oxygen requirements of bacteria
what are the two main parts of the gaspak apparatus
- Gas Generator Sachet - Anaerobic Indicator Strip
what is a TSA shake
- agar is kept in its liquid state by placing the tubes in a warm waterbath.
what are the three things in the gas generator sachet
-contains sodium borohydride -sodium bicarbonate -palladium which acts as a catalyst
describe the oxygen placement in a TSA deep
-gradient; more oxygen at top, less at bottom
describe oxygen movement in the creation of a TSA deep
-oxygen removed during the autoclaving; diffuses back as agar cools and solidifies
what is a TSA deep
-solid agar
what do you do after the inoculation of bacteria into a TSA shake and why
After inoculation, the tube is gently rolled between the palms to disperse the bacteria, and then it is allowed to solidify.
when is the oxygen gradient in a TSA shake created
As the media solidifies, the oxygen gradient is created.
what are the three microorganisms used in this lab
Clostridium sporogenes Escherichia coli Micrococcus luteus
where is micrococcus luteus found
M. luteus is found in soil, dust, water, air, and on human skin
what is Fluid Thioglycollate Medium and what does it contain
a basic medium that contains sodium thioglycollate, Lcystine, resazurin, agar and gelatin; an oxygen gradient is formed in the media during cooling
purpose of Resazurin
a dye used as an oxygen indicator in the presence of oxygen - the dye becomes pink in the absence of oxygen - the dye is straw-colored (yellowish) (the natural color of the media)
Aerotolerance is
ability or inability to live in the presence of oxygen
upon exposure to air, the sachet becomes ________________
activated -the hydrogen from the sodium borohydride combines with the oxygen inside the jar in the presence of the palladium catalyst to form water -the sodium bicarbonate produces carbon dioxide gas
name the five oxygen classes of bacteria
aerobes facultative anaerobes anaerobes microaerophiles capnophiles
where is e coli found
all over in the environment and in the intestinal tract
how are TSA deeps inoculated
an inoculating needle to obtain the bacteria and stab into the agar deep.
what are anaerobes?
cannot grow in the presence of oxygen • must grow in an oxygen-free environment since oxygen is inhibitory or in some cases, lethal • lack the enzymes to deal with the harmful products of oxygen metabolism
what color is Escherichia coli
cream colored
what does clostridium sporogenes form
endospores
What are facultative anaerobes?
grow in the presence or absence of oxygen but prefer the presence of oxygen - have the ability of utilizing free oxygen in the environment -in the absence of oxygen, however, they can obtain oxygen from an alternative source such as nitrate
how are the TSA plates
incubated aerobically and anaerobically (through the use of a GasPak apparatus)
C. sporogenes- the absence of growth at the top indicates that it is anaerobic E. Coli- growth throughout the entire tube with an emphasis on the top of the tube indicates facultative M. Luteus- absence of growth at the bottom indicates that the bacteria is aerobic
interpret the results
These are AGAR DEEP stabs C. sporogenes- has a crack in the agar meaning that gas was produced. Bacterial growth is in the bottom of the agar which indicates it is anaerobic. E. Coli- is able to grow throughout the entire media meaning that it is facultative M. luteus- is able to grow on the top of the media only meaning that it is aerobic
interpret the results
This is an AGAR SHAKE C. sporogenes- has a crack in the agar meaning that gas was produced. Bacterial growth is in the bottom of the agar which indicates it is anaerobic. E. Coli- is able to grow throughout the entire media meaning that it is facultative. it also has a crack indicating that gas was produced. M. luteus- is able to grow on the top of the media only meaning that it is aerobic
interpret the results
aerobic. Only E coli and the micrococcus luteus are able to grow. C sporogenes is not able to grow because it anaerobic anaerobic. C sporogenes and E coli are able to grow. Micrococcus luteus is not able to grow because it is aerobic. E coli is able to grow because it is facultative
interpret the results
how can clostridium contamination occur
it can occur in food which is improperly canned or for which the can has been damaged during storage causing the lid and/or bottom of the can to become swollen due to the production of gas.
What are capnophiles?
microaerophiles that require elevated levels of carbon dioxide to survive
how are the TSA shakes inoculated
obtained from the waterbath. Students will inoculate the media while it is still in its liquid state using the appropriate bacteria and their inoculating loops. After the media is inoculated, the tube should be gently rolled between the palms of your hands to disperse the bacteria. DO NOT VORTEX!
what is the Anaerobic Indicator Strip
paper strip containing resazurin •The strip is pink in the presence of oxygen •The strip is colorless (white) in the absence of oxygen
what is micrococcus luteus used for
pharmaceutical environmental monitoring, and its presence is a sign of improper decontamination.
what is the purpose of Agar and Gelatin
present in small quantities; helps to localize the organisms as they grow
what are microaerophiles
require free oxygen, but only in limited amounts
what are aerobes
require oxygen because they use oxygen as the final electron acceptor
how are FTM tubes inoculating
use an inoculating loop. Do not shake the tube, otherwise you will introduce oxygen into the media. Work quickly
purpose of Thioglycollate and L-cystine
used as reducing agents to reduce oxygen present in the media to water
what is the purpose of a GasPak apparatus
used to create an anaerobic environment
what color is clostridium sporogenes
white cream colored
how are the TSA plates inoculated
with a straight line streak of each microorganism
what color is micrococcus luteus and why
yellow because of carotenoid pigments
