9, 11, 12 Lab Exercise

Corresponds with Ch.10 (susceptibility testing), Ch.5 (heat), Ch.20 (antibiotics)

Sterilization

Total, irreversible destruction of living cells

heat

[__] is an excellent sterilizing agent, when applied at high enough temperatures for an adequate amount of time

» coagulates cellular proteins

» oxidizes cell components

moist heat

[__] is much better at sterilizing than dry heat, as in the dry state, protein structures are much more stable.

» why dry heat must be raised much higher and maintained longer

thermal death point

Lowest temperature required to sterilize a standardized pure culture of bacteria within a given time

thermal death time

Time required to sterilize the culture at a stated temperature

Autoclave

[__] uses Moist heat. In a closed chamber, utilizes the trapped steam to be further pressurized to generate more energy (pressure and temperature are proportional). Uses sealed ampules of endospores to test functionality

» does the same in a dry oven, except only uses dry gas

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Dry ovens are dry heat/no steam→ not able to penetrate (nor condense) as much → touches less surfaces and thereby takes longer time and hotter temperatures to sterilize as [__]

→ uses paper-strip endospore tests to determine functionality

Incineration

Sterilization by dry heat

eg. using a bunsen burner and sterilizing inoculating loop by [__]

-cidal

Suffix meaning “lethal.” Describes disinfectants that must kill pathogens while it is in contact with them such that they cannot grow again once it is removed

» bactericidal → kills bacteria

» germicidal → kills germs

-static

Suffix meaning “arrest.” Describes disinfectants inhibits the organisms/prevents growth while it is still in contact with them such that they can grow again once removed

» bacteriostatic → inhibits bacteria growth

» fungistatic → inhibits fungal growth

factors

[__] that influence the effectiveness of a disinfectant are:

» type of microbe present in contaminated material

» exposure time

» concentration required to kill microorganism

» temperature and pH for optimal activity

» concentration of microorganisms present

» toxicity of agent (effect on materials to be disinfected and on skin)

resistant

Some microbes are more susceptible to disinfectants (Gram-positive and vegetative cells) because they do not have the properties of more [__] microbes, such as:

• Clostridium and Bacillus species → endospore-forming (dormant) bacterium

• Mycobacterium → waxy cell walls (mycolic acid)

• Mycoplasma → bacteria that lack cell wall (antibiotics based on degrading cell walls can’t harm them)

• Gram-negative bacteria → outer membrane

Antimicrobial agent

[__] are chemicals that are naturally produced by organisms (typically bacteria and fungi) or synthesized in the lab that have inhibitory or lethal effects on pathogenic organisms

» used for therapeutic treatment

minimum inhibitory concentration

Lowest concentration of an antimicrobial drug needed to prevent growth of a specific organism

disk diffusion assay

Filter paper disk agar diffusion method or Baur-Kirby method.

Tells whether or not antimicrobial works; qualitative result (Y/N); doesn’t tell you at what concentration it is effective (minimum inhibitory concentration)

Process:

» have a lawn of bacteria, with a number of small sterile filter papers impregnated with antimicrobial agents scattered on top

» paper will generate zone of inhibition (area that clears) according to the antibiotic’s compatibility with microbes in lawn

» Measure zone of inhibition to determine susceptibility

→ R = resistant (no inhibition/zone)

→ I = intermediate susceptibility (medically means that organisms tested may be inhibited provided higher doses are given, or that the infection is at a site where the drug is heavily concentrated)

→ S = susceptible (zone of inhibition falls within acceptable range deeming microbe susceptible)

Broth dilution test

Test done to determine quantitative result → gives MIC

» prepare 9 tubes filled with nutrient broth (0.5 mL)

» antimicrobial broth is serially diluted (by 0.5 mL) in 7 tubes

→ 0.5 mL + 0.5 mL = 1 mL total. by serial dilution, transferring 0.5/1mL total, it is decreasing the ampicillin conc. by ½ per dilution.

→ 1st tube has 64 microliters of ampicillin; gets divided by 2⁶ at 7th tube

» set two controls aside

→ 8th tube = growth control: shows normal growth of bacteria without antimicrobial

→ 9th tube = negative control: shows how nutrient broth is supposed to look like prior to inoculation… sterile!

» using a pipette, prepare bacteria to inoculate tubes with → 0.1 mL of bacteria to a tube containing 9.9mL of saline

» fill tubes 1-8 with the bacterial solution (again, making sure 9th tube is sterile)

» incubate, and note where the turbidity grows→ conc. prior to where the turbidity grows is the MIC