Exam 1 Super review Flashcards
- Talk about why microbes are essential
They're everywher producers decomposers drugs/chemicals recyclers damage understand higher forms of life
i. Provides contrast between the specimen and its background
ii. Depends on lens quality
iii. Improved with certain magnification and staining
Definition
i. The capacity to distinguish or separate two adjacent objects
ii. Depends on the wavelength of light that forms image
1. shorter wavelength, or use of electrons, increases the resolution
iii. Also improved with oil immersion (immersion has the same refractive index as glass)
Resolution
Heat-dried
Capsule stain
Air dried
Negative Stain
Bacteria spread out in ink
Negative and Capsule
Heat Fixed
Positive Stain
bacteria spread out in water, then air dried
Positive Stain
kills the bacteria; makes the bacteria adhere; helps the bacteria absorb stain
Heat Fix
one dye is used; reveals shape, size and arrangement (Methylene Blue in class)
Simple Stain
uses a primary stain and a counterstain to distinguish cell types or parts
Differential Stain
Gram stain
acid fast stain
endospore stain
capsule stain
are examples of ____ stain
Differential Stain
surfaces of microbes are negatively charged and attract basic dyes
Positive staining
these dyes are cationic, with positively charged chromophores
Basic dyes
Reduces the refractive loss of light
Oil Immersion
i. Aseptic Technique to prevent contamination.
ii. To transfer bacteria from broth culture to an agar surface - whenever you’re transferring a liquid
Inoculating Loop
i. Used whenever you are transferring bacteria from solid media.
Inoculating Needle
to dilute bacteria so you can isolate into pure culture where each colony comes from one bacteria that is isolated.
Quadrant Streak Plate
tube where liquid agar was cooled while tube was lying down on angle tocreate a sloped agar surface, so there’s more surface area where bacteria can grow.
Nutrient Agar Slant
provides the surface where your smear will be located.
Glass Slide
for smears prepared with positive stains, used to make a target circle on the bottom of the glass slide.
Wax Pencil
stains the background
negative
stains bacteria itself
positive stain
only one dye used
simple stain
more than one dye used
differential
i. improve definition
ii. negative stains background
iii. positive stains bacteria itself
staining
- nigrosine toward one end; transfer small amount to ink and mix; smear with second slide
air dry for (-) stain
heat dry gently if capsule
little shrinkage and bacterial shape/size can be more reliably interpreted.
benefits of air-dry
Looks for glycocalyx in forms of capsule - a protective covering sometimes used for attachment and nutrient reserve
Capsule Stain
- Place heat dried smear in staining rack resting over sink
- Flood smear with enough crystal violet to cover for 1 minute
- Rinse with distilled water
- Blot slide within pages of your bibulous paper tablet
- Oil immersion.
Capsule Stain
The smear with nigrosine air dried is it
Negative stain
i. One stain used-Typically a single positively charged stain+smear is good (methylene blue is good)
ii. give information about shape/arrangement of bacteria
iii. heat fix smear
iv. chromophores
Simple stain
- Place heat fixed smear on staining rack over your sink.
- Flood with methylene blue for 1 minute
- Rinse slide with distilled water
- Blot with bibulous paper
- View with oil immersion.
Simple stain
- makes bacteria stick to slide, kills bacteria, allows bacteria to absorb stain more easily.
Pros for heat smear
The most important stain
Gram
- Gram negative have thin peptidoglycan surrounded by outer membrane with phospholipids and lipopolysaccharide RED/PINK
- Gram positive has thick layer of peptidoglycan and no outer membrane. PURPLE
Gram Stain
- Apply crystal violet for 1 minute
a. primary stain = crystal violet - Wash off stain with distilled water
- Apply Gram’s iodine for 1 minute
a. iodine = mordant = combiens with primary stain to form insoluble crystalline compound. crystals get trapped in thick peptidoglycan but fail to be so where it is thin. - Wash off iodine with distilled water
- Apply 95% alcohol, drop by drop until alcohol runs clear (no more than 3-4)
a. crystal violet is washed out of cells with thin peptidoglycan; destroys outer membrane of gram negatives. - Wash off the alcohol with distilled water
- Apply safranin for 20 seconds.
a. safranin is the counterstain. - Wash off the stain with distilled water.
- Blot dry with bibulous paper
Gram Stain
i. Negative stain + gentle heat fix + adding crystal violet –> differential stain
ii. uses ink to smear/colorb ackground + stain to color the background itself.
iii. Differentiates the capsule from rest of cell
Capsule stain = negative + simple positive stain
Coat is made of keratin and spore specific proteins makes acid/radiation/chemical/disinfectants/dyes/antibiotics difficult to penetrate
Endospores
Interior has high concentrations of calcium and dipicolinic acid which makes _ heat resistant by displacing water and making dehydrated _ non metabolic
Endospores
- Take one of heat fixed smears and place it on screen over steaming water
- Put small piece of paper towel on top of the smear and add enough malachite green to saturate the paper.
a. malachite green primary stain - Steam for 5 minutes while keeping the paper moist with additional stain as needed.
a. since spores resistant to staining. - To avoid stain get on bottom of slide, hold slide with clothespin over steam rather than let the slide sit there.
- remove slide from screen and let it cool; rinse with distilled water for 30 seconds.
- Place the slide on staining rack and counterstain with sfranin for 20 seconds
a. This is the counterstain that stains the vegetative cells. - Rinse the slide and blot dry.
a. Endospores should be green; vegetative cells should be red/pink.
Endospore Stain
i. Contain negatively charged chromophores; typically stain proteins
ii. waxy mycolic acid of wall of bacteria make it difficult to stain with usual dyes, but steaming acilitates the stain entertaining the cell wall.
iii. Typically include cells of genus Mycobacterium
iv. “Acid fast” means that once the stain has entered, subsequent washing with acid-alcohol won’t remove the stain.
Acid Fast Stain
- Add mycobacterium mix it with water. Also add staphylococcus
- Air dry and heat fix smears.
- Place heat fixed smear on screen over steaming water.
- Apply carbolfuchin to cover smear; steam for 5 minutes.
a. primary stain - used to color acid-fast cells; when absorbed, will give pink/red color to acid fast cell wall. - Remove slide from screen and let it cool; rinse with water for about 30 seconds.
- Rinse drop by drop with acid alcohol until run off clear
- Briefly rinse with water
- Place slide on staining rack and counterstain with methylene blue for 30 seconds.
a. counterstains colors any non acid fast cells - Dry with bibulous paper.
Acid Fast Stain
Branched apart 3.5 bya
prokaryotes
Branched apart ~1.5 bya
eukarya from archaea
- Schwann: all animal tissues composed of cells
- Schleiden: All plant tissues composed of cells
- Virchow: All cells only arise from pre-existing cells
Cell theory
- Pasteur and Koch were leading contributors
- The belief that many diseases are caused by the growth of microbes int he body, not by sins, bad character, or poverty.
germ theory of disease
- THe microrganism or other pathogen must be present in ALL cases of the disease
- Thep athogen can be isolated from the diseased host and grown in pure culture.
- The pathogen from the pure culture must cause the disease when inovulcated into a healthy, susceptible lab animal.
- The pathogen must be reisolated from the new host and shown to be the same as the originally inoculated pathogen.
Koch’s Postulates
The four criteria established by Koch to identify the causative agent of a particular disease
i. discovered first antibiotic, penicillin
ii. isolated in 1939 by Ernest Chain and Howard Florey
iii. Extracted from Penicillum mold
Alexander Fleming 1929
i. Dutch linen merchant
ii. First to observe living microbes
iii. Single=lens magnified up to 300X
iv. Very protective of his work
v. father of microscopes
vi. saw animalcules (algae and protozoa)
Anton van Leeuwenhoek 1632-1723
i. Allowed heated air to enter abroth filled flask through a coiled tube
ii. Broth stayed clear, he concluded that microbes can not spontaneously generate from broth.
iii. Opponents claimed he killed the “vegetative force” in the air by heating it.
iv. Air inlet - flame heated air - previously sterilized infusions remain sterile.
Schultze & Schwann, 1839
i. Refuted spontaneous generation of macroscopic organisms
ii. demonstrated maggots don’t generate from meat.
iii. Meat with gauze had no maggots; meat open had maggots hatching into flies.
Francesco Redi Italian mid 1600’s
i. supported spontaneous generation
ii. assumed boiling kills everything
iii. when boiled mutton brother produced large quantities of bacteria he concluded that they spontaneously generated from the broth
iv. also left the lid open for awhile.
f. John Needham 1748 England
i. Boiled the broth longer, sealed the flask, nobacteria grew.
ii. argued that he destroyed the “vegetative force” of the broth and degraded the small amount of air that was there.
iii. Gravy boiled + lid –> w/o lid? bacteria growth. w/lid? no growth.
g. Lazzaro Spallanzani 1765 Italian
i. Used swan-necked flask to demonstrate the dust is associated with microbes in the air
ii. 1861 paper tried to persuade readers that mcirobes do not spontaneously generate
iii. still had some results that were contrary to the idea.
iv. Developed pasteurization
v. Demonstrated what is now known as the Germ Theory of Disease
h. Louis Pasteur, 1859 Frenchman
i. demonstrated thatif dust removed from the air, bacteria don’t grow
ii. demonstrated the presence of heat resistant forms of some microbes
iii. Developed Tyndallization, intermitten boiling that eliminates what we now know to be the endospores that caused Pasteur to have inconsistent results
iv. Explaiining Pasteur’s results (which were sprouting endospores) end belief in spontaneous generation.
i. John Tyndall England 1859
i. Contributed the most to the development of pure culture techniques
ii. Was the first to offer convincing proof that microbes were associated with disease
iii. Developed Koch’s Postulates, a method foas associating a particular organism with a particular disease. (ahtrax, cholera, tb)
iv. Developed pure culture methods
j. Robert Koch, Germany 1870’s
i. Connected infection with microbes - Savior of the Mothers
ii. Failed to convince doctors to wash their hands.
iii. Pioneer of Antiseptic procedures
k. Philipp Semmelweis (1840’s)
i. Pioneer of Antiseptic Surgery
ii. washed hands and heated equipment with phenol and found that it greatly reduced infection
l. Jospeph Lister
i. Discovered endospores
ii. Resulted in final overthrow of Spontaneous Generation
m. Ferdinand Cohn, 1876
i. early belief that some forms of life could arise from vita forces present in nonliving or decomposing matter (flies from manure)
a. Spontaneous Generation
i. the idea that living things can only arise from other living things
Biogenesis
spherical
Coccus
one
singular
spheres in pairs
diplococci
groups of 4 spheres
tetrads
irregular clusters
staph
chains
strpt
packets
cubical packets
rods
bacillus
rods in pairs
diplobacilli
chains
strept
rods laying side by side
palisade
curved rod
vibrio
flagella on outside
spirillum
flagella on inside
spirochete
which bacteria shapes are always solitary
vibrio, spirillum, spirochete
Sizes of bacteria
1 um to 200 nm -ish
single flagellum at one end
small bunches emerging from the same site
flagella at both ends of the cell
flagella dispersed all over the cell
monotrichous lophotrichous amphitrichous peritrichous Flagellar Arrangements
3 parts
filament
hook
basal body
flagella components