Lecture 2 Demonstration of Microorganisms Flashcards
Pathogenic organisms can often be directly visualized by microscopic examination of patient specimen
Sputum
Blood
Urine
Cerebrospinal fluid
Gives rapid and inexpensive results without waiting for culture
Microscopy
Can provide 1st screening step in arriving at a specific identification
Microscopic morphology and staining characteristics
Dye that binds to a cellular structure and gives it color
Unstained bacteria may be difficult to detect with light microscope
Stain
Negatively charged, binds to positively charged structures like proteins
E.g. acid fuchsin, eosin, picric acid, nigrosine, India ink
Acidic dyes
Positively charged
Since surface of bacterial cells msre negatively charged, _ commonly used in bacteriology
E.g. methylene blue, crystal violet, safranin, malachite green
Basic dyes
Classification of stains
Simple stains
Differential stain
Special stain
Uses a single dye
Size, shape, arrangement of cells
E.g. methylene blue, crystal violet
Simple stains
Uses two or more dyes to distunguish different types or structures of bacteria
Distinguishes gram + and gram -
E.g. mycobacteria, nocardia
Differential stain
Identifies specialized structures
Exhibits the presence of flagella, endospores, capsule
E.g. Shaeffer-Fulton method, India ink
Special stain
The most common and useful staining procedure
Separates bacteria into 2 classifications according to composition of cell walls
Most, but not all, bacteria are stainable and fall into one of these two groups
E.g. mycoplasma - lacks cell walls; gram-variable bacteria
Gram Stain
Applications of Gram Stain
Important therapeutically
- gram + and gram - bacteria have differing susceptibilities to antibiotics
- guide to initial therapy until definitive ID
Sometimes diagnostic
- morphology of stained bacteria
- e.g. spore-forming bacilli in blood of cattle
Applications of Gram Stain (2)
Blood smear of anthrax case - Gram + bacilli are seen
Aids interpretation of culture results
- e.g. specimen shows organisms under the microscope but appear sterile on culture media which suggests:
1. Fastidious organisms - bacteria with complex nutrient requirement
2. Fragile organisms - may not survive transport
May provide only clue to nature, variety, and relative number of infecting organisms
Limitations of the Gram Stain
High number of microorganisms required
Liquid samples with low numbers of microorganisms (CSF)
- requires centrifugation to concentrate pathogens
- pellet then examined after staining
Used to identify organisms that have waxy material (mycolic acids) in cell wall
Acid-fast stain
Most clinically important acid-fast bacteria
Mycobacterium tuberculosis
Also occurs in animals and may have clinical importance
Nocardia sp.
One of the simplest microscopic methods
Background is stained while organisms remain clear
Determines if organism has gelatinous capsule
Able to visualize bacteria as large, transparent capsules that displace II particles
Useful in detecting cryptococcus neoformans in CSF
India ink
Stained background
Capsule appears transparent/translucent
Eosin, nigrosin, india ink can be used to stain background
Crystal violet to stain bacterial cells
India ink
Technology producing images of very small things with high clarity and magnification
Microscopy
Two broad categories of microscopy
Light microscopes
Electron microscopes
Magnification should be coupled with
Good resolution
Distance at which two objects can be viewed as separate or distinct objects
Resolution
Units in Microscopy
Micrometers or microns (mu m) = 10^-6 m
Nanometers (nm) = 10^-9 m
Angstrom (A) = 10^-10 m
Involves use of optical lenses and light radiations
Light microscopy
Light microscopy can be further categorized as
Bright field m
Dark field m
Phase contrast m
Fluorescence m
Invented by Anton van Leeuwenhoek
Single lens, bi-convex
Not common nowadays, used for dissection purposes
Simple microscopes
Multiple lenses
Credited to Zaccharias Janssen
Uses visible light as source of illumination
Compound microscope
Brightly illuminated by focused natural sunlight or white light
Results in bright field image of a stained specimen
Bright field m
For examining live microorganisms sudpended in liquid
Only light reflected by specimen enters objective lens
Dark field m
Internal structures of living microorganisms can be examined without fixing or staining
Enhanced contrast
Phase contrast m
Uses UV light
Specimen can be stained using fluorescent dyes
Useful for diagnostic and ID purposes
Fluorescence m
Phenomenon in which fluorescent substance absorbs short wavelengths of light and emits a light of longer length
Fluorescence
Uses electron beam (instead of light) and electromagnets (instead of lens)
Provides high resolution
Electron microscopes
Two types of electron microscopes
Transmission e m
Scanning e m
Objects can be seen to order of a few angstrom
Ability to study small details in the cell, near atomic levelsuses electromagnetic lenses to focus electrons into a very thin beam
2d, black and white images
Transmission e m
Relies on electron interactions at the surface
Produces image of the surface
Creates 3d images
About 50,000x magnification
Scanning e m
