lab 7 8 9

Question 1

most media is made by dissolving dry ingredients in water, then autoclaving the mixture for 15 minutes to sterilize however

Answer 1

some media ingredients are temperature sensitive and cant be sterilized in an autoclave. these components are normally sterilized separately by membrane filtration and then added to media as sterile stock solutions just before the media is used

Question 2

the autoclave 15

Answer 2

• used to sterilize media and equipment
• acts like a large pressure cooker
• water is heated to boing temperatures and the resulting steam is injected into the chamber containing your materials to be sterilized.
• the chamber is under pressure (about 15 pounds per square inch), this allows the temperature of steam to reach 121 degrees c
• all cells and endospores in a small volume of liquid will be killed within a short period of time (usually 20 min for a litre of media)
• time-temperature relationship is important, especially when using larger volumes or the autoclave is packed full.
• bottles with screw caps must be loose before autoclaving, otherwise pressure can build up and they will explode
• bottles or flasks cant be filled to the top with liquid bc they will boil over
• must be check periodically to ensure that sterilization is occurring

Question 3

filtration

Answer 3

• many components that are added to media are heat-sensitive, and cannot be sterilized using the autoclave
• antibiotics, which are used quite frequently in microbiology, can be denatured by heat. solutions that are aded after autoclaving must be sterilized, otherwise you may contaminate your fresh media
• filtration is sterilizing without heat
• solutions are passes through a membrane with a pore size or 0.22mumeters which will exclude bacteria
• the apparatus used to hold the membrane can be autoclaved and the membranes themselves are packaged individually and sterilized. the filtered solution is collected in an autoclaved container

Question 4

laminar flow hood

Answer 4

-you can work in a relatively sterile environment without worrying that the air is contaminating your media
-a gentle flow of air from the back of the hood pushes out any contamination
-the air is filtered through a HEPA (high efficiency particulate air) filter at the back of the hood, so the air flow is sterile
a HEP filter has 99.97% efficiency at removing particles of 0.3mumeters or larger
-do not remove volatile gases only particulate matter

Question 5

Biosafety cabinets

Answer 5

• have additional feature that protects workers from aerosols that might be blown into their face in a regular flow hood
• glass door that can be pulled down
• the airflow is also directed straight down rather than toward the worker
• work towards the back of the hood. the closer that you get to the opening, the less sterile the environment
• -your materials should be at least 10cm into the cabinet

Question 6

one of the most important ingredients necessary for growth is carbon

Answer 6

• autotrophs will use CO2 from the air as their sole source of carbon, so no additions C source is needed in the medium
• heterotrophs, (most bacteria) require preformed organic carbon to be supplied
• glucose is often used bc it is readily utilized by most heterotrophs
• however many heterotrophs are able to consume a wide variety of other organic carbon sources if glucose is not available

Question 7

growth factors for heterotrophic bacteria

Answer 7

wide range of growth factor requirements

Question 8

if growth factors not supplied

Answer 8

only bacteria that can make the growth factors themselves will be able to grow
-some heterotrophic bacteria can make all of the growth factors from scratch, and are said to have no growth factor requirements

Question 9

some heterotrophs are picky eaters

Answer 9

must obtain virtually all of the growth factors from their surroundings

Question 10

fastidious

Answer 10

even with complex media some bacteria are difficult to grow in the lab
require special media or special incubation conditions
-many human pathogens (can only be cultures on enriched media )

Question 11

just about any substance known whether natural or synthetic, can be metabolized by certain bacteria

Answer 11

this is why bacteria can be used to clean up toxic waste dumps and oil spills in the process known as bioremediation

Question 12

metabolic processes are very useful in

Answer 12

classification and identification of different bacteria

Question 13

extracellular enzymes or exoenzymes examined with

Answer 13

starch and DNase plates and nutrient gelatin tubes

Question 14

fermentation of carbohydrates examined with

Answer 14

purple broth base (PBB) tubes

Question 15

breakdown or urea examined with

Answer 15

urea broth

Question 16

oxygen use was examined using

Answer 16

catalase test

Question 17

starch plates

Answer 17

• allows us to detect production of extracellular amylase enzymes .
• after incubation the plates were flooded with iodine, which reacts with any starch remaining in the plate to give a blue-black color. if the bacteria growing on the plate were producing extracellular amylases, we should see a zone of clearing after iodine treatment, due to the absence of starch ie any starch near the bacteria was broken down by the enzymes and is not available to react with iodine

Question 18

bacillus subtilis on starch plate

Answer 18

grows very well on the starch agar, after flooding with iodine we see that the starch has gone missing from the region directly around the bacteria
b. su has excreted amylase to break the starch into sugars which can then be consumed for carbon and energy

Question 19

micrococcus luteus on starch plate

Answer 19

does not degrade starch, but still grows well using other components of the medium

Question 20

DNase agar

Answer 20

• tells us whether or not our bacteria are producing extracellular nucleases
• the bright blue-green color of the plate comes from DNA in the medium complexed to the dye methyl green
• as extracellular enzymes break down the DNA, the green color fades allowing us to see a zone of clearing around the bacteria that actively excrete nucleases
• the results of the DNase agar can be very difficult to see

Question 21

s. au on DNase plate

Answer 21

surrounded by a faint zone of clearing indicating that DNA in the plate has been degraded

Question 22

s. ep on DNase plate

Answer 22

not surround by a zone of clearing

Question 23

nutrient gelatin tubes

Answer 23

-were solidified with gelatin (a protein) instead of agar
-those bacteria that produce extracellular proteases will degrade the gelatin and liquify the tube
-the trick; put gelatin tubes in refrigerator 15 min before reading the results, this way we could be sure that any liquification observed was due to enzymatic activity and not just because gelatin melts about room temperature
(gram positive liquifies??)

Question 24

purple broth base tubes (PBB)

Answer 24

• fermentation of carbohydrates are observed
• the tube contained a carbohydrate (glucose) as well as the pH indicator bromocresol purple, which turns yellow below pH 6.8. the durham vials were small glass tubes placed upside down inside the test tube to trap any gas that was produced by fermentation
• if it turns yellow it ferments glucose
• if there is a bubble inside the durham vile this tells us that this bacterium uses a fermentative pathway that produces a gaseous end product

Question 25

Urea tube

Answer 25

• determines if our organisms could produce the enzyme urease, which cleaves urea into ammonia and carbon dioxide
• the ammonia can then be used as a source of N for the production of amino acids
• the urea broth contains a pH indicator that turns pink-red at alkaline pH
• as ammonia accumulates in the medium, the pH rises resulting in a bright pink color exhibited by bacteria capable of producing urease
• p. vu capable of producing urease

Question 26

catalase test

Answer 26

-used to determine our organisms ability to use Oxygen
-aerobic organisms produce enzymes to protect them from reactive oxygen species that may be formed during aerobic electron transport
-ex. catalase which converts 2 molecules of H2O2 into molecules of H2O and O2 gas
in the catalase test, we place a single drop of 3% H2O2 onto a pile of bacteria. if the catalase enzyme is present, O2 gas will be produced and will be visible as bubbles.
-so if it bubbles that means it produces catalase and most likely performs aerobic respiration to generate its energy

Question 27

e.coli PBB tube

Answer 27

yellow

gas

Question 28

s.ep urea tube

Answer 28

hot pink

Question 29

l.la

Answer 29

fastidious did not grow on any plate