MI02: Microbial Growth and Sterilization

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What are two aspects to bacterial growth?

1. Binary fission (individual cell): cell increases in size, doubles DNA, and divides.

2. Generation time (whole population): time it takes for a population to double in number.

What is exponential growth and what does it determine?

Binary fission of all cells lead to doubling of the population at each division.

Determines: pattern of culture growth and time it takes colony to form.

What is a pure culture? Where are they usually found?

A population that descended from a single cell which contains only one species. These cultures are usually found in a lab because in nature bacteria species are often mixed together (not pure).

Who emphasized the need for pure cultures?

Koch

What are plate counts?

A counting method that measures viable cells since a viable cell will produce a single colony. This can be achieved by diluting cells to get a good number (e.g. serial dilutions).

What environmental conditions affect bacterial growth rates?

What are the growth phases of bacterial growth?

1. Lag phase

2. Exponential/log phase

3. Stationary phase

4. Death phase

Define mesophiles.

Organisms that can live near body temp. (37 C/ 98.6 F)

Define thermophiles.

Bacteria that can grow at temperatures above 100 C (above boiling)

• Hyperthermophiles: thrive at extreme temperatures

Define psychrophiles.

Bacteria that contain more unsaturated fatty acids so the membrane is more fluid at lower temperatures (-5 C to 20 C)

What are obligate aerobes?

Require oxygen to grow (has enzymes to detoxify toxic-side products of oxygen metabolism)

What are microaerophiles?

Bacteria that grow in reduced levels of oxygen (e.g. C. jejuni, H. pylori).

What are obligate anaerobes?

Bacteria that require the absence of oxygen to grow and live via fermentation.

What are facultative anaerobes?

They can live in the presence of oxygen and ferment in its absence. (Growth is more efficient w/ oxygen)

What are acidophiles?

Organisms that prefer low pH

What are alkaliphiles?

Organisms that prefer high pH

What pH do medically important bacteria prefer?

Intermediate pH (~7 pH)

What is osmotic pressure?

Salt concentration must be suitable for growth.

What are halophiles?

Bacteria that require high salt concentrations.

What is complex media?

Media that are composed of extracts of biological materials (e.g. yeasts, meats, plants).

What are synthetic/defined media?

Media that contain known amounts of pure compounds.

What is a selective media?

Allows some bacteria to grow while not allowing others (restricted). (Ex. SS agar)

What is a differential media?

Allows two different kinds of bacteria to grow on the same plate but growth rate is the same for both. (Ex. MacConkey-lactose plates)

What components need to be included in a growth media?

• Sources of carbon, nitrogen, phosphate, sulfur

• Minerals (Mg, Ca, etc.)

• Organic compounds (GFs, vitamins, amino acids, purines/pyrimidines, etc.)

What are standard precautions and who do they protect?

Precautions that state to treat all patients and all body fluids (except sweat) as potentially infectious.

Protects patients, employees, and prevent cross-contamination.

What is the difference between sterilization and disinfection?

Sterilization: removal of all living organisms.

Disinfection: process of removing or killing most microorganisms on or in a material.

What is the clinical significance about the adenovirus in optometry?

It can survive well in eye clinics and has been associated with outbreaks linked to inadequately disinfected ophthalmic equipment.

What are some routes of transmission of microorganisms?

• Direct contact (w/ infectious lesions, saliva, or blood)

• Needlesticks

• Indirect contact w/ formites

• Spatter of body fluids and secretions directly onto the skin or mucosa

• Aerosolization (airborne transfer of microbes)

How can we control microbial growth?

• Physical methods: use heat or other physical methods to kill microbes

• Chemical methods: kill bacteria in place.

• Antibiotics: kill/stop growth of infecting bacteria in patient but allows host to survive.

What are some things that are used to sterilize instruments and media (liq. and solid)?

Dry heat (oven) and steam heat (autoclave)

What is mechanical sterilization?

It’s when filtration occurs through small pore filters to filter out bacteria in liquids (is not solid media). Does not filter out viruses (bc they are smaller than bacteria).

What is chemical sterilization?

The use of peroxides, aldehydes, and ethylene oxide.

How does radiation for sterilization work?

It damages DNA without leaving a residue.

What are two types of disinfection that are used to control bacterial growth?

Bacteriostatic: stops growth

Bacteriocidal: kills

What are resistant to disinfecting chemicals?

Spores (very resistant)

Stationary phase cells (relatively resistant)

What is pasteurization?

It is not a complete sterilization method, where some bacteria are killed and others aren’t. It is also used to protect from some diseases (e.g. tuberculosis, typhoid fever, and brucellosis)

What are three ways to pasteurize?

• Old method: 62 C for 30 mins.

• Flash pasteurization: 72 C for 15 secs

• Ultrahigh temperature pasteurization: 150 C for 1-3 seconds

What are parameters that affect the efficiency of killing by a particular treatment?

• Number of microbes

• Nature of microbes in population

• Temp and pH of environment

• Concentration or dosage of agent

• Mode of action of the agent

• Presence of solvents, organic matter, or inhibitors

What temp, pressure, and time interval does the gravity displacement autoclave (moist heat) require to operate?

121 C, 15 psi, and 15 mins

What temp, pressure, and time interval does dri-clave (dry heat oven) require to operate?

160 C, 2 hrs

170 C, 1 hr

What temp, pressure, and time interval does chemiclave require to operate?

131 C, 20 psi, 30 mins

What temp, pressure, and time interval does prevacuum autoclave require to operate?

3.5-10 minutes, 132 C (psi)

What are the advantages/disadvantages of moist (steam) heat sterilization?

Adv: Short cycle time

Disadv: Can’t use on heat-sensitive materials and dull unprotected sharp edges.

How does steam sterilization work and what is it intended for?

It uses air evaculation via gravity displacement and prevacuum (faster). Intended for heat-stable instruments.

What is the benefit of a prevacuum steam sterilizer?

It is faster and more thorough penetration of steam throughout load.

What are the advantages/disadvantages of dry heat sterilization?

Adv: Does not dull sharp edges

Disadv: Require long cycle time and can’t use on heat sensitive materials

How does a chemiclave sterilize items?

• It uses chemical vapor to be removed prior to opening the door.

• It uses adequate ventilation along with chemicals such as alcohol, formaldehyde, ketone, acetone, and water.

What are some advantages/disadvantages of the chemiclave?

Adv: Short cycle time and doesn’t dull sharp edges and corrode steel.

Disadv: Can’t work on heat sensitive items and release chemical odor.

What are some advantages/disadvantages of rapid dry heat sterilization?

Adv: 190 C 12-15 mins and very useful for practices with rapid instrument turnover.

Disadv: relatively small chamber capacity.

What are the sterilization cycle components?

• Heat-up period: reach temp

• Exposure interval: time required for sterilization of load

• Cool down period: allow cooling for handling and removes excess moisture

Why is sterilization monitoring important? How is it done?

• It’s important to ensure the machine works properly.

• Done using chemical indicators and biological monitoring.

What are the two types of chemical indicators?

• Rapid-change indicator: Confirms exposure to process but doesn’t confirm sterilization.

• Slow-change or integrated indicator: Requires exposure to temp for a period of time. Confims that met sterilizing conditions but not necessarily sterilized.

What are examples of biological monitoring?

• bacterial spores

• spore strips

• glass vials

What does biological monitoring like spore strips confirm?

If there is no growth with 1 week incubation. They confirm sterilization after one day of reading the result.

What type of indicator is autoclave tape?

It is a chemical indicator.

Why do cold temperatures prevent food spoilage?

It is microbiostatic which means it can slow down the growth of microbes. (refrigeration 0-15 C & freezing <0 C)

What are two types of radiation used for disinfection/sterilization?

Ionizing radiation: breaks DNA and use to sterilize medical supplies and food products.

Nonionizing radiation: that use little penetrating power that has to be directly exposed.

How can UV light interfere with replication?

UV light creates thymine dimers. UV light reaches sterilization with long enough exposure time and close proximity of light source. Bacterial spores are more resistant to UV light than growing bacterial cells.

When is filter sterilization preferred?

When there is a need for the physical removal of microbes by passing a gas or liquid through filter. It is used to sterilize heat sensitive liquids and disinfect air in hospital isolation units and industrial clean rooms.

What organisms can be filtered out of liquids?

Bacteria only

What organisms can be filtered out of air?

Bacteria and viruses (via HEPA filters)