Microbiology Techniques and Antibiotic Testing

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Questions and Answers

What does a small inhibition zone diameter indicate when comparing with the reference value?

Intermediate susceptibility

No significant growth

Susceptible

Resistant (correct)

Which of the following techniques is NOT classified as a genotypic method of microbe identification?

rRNA analysis

PCR

Plasmid fingerprinting

Antibiotic susceptibility testing (correct)

In what year was the Polymerase Chain Reaction (PCR) developed?

1990

1995

1983 (correct)

1981

Which method allows for the detection of microorganisms even in low cell populations?

Polymerase Chain Reaction (PCR) Signup and view all the answers

What is primarily measured to determine the effectiveness of an antibiotic during susceptibility testing?

Diameter of the inhibition zone Signup and view all the answers

What color indicates the presence of cytochrome c oxidase when the reagent is oxidized?

Purple Signup and view all the answers

What is the purpose of the API test strips?

To detect enzymatic activity related to carbohydrate fermentation Signup and view all the answers

Which of the following describes a key step in the Kirby–Bauer method?

Placement of antibiotic discs on the agar plate Signup and view all the answers

What does a colorless result indicate when the reagent is tested for the presence of cytochrome c oxidase?

The enzyme is absent Signup and view all the answers

What is the first step in performing the Kirby–Bauer susceptibility test?

Picking a colony to inoculate sterile water Signup and view all the answers

What does the MacFarland standard measure?

The turbidity or cloudiness of bacterial suspension Signup and view all the answers

Why is it important to test pathogens for susceptibility to individual antimicrobials?

To ensure appropriate chemotherapy Signup and view all the answers

What is used to rehydrate each well in the API test strips?

A bacterial suspension Signup and view all the answers

What does high sensitivity in immunological tests primarily prevent?

False negatives caused by low antigen presence Signup and view all the answers

Which method involves the interaction between a soluble antigen and a soluble antibody to form an aggregate?

Precipitation Signup and view all the answers

Which of the following statements regarding specificity in immunological tests is true?

Specificity ensures the correct identification of particular antigens Signup and view all the answers

What is one of the primary applications of serological tests?

To evaluate the interaction between antigens and antibodies Signup and view all the answers

What occurs when there is a reaction in an immunological test but antigens or antibodies are absent?

False positive Signup and view all the answers

Which of the following techniques is NOT classified under immunological methods?

Gas chromatography Signup and view all the answers

What does the use of EtBr in agarose gel electrophoresis indicate?

It stains the gels to visualize DNA Signup and view all the answers

Which factor is NOT associated with the sensitivity of an immunological test?

Capacity to distinguish between similar antigens Signup and view all the answers

Which method is NOT part of the classic microbiological route?

Amplification Signup and view all the answers

What is the primary function of the catalase enzyme in microorganisms?

To neutralize hydrogen peroxide by breaking it down Signup and view all the answers

Which of the following steps involves the introduction of bacteria into a growth medium?

Inoculation Signup and view all the answers

Which characteristic is NOT typically associated with anaerobes concerning catalase?

They thrive in oxygenated environments Signup and view all the answers

Which component is essential for quickly transporting the specimen to the lab?

Appropriate temperature control Signup and view all the answers

What is the outcome of the oxidase test?

It identifies bacteria that produce cytochrome c oxidase. Signup and view all the answers

What is the primary purpose for using proper aseptic techniques during specimen handling?

To prevent contamination Signup and view all the answers

Which of the following steps comes after incubation in the classic microbiological route?

Isolation Signup and view all the answers

What is the role of primers in the PCR process?

Bind to the DNA template to initiate synthesis Signup and view all the answers

At which temperature does the denaturation step of PCR occur?

94ºC Signup and view all the answers

Which component of the PCR reaction is essential for the extension of the new DNA strand?

DNA polymerase Signup and view all the answers

How many copies of DNA are produced after 20 cycles of PCR starting with a single molecule?

1 million Signup and view all the answers

What characteristic of Taq polymerase makes it suitable for PCR?

It remains stable at high temperatures Signup and view all the answers

What is the result of the extension step in PCR?

New DNA strands are synthesized Signup and view all the answers

Which component stabilizes the DNA polymerase and nucleotides during the PCR process?

Buffer Signup and view all the answers

What happens to the amount of DNA after 30 cycles of PCR if starting from one molecule?

It increases to about 1 billion copies Signup and view all the answers

Which method involves direct interaction between a fluorescent antibody and a surface antigen of an organism?

Direct fluorescent antibody method Signup and view all the answers

What is the main advantage of using passive agglutination tests in screening?

They are simple and suitable for large scale screening. Signup and view all the answers

How does direct agglutination occur?

Through interaction with a surface antigen of a cell. Signup and view all the answers

What type of agglutination test is utilized for blood typing?

Direct agglutination test Signup and view all the answers

In which type of agglutination does soluble antigen become linked to an inert carrier?

Passive agglutination Signup and view all the answers

Which chemical modification allows antibodies to emit bright light in fluorescent antibody testing?

Coupling with dye molecules Signup and view all the answers

Which of the following is NOT a benefit of agglutination tests?

Time-consuming Signup and view all the answers

What occurs during the indirect method of fluorescent antibody procedures?

Non-fluorescent antibodies bind before fluorescent antibodies. Signup and view all the answers

Which technique utilizes a second antibody that has an enzymatic activity linked to it?

ELISA Signup and view all the answers

What is the primary function of the ImmunoXpert tm diagnostic test?

To distinguish between bacterial and viral infections Signup and view all the answers

Which microorganisms can be detected using the ELISA technique?

S.aureus, E.coli, and Salmonella Signup and view all the answers

What distinguishes direct fluorescent antibody testing from indirect methods?

Direct binding to an antigen Signup and view all the answers

What role do the immune response proteins play in the ImmunoXpert tm test?

They help in distinguishing infection type Signup and view all the answers

Which step of the classic microbiological route focuses on keeping microorganisms free from contaminants?

Inoculation Signup and view all the answers

What is the main outcome of successful isolation in microbiology?

A pure species of microorganism Signup and view all the answers

What characterizes the catalase test in identifying microorganisms?

Bubbles indicate catalase-positive microorganisms. Signup and view all the answers

What is primarily being measured when conducting an oxidase test?

The presence of cytochrome c oxidase activity. Signup and view all the answers

What does the term 'pure culture' refer to in microbiology?

A culture derived from a single type of microbe. Signup and view all the answers

Which condition is NOT essential for the incubation step in microbiological methods?

Presence of toxic substances. Signup and view all the answers

What role does the Bunsen burner play in microbiological practices?

It aids in inoculation through sterilization. Signup and view all the answers

Which of the following methods is not a category used in microbial identification?

Electrophoresis techniques Signup and view all the answers

What is a key characteristic of real-time PCR compared to traditional PCR?

It continuously monitors amplification through fluorescent signals. Signup and view all the answers

Which enzyme is essential in the reverse transcriptase-PCR process?

Reverse transcriptase Signup and view all the answers

What is the primary purpose of plasmid fingerprinting in microbial identification?

To identify the number of plasmids and their molecular weight Signup and view all the answers

In the context of DNA sequencing, which part of the DNA is most commonly targeted for microbial identification?

The 16S rRNA gene sequence Signup and view all the answers

What differentiates DNA viruses from RNA viruses in terms of persistence within a host?

DNA viruses tend to remain dormant within the host. Signup and view all the answers

When separating DNA by size using an electric field, how does the size of the DNA fragments affect their migration?

Larger DNA fragments migrate closer to the starting point. Signup and view all the answers

Which statement accurately describes the output of a PCR amplification plot in real-time PCR?

It indicates the quantity of DNA at each cycle. Signup and view all the answers

What is the significance of using small amounts of DNA for microbial identification?

It enables the identification of rare microbial species. Signup and view all the answers

What is the primary purpose of using agarose gel electrophoresis in microbiological studies?

To separate plasmids Signup and view all the answers

Which of the following best describes sensitivity in immunological tests?

Ability to detect traces of antigens or antibodies Signup and view all the answers

What type of immunological test involves the interaction of soluble antigen with soluble antibody to form an insoluble complex?

Precipitation Signup and view all the answers

Which term describes a situation where there is no reaction in an immunological test, although antigens or antibodies are actually present?

False negative Signup and view all the answers

In serology, what does specificity primarily ensure in immunological tests?

Detection of a single target antigen among various others Signup and view all the answers

Which immunological method is commonly used for the identification of microorganisms through the interaction of antigens and antibodies?

Enzyme-linked immunosorbent assay (ELISA) Signup and view all the answers

What type of error occurs when there is a cross-reaction with another molecule in immunological testing?

False positive Signup and view all the answers

Which method is NOT typically associated with immunological testing of microbial antigens?

Electrophoresis Signup and view all the answers

What is the expected outcome when tetramethyl-p-phenylenediamine is oxidized by cytochrome c oxidase?

It produces a purple color. Signup and view all the answers

Which of the following statements accurately describes the role of API test strips?

They evaluate enzyme activity related to catabolism. Signup and view all the answers

In the Kirby–Bauer method, what is the purpose of placing discs containing antibiotics on the agar plate?

To create a gradient of antibiotic concentration. Signup and view all the answers

What does a bacterium's classification as 'intermediate' indicate in antimicrobial susceptibility testing?

The bacterium can grow but only at higher concentrations of the antibiotic. Signup and view all the answers

What is the significance of achieving a bacterial culture density equivalent to 1 MacFarland standard in susceptibility testing?

It ensures consistency in assessing antibiotic effects. Signup and view all the answers

During the Kirby–Bauer method, which media is typically used to incubate the antibiotic discs?

Muller-Hinton agar plate Signup and view all the answers

What happens when cytochrome c oxidase is absent during the reagent test?

The reagent remains colorless. Signup and view all the answers

What does the presence of specific antimicrobial susceptibility testing results imply for pathogen treatment?

They ensure the chosen antibiotic will be effective. Signup and view all the answers

What is the optimal temperature for the denaturation step in the PCR process?

94ºC Signup and view all the answers

What role do primers play in the PCR process?

They start the synthesis of new DNA strands. Signup and view all the answers

After how many cycles of PCR would you expect to obtain approximately one billion copies of DNA starting from a single molecule?

30 cycles Signup and view all the answers

Which component of the PCR reaction is critical as a co-factor for the DNA polymerase?

Mg++ ions Signup and view all the answers

What is the ideal temperature for the extension step in PCR?

72ºC Signup and view all the answers

Taq polymerase is derived from which organism that allows it to withstand high temperatures during PCR?

Thermus aquaticus Signup and view all the answers

What happens to the configuration of DNA after the extension step in PCR?

Two new double helices are formed. Signup and view all the answers

Which component is used to stabilize the reaction in PCR?

Buffer Signup and view all the answers

Study Notes

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.

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Description

Test your knowledge on microbiological techniques, particularly focusing on antibiotic susceptibility testing and microbe identification methods. This quiz covers important historical developments like the PCR and critical concepts in microbiology. Challenge yourself to see how well you understand these key topics!