Chapter 3: Observing Microorganisms Through A Microscope Flashcards
Total magnification
Objective lens x ocular
Parfocal
Little refocusing; having lenses w/ focal points that are all in the same plane
Resolution
Resolving power; ability of lenses to distinguish fine detail of specimen
Distinguish between 2 points that are a specified distance apart
Refractive index
Light-bending ability
Immersion oil is used to keep light bending away- improves resolving power; if not used poor resolution, fuzzy
Changes refractive index by staining them
Ocular lens (eyepiece)
Remagnifies and the image formed by the objective lens
Body tube
Transmits the image from the objective lens to the ocular lens
Stage
Hold the microscope slide in position
Condenser
Focuses light through specimen
Lens located below microscope that directs light rays through the specimen
Diaphragm
Controls the amount of light entering the condenser
Coarse
Focusing knob; 10x
Fine focusing knob
40x and 100x
Brightfield (light) microscopy
Light reflected off the specimen does not enter the objective lens
Which part of compound light microscope magnifies the specimen?
Ocular lens
Which part of compound light microscope magnifies the specimen?
Ocular lens
Signs and symptoms of active pulmonary TB (lower respiratory infection)
Signs- weight loss?, coughing up blood?, abnormal breathing
Symptoms- chest pain, no appetite, fatigue, night sweats, chills, fever
Discuss the cell wall acid fast bacilli
Contains waxy material- mycolic acid (creates a barrier-making this bacteria resistant to certain antibacterial agents as well as common staining methods, like gram stain, acid-fast staining overcomes this barrier-allowing for observation of acid-fast bacilli)
Acid fast bacilli’s most active member
Mycobacterium TB (causative of TB)
Steps of acid fasting staining
- Air dry and heat fix a thin film of microorganisms. Allow the slide to cool.
- Flood the slide with Carbolfuchsin. Steam the slide with a Bunsen burner over the sink. Let the slide set for 5 minutes. Rinse with water.
- Flood slide with Acid Alcohol for 30 seconds. Rinse with water.
- Counterstain by flooding the slide with Methylene Blue for 30 seconds. Rinse with water.
- Dry the slide by putting it between the pages of a book of Bibulous paper.
- View organisms using the oil immersion objective of your microscope.
Gram stain
Examined differences in bacterial cell wall, classifying bacteria into 2 groups:
1)gram-+. (Stain purple)
2)gram- - (does not retain stain purple➡️ clear➡️ stain pink)
Type of differential stain
Acid fast stain
Identifies a specific group of bacteria- acid fast bacilli
Used to identify Mycobacterium species. Once stained with carbolfuschin and treated w/ acid-alcohol, remain pink or red b/c they retain carbolfuschin stain. Non-acid fast bacteria when stained and treated the same way and then stained w/ methylene blue, appear blue
Sputum
Phlegm
Clinical application of acid fast stain
To identify presence of bacteria like Myobacterium TB
Microscopy units
1) micrometer= 0.000001m (1x10^-6)
2)nm= 0.00000000m (1x10^-9)
Compound light microscopy (LM)
Series of lenses and use visible light as its source of illumination
Best for stained bacterial smear and examining a clinical specimen like sputum smear
Max magnification: 1500x
Max resolution: 0.2micrometers
Objective lens
(In compound light microscope)lenses closet to specimen
The primary magnifying lenses located on the revolving nosepiece, providing different levels of magnification depending on which lens is selected.
Ocular lens
(In compound light microscope) closest to the viewer; “eyepiece”
Scanning power
x4
Scanning power
x4
Brightfield microscope description
Dark objects are visible against a bright background
Light reflected off the specimen does not enter the objective lens
Which part of the compound light microscope magnified the specimen?
Ocular lens
Darkfield microscopy
-light objects are visible against a dark background
-examine live microbes; distorted by staining
-special condensers
-dx ex. Very thin spirochetes(spiral-shaped bacteria): Syphilis- Treponema palladium; Lyme disease- Borrelia burgdorferi- bx of rash cells
Best for unstained bacterial cells: the cells are small, and no detail is needed
Fluorescence microscopy
-absorbs UV light energy and emit visible light
-cells are stained w/ fluorochromes
-rapidly detect and identify microbes in tissues or clinical specimens
-sputum specimen- add fluorescence dye in detection of TB- Mycobacterium TB- if + fluorescence will appear
-3-6 wks to culture
-immunofluorescence
Immunofluorescence
-infection occurs➡️ pt will develop antibodies (defense proteins) made specifically in response to foreign cells (antigens)
-antibodies attach to antigens
-ex. In detection of
-Treponema pallium
-Legionella pneumophila (common in hotels and nursing homes
-7-14 days to culture
-rabies dx- saliva or brain tissue
-causes fatal encephalitis
e- microscopy
-shorter wavelength of e-‘s (energy source)
-electromagnetic lenses
-resolve objects <0.2micrometers
-used to observe ribosomes
Best for Intracellular detail of a cell that is 1micrometers long
Max magnification: 10,000,000x
Max resolution: 10pm
Transmission e- Microscopy (TEM)
-ultra thin sections of specimens stained w/ heavy metals
-light passes through specimen, then an electromagnetic lens, to a screen or film
Best for confirming 9 + 2 microtubule arrangement in eukaryotic flagellum, viewing cross section of poliovirus, viewing ribosomes, viewing layers of gram-(-) cell wall cross section
Scanning e- Microscopy (SEM)
-an e- gun produces a beam of e-‘s that scans the surface of a whole specimen
-secondary e-‘s emitted from the specimen produce the image
Simple vs. differential stain
simple stain uses only one dye to color a specimen, providing basic information like cell shape and arrangement, while a differential stain uses multiple dyes to differentiate between different types of microorganisms based on their cellular properties, allowing for more detailed identification of specific cell structures or species
e- microscopy vs light microscopy
electron microscope uses electrons as the source of illumination instead of light. The beam has a short wavelength. The focusing element is electromagnets. It’s resolving power is 2nm instead of the light microscope’s 0.2um. It is used a lot for viruses and internal cell structures
Arm
The vertical support that connects the stage to the eyepiece
Preparation of specimens for light microscopy
-Smear: a thin film of a solution of microbes on a slide
-usually heat-fixed to attach the microbes to the slide
-staining: coloring the microbe w/ a dye that emphasizes certain structures and provide contrast
Heat-fixation methods
Bunsen burner
Slider warmer
Simple stain
-single dye to observe cell morphology (
-shape (bacilli (rod), cocci, or spiral) and arrangements
-methylene blue- basic dye (+ charge)
Differential stains: gram stain
1) gram-(+) bacteria tend to be killed by penicillin and are sensitive to detergents
-stain purple
-many layers of peptidoglycan
2)gram-(-) bacteria tend to be more resistant to penicillin and detergents
-stain pink
-⬆️’d lipid in cell wall
-CV-I complex(insoluble colored compound formed when crystal violet and iodine are combined) is rem’d
Gram stain procedure
1) application of crystal violet (purple dye)- 1 min (both purple)
-water rinse
2) application of iodine (mordant- intensify crystal violet staining and forms complex w/ crystal violet dye)- 1 min (both purple)
-water rinse
3) alcohol wash (decolonization- rem crystal violet and iodine complex from certain cell➡️ clear) ((+)- purple, ((-)- clear)
-water rinse
4) application of safranin (counterstain- stains cell that are decolonized (clear; gram-(-))pink)- 30 sec
End result: pink gram-(-) vs. purple gram-(+)
Why is the age of bacterial culture important in gram stain?
24-48 hrs (young cultures) of growth best time to perform gram stain b/c as cells age, cell wall begins to break down and is unable to hold stains
What is the basis of gram stain reaction?
-gram-(+)- peptidoglycan
-gram-(-)- lipid (alcohol dissolves lipids)
Bacillus species
-gram stain reaction is based upon differences in the cell structure
-as Bacillus cells age, their cell walls breakdown and their Gram reaction is variable
Importance of Gram stain
-1st step in identification of bacterial wall
-provide a preliminary for a presumption dx
-guid antibiotic therapy
Differential stain: acid-fast stain
-useful in dx of TB, leprosy, cryptosporidiosis
-steps of acid-fast stain:
1)carbolfuchsin dye (primary stain- stains mycolic acid in cell wall)
-water rinse
2)acid-alcohol (decolonizer)- 2min
-water rinse
3)methylene blue (counter stain)- 2min
-acid-fast (-)- stain blue
-acid-fast (+)- stain red
What disease can be rapidly dx’d?
Leprosy (M. leprae)- red bacilli (rod) present
Acid-fast stain for dx
A rapid, reliable, and inexpensive method to dx TB in a clinical specimen (sputum used)
What color would bacterial cells appear if the pt has TB?
Stain red
(-) staining demonstrating presence of capsules
-virulence factor (makes it easier to cause disease)
-able to adhere to host (lungs)
-prevent phagocytosis (clear halo outside of the cell
-pneumonia dx- acquired from days in the hospital
-Klebsiella pneumoniae
-Streptococcus pneumoniae
Endospore staining
-dormant, resting structures (internal to plasma membrane and cell wall)
-inactive
-gram- (+) bacteria (ex. Bacillus, Clostridium)
-survival in harsh environment
-dye: malachite green
-counterstain: safranin
-location of endospore (ex. Terminal, central, etc.)
Special stains: flagella staining
-gram (-) (ex. Pseudomonas, E. Coli, Proteus)
-look at # of and arrangement of flagella (ex. Polar)
-motile
Unstained preparation: wet mounts
-stain is rarely used
-live organisms are seen high dry power (40x) objective
-lower the condenser to reduce light
-observe motility and cell size
Gram stain is an example of a _ stain
Differential b/c the process uses 2 contrasting stains to separate bacteria on the cell wall comp
Gram-(+) cells have a thick _ layer in their cell wall, gram-(-) cells have a thin layer, surrounded by an addition lipolysaccharide layer.
Peptidoglycan
Basic stains used in gran staining
Crystal violet and sarfain
Decolorizingagent in gram stain
Ethly alcohol
After performing gram stain, gram-(-) cells appear _, while gram-(+) cells appear?
Pink or red; purple
What is the purpose of using Gram’s iodine during the Gram staining procedure?
Gram’s iodine is a killing agent, binds to crystal violet, and serves as a mordant.
Acid-alcohol acts as an
Acid-alcohol—decolorizing agent. Acid-alcohol disrupts non-acid-fast bacteria, allowing for the removal of stain. Acid-fast bacteria are resistant to the actions of acid-alcohol, allowing these cells to retain the primary stain carbolfuchsin.
Acid-fast staining is used to detect members of which bacterial genus?
Mycobacterium. Members of the genus Mycobacterium are considered acid-fast bacilli due to the presence of mycolic acid in their cell wall. The presence of Mycobacterium tuberculosis in acid-fast-stained sputum samples is definitive for
Max resolution of a compound light microscope
0.2micrometers
Simple stain
Methylene blue, carbolfuchsin, crystal violet, safranin
Used to highlight microorganisms to determine cellular shape and arrangements. (Aq) or alcohol solutions of a single basic dye cells (sometimes a mordant is added to intensify stain)
e- microscope
-Shorter wavelength of e-’s (energy source)
-Electromagnetic lenses
-Resolve objects <0.2um- greater resolution
-Used to observe ribosomes
Differential stain
Used to distinguish different types of bacteria
Special stain
Used to color and isolate various structures
Ex. Capsules, endospores, and flagella
Sometimes used as dx aid
Negative stain
Type of special stain
Used to demonstrate presence of capsules. B/c capsules do not accept most stains, they appear as unstained halos and bacterial cells and stand out against a contrasting background
Endospore stain
Used to detect the presence of endospores in bacteria. When malachite green is applied to a heat-fixed smear of bacterial cells, the stain penetrates the endospores and stain them green. When safranin (red) is then applied, it stains the remainder of the cells red or pink
Flagella stain
Special stain
Used to demonstrate presence of flagella. A mordant is used to build up the diameters of flagella until they become visible microscopically when stained w/ carbolfuschin
One advantage of SEM over TEM?
Seeing 3-D detail
Why is mordant used in Gram stain? In the flagella stain?
In gram stain, the mordant combined w/ basic dye to form a complex that will not wash out of gram-(+) cells.
In flagella stain, the mordant accumulates on flagella so they can see w/ a microscope
What is the purpose of a counter stain in the acid-fast stain?
The counterstain stains the colorless non-acid fast cells so that they are easily see through a microscope
What is the purpose of a decolorizer in the gram stain? In the acid-fast stain?
In gram stain, decoloizer removes the color from gram-(-) cells.
In acid-fast stain, the decolorizer removes the color from non-acid-fast cells
A thick waxy layer of what substance in the cell wall constitutes the major portion of the mycobacterial cell wall, separating it from the other microorganisms?
Mycolic acids (lipoidal)
What color do you expect E. coli to be after performing acid-fast stain?
Blue
What color do you expect Mycobacterium smegmatis to be after performing the acid-fast stain?
Red
Gram-(+) cell wall
Thick peptidoglycan
Gram-(-) cell wall
Thin layer surrounded by lipopolysaccharide layer
Which waxy molecule, found in the cell wall of acid-fast bacteria, prevents these cells from being Gram stained?
Mycolic acid
Why is a specimen smaller than 200nm not visible w/ a light microscope?
Anything smaller than 200nm cannot interact w/ visible light
Why do e- microscopes have a higher resolving power than light microscopes?
e-‘s have a smaller wavelength than visible light, leading to higher resolution
Which type microscope would allow the viewer to see ribosomes inside a cell?
TEM
