BI203A chapter 3 observing microbes

Light microscopy

the stained specimen absorbs light and appears dark against a bright background. The specimen is dead and attached to the slide.

Fluorescence microscopy

the background and specimen appear dark except for the fluorophores. Fluorophores are molecules that fluoresce. Only labeled structures are visible.

Scanning electron microscopy

beams of electrons scatter from the stained surface of a specimen. The image shows much greater detail than light microscopy but only the specimen’s external surface is enhanced

Transmission electron microscopy

electrons pass through a stained section. Very fine details may appear, such as individual ribosomes, but only a small fraction of the whole cell may be seen.

simple stain

adheres to cells but not the external medium surrounding the tissue

• methylene blue

differential stain

stains two kinds of cells differently

• gram stain

The Gram stain distinguishes between

gram-positive cells and gram negative cells

gram positive cells

appear purple

they have cell walls with lots of peptidoglycan (multiple layers) that retains crystal violet

thick cell wall

no outer membrane, no LPS/endotoxin

• Stpahylococcus aureus

• streptococcus species

gram-negative cells

appear pink

they have little peptidoglycan (single layer) so they dont retain crystal violet

thin cell wall

outer membrane containg LPS/endotoxin

• escherichia coli

• proteus species

lipopolysaccharide (LPS) = polysaccharides & lipid A

—Endotoxin that is an integral part of the cell wall released after cell death 

—Causes fever, dilates blood vessels, etc

—Antibiotics given late in infection can cause symptoms to worsen & may even cause death

—Do not retain crystal violet, but counter stain w/ safranin (red)

Mycobacteria

Considered Gram- positive,  but their envelopes are exceptionally thick and complex,  including extra layers not found in other Gram-positive cells.

acid-fast stain

laboratory test used to determine bacteria that have waxy cells

Carbol fuchsin stains mycolic acid only found in cell walls of

Mycobacterium tuberculosis other Mycobacterium species.

spore stain

Malachite Green is added to a heat-fixed slide and heated so the dye enters the hard spore coat.

  Bacillus and Clostridium species are spore formers.

negative stain

Capsule-forming bacteria can be stained with dyes that darken the background, allowing the capsule to be visible.

antibody stain

Specialized stains that use antibodies to detect specific molecular components of the cell.

who is jared

a 48-year-old, previously healthy man, was admitted to a hospital in Columbus, OH, after 6 days of increasing dyspnea (shortness of breath) and watery diarrhea.

Jared also complained of a dry cough, myalgia, (muscle pain), nausea, and vomiting. He appeared flushed and had dry mucous membranes.

what was jared vitals

His temperature was 39°C (102.2 °F), and his pulse rate 103 beats per minute

what did chest radiographs show for jared

Chest radiographs showed pneumonia with consolidation (formation of a firm mass) of the left lower lobe.

Jareds C-reactive protein (CRP, a blood protein produced by the liver) showed what

greater than 220mg/l, a high level that indicates inflammation

what did jared do to get sick

he cleaned the filter of an outdoor whirlpool spa

what did culture from jareds sputum and spa filter reveal?

gram-negative bacilli consistent with Legionella Pneumophila

how was Jared diagnosis confirmed

Fluorescence microscopy usig direct fluorescent antibody (DFA) stain.

what was Jared prescribed to overcome the disease

He received IV amoxicillin and clarithromycin antibiotics for 8 days before his condition returned to normal.

Fluorescence Microscopy

A powerful tool to detect specific microbes and subcellular structures while avoiding signals from dust and other nonspecific particles.