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Diagnostic Microbiology Identification of Microbes Dr. Chhaya Sawant Lecture 3  April 2010
Scope of the present lecture ,[object Object],[object Object]
The importance of studying microorganisms ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Identification of Microorganisms ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Phenotypic Methods ,[object Object],[object Object],[object Object]
Phenotypic Methods ,[object Object],[object Object],[object Object]
Immunological Methods ,[object Object],[object Object],[object Object]
Genotypic Methods ,[object Object],[object Object],[object Object]
Microbe Identification Scheme
Bacterial Classification
Process ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Microbe Identification ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Standard Precautions ,[object Object],[object Object],[object Object],[object Object],[object Object]
Standard Precautions (cont.) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Specimen Collection
[object Object],[object Object],[object Object],[object Object],[object Object]
Aerobic/Anaerobic Blood Culture Bottles AFB Blood Culture Bottle Wire Swab
Suprapubic Aspiration
Transportation ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Microbe Identification ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Different Identification methods  ,[object Object],[object Object],[object Object],[object Object],[object Object]
Staining Reactions ,[object Object],[object Object],[object Object],[object Object],[object Object]
Staining Reactions ,[object Object],[object Object],[object Object],[object Object],[object Object]
Cultural Characteristics ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
MacConkey’s Agar
Plates showing differentiating characteristics
Resistance ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Metabolism  ,[object Object],[object Object],[object Object],[object Object],[object Object]
Biochemical properties ,[object Object],[object Object],[object Object],[object Object],[object Object]
Biochemical properties ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Stormy Fermentation of Litmus Milk.  The tube on the left shows fermentation; the tube on the right is negative for stormy fermentation.  Used for the identification of  Clostridium  species.
Sugar fermentation  Acid and gas production
Biochemical properties ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
MR and VP test This is a qualitative test of the acidity produced by bacteria grown in MR-VP broth. Citrate utilization
Biochemical properties ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Indole test Phenylalanine Deamination Test. Gelatin Hydrolysis or Liquefaction . Test for Amino Acid Decarboxylase.  The tube on the left is an uninoculated control; the second tube from the left is lysine decarboxylase negative; the third tube is lysine decarboxylase positive; and the tube on the right is lysine deaminase positive
Biochemical properties ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
cogulase test Urease Tube and slide Catalase Test.
Oxidase test
Biochemical properties ,[object Object],[object Object],[object Object],[object Object]
Nitrate reductase test
Biochemical properties ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Triple sugar iron agar
Differentiation of two organisms
Biochemical properties ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
API Strips - Rapid Tests  Commercial miniaturized biochemical test panels - Cover a significant number of clinically-important groups of bacteria, as well as food- and water-associated microorganisms.  The earliest, is the Analytical Profile Index (API) panel.  Different test panels are prepared in  dehydrated forms  which are reconstituted upon use by addition of bacterial suspensions. After incubation, positive test results are scored as a seven-digit number (profile). Identity of the bacterium is then easily derived from the database with the relevant cumulative profile code book or software. Identification of Enterobacteriaceae using API 20E, a standardized microplate method. Positive and negative reactions are shown by color reactions.
Rapid Tests  ONPG ( β  galactosidase); ADH (arginine dihydrolase); LDC (lysine decarboxylase); ODC  (ornithine decarboxylase); CIT (citrate utilization); H 2 S (hydrogen disulphide production); URE (urease); TDA ( tryptophan deaminase); IND (indole production); VP (Voges Proskauer test for acetoin); GEL ( gelatin liquefaction); the fermentation of glucose (GLU), mannitol (MAN), inositol (INO), sorbitol (SOR), rhamnose (RHA), sucrose (SAC); Melibiose (MEL), amygdalin (AMY), and arabinose (ARA); and OXI (oxidase). positive negative
Rapid Test Results In Other System, the use of Paper discs impregnated with biochemical substrates (e.g. Minitek, Bioquest)  OXI--0 - ARA-2  2 + AMY-0 - MEL--4 + SAC-2  7 + RHA-1 + SOR--4 + INO-0  5 - MAN-1 + GLU--4 + GEL-0  4 - VP--0 IND--4 + TDA-0  4 - URE-0 - H 2 S--0 - CIT-0  1 - ODC-1 + LDC--4 + ADH-0  5 - ONPG-1 + normal 7 digit code 5 144 572 =  E. coli
Immunologic Techniques ,[object Object],[object Object],[object Object]
serology ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Precipitation Reactions ,[object Object],[object Object],[object Object],[object Object]
Agglutination Reactions ,[object Object],[object Object],[object Object],Direct agglutination  occurs when a soluble Ab results in clumping  by interaction with an Ag which  is part of a surface of a cell.  E.g. Blood typing and detection of  mycoplasma pneumonia. Indirect (passive) agglutination . Ab/Ag is  adsorbed or chemically coupled  to the cell, latex beads or charcoal particles which serves as an inert carrier. The latex beads can be used to detect  for surface Ag.
Direct/Indirect Agglutination Commercial suspension of latex beads are available for the detection of  Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenza  and  Camplyobacter  spps .  A similar technology is used for  urine pregnancy tests . Hemagglutination   usually results from antibodies cross linking red blood cells through attachment to surface antigens and is routinely used in blood typing.  The hemagglutination inhibition test is widely used to diagnose influenza, measles, mumps,  mononucleosis, and other viral infections
[object Object],[object Object],[object Object]
Using Fluorescent Antibodies ,[object Object],[object Object],[object Object],Direct method   Indirect method
Fluorescent Antibodies ,[object Object],[object Object],Fluorescent Ab  can be used to detect microorganisms  directly in tissue , long before a primary isolation technique yield the suspected pathogen.  Fluorescent Ab has been used for the detection of  Bacillus anthracis  and HIV virus.
ELISA - Enzyme-linked immunosorbent assay ,[object Object],[object Object],[object Object]
ELISA ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Immunoblot/Western Blot ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Immunoblot
Immuno/Western Blot ,[object Object],[object Object],[object Object],[object Object]
HIV Immunoblot Test ,[object Object]
HIV Immunoblot Test ,[object Object],[object Object],[object Object],[object Object]
A technique used to measure the concentration of hormones, Drugs, enzymes, viruses, bacterial antigens and other organic substances of biological interest found in Blood, Tissues and other biological fluids  Radio immuno assay  Gamma Counter ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Advantages & Disadvantages of RIA ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Immuno-electron microscopy Electron Microscopy or EM can be used to study the detailed microarchitecture of tissues or cells. Immuno-EM allows the detection of specific proteins in ultrathin tissue sections.  Antibodies labelled with heavy metal particles (e.g. gold) can be directly visualised using Transmission Electron Microscopy .  While powerful in detecting the sub-cellular localisation of a protein, immuno-EM can be technically challenging, expensive, and require rigorous optimisation of tissue fixation and processing methods.
 
Problems With Traditional Methods Cultivation-based methods insensitive for detecting some organisms. Cultivation-based methods limited to pathogens with known growth requirements. Poor discrimination between microbes with common behavioral features. Failure to detect infections caused by uncultivated (e.g., novel) organisms, or organisms that fail to elicit a detectable host immune response. Visual appearance of microorganisms is nonspecific. Examples of Failures With Traditional Approaches Detection and speciation of slow-growing organisms takes weeks  (e.g.,  M. tuberculosis ). A number of visible microorganisms cannot be cultivated (e.g.,  Whipple bacillus ). Diseases presumed to be infectious remain ill-defined with no detected microorganism (e.g., abrupt fever after tick bite).
Genotypic methods ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Nucleic acid probes ,[object Object],[object Object],[object Object],[object Object]
Nucleic Acid Probes ,[object Object],[object Object],[object Object]
Two Component Probes ,[object Object],[object Object],[object Object],[object Object],[object Object]
Two Component Probe
Advantages of Nucleic Acid Probes ,[object Object],[object Object],[object Object],[object Object],[object Object]
Polymerase Chain Reaction (PCR) ,[object Object],[object Object],[object Object],[object Object]
Limitations of End-Point PCR Agarose gel results are obtained from the end point of the reaction. Endpoint detection is very time consuming. Results may not be obtained for days. Results are based on size discrimination, which may not be very precise. The end point is variable from sample to sample. Gels may not be able to resolve these variability in yield, real-time PCR is sensitive enough to detect these changes. Agarose Gel resolution is very poor, about 10 fold. Real-Time PCR can detect as little as a two-fold change! Some of the problems with  End-Point Detection: Poor Precision Low sensitivity Short dynamic range < 2 logs Low resolution Non - Automated Size-based discrimination only Results are not expressed as numbers Ethidium bromide for staining is not very quantitative Post PCR processing
Real Time PCR ,[object Object],[object Object],[object Object],[object Object]
Real-time PCR ,[object Object],[object Object],[object Object]
RT-PCR  (reverse trancriptase PCR ,[object Object],[object Object]
RT-PCR
RAPD Profile ,[object Object],[object Object],Standard PCR is used to amplify a known sequence of DNA.  The scientists chooses the sequence he or she wants to amplify, then designs and makes primers which will anneal to sequences flanking the sequence of interest.  PCR leads to the amplification of a particular segment of DNA.
RAPD analysis ,[object Object],[object Object],[object Object]
RAPD-PCR ,[object Object],[object Object],[object Object],[object Object],[object Object]
DNA sequencing ( 16s rDNA) ,[object Object],[object Object],This work was pioneered by Carl Woese, who proposed the  three Domain system of classification - Archaea, Bacteria,  and  Eucarya  - based on such sequence information.
DNA Sequencing ,[object Object],[object Object],[object Object]
Restriction Fragment Length Polymorphism   ,[object Object],[object Object],[object Object],[object Object]
RFLP of  M. tuberculosis  from 17 patients
Plasmid fingerprinting ,[object Object],[object Object]
Plasmid fingerprinting ,[object Object],[object Object],[object Object],[object Object]
Gas-liquid chromatography ,[object Object],[object Object],Volatile Fatty Acid Profiles from Different Bacteria.
Bacteriophage Typing ,[object Object],[object Object],[object Object],[object Object],[object Object],Heavily Inoculated Plate
Bacteriophage Typing ,[object Object],[object Object],[object Object],[object Object],A bacterial lawn inoculated  with a range of bacteriophage
Unculturable Organisms ,[object Object],[object Object]
Flow Cytometry ,[object Object],[object Object],[object Object]
Flow Cytometry ,[object Object],[object Object]
Computer and Bacteria Identification ,[object Object],[object Object]
Bacterial species ,[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Identification  ,[object Object],[object Object]
Synthetic Peptides in Diagnosis of Viral Infections ,[object Object],[object Object]
How to Choose? There are a variety of identification technologies available Bear in mind the strengths, and weaknesses, of the various methodologies.  E.g., the recently released aseptic processing guidance document (FDA 2004) recommends the use of genotypically based methods. In PCR based methods or DNA sequencing - an associated cost in facilities, labor (highly skilled technicians) and maintenance that is not present with the more traditional methods. The most direct approach to decide - based on an understanding of what your requirements may be. Develop User Requirements Specification (URS) document to drive this process. This is a formal Quality document, similar in concept to a Design Qualification document. Different companies will have different formats for these documents, but the essential features of the document will be that it has the essential requirements and that it has upper management sign-off (for a variety of reasons it is a good idea to document upper management commitment).
A partial list of topics to be covered in any URS designed for an identification system should include: Assay Throughput  - How many samples a day? Assay Time-to-Completion  - How quickly? Cost of Consumables  - How much? Frequently the cost of consumables can soon dwarf the capital expense. Labor Requirements  - Including the technological sophistication of the operators—can your technicians actually operate the equipment reliably?  Size and Composition of Microorganism Identification Database  - A major consideration. If you purchase two systems to cover identifications of unknowns, it is imperative to ensure that the databases are large and complementary; that is they both don’t have the same organisms in them, but that they include many different ones as well. Facility Requirements  - Obvious stuff like electrical and plumbing, but also less obvious concerns about RNA/DNA contamination and clean room issues. Compatibility with Existing Systems  (LIMS, workflow, etc.) Need for Physiological Information  - Do you need to know if the organisms are capable of degrading your product components? You may want to use a system that will help determine this. Purpose  - Do you plan to use this for routine identifications or for investigations?  The use of the system may be different for different systems. A good system for routine work may not be the best for investigations, and vice versa. In short, there are a wide variety of choices available to help with the identification of unknown organisms. It is important to define your specific requirements and to purchase the appropriate system to meet those needs.

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Diagnostic microbiology - Traditional and Modern Approach

  • 1. Diagnostic Microbiology Identification of Microbes Dr. Chhaya Sawant Lecture 3 April 2010
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  • 17. Aerobic/Anaerobic Blood Culture Bottles AFB Blood Culture Bottle Wire Swab
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  • 31. Sugar fermentation Acid and gas production
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  • 33. MR and VP test This is a qualitative test of the acidity produced by bacteria grown in MR-VP broth. Citrate utilization
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  • 35. Indole test Phenylalanine Deamination Test. Gelatin Hydrolysis or Liquefaction . Test for Amino Acid Decarboxylase. The tube on the left is an uninoculated control; the second tube from the left is lysine decarboxylase negative; the third tube is lysine decarboxylase positive; and the tube on the right is lysine deaminase positive
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  • 37. cogulase test Urease Tube and slide Catalase Test.
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  • 45. API Strips - Rapid Tests Commercial miniaturized biochemical test panels - Cover a significant number of clinically-important groups of bacteria, as well as food- and water-associated microorganisms. The earliest, is the Analytical Profile Index (API) panel.  Different test panels are prepared in dehydrated forms which are reconstituted upon use by addition of bacterial suspensions. After incubation, positive test results are scored as a seven-digit number (profile). Identity of the bacterium is then easily derived from the database with the relevant cumulative profile code book or software. Identification of Enterobacteriaceae using API 20E, a standardized microplate method. Positive and negative reactions are shown by color reactions.
  • 46. Rapid Tests ONPG ( β galactosidase); ADH (arginine dihydrolase); LDC (lysine decarboxylase); ODC (ornithine decarboxylase); CIT (citrate utilization); H 2 S (hydrogen disulphide production); URE (urease); TDA ( tryptophan deaminase); IND (indole production); VP (Voges Proskauer test for acetoin); GEL ( gelatin liquefaction); the fermentation of glucose (GLU), mannitol (MAN), inositol (INO), sorbitol (SOR), rhamnose (RHA), sucrose (SAC); Melibiose (MEL), amygdalin (AMY), and arabinose (ARA); and OXI (oxidase). positive negative
  • 47. Rapid Test Results In Other System, the use of Paper discs impregnated with biochemical substrates (e.g. Minitek, Bioquest) OXI--0 - ARA-2 2 + AMY-0 - MEL--4 + SAC-2 7 + RHA-1 + SOR--4 + INO-0 5 - MAN-1 + GLU--4 + GEL-0 4 - VP--0 IND--4 + TDA-0 4 - URE-0 - H 2 S--0 - CIT-0 1 - ODC-1 + LDC--4 + ADH-0 5 - ONPG-1 + normal 7 digit code 5 144 572 = E. coli
  • 48.
  • 49.
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  • 52. Direct/Indirect Agglutination Commercial suspension of latex beads are available for the detection of Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenza and Camplyobacter spps . A similar technology is used for urine pregnancy tests . Hemagglutination usually results from antibodies cross linking red blood cells through attachment to surface antigens and is routinely used in blood typing. The hemagglutination inhibition test is widely used to diagnose influenza, measles, mumps, mononucleosis, and other viral infections
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  • 64. Immuno-electron microscopy Electron Microscopy or EM can be used to study the detailed microarchitecture of tissues or cells. Immuno-EM allows the detection of specific proteins in ultrathin tissue sections. Antibodies labelled with heavy metal particles (e.g. gold) can be directly visualised using Transmission Electron Microscopy . While powerful in detecting the sub-cellular localisation of a protein, immuno-EM can be technically challenging, expensive, and require rigorous optimisation of tissue fixation and processing methods.
  • 65.  
  • 66. Problems With Traditional Methods Cultivation-based methods insensitive for detecting some organisms. Cultivation-based methods limited to pathogens with known growth requirements. Poor discrimination between microbes with common behavioral features. Failure to detect infections caused by uncultivated (e.g., novel) organisms, or organisms that fail to elicit a detectable host immune response. Visual appearance of microorganisms is nonspecific. Examples of Failures With Traditional Approaches Detection and speciation of slow-growing organisms takes weeks (e.g., M. tuberculosis ). A number of visible microorganisms cannot be cultivated (e.g., Whipple bacillus ). Diseases presumed to be infectious remain ill-defined with no detected microorganism (e.g., abrupt fever after tick bite).
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  • 74. Limitations of End-Point PCR Agarose gel results are obtained from the end point of the reaction. Endpoint detection is very time consuming. Results may not be obtained for days. Results are based on size discrimination, which may not be very precise. The end point is variable from sample to sample. Gels may not be able to resolve these variability in yield, real-time PCR is sensitive enough to detect these changes. Agarose Gel resolution is very poor, about 10 fold. Real-Time PCR can detect as little as a two-fold change! Some of the problems with End-Point Detection: Poor Precision Low sensitivity Short dynamic range < 2 logs Low resolution Non - Automated Size-based discrimination only Results are not expressed as numbers Ethidium bromide for staining is not very quantitative Post PCR processing
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  • 85. RFLP of M. tuberculosis from 17 patients
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  • 101. How to Choose? There are a variety of identification technologies available Bear in mind the strengths, and weaknesses, of the various methodologies. E.g., the recently released aseptic processing guidance document (FDA 2004) recommends the use of genotypically based methods. In PCR based methods or DNA sequencing - an associated cost in facilities, labor (highly skilled technicians) and maintenance that is not present with the more traditional methods. The most direct approach to decide - based on an understanding of what your requirements may be. Develop User Requirements Specification (URS) document to drive this process. This is a formal Quality document, similar in concept to a Design Qualification document. Different companies will have different formats for these documents, but the essential features of the document will be that it has the essential requirements and that it has upper management sign-off (for a variety of reasons it is a good idea to document upper management commitment).
  • 102. A partial list of topics to be covered in any URS designed for an identification system should include: Assay Throughput  - How many samples a day? Assay Time-to-Completion  - How quickly? Cost of Consumables  - How much? Frequently the cost of consumables can soon dwarf the capital expense. Labor Requirements  - Including the technological sophistication of the operators—can your technicians actually operate the equipment reliably?  Size and Composition of Microorganism Identification Database  - A major consideration. If you purchase two systems to cover identifications of unknowns, it is imperative to ensure that the databases are large and complementary; that is they both don’t have the same organisms in them, but that they include many different ones as well. Facility Requirements  - Obvious stuff like electrical and plumbing, but also less obvious concerns about RNA/DNA contamination and clean room issues. Compatibility with Existing Systems  (LIMS, workflow, etc.) Need for Physiological Information  - Do you need to know if the organisms are capable of degrading your product components? You may want to use a system that will help determine this. Purpose  - Do you plan to use this for routine identifications or for investigations?  The use of the system may be different for different systems. A good system for routine work may not be the best for investigations, and vice versa. In short, there are a wide variety of choices available to help with the identification of unknown organisms. It is important to define your specific requirements and to purchase the appropriate system to meet those needs.