107 terms

Biol 2042 Midterm Practical

simple stain
used to observe the morphology or size, shape, and arrangement
simple stain (primary stain)
methylene blue
simple stain procedure
1. smear prep, air dry, heat fix
simple stain organisms used
E. coli, B. meg, S. spp, P. fluorescens
gram stain
cell wall properties; differentiates between gram negative and gram positive (violet=gram positive, pink=gram negative)
gram stain (stains used)
primary stain, mordant, decolorizing agent, counter stain
gram stain (primary stain)
crystal violet
gram stain (mordant)
grams iodine
gram stain (decolorizing agent)
95% ethyl alcohol
gram stain (counter stain)
gram stain (procedure)
1. smear prep, air dry, heat fix
2. stain with crystal violet (2 min)
3. add grams iodine or mordant (1 min), rinse with water
4. use ethyl alcohol for 5-10 seconds and rinse
5. add safranin (1 min) rinse and blot dry
gram stain (organisms used)
E. coli, B. meg, S. spp, P. fluorescens
negative staining
used to see bacterial cell morphology, size, capsules. bacteria appear bright in a dark background
negative staining (stains used)
negative staining (procedure)
1. put inoculum on a slide
2. add one drop of nigrosin and mix it with a loop
3. use another slide to make a smear
4. dry and observe, no rinsing
**no heat applied
negative staining (organisms used)
B. meg, Paenibacillus velai
endospore staining schaeffer fulton
endospores will appear green and the vegetative cells red/pink
**heat is applied so that it can help the malachite green to penetrate the thick endospore
endospore staining schaeffer fulton (stains used)
primary stain, second stain
endospore staining schaeffer fulton (primary stain)
malachite green
endospore staining schaeffer fulton (second stain)
endospore staining schaeffer fulton (procedure)
1. prep smear, air dry, heat fix
2. put the slide on a beaker containing boiling water
3. add malachite green cont. for 5 min by putting a piece of paper towel on the slide
4. remove the paper towel and add safranin for 1 min
5. dry and observe
endospore staining schaeffer fulton (organisms used)
B. meg (3 days old and days old)
acid fast, ZN or kinyoun stain
mycobacteria will appear pink in a blue background
**can not perform gram stain as mycobacteria has mycolic acid which is waxy (lipid substance) the phenol in carbol fuchsin is lipid soluble
acid fast, ZN or kinyoun stain (stains used)
primary stain, decolorizing agent, secondary stain
acid fast, ZN or kinyoun stain (primary stain)
carbol fuchsin
acid fast, ZN or kinyoun stain (decolorizing agent)
acid alcohol
acid fast, ZN or kinyoun stain (secondary stain)
methylene blue
acid fast, ZN or kinyoun stain (procedure)
1. smear prep (with saliva)
2. add carbol fuchsin for 5 min (no heat)
3. rinse with acid alcohol
4. add methylene blue
acid fast, ZN or kinyoun stain (organisms used)
Mycobacterium smegmatis
the ability to distinguish 2 points as separate entities
stays in focus when magnification is changed
4 basic requirements for growing microorganisms
1. macronutrients
2. micronutrients
3. growth factors
4. water
C, O, N ,H, make up 96% of cell; required nutrients
trace elements
growth factors
things that microorganisms cannot synthesize (auxotroph and prototroph)
cannot produce growth factors; have requirement
produce their own growth factors; do not have requirement
water (4 basic requirements for...)
require light and CO2
require light and organic compounds
require CO2
require organic compounds
classification of culture media
based on chemical composition, agar concentration, and purpose
based on chemical composition
synthetic or defined media, complex or rich media (chemically undefined)
synthetic or defined media
all of the chemical composition of the media is known; ex. burks media
complex or rich media
chemically undefined media; ex. beef extract
solidifying agent; kind of algae; polysaccharide; 45C solid; organims cannot break it down
agar concentration
solid media (1-2%)
semisolid media (0.4-0.6%)
liquid media (0%)
purpose of media
general media, selective media, differential media
general media
nutrient rich to allow a large range of microorganisms to grow on it
selective media
selective for a specific organism (gram positive or gram negative); ex. MSA
mannitol salt agar; supresses growth of all bacteria except Staph; 7.5% mannitol salt; phenol red to yellow; ph drops; color changes; S. spp makes yellow colonies and ferments mannitol; S. epi grows but does not ferment mannitol so does not make media yellow
differential media
helps to differentiate between organisms; ex. color changing
colistin nalidixic acid; selective for gram positive; inhibitory for gram negative
selective for gram negative and differential for lactose fermentors; inhibitor is bile salts; indicator is neutral red (ph drop around colony causes color change); pink or white colonies with or without a zone of precipitation if lactose fermenters; if lactose nonfermenters they are clear colonies
alpha hemolysis
green pigment
beta hemolysis
clearing on plate
gamma hemolysis
no hemolysis
blood agar
selective for hemolysis
colony morphology
size, shape, pigments, opacity
cell morphology
size, shape, arrangement
thermal death point; the minimum temperature required to kill bacterial suspension in 10 min
thermal death time; the minimum length of time required to kill a bacterial suspension at a give temperature
have endospores so they survive at higher temps
strict aerobes
require oxygen; ex. pseudomonas
strict anaerobes
cannot survive in oxygen; ex. clostridium
facultative anaerobes
can grow in oxygen or without oxygen; ex. E. coli
can survive in oxygen but growth is no better with or without oxygen; ex. rhizobium
fluid thioglycolate media
pink on top; indicator is resazurin; indicates presence of oxygen
biobag 3 components
gas pack, catalyst, indicator
biobag gas pack
potassium barohydride, sodium bicarbonate, HCl
biobag catalyst
biobag indicator
direct methods of counting bacteria
microscope count, filtration, MPN, spread plate
indirect methods of counting bacteria
turbidity, metabolic activity
spread plate
main advantage is you can see viable cells to make spread plate; we use a technique called serial dilution
too few to count (less than 30)
too numerous to count (more than 300)
some bacteria calulation
number of colonies X 1/10^n or DF
one is unharmed but the other is harmed
water agar
no nutrients, just water and agar
kills bacteria
stops growth
inhibits cell wall synthesis
inhibits plasma membrane
inhibits protein synthesis
inhibits protein sythesis
inhibits nucleic acid synthesis
water agar isolates...
gives earthy smell in streptomyces
3 domains
eubacteria, archae, and eukarya
cold loving
erroneously called blue green algae; found in aquatic lakes and oceans; some fix nitrogen in heterocysts (important fertilizer providers); oxygenic photosynthetic (in thylakoids) bacteria; unicellular; colonial; filamentous;
types of cyanobacteria
anabaena, oscillatoria, spirulina, gleocapsa
tryptic soy agar
nurient agar
serratia produces red pigment...
called prodigiosin at 25C due to enzyme activity
dangerous wave length
260nm damages DNA
gram negative, aerobic, rods
gram positive clustered cocci
gram positive chained cocci
endospore forming gram positive nonfilamentous rods; strict anaerobe
endospore forming gram positive nonfilamentous rods; strict anaerobe
weakly gram positive nonsporlating acid fast slender rods; acid fast growth
gram positive filamentous rods
S. spp
catalase +; mannitol ferm -