[01.14a] Didactics 1_ Principles of Diagnostic Microbiology V2

Diagnosis of infection

What is Diagnostic Microbiology concerned with etiologically?

Prompt diagnoses vital in controlling spread of COVID-19

Why is diagnostic microbiology important during a pandemic?

RT-PCR swab test, Saliva test, Rapid antigen test, Rapid antibody test

What are the four tests mentioned for diagnosing COVID-19?

Morphologic Identification, Culture Isolation, Detection of the Agent in Patient Specimens by Antigen Testing, Demonstration of Meaningful Antibody or Cell-Mediated Immune Responses to An Infectious Agent

What are the four basic clusters of lab procedures used in diagnosing infectious disease?

Examination of Stained Slides

What is the primary method for morphologic identification?

Traditional way identifying etiologic agent

How is morphologic identification generally characterized in terms of its newness?

Shape, Color, Arrangement

What three characteristics are used to characterize microbes in morphologic identification?

Gram-negative (red) cocci (spherical) in pairs

What are the morphologic characteristics of Neisseria gonorrhoeae?

Production of precipitate, Motility of the microorganism, Colony color, Arrangement

What are some culture characteristics integral to identifying etiology?

Sensitivity studies

What important studies are done using culture isolation?

Antibiotic resistance

What specific resistance is checked for in sensitivity studies?

Microorganisms or the toxins they produce

In the context of microbiology, what do "antigens" refer to?

Molecular level

How does antigen testing differ from morphological identification, especially with methods like RT-PCR?

Serologic antibody testing

What type of testing focuses on the body's reaction towards an antigen and the specific antibody produced?

Parasitic infections

What type of infections does this method generally not include the diagnosis of?

Technical proficiency of the laboratory personnel and microbiologists

What do most diagnostic tests rely on primarily?

Single most important step

What is considered the most important step in the diagnosis of infection?

Selection, Timing, Method of collection of specimens

What three factors determine the results of diagnostic tests for infectious diseases?

Representative of infectious process

What must an ideal specimen be to ensure proper diagnosis?

Site most likely to yield the agent at that particular stage of illness

From where must the specimen be obtained?

Sputum

What type of specimen would be submitted to test for pulmonary tuberculosis?

Urethral discharge

For a female patient diagnosed with an STI, what would be the best sample?

Quantity must be adequate

What is a key requirement for the amount of specimen collected?

Avoid contaminant

What is crucial to prevent when collecting specimens, often involving sterile equipment and aseptic technique?

Touching the inside of the sample bottle for urinalysis

What is an example of negligence during specimen collection that can lead to contamination?

Normal flora of the skin (Staphylococcus)

What type of organisms might contaminate a urinalysis sample if proper technique is not followed?

Midstream

What technique is recommended for urine collection to remove normal flora?

Prompt transport

What must be done quickly with specimens after collection?

Before antimicrobial drugs are administered

When must meaningful specimens to diagnose infections ideally be secured?

Empiric treatment

What kind of treatment may be started if results take too long, based on a "guessed" etiologic agent?

3-5 days or even longer

How long can culture results take?

Escherichia coli

What is the most common bacterium producing UTI that may be targeted by empiric treatment?

GI tract

Where is E. coli normally found?

Urethral orifice

In females, what anatomical feature is very near the GI tract, making UTI more common?

Optical methods of microscopy

What does microscopic examination refer to?

Relatively simple and inexpensive

What are two general characteristics of microscopic examination?

Less sensitive than culture

How does microscopic examination compare to culture for detecting small numbers of bacteria?

Light Microscope, Electron Microscope, Confocal Scanning Laser Microscope, Scanning Probe Microscope

What are the four types of microscopes mentioned?

Bright Field Microscope

What is the most common method of light microscopy?

Bright visible light from a bulb

What does bright field microscopy use to illuminate the specimen?

Ocular Lens and Objective Lens

What two series of lenses does a compound light microscope have?

10x magnification

What is the magnification of the ocular lens?

Objective Lens

What are the cylindrical lenses found below the nose piece?

10x magnification (bacteria seen as dots)

What is the magnification and use of the Low Power Objective (LPO)?

40x magnification (can see the shape of bacteria)

What is the magnification and use of the High Power Objective (HPO)?

100x magnification (can clearly see the rod shape)

What is the magnification and use of the Oil Immersion Objective (OIO)?

Cedar oil

What is needed to use the OIO?

Multiplying ocular lens magnification by objective lens magnification

How is total magnification computed?

400x

What is the total magnification if the ocular lens is 10x and the HPO is 40x?

Differences in contrast between them and the surrounding medium

Why are specimens visible under a bright field microscope?

Dyes/stains

What can be used to increase contrast under a bright field microscope?

Viruses

What microorganisms are unable to be viewed under a light microscope due to their small size?

Improves contrast between cells and their surrounding medium

What is the main advantage of phase contrast microscopy?

Living cells without staining them

What can be seen using phase contrast microscopy that is important because dyes can kill microorganisms?

Phase plate and Condenser annulus

What two plates are added to a light microscope to make it a phase contrast microscope?

Changes to the phases of light waves

What does the phase contrast microscope detect?

Reaches the specimen from the sides only

How is the lighting system modified in dark field microscopy?

High

What is the resolution quality of dark field microscopy?

Treponema pallidum or other spirochetes (e.g., Leptospira)

What specific organisms is dark field microscopy used to detect?

Dark background

What kind of background does dark field microscopy produce?

Fluoresce

What type of specimens does a fluorescence microscope visualize?

Absorb short wavelengths (UV) and give off light at a longer wavelength (visible light)

What is fluorescence?

Chlorophyll

What natural substance causes some microorganisms to fluoresce?

Fluorochromes

What are used to stain non-naturally fluorescing organisms to make them glow?

Mycobacterium tuberculosis

What specific microorganism is detected using fluorochrome auramine O?

Glows yellow when exposed to UV light

What color does auramine O glow when exposed to UV light?

Fluorescent-antibody (FA) technique or immunofluorescence

What is the principal use of fluorescence microscopy in clinical diagnostics?

Fluorochrome

In immunofluorescence, what are specific antibodies chemically labeled with?

Direct fluorescent microscopy

What type of immunofluorescence involves the primary antibody, bound to the fluorophore, directly binding to the target antigen?

Indirect fluorescent microscopy

What type of immunofluorescence involves a primary and secondary antibody, with the secondary antibody linked to the fluorophore?

Adds to the sensitivity of the test

What is the advantage of indirect immunofluorescence?

Polarized light

What type of light do DIC microscopes employ using a polarizer?

Condenser Wollaston/Nomarski Prism and Objective Wollaston/Nomarski Prism

What are the two prisms used in DIC microscopy?

Structures may appear 3D, Acts as a phase contrast, Can detect waves (phases of light) showing a much higher resolution

What are the advantages of DIC microscopy?

Most powerful

How is the electron microscope characterized in terms of power?

Very high resolving power

What is the key feature of the electron microscope that allows observation of detailed cell structures?

Electrons having much shorter wavelengths than photons of white light

Why do electrons provide very high resolving power?

Electron beam

What does an electron microscope use instead of a light source?

Transmission electron microscope (TEM) and Scanning electron microscope (SEM)

What are the two types of electron microscopes?

0.001µm

What is the resolving power of a TEM for particles?

0.01-0.2µm

What is the diameter range of viruses easily resolved by TEM?

Spikes protruding away from the surface of the virus

What distinctive feature of the SARS-CoV-2 virus is visible under TEM, resembling a corona?

Lower resolving power

How does the SEM compare to the TEM in terms of resolving power?

Providing 3D images of the surface of microscopic objects

What is the main advantage of SEM?

Violet circles

What color are the virus representations under the SEM in the source image?

Not readily seen

Are the spikes of the SARS-CoV-2 virus readily seen under SEM?

Confocal Scanning Laser Microscope

What type of microscope couples a laser light source to a light microscope?

Scanning Probe Microscopes

What type of microscope measures surface features by moving a sharp probe over the object's surface?

10% KOH (potassium hydroxide)

What solution is used to treat fungi for unstained microscopic examination?

Combine chemically with the bacterial protoplasm to greatly improve contrast against background

How do stains work to improve contrast?

Kill it

What happens to a living cell during the staining process?

Crystal Violet

What is the primary stain in Gram staining?

Iodine

What is the mordant in Gram staining?

Alcohol

What is the decolorizer in Gram staining?

Safranin

What is the counterstain in Gram staining?

Decolorizer

What differentiates Gram-positive from Gram-negative bacteria in Gram staining?

Gram-positive (purple/blue)

What color are Gram-positive bacteria after Gram staining?

Gram-negative (red/pink)

What color are Gram-negative bacteria after Gram staining?

Crystal Violet > Iodine > Alcohol > Safranin

What is the proper sequence of Gram staining steps?

Carbolfuchsin

What is the primary stain in Ziehl-Neelsen Acid Fast Stain?