Gnotobiotics

Question 1

What were initial barriers to development of gnotobiotic mice?

Answer 1

1. necessity of deriving germ free animals via hysterectomy or hysterotomy and hand-rearing
2. expense of complex, custom stainless steel equipment

Question 2

How was the initial barrier of hand-rearing hysterectomy-derived germ free animals overcome?

Answer 2

development of nutritionally adequate autoclavable diets based on role of microbiota in animal nutrition.

Question 3

How was the barrier of expensive stainless steel equipment overcome?

Answer 3

development of the flexible film isolator and the use of peracetic acid for sterilization

Question 4

What are the major considerations of a gnotobiotic facility?

Answer 4

1. budget - equipment, personnel, per diem
2. space - large isolators, many isolators per experiment, lower productivity of breeders
3. autoclave -
4. personnel
5. SOPs
6. work area - separate from isolators
7. microbiology lab support - monitor for containment

Question 5

Describe the primary components of a gnotobiotic isolator

Answer 5

1. Chamber
2. air supply
3. transfer port
4. gloves

Question 6

What are the advantage and disadvantages of each of these types of isolator chambers:

1. flexible film
2. semirigid polypropylene
3. rigid stainless steel

Answer 6

1. A - inexpensive, fully welded seams, accommodates smaller people. D - may puncture, cannot be stacked, requires rigid work surface.
2. A - can use negative pressure, can stack. D - may be prone to failure at gasket seals, requires non-autoclavable cartridge filters.
3. A - negative pressure operation. D - costly

Question 7

What are the major components of a gnotobiotic isolator air supply system?

Answer 7

power supply
blower
inlet filter
damper +/-
outlet filter (biohazards)

Question 8

What is the difference between a cylindrical port and a rectangular port?

Answer 8

cylindrical ports allow the use of transfer sleeves, where rectangular ports require the use of an adapter for a transfer sleeve or that a sterilizable container be used.

Question 9

What is the most vulnerable component of an isolator?

a. chamber
b. air supply
c. transfer port
d. gloves

Answer 9

d. gloves

Question 10

Name commonly used liquid chemical sterilants and their contact times

Answer 10

1. peracetic acid 1%; 30 minutes
2. per acetic acid 0.08% and H2O2 1%; 1 hour
3. Chlorine dioxide; 30 minutes

Question 11

Name commonly used methods of sterilization

Answer 11

1. liquid chemicals
2. gas/vapor chemical
3. steam 270 F
4. dry heat
5. ionizing radiation
6. filtration

Question 12

What is the maximum dose of gamma radiation allowed by US federal regulations for sterilizing feed?

Answer 12

50 kGy

Question 13

what are the most common sources of contamination to a gnotobiotic isolator?

Answer 13

damaged gloves

incomplete sterilization of chow

Question 14

Name some characteristic traits of germ free mice

Answer 14

cecum enlarged and prone to volvulus
poor reproduction - prolonged estrus, decreased implantation rates due to decreased enterohepatic estrogen recirculation
prone to colonization with opportunistic pathogens

Question 15

What is Altered Schaedler Flora?

Answer 15

limited microbiota cocktail consisting of facultative anaerobes, oblige anaerobes 8 in total

Question 16

What are the NCI criteria for a standard defined microbiota?

Answer 16

1. anaerobic organisms only
2. reduce cecal volume
3. no more than 8 bacteria

Question 17

Which enteric bacterium induced colitis when inoculated alone in germ free mice?

Answer 17

Enterococcus fecalis, exacerbated by presence of E. coli