Lab Two - staining and microscopy

“founder” of the microscope

antonie van leewenhoek

light (compound) microscope

uses multiple lenses to increase magnification

dark field microscope

uses scattered light to illuminate sample, blocks direct light to increase contrast

phase contrast microscope

uses the change in phase of light passing through the sample to increase contrast, caused by having different refractive index

fluorescent microscope

uses fluorescent dyes or proteins, highly sensitive with high contrast

electron microscope

uses electrons instead of light giving extremely high resolution (about 0.1 nm)

Scanning EM or Transmission EM

objective lens

lens close to specimen, can be 10x, 40x, 100x

ocular lens

lens closer to the eye, typically 10x or 20x

stage

part of microscope that holds sample

light source

the light that illuminates the sample in a microscope

condensor

to focus light on sample for brightness, contrast, and resolution

resolution

minimum distance two objects must be apart to be able to differentiate the objects in the image (visible light has 200 nm)

contrast

the difference between light and dark regions

oil immersion lens

oil is placed between the sample and the lens, oil has a similar refractive index to the class so it collects more light and gives better resolution and higher magnification, oil stops dispersion of ligth through refraction by air giving greater clarity of image

basic dyes

most common, have a positive charge and bind to the negatively charged bacteria surface or to negatively charged DNA and proteins

acidic dyes

have a negative charge so bind to the media and give contrast to unstained cells

downsides of staining

stains kill the microbes they are used on, which limits what we can observe with stains

methylene blue

simple stain, cationic, used to observe cell morphology, increases contrast to visualize bacterial cells

cell morphology

shape and arrangement of bacteria

Gram Stain

routine primary diagnostic test to identify causative agent of an infection, separates cells into gram positive and gram negative

Gram Stain methodology

1. stain with crystal violet (primary stain)

2. stain with iodine

3. wash with an alcohol (ethanol)

4. stain with safranin (counterstain)

use of iodine in gram staining

iodine forms a complex with the crystal violet stain that adheres to peptidoglycan cell walls

use of alcohol in gram staining

dehydrates thick peptidoglycan walls in gram positive bacteria trapping crystal violet-iodine complexes, removes outer LPS on gram negative bacteria making the cell permeable to the crystal violet-iodine complex and leading to the washing away of the stain

myobacterium tuberculosis

causative agent of tuberculosis, cannot be stained by typical dyes or gram staining; has a specialized outermembrane with an inner layer of mycolic acids and an outer layer of free lipids that blocks stains from reaching it

staining myobacterium tuberculosis

primary stain with carbol fuschin (red), wash with an acid wash, then counterstain with methylene blue

enterococci

UTIs, endocarditis

streptococci A, B, C, D, G

neonatal sepsis

streptococcus pneumonaie

community pneumonia, septic shock, meningitis

staphylococcus aureus

furunculosis, cellulitis, septic shock, meningitis, endocarditis

E. Coli

UTI, septic shock, hemorrhagic colitis

Klebisella spp.

UTI, septic shock, pneumonia

neisseria meningitidis

septic shock, meningitis

hameophilius influenzae

respiratory tract infection

ring

surface growth adhering to the glass

pellicle

surface growth forming either continuous or interrupted sheet over culture liquid

flocculent

surface growth or sediment made up of small adherent masses of various shapes floating in culture liquid

membranous

surface growth that is a thin coherant membrane like surface

uniform turbidity

even growth in media

compact

sediment that is a single fairly tenacious mass

granular

sediment that is composed of small granules

flaky

sediment that is in the form of numerous separate flakes

viscid

sediment that on shaking rises in a coherent swirl