Lab Exam 1 Flashcards
1
Q
CFU/PFU
A
- colony forming unit - the beginning cell/cells that started the colony. Often unknown how many cells started the colony
- plaque forming unit - unknown how many phages began the plaque
2
Q
Oil Immersion
A
- oil has the same refractive index as glass. Unrefracted light rays
- only used for 100X lens
- increases resolution/resolving power
3
Q
Aseptic technique
A
- flame tube neck
- flame sterilize innoculating loop/needle
- minimize contamination of sample
- maintain pure culture (needed for Koch’s postulate)
4
Q
ubiquitous
A
-bacteria are everywhere
5
Q
Koch’s Postulate
A
germ theory
- association - microbe in diseased animal but not healthy animal
- isolation - microbe isolated in pure culture, requires aseptic technique. One disease causing agent in culture
- causation - animal innoculated with isolated microbe to and causes same disease
- reisolation - isolate same microbe from diseased animal
6
Q
Colony Morphology
A
- configuration
- margins
- elevation
- texture (moist mucoid dry)
- color (shiny, dull, opaque, translucent)
may be effected by media, temperature, age
-size
7
Q
Aseptic technique method (quadrant streak)
A
- label the agar side initials, date, procedure
- light bunsen burner
- sterilize innoculating loop
- swirl tube
- take lid off and flame neck
- dip loop into tube
- streak plate several times
- sterilize loop each time and streak 3 more times
8
Q
Parts of Microscope
A
- ocular
- nose piece
- objective lens
- stage
- condenser
- diaphragm
- coarse and fine knobs
- light control
9
Q
resolution
A
Resolution - clarity
Resolving power - number used to determine smallest distance between two distinguishable objects
Resolving power = 0.61 (wavelength) / numerical aperture
-normally nm unit. Lower wavelength is better for human eyes
10
Q
parcentric/parfocal
A
parcentric - keeps centered through objectives
parfocal - remains relatively focused through objectives
11
Q
primary/secondary containment
A
- primary - protect people in the lab
- secondary - protect the environment
12
Q
BSL 1-4
A
Bio Safety Level
- dependent on pathogenicity
1. not known to cause diseaes in healthy people
2. easily contained. can cause disease to healthy people
3. can cause severe disease, inhalable
4. highly virulent, extreme risk, inhalable
13
Q
Microscope light path
A
light source
condenser
diaphragm
specimen
objective lens
ocular lens
eyes
14
Q
Types of stains
A
- differential - use to determine the difference in structures, shape, size, arrangement (gram stain)
- negative - stain background and improve contrast (capsule stain)
- pro to negative staining is no heat fixed slide. No heat shrinkage of cells
- simple stains - cells stained and contrast to light background
- structural stain - highlights a specific structure
15
Q
Colonies
A
-result of a single cell/CFU dividing numerous times in order to produce millions of cells
16
Q
Pure culture
A
- required for Kochs 2nd postulate
- only one disease causing agent on the culture
-
17
Q
Cell arrangements
A
basilli - rods
cocci - spheres
vibrio - curved rods
spirilla - spirals
diplo - 2
strepto - line chain
staphylococci - irregular cluster of spheres
sarcina - organized cluster of spheres (90 deg angles)
palisades - parallel rods
pleomorphic - many shapes
18
Q
Gram Stain Procedure
A
- flood crystal violet for 30 sec
- wash lots of water
- iodine for 1 min
- EtOH till colorless
- rinse water immediately
- flood safranin
- gentle wash water
- blot dry
19
Q
Gram Stain theory
A
- crystal violet is the primary stain
- iodine is the mordant - fixes dye into place by forming complex with the CV in the thick PGN of Gram pos
- Decolorized with EtOH - dissolves lipids on outer membrane (LPS) of gram neg. CV-iodine escapes
- Safrinin as counterstain to stain the colorless Gram neg cells
20
Q
Capsule Stain Theory
A
- negative charge of the stain repels the negative charge of the cell
- do not heat spear the bacteria because they will shrink and cause a false positive
- cell is violet and capsule is a clear halo
21
Q
Capsule Advantages
A
- adhere to surfaces, lead to biofilms
- avoid detection by phagocytes - hided thick PGN, TA, LTA
- Avoid phagocytosis
- resistant to dehydration
22
Q
Acid Fast Stain Procedure
A
- heat fix slide
- saturate with carbolfuschin
- over boiling water
- rinse 8-12 sec acid alcohol
- water to decolorize
- methylene blue 30 sec
- rinse and blot
23
Q
Acid stain theory
A
- differential stain - stained based on structure
- most acid fast bacteia are mycobacterium and there are few species
- leprosy and TB (mycobacterium)
- mycolic acid in the cell walls makes them hard to penetrate. Heat must be used to allow the carbol fuschin (pink/red) to enter the cell
- counterstain is blue (nonacid fast)
- Ziehl Neelsen Method
- hard for antibiotics to enter
- use acid alcohol in order to decolorize the strong stain of carbol fuschin in non acid fast cells
- strong stain and heat required in order to get CF into acid fast cells
24
Q
Endospore Stain Procedure
A
- heat fix slide
- saturate with malachite green
- steam 5 min
- rinse with water
- counterstain with safranin
- rinse and blot
25
Endospore Theory
- much more likely in gram pos
- structural stain
- chlosteridium botulinum/difficile, bacillis anthrecis
- malachite green is water soluble (no alcohol decolorizing)
- thick endospore wall keeps in stain
- endospores are green and regular cells are pink
- sporangium contain a developing endospore. Will be pink surrounding green center
- diplicolonic acid protects DNA
26
Motility procedure
- use aseptic technique
- stab agar tube with needle
27
Motility Theory
* agar is 0.4% instead of 2%
* softer agar allows for mobile bacteria to move. Normal agar would restrict them
* TTC - triphenyltetrazolium chloride - turns red when oxidized by bacteria. Whole tube is red then bacteria is motile.
* TTC can be a motility inhibitor. Could cause a false negactive. Conclusion could not be made
28
Kirby Bauer Method
* cover plate with bacteria to make a confluent lawn
* 5 antibiotic discs per plate
29
Kirby Bauer Theory
* goals - test sensitivity and resistance
* cannot determine MIC or selective toxicity (cannot determine effect on commensals)
* advantage - quick, easy, test multiple antibiotics
* measure zone of inhibition to determine effectiveness
* MIC - minimal inhibitory concentration. limit the side effects of the antibiotics
* Sensitive, Intermediate, Resistant
* factors for size of zone - media, conc of bacteria, molecular weight of antibiotic
* bacteria motility is NOT a factor
30
Phage Dilution method
- 1mL of stock phage in 9mL of media. (10 fold)
- 1mL of ten fold into second tube of 9mL media (100 fold)
- 1mL of 100 fold into third tube of 9mL media (1000 fold)
- 1mL of dilution, 1 mL E. Coli B into warm agar. Pour onto plate
31
Phage Dilution Theory
- Used to determine phage conc in the original tube
- host range determines what bacteria can be infected by the phage. Can be used to limit possible identity of unknown
- PFU - plaque forming unit
- tail fibers determine host range
- phage uses DNase to kill host DNA. Prevents launch of defense mechanisms. Reduce competition for DNA mechanisms
- use of the equation is called phage titering
conc = # of plaques x dilution factor / vol of phage added
unit is PFU/mL
30-300 rule - use a plate with between 30 and 300 plaques for the equation
32
Antibiotic resistance
- random genetic mutations that are advantageous survive
- horizontal gene transfer - spread resistance genes
--conjugation (sex pilus)
--transformation (pick up genetic info from outside)
--bacteriophage - transduction
Causes - misuse, over use, prophylactic use in livestock
--kill selective bacteria and select mutations that are resistant
Compare DNA from resistant to sensitive DNA to determine source of resistance
33
About Phages
- complex symmetry
- tail fibers determine host range
- use lytic phage replication - inject DNA, proteins destroy host DNA, synthesize head and other structural parts, use lysin to escape the cell
- lysogenic phage replication - inject DNA, phage DNA into host DNA, all DNA replicated, binary fission, both cells have phage DNA in their chromosome, proteins destroy host DNA, make phage parts, lyse cell to escape
34
Palisades
almost parallel bacilli
