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Gnotobiotics Flashcards

1
Q

Define gnotobiotics.

A

Rearing of animals that are free of all microorganisms, or associated only with known species.

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2
Q

Define axenic or germfree.

A

Free of all foreign organisms

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3
Q

Define define microflora.

A

AKA Associated animal. Maintained in isolated, intentionally associated with one or more microorganisms.

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4
Q

Define monoxenic/dixenic.

A

Associated with one or two species of microorganisms.

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5
Q

Define restricted flora/microbiota.

A

Associated with a defined microbiota. Removed from an isolator and maintained in a barrier room. Continues to be free of both primary and opportunistic pathogens (highest level of SPF).

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6
Q

Define ex-germfree.

A

Born in germfree conditions but removed. Has unknown microbiota.

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7
Q

Define specific pathogen free.

A

Free from a specified list of pathogens, but otherwise has undefined microflora.

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8
Q

Define conventional.

A

Unknown microbiota and disease status.

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9
Q

Define microbiota and microbiome.

A

Microbiota - Community of microorganisms harbored by a host.
Microbiome - Collective genome of a host’s microbiota.

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10
Q

Define pathobiont.

A

Organism associated with the host that has the potential to cause dysregulated inflammation and disease under certain environmental conditions.

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11
Q

Define symbiosis. What are three types?

A

Close biological relationship between organisms.
Commensalism - One benefits, one is unaffected.
Mutualism - Both benefit
Parasitism - On benefits, one is harmed

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12
Q

Are larger or smaller isolators preferred?

A

Large number of smaller isolators. Allows separate isolators for each population with less risk of loss due to contamination. Use two breeding isolators for each stock.

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13
Q

What is the single most important item of equipment for a gnotobiotic facility?

A

Autoclave

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14
Q

When are rigid isolators typically used? Why?

A

Biohazardous work. Are amenable to negative pressure for containment, but costly.

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15
Q

Describe semirigid isolators.

A

Panels of natural polypropylene welded to a clear flexible film front panel with gloves attached. Can be operated at negative pressure and stacked to save space. May have greater risk of contamination due to gasket seals.

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16
Q

Describe flexible isolators.

A

Optically clear PVC film joined by thermo-welded seams. Least expensive, easier to reach all areas. Tough, easily repaired. Requires support for floor of chamber, isolator, port, blowers, and filters.

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17
Q

Why is emergency power critical for isolator air supply?

A

Prevents suffocation if power is lost.

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18
Q

Most isolators have how many air changes per hour?

A

30 air changes/hour

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19
Q

Why is plastic preferred for transfer ports?

A

Liquid sterilants are corrosive to stainless steel.

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20
Q

Which styles of isolators use cylindrical and rectangular ports?

A

Cylindrical - Flexible or semi-rigid.
Rectangular - Has clear plastic doors that seal and have latches that apply clamping pressure when closed. Requires an adaptor. Semirigid.

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21
Q

What is the most vulnerable part of the isolator? What is the most common cause of contamination?

A

Gloves!

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22
Q

How often should gloves be changed?

A

At least annually

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23
Q

Describe peroxyacetic acid use as a sterilant.

A

Effective in vapor and liquid. Optimal activity at 80% humidity in the vapor phase with 30 min contact time. Degradation products nontoxic and non-carcinogenic. Does not penetrate parasite cysts and arthropod eggs. Use immediately, with half-strength in 24 hours. Human health hazard.

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24
Q

Describe peracetic acid with hydrogen peroxide.

A

Spor-Klenz. Contact time of 1 hour.

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25
Q

Describe chlorine dioxide sterilants.

A

Most commonly used in gnotobiotics. Effective against all micro-organisms in vapor and liquid phase. 30 minute contact time. Clidox-S.

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26
Q

What gas and vapor chemical sterilants exist?

A

Ethylene oxide - Isolator chambers and bedding. Usually reserved for non-autoclavable items. Carcinogenic and hazardous.
Chlorine dioxide gas
Vaporized hydrogen peroxide
Formaldehyde gas generators

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27
Q

What is the chief method for sterilization of supplies and materials?

A

Steam.

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28
Q

What temperature, pressure, and quality of steam is used for sterilization?

A

Temp: 121C (250F) and 132C (270F)
Pressure: Necessary to maintain temp
Quality: Dry, saturated steam. <3% entrained water.

29
Q

What are the advantages and disadvantages of steam sterilization?

A

A: Effective
D: Sufficient time and conditions for entire load. Trapped air inhibits sterilization. Time depends on method of replacing air with steam, nature of materials, and how materials are packed.

30
Q

What type of autoclave is not suitable for gnotobiotic work? What do these systems require to work effectively?

A

Gravity displacement autoclave. Valves cannot be obstructed and chamber must not be overfilled.

31
Q

What is the Bowie-Dick test?

A

Evaluates air removal from test of challenge packs.

32
Q

What organisms are used as biological indicators in sterilization? Where are they placed?

A

Geobacillus stearothermophilus or Bacillus atrophaeus
Place in most challenging location - Center/densest part or coolest area near steam discharge.

33
Q

What is the browning process that can occur with steam sterilization of diets?

A

Altered structure of amino acids, especially methionine, cysteine, and lysine. Can diminish protein bioavailability.

34
Q

What vitamins are sensitive to degradation due to steam autoclaving?

A

Sensitive: B1 (Thiamin), B5 (pantothenic acid), B6, B12
Moderate: Vitamins A, D3, E, folate

35
Q

How can steam sterilization affect diet structure?

A

Can make pellets harder. Offset by extruded pellets.
Can cause clumping. Offset by coatings.

36
Q

What is the most cost effective way of sterilizing rodent feed for larger operations?

A

Steam autoclave.

37
Q

What form of ionizing radiation is used to sterilize rodent feed? At what dose?

A

Gamma irradiation. Dose of 30-59 kGy, US is limited to 50 kGy. Typical rodent diets are 20-25 kGy.

38
Q

What are the advantages and disadvantages of gamma irradiation of feed?

A

A: Economic, convenient, more reliable, and available commercially.
D: Need minimum dose of farthest package. Bacterial resistance possible. Vitamin deficiencies at higher doses. Free radical-induced oxidation of fats producing peroxidases. More expensive.

39
Q

For what products is dry heat sterilization used? What are the disadvantages?

A

Oils, greases, and powders, which are not suitable for an autoclave.
Microorganisms more resistant to dry heat, so can require 160C for up to 4 hours.

40
Q

What system can eliminate the need to autoclave water?

A

Hydropac system. Water is sterilized by filters and 5-10 ppm free chlorine.

41
Q

What level of filtration should HEPA filters meet?

A

Remove 99.7% of particles 0.3 um or larger in diameter.

42
Q

How thick should the gloves be in a gnotobiotic isolator?

A

> 15 mm

43
Q

What is the advantage of a 2-piece glove and sleeve set-up for an isolator?

A

Can replace the glove while the isolator is in operation.

44
Q

How is an isolator tested for leaks prior to setup?

A

Use of a gas detector and duster gas.

45
Q

What device is used to apply the liquid sterilant?

A

Air atomizer

46
Q

Why is extruded food easier to autoclave?

A

More porous with fewer viable bacteria.

47
Q

What biologic indicator is used to monitor water sterility?

A

G. stearothermophilus

48
Q

In addition to gloves, what is another common source of contamination?

A

Incomplete sterilization of supplies, especially food.

49
Q

What are the most common contaminating organisms?

A

Aerobic bacteria that grow well on standard media.
If spore-forming - Inadequately autoclaved food/bedding
Fungi - Food or growth on filter media
Non-spore forming - Gloves, inadequate sterilant coverage or contact time

50
Q

What would cause repeat contamination of gnotobiotic isolators?

A

Autoclave malfunction, poor steam supply

51
Q

Do germfree mice produce ammonia?

A

No

52
Q

What is unique regarding the cecum of germfree mice?

A

Very large cecum. Majority of those found dead probably will have cecal torsion.

53
Q

What does MacConkey agar select for?

A

Gram negative organisms

54
Q

What does Sabouraud’s agar select for?

A

Fungal organisms

55
Q

The risk of viral contamination in gnotobiotic animals is low except:

A

Newly derived mice from stock of unknown health status or one known to harbor virus that is transmitted vertically.

56
Q

How often are gnotobiotic isolators tested for breaks?

A

Monthly is typical

57
Q

What is the risk of a new contamination each week?

A

2%

58
Q

What are the primary methods by which mice are rederived? Describe each.

A

Hysterectomy. Can be done by any facility, but need germfree foster mice. Risk of transplacental microorganisms.
Embryo transfer. No risk of transplacental tx. More difficult under sterile conditions, commercial services expensive.

59
Q

What is a unique need of newly derived mice?

A

Intensive monitoring for contamination.

60
Q

What is the difference between a hysterotomy and hysterectomy?

A

Hysterotomy - Abdomen of donor female in direct contact with plastic membrane. Pups delivered into isolator.
Hysterectomy - Uterus removed, passed into isolator after immersion in dip.

61
Q

How can estrus in mice be synchronized?

A

Progesterone and cloprostenol. Delay parturition with progesterone.

62
Q

What percent of transferred embryos should be expected to survive to weaning?

A

50%

63
Q

Why are gnotobiotic mice poor reproducers? (2)

A
  1. Enlarged cecum reduces abdominal space for gravid uterus
  2. Enterohepatic estrogen recirculation
64
Q

Describe enterohepatic estrogen recirculation.

A

Glucuronidated estradiol is recycled after secretion into intestine, depending on intestinal bacterial glucuronidases. Altered estrogen peaks impacts secretion of LH and FSH. Contributes to prolonged diestrus, reduced frequency of estrus and copulation, and decreased implantation rates.

65
Q

What is a concern when gnotobiotic mice are removed from the isolator?

A

Colonization with opportunistic bacteria.

66
Q

What are most flora in the GI tract?

A

Facultative and obligate anaerobes.
Lactobacillus, enterococcus, enterobacter, bacteroides, and clostridium. Fusiform or tapered rods that could not be cultured.

67
Q

Describe Schlaedler flora.

A

Developed to inoculate germfree mice in the mid-1960s. Reduce cecal volume to eliminate cecal torsion and restore repro performance.

68
Q

How are mice inoculated with ASF or defined flora?

A

From cultured organisms or via fresh feces in drinking water.

69
Q

What agents are in ASF? (8)

A
  1. Lactobacillus murinus
  2. Lactobacillus intestinalis
  3. Parabacteroides goldsteinii
  4. Clostridium
  5. Mucispirillum schaedleri
  6. Pseudoflavonifractor sp.
  7. Clostridium
  8. Eubacterium plexicaudatum

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