Basic Bacteriology Flashcards
general principles to consider in visualization
- wavelength of radiation
- magnification
- resolution
- contrast
______ act as waves with wavelengths dependent on the ____ of the electron beam
moving electrons, voltage
True or False
Radiation of smaller wavelengths results in enhanced microscopy
True
the apparent increase in size of an object, indicated by a number and the letter x
Magnification
How does magnification occur?
When a beam of radiation refracts as it passes through a lens
Analogy
Curved glass lenses: Light
Magnetic fields: _____
Electron beams
the ability of an instrument to distinguish between objects that are close together, as close as 0.2 µm
Resolution
resolution distance is dependent on ____
- wavelength of light/electron beam
- numerical aperture of lens
modern microscopes use _____ wavelength radiation and lenses with ____ numerical apertures
Shorter, larger
the differences in intensity between two objects or between an object and its background
Contrast
How is contrast achieved?
Using stains
5 types of light microscopy
- Brightfield
- Darkfield
- Phase Contrast
- Confocal
- Fluorescent
the phenomenon wherein UV light causes specimens to radiate visible light
Fluorescence phenomenon
2 categories of staining under fluorescent microscopy
- fluorochroming
- immunofluorescence/fluorescent antibody technique
the reaction between the fluorescent dye and the specimen (everything will be stained)
Fluorochroming
technique involving enhanced staining wherein the antibody binds to the fluorescent dye and only the part with the corresponding antigen is stained
Immunofluorescence
2 types of electron microscopy
- Transmission (TEM)
2. Scanning (SEM)
electron beams pass through the object and allows visualization of internal structures
Transmission (TEM)
electron beams scan the surface of objects and provide a 3D view of surface structures
Scanning (SEM)
2 ways by which bacteria can be examine
- unstained, living state
- fixed, stained state
3 techniques to examine unstained, living bacteria
- Direct wet mount prep
- Hanging drop prep
- Intravital staining
preparation used to detect motile bacteria such as Campylobacter jejuni and Vibrio cholerae
Direct wet mount prep
microscopes used to view a direct wet mount prep of bacteria
- dark field
- phase contrast
- light microscope w/ partially closed diaphragm (if previous 2 are unavailable)
differences between direct wet mount prep and hanging drop prep
- morphology in hanging drop is less distorted
- motility in hanging drop is better appreciated
technique in examining unstained living bacteria using a diluted dye
Intravital staining
What should be done before fixing and staining bacteria?
Smear preparation
4 steps for fixed, stained state
- smear prep
- air-drying
- fixation
- staining
purpose of air-drying after smear prep
- preserve bacterial morphology
- allow smear to adhere to slide
most common method of fixation
Heat fixation
How do you heat fix a slide?
Pass the slide with smear side up over a flame 3-5 times
fixative used during chemical fixation
Alcohol and formalin
3 types of staining
- simple
- differential
- special
type of staining wherein a single dye is employed so that all the cells will have the same color
Simple staining
examples of simple stains
- dilute carbol fuchsin
- methylene blue
2 types of simple staining
- positive staining (stained spx)
- negative staining (stained bg)
- type of staining wherein more than one dye is employed so that more than one color is observed
- where contrast is most commonly observed
Differential staining
commonly used differential stains
- Gram stain
- acid-fast stain
type of staining employed to isolate a structure of the bacterial cell so that it can be seen
Special staining
devised Gram staining
Dr. Hans Christian Gram
2 large groups divided by Gram stain and their colors
Gram (+) - deep-violet/purple
Gram (-) - pink/red
general rules for Gram (+) bacteria
All cocci are + except:
- Neisseria grp.
- Moraxella catarrhalis
- Veillonella
general rules for Gram (-) bacteria
All bacilli are - except:
- acid fast organisms (including Mycobacterium, Nocardia)
- spore formers (Bacillus, Clostridium)
- Corynebacterium sp.
Spiral organisms are also - when stained
primary stain in Gram staining
Crystal violet
mordant in Gram staining
Gram’s iodine
the large insolube complex formed after adding the mordant
Crystal violet iodine (CV) complex
When does differentiation start during Gram staining and acid fast staining?
In the decolorization step
decolorizer in Gram staining
Absolute alcohol or a mixture of acetone and alcohol
after decolorization, what happens to all gram (-) cells?
Become colorless
after decolorization, what happens to all gram (+) cells?
Remain violet
counterstain in Gram staining
Safranin
What does safranin do in Gram staining?
Give color to gram (-) cells
Steps in Gram staining (Hucker’s method)
- Smear
- Crystal violet
- Gram’s iodine
- Absolute alcohol
- Safranin
*washing is done in between steps and after the last step
3-step variation in Gram staining
Incorporate Safranin w/ Alcohol so that when the mixture is added, it will both decolorize and counterstain
3 bases on the principles behind Gram staining reaction
- composition of cell envelope
- presence of magnesium ribonucleate (protein complex)
- loss of cell wall integrity
explain the principle behind Gram staining based on cell envelope composition
Gram (+): cell wall has more proteins than lipids. Decolorizer coagulates proteins, thereby narrowing pore sizes, decreasing permeability, and trapping CVI complex in cells - retaining violet color.
Gram (-): cell wall has more lipids than proteins. Decolorizer dissolves lipids, thereby widening pore sizes, increasing permeability, and releasing CVI complex from cells - losing violet color.
What is the substance responsible for an organism’s gram positiveness?
Magnesium ribonucleate
What causes the loss of cell wall integrity?
- old age
- action of autolytic enzymes
- antibiotic treatment
meaning of ‘acid fast’ organisms
organisms with long chains of fatty acids in cell walls that make them resistant to decolorization by acid alcohol
What is the substance responsible for the acid fastness of an organism?
Long chain mycolic acids
2 methods of staining acid fast organisms
- Ziehl-Neelsen
2. Cold Kinyoun
primary stain in acid fast staining
Carbol fuchsin (red)
decolorizer in acid fast staining
Acid alcohol