Biol 2042 Midterm Practical

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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)

safranin

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)

nigrosin

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)

safranin

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

resolution

the ability to distinguish 2 points as separate entities

parfocal

stays in focus when magnification is changed

4 basic requirements for growing microorganisms

1. macronutrients
2. micronutrients
3. growth factors
4. water

macronutrients

C, O, N ,H, make up 96% of cell; required nutrients

micronutrients

trace elements

growth factors

things that microorganisms cannot synthesize (auxotroph and prototroph)

auxotroph

cannot produce growth factors; have requirement

prototroph

produce their own growth factors; do not have requirement

water (4 basic requirements for...)

70-90%

photoautotrophs

require light and CO2

photoheterotrophs

require light and organic compounds

chemoautotrophs

require CO2

chemoheterotrophs

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

agar

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

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

CNA

colistin nalidixic acid; selective for gram positive; inhibitory for gram negative

macconkey

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

TDP

thermal death point; the minimum temperature required to kill bacterial suspension in 10 min

TDT

thermal death time; the minimum length of time required to kill a bacterial suspension at a give temperature

bacillus

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

aeotolerant

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

palladium

biobag indicator

resazurin

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

TFTC

too few to count (less than 30)

TNTC

too numerous to count (more than 300)

some bacteria calulation

number of colonies X 1/10^n or DF

antagonism

one is unharmed but the other is harmed

water agar

no nutrients, just water and agar

bacteriocidal

kills bacteria

bacteriostatic

stops growth

penicillin

inhibits cell wall synthesis

polymysin

inhibits plasma membrane

streptomycin

inhibits protein synthesis

tetracyline

inhibits protein sythesis

ciproflaxin

inhibits nucleic acid synthesis

trimethoprin

antimetabolite

water agar isolates...

streptomyces

geosmine

gives earthy smell in streptomyces

3 domains

eubacteria, archae, and eukarya

psychrophile

cold loving

cyanobacteria

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

TSA

tryptic soy agar

NA

nurient agar

serratia produces red pigment...

called prodigiosin at 25C due to enzyme activity

dangerous wave length

260nm damages DNA

Pseudomonas

gram negative, aerobic, rods

Staphylococcus

gram positive clustered cocci

Streptococcus

gram positive chained cocci

Bacillus

endospore forming gram positive nonfilamentous rods; strict anaerobe

Clostridium

endospore forming gram positive nonfilamentous rods; strict anaerobe

Mycobaterium

weakly gram positive nonsporlating acid fast slender rods; acid fast growth

Streptomyces

gram positive filamentous rods

S. spp

catalase +; mannitol ferm -

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