Micro Lab 5 Pour Plate and Streak Plate

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Beck

pure culture

a culture contains a single species of organism only.

colonies

separate and distinct masses
each colony arises from the growth and reproduction of a single bacterium
all the cells are genetically identical

basic principle for streak and pour plate technique

mixture of bacteria gradually thinned out in growth medium
so that individual organisms have enough room to form separate distinct colonies

streak plate technique

a rapid moving method for separating bacteria in a mixed population
standard practice in many research, industrial and clinical labs

pour plate technique

mixed population of bacteria is diluted into tubes of nutrient agar
medium is then poured into petri dish for incubation
colonies are formed on surface and in the medium
must be performed quickly because the liquid agar will solidify quickly in the tubes

Pour Plate Steps

label bottom
melt agar in boiling bath
after melting, cool to 45C
note that agar will solidify at 42C
Wipe tube to remove any superficial moisture
inoculated the melted agar deep in broth culture
mix contents by twirling tube in palms of hands
lift lid on petri dish just enough to pour contents
rotate dish to distribute agar
agar cooled in about 15 minutes
incubate the petri dish, in an inverted position in canister
next week make gram stain

Streak Plate technique

follow procedure to pour plate without inoculating bacteria
allow agar to cool and solidify in petri dish
remove loopful of mixed broth culture using aseptic technique
rotate plate on desk covering quarter of dish
rotate dish, bring loop close to 1st streak once or twice
continue streaking without touching first inoculated area
repeat steps 2 more times
incubate dish in inverted position
next week 2 different colonies and gram stain

Serratia marsescens

gram negative rod
opportunistic pathogen: urinary tract, VAP
but bugs in air over San Francisco
antibiotic pathogen
produces red orange pigment at 22C
grows in a large mucoid or sloppy pile
found on communion wafers
grows in dark, moist places
soil loving bug
storing at 22C so won't overgrow
will grow faster than other species

Micrococcus luteus

gram positive organism
tight, little yellow colony
halophile - loves salt
lives on human skin
causes BO
gram positive cocci
stains purple
forms tetrad - 4's
forms sarcines - 8's
resonsible for nosocomial infections

5 I's of bacteriology

Inoculation, isolation, incubation, inspection, identification

Pour Plate

visible number of colonies
mixed with agar
used for water sampling
industrial beer and wine

streak plate

each quad gets thinner
4th quad is individual bug
4th quad best isolation
thin it out so 1 single bacterium could give rise to a pure colony
only on surface of agar

What temperature does agar solidify at?

42C

Growth Curve phases

Lag, Log, Stationary and death

Lag Phase

bug getting used to neighborhood: air and food

Logarithmic Phase

steep climb
where we want to do all our staining
fat
happy
metabolizing

Death Phase

Die out

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