Microbiology Techniques and Antibiotic Testing

Diagnostic Microbiology

• Diagnostic microbiology aims to identify microbes.
• Accurate identification depends on proper aseptic techniques, specimen collection, and rapid transport to the lab.
• Successful microbial identification relies on proper aseptic techniques, correct specimen handling, and rapid transport.

Microbe Identification: Arrival to the Lab

• Accurate identification of microbes relies on:
  • Proper aseptic technique.
  • Correct specimen acquisition and handling.
  • Speedy specimen transport to the lab.
• The successful identification of microbe depends on using the proper aseptic techniques, correctly obtaining and handling the specimen, and quickly transporting the specimen to the lab.

Identification of Microorganisms

• Microbiologists use three main categories of methods:
  • Classic microbiology.
  • Molecular microbiology (genetic tests).
  • Immunological analysis.

Where do we start?

• Initial steps depend on suspected infectious disease and route:
  • Immunological route: Blood sample analyzed for antibodies against the suspected pathogen. Antibody assays (agglutination, RIA, ELISA, etc.) are performed.
  • Microbiological route: Blood, feces, urine, tissue, or mucosal swabs are collected and processed conventionally. Enrichment cultures, selective media, differential media, and pure culture isolation are used.

Classic Microbiology

• Classic microbiology involves 5 steps (the five I's):
  • Inoculation: Making a pure culture of the microbe.
  • Incubation: Growing the microbe under appropriate conditions (e.g., optimal temperature and oxygen).
  • Isolation: Separating individual microbes to obtain a pure culture.
  • Inspection: Observing and recording characteristics (e.g., morphology).
  • Identification: Using data to identify the organism to species level (e.g., biochemical tests).

Inoculation

• Introducing bacteria to a growth medium using aseptic technique (e.g., Bunsen burner, platinum loop).

Incubation

• Allowing microbes to grow under optimal temperature and oxygen conditions. Methods include the use of incubators, methyl blue indicator strips, and GasPak jars.

Isolation

• Separating individual colonies on media to obtain a pure culture.

Identification

• Determining characteristics (e.g., shape, size, color, biochemical properties) to identify an organism to species level.

Identification in selective media

• Table (with limited details since format does not support tables). Shows different colony characteristics on various media useful for identifying microbes. Specific examples of media (e.g. Blood agar, Enteric agar, CA, MTM, ANA) and their use for different specimens are illustrated in the table.

Confirmation tests: catalase

• Catalase is an enzyme produced by microorganisms in oxygen-rich environments. It neutralizes hydrogen peroxide and helps protect them from pathogens. Anaerobes generally lack this enzyme.
• A positive test displays evidence of foaming.

Confirmation tests II: oxidase

• The oxidase test identifies microbes that produce the enzyme cytochrome c oxidase.
• The reagent (tetramethyl-p-phenylenediamine) turns purple if the enzyme is present.
• The absence of color indicates the absence of the enzyme.

Confirmation tests III: API systems

• API test strips contain dehydrated substrates that react with microbial enzymes to create a color change. This reveals data about their metabolic and enzymatic activity.
• A bacterial suspension rehydrates the wells for testing, and incubation helps organisms react with the strips.

Antimicrobial susceptibility testing

• Antimicrobial drugs treat infectious diseases. Testing pathogens' susceptibility is vital for appropriate chemotherapy.
• The Kirby-Bauer method is a standard antimicrobial susceptibility test procedure.

The Kirby-Bauer method

• A picked colony is inoculated into a tube with sterile water, achieving a specific density (e.g., MacFarland standard 1).
• A swab is dipped into the liquid culture and streaked evenly over a plate of sterile agar (e.g., Muller-Hinton agar), which allows for proper antibiotic diffusion.
• Discs with different antibiotics are placed on the plate; after incubation, the inhibition zones give information regarding microbial resistance to those antibiotics.

Molecular Microbiology

• Molecular methods examine the genetic material of microorganisms.
• Techniques have become dominant in identifying microbes due to speed and accuracy.

Genotypic methods

• PCR, real-time PCR, reverse transcriptase PCR (RT-PCR), rRNA analysis, and plasmid fingerprinting are used in genotypic methods of microbe identification.

Polymerase Chain Reaction (PCR)

• A technique widely used in microbe identification and pathogen detection.
• Specific primers amplify target DNA/RNA sequences, enabling even single-cell detection.
• Primers for food monitoring exist to detect Salmonella and Staphylococcus.

PCR Reaction Components

• Necessary components of PCR, including: water, buffer, DNA template, primers, nucleotides (dATP, dGTP, dCTP, dTTP), Mg++ ions and an appropriate thermostable DNA polymerase (e.g., Taq polymerase).

How PCR Works

• Denaturation (94°C): DNA is heated to separate DNA strands;
• Annealing (60°C): Primers bind to complementary strands ;
• Extension (72°C): DNA polymerase copies the template.

PCR: first cycle

• The steps involved in the first cycle of a PCR reaction: denaturing, annealing, and extension.

PCR overview

• PCR cycle results in exponential growth of target DNA, allowing for millions or even billions of copies in a matter of hours.

Taq polymerase

• A heat-stable DNA polymerase from the bacterium Thermus aquaticus, commonly used in PCR.

PCR: Visualizing results

• After cycling, separated DNA fragments are loaded onto an agarose gel, stained (e.g., with ethidium bromide) and visualized as distinguishable bands.

Real Time PCR

• Real-time PCR monitors the amplification of PCR products in real time through fluorescence.

Reverse Transcription-PCR (RT-PCR)

• Using RNA as template to produce cDNA. RT-PCR useful to detect HIV (RNA virus).

DNA sequencing

• DNA sequence analysis identifies microbes by sequencing the 16S rRNA gene.

### Plasmid fingerprinting

• Analyzing the plasmid profiles in bacteria for identification. This is an accurate method used for species and strain recognition.
• Plasmid fingerprinting typically entails isolating plasmids, separating them via agarose gel electrophoresis, and staining for visualization; comparative data for similarities and differences aid in identification.

Immunological Route

• Diagnoses focus on testing for the immune response to microbial infections. Antibody assays are performed (e.g., agglutination, RIA, ELISA).

Immunological methods

• The methods of interaction of microbial antigens with antibodies produced by the host's immune system.
• Tests for detecting microbial antigens or antibodies production are available for identifying numerous microbes via testing. This method is a very useful tool for confirming infection.

Immunological Methods (II)

• Serology tests the reactions of antibodies to antigens.
• Useful serological tests are based on parameters of sensitivity (ability to detect small amounts of antigen/antibody) and specificity (ability to detect particular antigens/antibodies without cross-reactions with similar molecules).

False Negatives/Positives

• High sensitivity reduces false negative results. False negatives happen when there is no reaction despite the presence of the antigen or antibody.
• High specificity reduces false positive test outcomes. False positives result from cross-reactions with similar molecules.

Immunological tests

• Includes precipitation, agglutination, fluorescent antibody techniques, ELISA, and Immunoxpert™ tests.

Precipitation Reactions

• A reaction that involves the interaction of soluble antigens and antibodies to form an insoluble complex (precipitate).

Agglutination Reactions

• Microbes are clumped together by the interaction of an antigen and antibody. This is a rapid and accurate approach to diagnosis and identification (e.g., blood typing).

Direct Agglutination

• Direct interaction of antibodies and antigens to form visible clumps and used in blood typing and identifying Mycoplasma pneumoniae.

Indirect or Passive Agglutination

• Measuring pathogens or antibodies using soluble antigens as targets adsorbed or coupled to reagents (latex beads/charcoal). This facilitates a large-scale screening method.

Fluorescent Antibodies

• Fluorescent antibodies are used to detect microorganisms directly in tissue or fluid samples, even before isolation, allowing early organism detection.

ELISA (Enzyme-Linked Immunosorbent Assay)

• ELISA tests involve detecting microbes using antibodies linked to enzymes, producing color changes, which are measured, useful in assessing many microbes (e.g., S. aureus, E. coli, Salmonella).

Immunoxpert™

• This is a pioneering in-vitro diagnostic test that can accurately distinguish between bacterial and viral infections using an algorithm based on measurements of three host immune response proteins in human serum.