Chapter 1 (Microscopes & Stains) Flashcards
Part 2
Microscopes for visualization
Brightfield
Fluorescent
Phase Contrast
Darkfield
Electron
What microscope visualize most fungi, parasites and bacteria?
Brightfield
T/F: Visible light passes through the specimen and then through a series of lenses that reflect light in a manner that results in magnification of the organism present in the specimen
True
T/F: The total magnification of the organism present in the specimen
True
The extent to which detail in the magnified object is maintained
Resolution / Resolving Power
Fill the space between the objective lens and the glass slide onto which the specimen has been affixed.
Enhances resolution by preventing light rays from dispersing and changing wavelengths after passing through the specimen
Oil Immersion
Needed to make objects stand out from background.
It is commonly achieved by staining which highlights organisms and allow them to be differentiated from one another and from background material and debris
Contrast
Can be raised to a higher energy level after absorbing - UV (excitation) light
Fluors/ fluorochromes
The excess energy when the dye molecules return to normal, low energy state
Visible (fluorescent) light
The process when the dye molecules return to normal (low) releasing excess energy in the form of visible (fluorescent) light
Fluorescence
T/F: Color of the fluorescent light depends in the dye and light filters used
True
General categories of Fluorescent
Fluorochroming
Immunofluorescence
Fluorescent dye is used alone
Interaction bet. de and cell component
Enhances contrast and amplifies the observer’s ability to defect stained cells compared to when using ordinary staining procedures
Fluorochroming
Fluorescent dyes have been chemically linked to specific antibodies
Combines the amplified contrast provided by fluorescence with the specificity of antigen-antibody binding
Immunofluorescence (Fluorescence Antibody Technique) [FAT]
In Phase Contrast microscope, Contrast is achieved ___________
Without the use of stains
Beam of light, pass through the specimen and are partially deflected by the different densities or thicknesses (i.e., refractive indices) of the microbial cells or cell structures in the specimens
Phase Contrast
The greater the refractive index
The more the beam of light is lowed down
(results in decreased light intensity)
Advantage of Phase Contrast
Permit observation viable microorganisms
Similar to phase contrast in that it involves alternation of microscopic techniques rather than the use of dyes or stains to achieve contrast
Darkfield
How does a darkfield microscope have a dark background?
The condenser does not allow light to pass directly through the specimen but directs light to hit the specimen at an oblique angle
(all other light that passes through the specimen will miss the objective)
Used in detecting bacteria directly in specimens of patients that, because of their thin dimension, cannot be seen by the light microscope and because of their physiology are difficult to grow in culture (spirochetes)
Darkfield Microscope
Uses electron beams instead of light
Instead of lenses, the electrons are focused by electromagnetic fields and form an image on a fluorescent screen
Electron microscope
Magnification in excess of 100,000 is achieved in electron microscope
Because of substantially increased resolution
Types of Electron microscopes
Transmission
Scanning
Passes electron beams through objects and allows visualization of internal structures
Transmission
Electron beams are used to scan the surface of objects and provide a three-dimensional view of surface structures
Scanning
Techniques for Microscopic Study of Microorganisms
Unstained, Living
Fixed, Stained
Unstained, Living state
Direct Wet Mount
Hanging Drop Preparation
Intravial Staining
Fixed, Stained state
Smear Preparation
Air Drying
Fixation
Staining
Used for detection of motile bacteria like C. jejuni or V. cholerae (darting motility); also, parasites
Viewed best through DF, PC & LM w/ a partially closed diaphragm
Direct Wet Mount Preparation
Advantage: morphology is less distorted and motility is barter appreciated
Viewed using the LM with a partially close diaphragm
Hanging Drop Preparation
A dye so dilute that it cannot exert any toxic effect or inhibitory action on the cell is employed
Intravital Staining
Either direct clinical specimen/ samples of growth from cultures, it is the most useful method for the presumptive ID of bacteria and the presence of certain viruses and for the definitive ID of most parasites and fungi
Fixed, Stained state
Preserve the morphology of the bacteria
Allow the smear to adhere to the slide
Air Drying
Heat
Alcohol (95% methanol)
Fixation
Coloring the microorganisms w/ a dye that emphasize certain structures
Staining
Staining types
Simple
Differential
Special
Employ a single dye
Most cells and most structures within each cell will stain the same hue
Simple stain
Organism is stained
Positive staining
Background is stained, not the organism
Negative staining
Consist of adding more than one dye added in several steps and teh stained structures are differentiated by color as well as shape
Based on teh relative affinity of different bacterial cells for the stains used
Differential
Used to color and isolate specific parts of microorganisms
Specific stains
Staining reactions (in ref. to diff. stains)
Gram stain (+) / (-)
Acid fast stain
Most useful, generally applied
Can be used effectively divide all bacterial species into 2 large groups
Gram stain
Gram stain devised by ________ on _______
Dr. Hans Christian Gram 1884
Those that retain the primary stain and are deep violet in color
Gram (+)
ALL COCCI ARE GRAM (+) except
Neisseria group
Moraxella (Branhamella) catarrhalis (and Veillonella)
Those that are decolorized and are stained pink or red in color
Gram (-)
ALL BACILLI ARE GRAM (-) except
the acid fast org (Mycobacterium, Nocardia)
sporeformers (Bacillus, Clostridium)
Corynebacterium spp.
Spirals are difficult to stain but when stained, they are Gram (__)
(-)
Steps in gram staining (Hucker’s method)
- Make a smear
- Add crystal violet
- 95% alcohol/ mixture of acetone and alcohol, colorizer
- The basic red dye safranin is applied as counterstain
Because it imparts color to all cells, it is referred to as the primary stain; cytoplasm of all cells will be purple
Crystal violet (basic purple dye)
The purple dye is retained by Gram (+) cells and is readily removed from Gram (-) cells
Gram (-) cells will be colorless at this stage
Decolorizer
Counterstain gives contrasting color to the primary stain
This stains Gram (-) cells while Gram (+) cells will remain purple
Counterstain (basic red dye safranin)
They have more protein in their cell envelope, particularly in the cell wall.
Gram (+) cell
This causes coagulation and dehydration of the proteins in Gram (+) cells retaining the primary stain
Decolorizer (95% alcohol/ mixture of acetone + alcohol)
They have more lipids in their cell envelope
Gram (-)
What happens when Gram (-) are added with decolorizer?
Dissolve the lipids upon the addition of alcohol leading to an increase in pore size, allowing the escape of the CVI complex from the cell
What protein complex is absent in Gram (-) bacteria?
Magnesium ribonucleate
5 steps in gram staining
- Fixation
- Crystal Violet
- Iodine Treatment
- Decolorization
- Counterstain w/ Safranin
What long chain fatty acids on the walls of a certain bacteria that resists decolorization of basic dyes by acid alcohol?
Long chain (50-90 C atoms)
fatty acid (mycolic acid)
Uses acid alcohol as decolorizer
Selective permeability
Acid fast stain
Acid fast organisms
Mycobacterium
Nocardia
coccidian parasite (Cryptosporidium)
They have a very hydrophobic surface which resists entry of dyes by the usual staining procedures
Mycobacteria
Employing heat to enhance penetration and retention of the dye
Ziehl Neelsen
Increasing the concentration of phenol
Cold Kinyoun technique
Another way of getting dyes into cells in acid fast stain
Inclusion of a detergent in the stain
Ziehl Neelsen acid fast staining steps
a. Make a smear
b. Carbol fuchsin (primary stain)
c. Acid alcohol (decolorizer)
d. Methylene blue (counterstain)
AFS: acid fast organisms
Colored Red
AFS: non-acid fast organisms
Colored Blue
