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Laboratory procedures employed in the identification of bacteria
1. 2. 3. 4. 5. 6. Isolation of organism in pure culture Bacterial colony morphology Microscopic morphology and Staining reaction Biochemical test Serological procedure 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 depends upon 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 and body orifices usually contains mixture of organism - Spread plate, streak plate, and pour plate are
techniques used to isolate pure culture
y Cultivation is the process of growing microorganisms by
taking bacteria from the infection site by some means of specimen collection and growing them in the artificial environment of the laboratory y For the in vitro environment of the bacteria, required nutrients are supplied in a culture medium y culture - organisms that grow and multiply in or on a culture media
which are used for growing microorganisms 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 .2 major types of growth media: .Culture Medium .microbiological culture.is a liquid or gel designed to support the growth of microorganisms .those used for cell culture. which use specific cell types derived from plants or animals .
Based on Chemical Composition Complex Media . so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections Defined or Synthetic Media -All components and their concentrations are known .Contain some ingredients of unknown composition and/or conc. beef.g.Nutrient media contain all the elements that most bacteria need for growth and are non-selective. .. yeast extract) .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. 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 . which turns brown and gives the medium the color for which it is named Selective media ..Support the growth of many microorganisms . Tryptic soy agar Enriched media . allowing only the growth of Gram (-) bacteria blood agar (used in strep tests).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.Functional Types of Media Supportive or general purpose media .E.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).
Alpha Hemolytic Streptococci Gamma Hemolytic Streptococci Beta Hemolytic Streptococci Incomplete lysis of RBC¶s Complete lysis of RBC¶s No lysis of RBC¶s .
Differential media Distinguish between different groups of microorganisms based on their biochemical characteristics growing in the presence of specific nutrients or indicators (such as neutral red. Blood agar differentiates hemolytic versus non-hemolytic bacteria MacConkey agar . which is differential for mannitol fermentation . eosin y. phenol red.lactose fermenters versus non-fermenters Eosin methylene blue (EMB). or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism Ex. which is differential for lactose and sucrose fermentation Mannitol Salt Agar (MSA).
the colony is the progeny of one. when inoculated into appropriate medium containing 2% agar and incubated 18-24 hours in a favorable atmosphere therefore a colony constitutes a clone of bacteria all genetically alike Ideally. 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. pigmentation. or at most. elevation. shape. texture.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. effect on growth medium .
To identify the following colonial characteristics/culture characteristics: WHOLE SHAPE OF COLONY EDGE/MARGIN OF COLONY .
translucent (almost clear. but distorted vision like looking through frosted glass iridescent (changing colors in reflected light) CONSISTENCY: butyrous (buttery). a granular suspension. Some pigments are contained within the cell (i. etc. opaque. red.e. probably not water soluble) Some pigments readily diffuse throughout the medium (i.e. hard to get off) brittle/friable (dry.Some bacterial species form an array of pigments: white. viscid (sticks to loop. water soluble) Some pigments fluoresce in UV light OPACITY OF COLONY: transparent (clear). purple. breaks apart) EMULSIFIABILITY OF COLONY: Is it easy or difficult to emulsify? Does it form a uniform suspension..ELEVATION OF COLONY (turn the place on end to determine height) CHROMOGENESIS (pigmentation) . or does not emulsify at all? .
mucoid/glistening.negative enterobacteria Ex.SURFACE OF COLONY: smooth.colonies gives the appearance of homogeneity and uniform texture without appearing as liquid or as mucoid colonies characteristically isolated from fresh wild type organism such as gram. Ex. pneumoniae. S.colonies exhibits a water-like glistening confluent appearance commonly seem among organism which from slime layer or capsule. usually produced by mutant strain that lacks surface protein and polysaccharide of freshly isolated wild-type parent organism . dull (opposite of glistening). pneumoniae Rough colonies are granulated and rough in appearance. Kleb. rough. Shigella Mucoid . Salmonella. rugose (wrinkled) Smooth .
which makes the study of the morphologic detail difficult when they are examined in the natural state Routinely used to determine: shape arrangement staining reaction .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 or biochemical Bacteria and other microorganism are usually transparent.
Bacterial Shape and Arrangement y Bacterial Shape determined by the configuration of the cell wall y detected by brightfield microscopy of stained smear Bacterial Arrangement y is the result of the number of plane division the organism may undergo and how the cell remain attached afterwards y divides only across their short axis y 3 conventional forms : Spherical (cocci) Rod (bacilli) Spirals .I.
Streptococcus 2. Pneumococcus 3. Coffee-bean shape .flat on one side. other end is flat Ex.one end is pointed.both sides rounded ends are pointed Ex. opposite side convex or appear as letter D form Ex. Lancet-shape . Ovoid shape . Neisseria . perfect sphere or globe y Variations : 1.Spherical (Cocci) y Shape: y round like a ball.
common with lancet-shaped and coffee-bean shaped spherical resulting from one plane division with daughter cell separating Ex.common among ovoid-form resulting from one plane division with daughter cells remained attached to one another to form a chain Ex. Singly occurs as a single spherical cell 2. Streptococcus pneumoniae Neisseria gonorrheae . Chain streptococci . Streptococcus pyogenes 3. Pairs diplococci .Arrangements: 1.
4. Micrococcus tetragenous 6. Staphylococcus aureus 5.result from 2 plane division with daughter cell separating from one another to form group of 4 cells Ex.common with spherical resulting from many plane division with daughter cell in grape-like agglomeration Ex. Sarcinae (Packets of 8) . Clusters staphylococci . Tetrads (Packets of 4) .results from many plane division producing cubical packets of 8 cells Ex. Sarcina lutea .
Clostridium diphtheriae/C. ends pointed . Bacillus anthracis 3. Corset-shape both sides swollen. Clubbed/drumstick shaped swollen on one end Ex.Rods (Bacilli) y Shape y 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 axis y Variations : 1. ends flat or concave Ex. Fusiform both sides parallel. tetani 2.
Packets of cigarette arrangement like bundles Ex. Corynebacterium diptheriae 5. Singly occurs as a single rod 2.Arrangements: 1. Serpentine commonly seen with virulent strain of Mycobacterium tuberculosis . Mycobacterium leprae 6. Chain result from one plane division with daughter cell remain attached to one another Ex. Palisade arrangement like fence due to slipping movement of daughter cells (side-by-side) Common among clubbed shaped rods Ex. Chinese-letter common with clubbed-shaped rods resulting from a snapping post division movement of the daughter cells (V shape) Ex. Mycobacterium tuberculosis 4. Bacillus anthracis 3.
when a rod is short & wide/ l .a entl cur e bacteria (comma-shaped) .it is an intermediate between a rod and a spiral Ex. r cella Vibrio .I t r i t f r Coccobacilli . Vibrio cholerae . aemophil s.these for is inter ediate between a s herical and rod Ex.
length. rigidity and amplitude of their coils y 2 types : 1. Spirochetes move by creeping y 2.Spirals y 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. Spirillum move by . Flexible spirals that can contract and relax movement Ex. Rigid spirals that cannot contract and relax rotation or corkscrew-like motion Ex.
Borrelia recurrentis . tightly coil w/ cork screw appearance Ex. Leptospira interrogans Genus Borrelia much less tightly coiled w/c has the appearance of extremely long undulating bacillary pores Ex.SPIRILLUM . Campylobacter jejuni SPIROCHETE whose long axis bends when in motion Genus Treponema char. Trepanema pallidum Genus Leptospira less tightly coiled w/ sharp hook-like bends Ex.whose long axis remains rigid when in motion Ex.
II.the largest unit of length used for measuring microorganism y micrometer µm .visible only with high powered microscope . Bacterial size y all linear measurements in microbiology are expressed in metric units the basic unit of the metric system is the meter m y centimeter cm (1/100th of a m) .4-2µm y Bacilli = 0.5-20µm in length y Spirals = 1-4µm in length y nanometer nm .unit of measurement most frequently used in microbiology y 1µm = 1/1000 of a mm y Cocci = 0.2-4µm in width by o.commonly used to measure virus y Angstrom smallest unit of measurement .
one of which is colored (chromophore color bearing ion). which imparts a color to cell or cell parts by becoming affixed to them through a chemical reaction Basic (cationic) Dyes . so are attracted to the positive chromophore of the BASIC DYE .III.salts composed of a positive and negative ion. Bacterial Staining Staining Reaction procedure that applies colored chemicals called dyes to specimen in order to facilitate identification Stains .chromophore is the negative ion dye Bacteria are slightly negative.chromophore is the positive ion dye Acid (anionic) Dyes .
y Preparing smears for staining 1.allow the smear to adhere to the slide 3.simultaneously kills the specimen and secures it to the slide . Smear preparation .preserve various cellular component in a natural state with minimal distortion 4. Bacteria are HEAT FIXED to the slide Heat Fixation . Stain is applied Staining coloring the microorganisms with a dye . if in liquid state spread the smear out . Air Dry .preserve the morphology of the bacteria .depends on the physical state.Bacteria on slide 2.
most common: methylene blue.sufficient to determine size. Simple Staining . crystal violet. carbol fuchsin.employs one dye .most cells will stain the same color with the dye used Positive Staining Appearance of organisms Colored by dye Negative staining Clear and colorless Background Not stained (generally white) Stained (dark gray or black) . shape arrangement .Types of Staining: 1.safranin .
differentiate acidfast from non-acidfast bacteria .2.differentiate gram (+) from gram (-) bacteria b) Acidfast staining .it is based on the relative affinity of different bacterial cells for the stains used . Differential Staining .enables microbiologist to differentiate one group from another a) Gram staining .employs the use of more than one dye added in several steps and stained structures are differentiated by color as well as shape .
Increase the affinity of a stain to the specimen y Decolorizer (ethanol is a good choice. mixture of acetone y Safranin (Counterstain) y alcohol) Counterstain gives contrasting color to the primary stain .intensifies the stain or coats a structure to make it thicker and easier to see after it is stained . which divided almost all bacteria into two large groups y The reagents needed: y Crystal Violet (Primary Stain) y Iodine Solution (Mordant) y Mordant .ram-staining y Hans Christian Gram (1884). accidentally stumbled on a method which still forms the basis for the identification of bacteria. a Danish doctor.
rinse with water. Then rinse with the water. 95% ethanol.Gram Staining STEP 1: Make a smear. STEP 3: flood the slide with the iodine solution (mordant) for 1min. The bacteria become deeply stained and appear deep purple in color due to crystal violet-iodine-complex formation Step 4: addition of the decolorizer. Rinse with water. Then rinse with water for 5 seconds. Gram (+) cells will incorporate little or no counterstain and will remain purple in appearance Gram (-) bacteria take on a pink/red color . 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. STEP 2: Flood the entire slide with crystal violet (primary stain) for 1min. Mounted and heat fixed.
y ram 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 and extracts enough lipid thus increasing the porosity further y alcohol more readily removes the deep purple CVI-complex from gram (-) bacteria thus becomes decolorized y The outer membrane is then permeabilized by the decolorizer.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 preventing loss of crystal violet-iodine-complex y When gram-positive bacteria are treated with alcohol. the alcohol causes coagulation and dehydrateion of the thick layer of peptidoglycan resulting in shrinkage of pores preventing CVI-complex from escaping and the bacteria remain deep purple y Reaction to Gram staining is also believed to be asso. and the pink safranin counterstain is trapped by the peptidoglycan layer . With protein complex Magnesium ribonucleate which is absent in Gram (-) org.
Divides bacteria into 2 groups y Gram (+) : violet y Gram (-) : red Dictome of Gram Staining y All COCCI are Gram Positive except Neisseria group. Sporeformers (Bacillus. Moraxella (Branhamella) catarrhalis and Veilonella y All BACILLI are Gram Negative except the acid fast organisms (Mycobacterium. they are Gram Negative . Clostridum) and Corynebacterium species y Spirals are difficult to stain but when stained. Nocardia) .
Acid Fast Staining y 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 decolorization of basic dyes by acid alcohol. to the inside of mycobacterial cells. thus called acid fast y The high lipid and wax content of the mycobacterial cell walls is thought 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 the penetration of the dye. this technique uses increasing the concentration of phenol or the inclusion of a detergent in the stain . carbol fuchsin. which are impermeable to basic dyes in routine stains like in Gram staining y Cold Kinyoun technique y Instead of heat.
y Divides bacteria into 2 groups y Acid . Decolorizer: Acid Alcohol 3. Counterstain: Methylene Blue .Fast organism: red y Non Acid Fast organism: blue y The reagents needed 1. Primary stain: Carbol fuchsin 2.
Methylene Blue Keep the counterstain on the slides for 1 minute.e. heat the slides slowly until they are steaming. STEP 4: Flood the slide with 3% acid-alcohol and allow to decolorize for 5 minutes. STEP 5: Flood with the counterstain. continue to flood the slides with 3% acid-alcohol until the slides are clear of stain visible to the naked eye. Throughout the 5 minutes. Mounted and heat fixed STEP 2: Flood the entire slide with Carbol Fuchsin. . Heat is used to enhance penetration and retention of dye Maintain steaming for 5 minutes by using low or intermittent heat (i. Rinse with water. STEP 3: Using a Bunsen burner.Fast Staining (Ziehl-Neelsen method) STEP 1: Make a smear.Acid . Acid fast organisms have a very hydrophobic surface which resist entry of dyes. by occasionally passing the flame from the Bunsen burner over the slides) Then rinse the slide with water. Rinse the slide thoroughly with water and then drain any excess from the slides.
Special Staining . inclusion granule.used to color and isolate specific structure of a microorganism like capsule. Positive Staining Capsule Negative staining Flagella Endospore . endospore and etc.3. flagella.
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 by metabolic test . metabolic product formation and sugar fermentation y Enzyme based test based on its reaction with a substrate y Catalase. indole.Biochemical Test y various species of organism exhibits characteristic pattern of substrate utilization. oxidase.
RIA. Western blot assay y Advantages: y Highly specific y Does not usually require the organism to be isolated into pure culture y Can be used to identify organisms that can t be grown on medium .Serological procedure y Antigen and antibody determination y 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 with antigens on the unknown organism. y Procedures: agglutination. precipitation test. ELISA. hemagglutination inhibition. complement fixation.
is a standard that has been used for years y . called the disc diffusion test.Antibiotic sensitivity y antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics y Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial 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 diffusion y the Kirby-Bauer test for antibiotic susceptibility.
or intermediate y Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug . 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 to give a result of sensititive.y The bacterium is swabbed on the agar and the antibiotic discs are placed on top y The antibiotic diffuses from the disc into the agar in decreasing amounts the further it 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 the concentration of the antibiotic. resistant.
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