Identification of prokaryotes•Identification of prokaryotes using phenotypiccharacteristics• Identification of prokaryotes using genotypiccharacteristics• Characterizing strain differences
A.Identification of prokaryotes using phenotypic characteristics1. Microscopic Analysis• An important step is to determine: • size, shape and staining characteristics of a microorganism.• Microscopic examination sometime gives information enough to make a presumptive identification. • Examples: • Trichomonas (protozoa) in vaginal secretion • Round worms eggs in stool can be identified based on their shape and size under the microscope.
Identification of prokaryotes using phenotypic characteristics•Gram stain is a differential method.•Gram stain of a specimen by itself - generally notsensitive and specific enough to diagnose the causeof most infection, •but very useful tool in narrowing the possible identities of an organism.• In certain cases, it gives enough information tostart appropriate antimicrobial therapy while waitingmore accurate identification.
Identification of prokaryotes using phenotypic characteristics•Certain microorganisms have unique characteristicsthat can be detected with special staining proceduree.g. – Fungus Cryptococcus neoformans (capsule staining) – Mycobacterium tuberculosis (acid-fast stain).
Metabolic differences1. Culture characteristics •Colony morphology: •can give initial clues for the identification of certain microorganism. – Colonies of Streptococci are generally fairly small relative to many other bacteria. – Pseudomonas aeruginosa often produces a soluble green greenish pigment which discolors the growth media and has a distinct fruity odor.
Metabolic differencesUse of selective and differential media •Blood agar media: differential media. – Beta-hemolytic colonies are characteristics of Streptococcus pyogenes. •MacConkey agar: both selective and differential media. – It inhibits the growth of most Gram positive bacteria and Gram negative cocci. – It has bile salts which inhibits the growth of most nonintestinal organisms thus usually it is used to select intestinal gram negative bacteria.
MacConkey agar: – It also differentiates lactose fermenting bacteria from nonlactose fermenter e.g. E. coli a lactose fermenter, forms characteristic pink colonies on MacConkey agar.
Biochemical tests•Culture characteristics can narrow the number ofpossible identities of bacteria. •but biochemical tests are generally necessary for a more conclusive diagnosis.Catalase: • nearly all bacteria which grow in the presence of oxygen are catalase positive. • Catalase positive bacteria break down hydrogen peroxide to release oxygen gas which cause bubbling.• Important exception are lactic acid bacteria whichinclude Streptococcus.
•Thus if a throat culture has beta-hemolyticcolonies on blood agar but are catalase positive,then Streptococcus pyogenes is ruled out. +ve -ve
Biochemical tests•Most biochemical tests rely on a pHindicator or chemical reaction that resultsin color change when a compound isdegraded e.g. • Fermentation of sugar results in acid production, which lowers the pH, resulting in a color change from pink to yellow and gas production. • No color change (central tube) indicate that sugar is not used.• A medium designed to detect ureaseenzyme that degrades urea to producecarbon dioxide and ammonia, utilizes adifferent pH indicator that turns brightpink in alkaline conditions.
Biochemical tests • The basic strategy for identifying bacteria based on biochemical test relies on the use of a dichotomous key, which is a flow chart of tests that give either a positive or negative result. • The biochemical tests are usually initiated simultaneously to speed identification.
Biochemical tests•In certain cases, biochemical test can be donewithout culturing the organism e.g. -Breath test which assays for the presence of urease is done to detect Helicobacter pylori•Commercial modifications of traditional biochemicaltests: •e.g. API test strip, enterotube and Biolog microtiter plate methods.
Serology•In some cases, proteins and polysaccharides present on thesurface of the bacterium are considered as identifying markers.•The most useful of these are the molecules that make upsurface structures including the cell wall, glycocalyx, flagellaand pili.•Antibodies directed against surface proteins andpolysaccharides are frequently used to identify variousbacteria.•Methods which use antibodies for the detection of antigens arecalled serology.•Some serological tests such as used to identify Streptococcuspyogenes are quite specific, simple and rapid.
Fatty acid analysis (FAME)•Bacteria differ in the type and relative quantity of fatty acidsthat make up their membranes. Thus cellular fatty acidcompositions can be used as an identification marker.•The bacterial cells are grown under standardized conditionsand then chemically treated with sodium hydroxide andmethanol to release fatty acids and to convert those acids totheir more volatile methyl ester form (FAME stands for fattyacid methyl ester).•FAME are analyzed by gas chromatography.•By comparing the pattern of peaks, or chromatogram, tothose of known species, an isolate can be identified.
Genotyping•Identification of prokaryotes using genotypic characteristics
•Genotypic characteristics are used in the identification ofmicroorganisms particularly which are difficult to cultivate.•Nucleic acid probes: are used to detect specific nucleotidesequences that characterize a particular species ofmicroorganism.•Fluorscence in situ hybridization (FISH) is increasingly beingused to identify intact microorganisms in environmental andclinical samples.•By using the rRNA specific probes, either specific species orgroups of related organisms can be identified
Polymerase chain reaction (PCR)• PCR can be used to amplify specific nucleotide sequences ofmicroorganisms from samples such as body fluids, soil, food,and water.• This technique can be used to detect microorganisms that arepresent in extremely low numbers as well as those can not begrown in culture.• In order to use PCR to detect microorganism of interest, asample should be first treated to release and denature DNA.• All ingredients needed for PCR along with specific primersknown and designed for particular microbe are then added.
• After ~30 cycles of PCR, sufficiently amplified DNA fragmentis visualized as discrete band on an ethidium bromide stainedagarose gel.• Alternatively, a DNA probe can be used to detect the amplifiedDNA.
Sequencing ribosomal RNA genes•Ribosomal RNA genes (DNA sequences) are highlyconserved, and can be used to identify organisms.•This method is particularly useful for identification of thoseprokaryotes which are difficult or currently impossible to growin culture.•Three different rRNAs: 5S, 16S, and 23S.•Some regions of 16S rRNA are virtually same in allprokaryotes whereas others have quite variable sequence andthis variable region is used to identify an organism.•In certain cases, 16S rDNA is used to identify uncultivableorganisms.
Characterizing strain differences• In some situations it is useful to distinguish among differentstains of bacteria especially when only certain strains causedisease. • Example: only certain strains of E. coli cause intestinal disease as only a few strains have the virulence factors such as toxin production etc.• Detecting strains differences is also helpful in tracing sourceof an outbreak.• The following methods are used for charactering variousstrains – Biochemical typing – Serological typing – Genomic typing – Phage typing – Antibiograms
Biochemical and serological typing1. Biochemical typing•Biochemical tests are mainly used to identify variousspecies of bacteria but they can also be used to distinguishstrains.•A strain that has characteristic biochemical pattern iscalled a biovar or biotype.
2. Serotyping•Serological procedures used to differentiate strains[serovars, serotypes] of microorganisms that differ in theantigenic composition of a structure or product.•Important in identifying a pathogen out of the group,primarily using cell wall antigens.Examples: Lancefield system to identify streptococci. –Serotypes are identified by a letter A-O and is based on specific antibody agglutination reactions with cell wall carbohydrate [Polysaccharide O}.
2. Serotyping –Further subdividing of Group A Strep based on specific M protein antigens can also be performed.•Other examples of serotyping: – Identifying specific antigen-antibody reactions involving Flagella [H] antigens, –Capusular [K] antigens, and –Cell wall [O] antigens.
2. Serological typing•Proteins and carbohydrates that vary among strains canbe used to differentiate strains. •Example: E. coli vary in the antigenic structure of certain parts of the LPS portion of the cell wall, the O antigen. The composition of the flagella, the H antigen, can also vary.•The strain designation of E. coli O157:H7 refers to thestructure of LPS and its flagella.•A strain that varies serologically from other strains issometime called a serovar or a serotype.
3. Genomic typing•Molecular methods can be used to detect genomic variationsthat characterize certain strains.•In some cases these differences include genes that encodefor toxins or other proteins related to disease. •Example: E. coli O157:H7. The toxin gene can be detected using a probe that consists of a specific nucleotide sequence unique to that gene.•Subtle differences in DNA sequences can be used todistinguish among strains that are phenotypically identical.This helps in tracing epidemics of foodborne illnesses.
3. Genomic typing•One method of genomic typing is to compare the pattern offragment sizes produced when the same restriction enzyme isused to digest DNA from each organism.•When the lengths of restriction fragments vary amongorganisms, it is termed ‘restriction fragment lengthpolymorphisms’ (RPLFs).
3. Genomic typing Common methods used to look for RFLPs :1. Pulse-field gel electrophoresis: •The bands can be visualized by staining gel with ethidium bromide.2. Ribotyping: •Uses a restriction enzyme that cuts genomic DNA into many small fragments. - As bacteria usually have several different rRNA genes, the probe hybridizes to several different restriction fragments, the pattern of which varies among strains. - Southern blot hybridization is then done using a probe that hybridizes to only those fragments that have sequences encoding ribosomal RNA.
Phage typing•Strains of a given species sometimes differ in theirsusceptibility to various types of bacteriophages.•The susceptibility of an organism to a particular type of phagecan be readily demonstrated in the laboratory.•The patterns of clearing around the bacteriophage spotindicate the susceptibility of the test organism to differentphages.•Different patterns are compared to determine straindifferences.•Bacteriophage typing is largely replaced by molecularmethods.
•Antibiotics susceptibility patterns or antibiograms, are alsoused to distinguish among different strains.•Again this method has largely been replaced by moleculartechniques.•Different strains will have different patterns of clearingaround antibiotics disks.