisms arenutrient broths and agar plates

y microbiological media currently are categorized as:





Based on Chemical Composition

y chemically defined medium

- the exact amount of pure chemicals used toformulate the medium is known

y Complex medium

- is composed of a mixture of proteins and extracts inwhich the exact amount of a particular amino

acid, sugar, etc. is not known- Nutrient media contain all the elements that most bacteria need for growth and are non-selective, so theyare used for the general cultivation and maintenance of bacteria kept in laboratory culture collections



Functional Types of Media

Supportive or general purpose media- Support the growth of many microorganisms- E.g. Tryptic soy agar

Enriched media- For fastidious organisms- contain nutrients ecologically favorable to the organism to be

isolated- 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 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- requirements for growth:

- specific pH- ionic strength- Chemical composition- may contain inhibitors- lack of nutrients for all but the organism in question

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



Differential media Distinguish between different groups of microorganisms based

on 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- defined as a visible mass of microorganisms

all originating from a single mother cell,when inoculated into appropriatemedium containing 2% agar and incubated12-24 hours in a favorable atmosphere

- ideally composed of the descendants of a single cell- Colony-forming unit (CFU) 2 or more daughter cells

that do not separate- gross characteristics: size, shape, texture, elevation,

pigmentation, effect on growth medium

- useful in bacterial identification



To identify the following colonial characteristics:



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 watersoluble) Some pigments readily diffuse throughout the medium (i.e, water soluble) Some pigments fluoresce in UV light

OP ACITY OF COLONY:transparent (clear), opaquetranslucent (almost clear, but distorted visionlike looking through

frosted glass)iridescent (changing colors in reflected light)

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



SURFACE OF COLONY:

Smooth (S colonies)- colonies gives the appearance of homogeneity

and uniform texture without appearing as liquid or as mucoidcolonies characteristically isolated from fresh wild type organismsuch as gram- negative enterobacteriaEx. Salmonella, Shigella, Proteus

Mucoid (M colonies) - colonies exhibits a water-like glistening confluent

appearance commonly seem among organism which fromslime layer or capsule. Ex. Klebsiella pneumoniae

Streptococcus pneumoniae

Rough (R colonies) 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 organism1. Bacterial morphology

2. Bacterial size3. Staining reaction

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 theyare examined in the natural state

Routinely used to determine: shape, arrangement, and

staining reaction



I. Bacterial Shape and Arrangement

y Bacterial Shape

determined by the configuration of the cell wall

detected by brightfield microscopy of stained smear usingoil immersion lens

y Bacterial Arrangement

y is the result of the number of plane division the organism may undergo

and how the cell remain attached afterwardsy divides only across their short axis

y 3 conventional forms :

pherical (cocci) od (bacilli) pirals



Spherical (Cocci)

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

y Variations :

1. Ovoid shape - both sides rounded ends are

pointedEx. Streptococcus

2. Lancet-shape - one end is pointed, otherend is flat Ex. Pneumococcus

3. Coffee-bean shape - flat on one side,opposite side convex or appear asletter D form

Ex. Neisseria



Arrangements:

1. Singly coccus

- occurs as a single spherical cell

2. Chain streptococci- common among ovoid-form resulting from one

plane division with daughter cells remainedattached to one another to form a chainEx. Streptococcus pyogenes

3. Pairs diplococci

- common with lancet-shaped and coffee-bean

shaped spherical resulting from one planedivision with daughter cell separatingEx. Streptococcus pneumoniae

Neisseria gonorrheae



4. Clusters staphylococci- common with spherical resulting from many

random plane divisions with daughter cell in

grape-like agglomerationEx. Staphylococcus aureus

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

cell separating from one another to formgroup of 4 cellsEx. Micrococcus tetragenous

6. Sarcinae (Packets of 8)- results from 3 plane divisions producingcubical packets of 8 cells

Ex. Sarcina lutea



ods (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 - bacillus

- occurs as a single rod

2. Chain streptobacilli- result from one plane division with daughter cell

remain attached to one another

Ex. Bacillus anthracis

3. Palisade arrangement like fence due toslipping movement of daughter cells(side-by-side)Common among club-shaped rods4. Chinese-letter common with clubbed-shapedrods resulting from a snapping post divisionmovement of the daughter cells (V shape)

Ex. Corynebacterium diptheriae

Mycobacterium tuberculosis



5. Packets of cigarette arrangement

like bundlesEx. Mycobacterium leprae

6. Serpentine commonly seen with virulent

strain of Mycobacterium tuberculosis



I t r i t f r f cilli

Coccobacilli- a rod is s ort & ide / l- t ese f or is i t er ediat e bet een a

s erical and rodEx. aemophil s, r cella

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

- it is an int ermediat e bet een a rod and 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 motion

Genus Treponema tightly coiled w/ cork screw appearanceEx. Treponema pallidum

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

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 apply 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

- Staining procedure was 1st introduced by Paul E hrlich



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 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



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 bluealternative: 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



Gram-staining

y Hans Christian Gram (1884), a Danish doctor, accidentallystumbled on a method which still forms the basis for theidentification of bacteria

y This staining procedure defines 2 bacterial group

y Those w/c retain the primary dye (gram-positive)y those which are decolorized (gram negative)

y The difference in dye retention is dependent on such physicalproperties as thickness, density, porosity and integrity of thebacterial cell wall, as well to some extent, the chemicalcomposition



y The reagents needed:

y Crystal Violet (Primary Stain)

y Iodine Solution/Grams Iodine (Mordant)

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

- Increase the affinity of a stain to the specimeny Decolorizer (ethanol is a good choice, mixture of acetone

alcohol)

y Safranin (Counterstain)

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% ethyl alcohol

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 Gram-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 dehydration of the thick layer of peptidoglycanresulting in shrinkage of pores preventing C VI-complex from escapingand 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 Gram Negative bacteria

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

y Alcohol readily penetrates the lipidrich 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 genus 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 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



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 substrate

y Catalase, oxidase, indole, ureasey 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 bybiochemical 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

is a term used to describe the susceptibility of bacteria toantibiotics

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

y Methods of testing:y Broth dilution

- The lower the dilution, the greater the antibiotic content

y Agar dilution

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

the disc diffusion test, is a standard that has beenused for years



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 antibioticsto which they are sensitive

y 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 sensitive, resistant, or intermediate



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Lecture 2 Classification & Identification