4. Lab Diagnosis

Light microscope

resolves to 0.2 um ad can see external morphology

purpose of microscopy

initial detection of microbes

scanning electron microscopy

high resolution view of cell surface —> useful in virology

transmission electron microscopy

provides high-resolution images of internal structures by transmitting electrons through thin specimens.

Most simple stains target

cell wall

example of negative stain

india ink (carbon particles)

What do negative stains do

they provide contrast by staining the background while leaving the specimen clear.

Antibody stain

is a technique that uses antibodies to bind to specific antigens in a specimen, allowing for precise visualization of targeted cells or structures.

Quellong reaction

is a test used to identify encapsulated bacteria, such as Streptococcus pneumoniae, by observing capsule swelling when mixed with specific antisera.

Quellung rxn has been replaced with modern

PCR to detect capsule dna

Capsule

mediates adhesion and increases virulence due to antiphagocytic property

A capsule may be identified after staining due to presence of

halo

first step is bacteria id

gram stain

During first step in ID, gram staining, is this specific of nonspecific

nonspecific because it is a broad classification method.

issue with antibiotics and gram staining

antibiotics affect cell wall integrity, which impacts staining outcome

Exceptions of gram staining

spirochetes
mycoplasma
mycobacteria

mycoplasma cannot be gram stained because

they lack a cell wall, making them resistant to the staining process.

mycobacteria cannot be gram stained because

they have a mycolic acid cell wall which is too waxy for the gram stain to penetrate.

acid fast stain

is a special staining technique used to identify bacteria with waxy cell walls, such as mycobacteria, by retaining the dye after washing.

Ziehl-Neelsen Method

older acid-fast staining procedure used to identify mycobacteria by applying a carbolfuchsin, heat, decolorizer, and methylene blue

Fluorescent Acid-Fast Staining

Fluorochrome dyes bind strongly to mycolic acid in bacterial cell wall —> makes them neon yellow

Kinyon “Cold” Method

acid fast staining that DOES NOT require HEATING —> positive is pink, neg is blue

Silver stain

use to stain difficult specimens like legionella, spirochetes, and fungi

why do you have to use silver stain for legionella

unique LPS content

why do you have to use silver stain for spirochetes

too thin

Primary purpose of liquid growth media

expanding an organism

Purpose of agar plates (semi solid growth)

pure colony isolation

Purpose of agar slants (semi solid growth)

for semi-long-term storage & transport

MALDI-TOF Mass Spectrometry

laser can identify bacterial species based on protein profile, by shooting laser at single colony

Total Laboratory Automation (TLA)

Automatically streak specimens onto agar plates then auto incubates them and take photos for high volume sample processing and analysis.

defined media (synthetic)

known chemical comp

rich media (complex)

Unknown chemical composition (e.g. beef heart broth)

selective media

Select for specific organism

differential media

Distinguish organisms with certain characteristics (e.g. enzyme production)

blood agar

differential media that shows which bacteria produce hemolysins (clear area around colonies)

Is blood agar selective, differential or both

Blood agar is considered differential because it reveals hemolytic activity, but it is not selective as it supports the growth of a wide range of bacteria.

Blood agar is strongly associated with which species

Streptococcus pyogenes

α-hemolysis

Incomplete, partial hemolysis (greenish)

β-hemolysis

Complete hemolysis, clear/yellow

γ-hemolysis

Non-hemolytic

MacConkey agar

A selective culture medium used to isolate and differentiate gram-negative bacteria, primarilyEnterobacteriaceae, based on lactose fermentation.

MacConkey agar selectivity

For Gram- bacteria due to bile salts presense

MacConkey agar-differential

Identify organisms that does lactose fermentation (+ is pink)

Urease production

some bacteria produce this enzyme which breaks down urea into ammonia and carbon dioxide, leading to an increase in pH.

Positive urease production often turns pH indicator agar which color

pink due to the alkaline environment. (increase pH)

Carbohydrate fermentation broth

detects acid production and gas production

If by-product of fermentation is acid, which lowers pH, causes indicator phenol red to turn

yellow in color.

Catalase test

detects the presence of the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen, resulting in bubble formation.

Catalase is strongly associated with which species

particularly staphylococcus

Why is molecular detection of genetic material more important than culture

most microbes cannot be cultured

Nucleic acid probes

short strips of DNA that hybridize to pathogen DNA or RNA, then give color reactions

Examples of genetic probes

southern blot, northern blot, western blost

Southern blot

uses genetic probes to detect DNA

Northern blot

uses genetic probes to detect RNA

western blot

uses genetic probes to detect proteins

In situ hybridization

is a technique used to detect specific nucleic acid sequences in fixed tissues or cells, using DNA probes

What modern technique replaced traditional in situ probes

modern spacial replacement (MERFISH/Spatial seq)

PCR

amplify DNA sequence using thermal cycling.

RT-PCR

amplify RNA after RTase converts it to DNA

Antibody

(immunoglobulin or Ig): Protective, Y-shaped protein produced by immune system to find, bind, and neutralize foreign invaders

Antigen

(antibody generator): Any substance that triggers your body's immune system to produce antibodies against it

Serology

study of serum meditated by Ag:Ab reactions

Common serology test are based on

precipitation and agglutination reactions.

zone of equivalence

insoluble, cross-linked lattice network that drops out of the solution as a visible solid —> Ab:Ag preceipitin complex

Radial immunodiffusion

old serological technique used to quantify the concentration of antigens by measuring the diameter of precipitin rings formed in a well containing Ab

Countercurrent electrophoresis

When Ag and Ab have opposite charges , they migrate towards each other in an electric field, allowing for the formation of a precipitate where they meet, facilitating the detection of antigens, rapid double diffusion technique

Agglutination

Antigen : Antibody cross-linking results in clumping of particles —> rapid and visible

Most efficient agglutinator

IgM

Latex agglutination

A serological test using latex beads coated with antigens, allowing for the visualization of agglutination when antibodies are present.

Viral latex agglutintination

A serological test that uses latex beads coated with viral antigens to detect the presence of corresponding antibodies in a sample, resulting in agglutination.

Negative result of viral latex agglutination

no clumping

Positive viral latex agglutination

Clumping because antibody is present in the blood

Hemoagglutination

used for blood typing to detect antigens on the surface of red blood cells, determining compatibility for transfusions.

Enzyme-Linked Immunosorbent Assay (ELISA):

Versatile, highly sensitive method to detect, quantify antigens and antibodies—> makes color product

Immunofluorescence

is a technique that uses fluorescently labeled antibodies to detect specific antigens in cells or tissues, allowing for visualization under a fluorescence microscope.

Conjugate enzyme reporter for ELISA

is an enzyme linked to an antibody or antigen that catalyzes a reaction, producing a measurable signal, typically a color change.

Direct ELISA

detects antigens directly in a sample by using antibodies that are conjugated to an enzyme, resulting in a color change for quantification.

Indirect ELISA

detects antibodies in a sample by using an enzyme-linked secondary antibody that binds to the primary antibody, producing a measurable signal.

Sandwich ELISA

can quantify antigen levels between two layers of antibodies, where the target antigen is sandwiched between a capture antibody and a detection antibody linked to an enzyme.

Competitive elisa: no glow result

No Ag binds to Ab

Competitive elisa: glow result

Ag can now bind with detection Ab

COVID 19 rapid antigen tests

are diagnostic tools designed to quickly detect the presence of specific proteins from the virus that causes COVID-19, typically providing results within minutes.

flow cytometry

can count type and number of cells within liquid suspension

in flow cytometry, more fluorescence means

more cells

Feluda

rapid covid test that uses CRISPR technology to detect viral RNA.

Saliva based testing

very common, noninvasive biomonitoring that uses modern laboratory techniques

Common examples of saliva based testing

drug tests
hormone testing
pathogen/infection testing

Problems with oral microbial testing

cannot distinguish these variations:

Pellicle – Biofilm – Dental plaque – Calculus