Biomedical Sciences: Microbial Growth and Sterilization (Lecture 2; Exam 1)

Binary Fission

(Bacterial Growth); Cell increases in size, doubles DNA, and divides.

Exponential Growth

Binary fission of all cells leads to doubling of the population at each division (1-2-4-8-16-32, etc).

• Determines the pattern of culture growth

• Determines time to form colony on plates

What are fomites?

Inanimate objects or surfaces that can carry and spread infectious germs like viruses, bacteria, or fungi.

Steam Sterilization

Indicated for heat-stable instruments.

What confirms if sterilization has taken place?

If there is NO growth with 1 WEEK incubation.

Autoclave Temperature

121C, 15psi, 15mins

Dry Heat/ Dri-clave (oven)

• 160C, 2hrs

• 170C, 1hr

Chemiclave Temperature

• 131C, 20psi, 30mins

Chemical vapor is removed prior to opening door.

Pure Cultures

Population descended from a single cell and therefore contains only one species.

• Emphasized by Robert Koch.

• Bacteria in nature are not pure; mixed with other bacteria.

Plate Counts

Measures viable cells since a viable cell will produce a single.

• Countable colony

• May have to dilute cells to get a good number on plate.

Doubling Time

Time for cells to double in number (generation time) during exponential growth.

• A measurement of bacterial growth rate.

Bacterial Growth Rates

• E. coli ~25mins

• M. tuberculosis ~12-24 hours

• Human fibroblasts ~20hours

Growth Phases

1. Lag

2. Exponential/Log

3. Stationary

4. Death

5. (Phase of prolonged decline)

Mesophiles

Microorganisms that thrive at moderate temperatures (20C-45C) and are medically important.

Thermophiles

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

• hyperthermophiles

Psychrophiles

Microorganisms adapted to colder regions; (-5C to -20C).

• Membranes adapt to contain more unsaturated fatty acids, so the membrane is more fluid at lower temperatures.

Neutrophiles

Species that find pH of ~7 optimal.

Obligate Aerobes

Must grow in oxygen.

Microaerophiles

Grows in reduced levels of oxygen.

Obligate anaerobes

Cannot grow in the presence of oxygen.

Live by fermentation; many are killed by oxygen. (some are aerotolerant)

Facultative anaerobes

Respire in presence of oxygen, ferment in its absence.

• Metabolism dependent on environment.

• Growth is more efficient with oxygen.

Acidophiles

Prefer low pH.

Alkaphiles

Prefer high pH.

Osmotic Pressure

Salt concentration must be suitable for growth.

• ~100mM pretty standard

Halophiles

Bacteria that require high salt concentration.

General features of Bacteriological Media Design

• Carbon source

• Nitrogen source

• Phosphate

• Sulfur

• Minerals

• Organic Compounds

Complex Media

Composed of extracts of biological materials.

• Yeasts, meats, plants

Synthetic or Defined Media

Have known amounts of pure compounds.

Specialized Media Types

• Enrichment culture

• Selective media

• Differential media

Enrichment culture

Allows one type of cell to grow better than others.

Selective Media

Grows some bacteria, but not others.

• Can see some bacteria grow out in presence of others.

Differential Media

Two different bacteria growing on same plate would look different, but growth rate is the same.

Infection Control

• Protection of patients

• Protection of employees

• Prevent cross-contamination (spread of disease)

Sterilization and Disinfection in Optometry

• Critical items

• Semicritical items

• Noncritical items

Critical Items

Contact with sterile tissue or enter the body.

• Sterilization

Semicritical Items

Touch mucous membranes or the ocular surface.

• Need at least high-level disinfection between patients.

• ex. Goldmann tonometer, gonio, diagnostic contact lenses

Noncritical Items

Contact intact skin only.

• Cleaning and low/intermediate-level disinfection

• ex. Trial frames, occluders, chin rests, forehead rests

Important Eye Infections to Prevent

• Adenovirus

• HSV

• Bacterial Conjunctivitis

• MRSA

• Hep C

• HIV

Routes of Tranmission of Microorganisms

• Direct contact

• Needlestick

• Indirect contact - fomites

• Spatter

• Aerosolization -airborne

Standard Precautions

Treat all patients and all body fluids (except sweat) as potentially infectious.

Control of Microbial Growth

• Physical Methods

• Chemical Methods

• Antibiotics

Mechanical Sterilization

Filtration through small pore filters.

• Does not filter out viruses!!!

Chemical Sterilization

• Peroxides

• Aldehydes

• Ethylene oxide

Radiation

Damages DNA without leaving a residue.

How does UV light interfere with replication?

By creating thymine dimers.

What kind of radiation is UV light?

Nonionizing radiation.

What are examples of ionizing radiation?

• Gamma rays

• X-rays

• Cathode rays

Antisepsis

Chemicals applied to body surfaces to destroy or inhibit vegetative pathogens.

Pasteurization

Not a complete sterilization method,

• (Protects from some disease, such as TB, typhoid fever and brucellosis).

Three ways to pasteurize:

• Old method, 62C for 30mins

• Flash pasteurization; 72C for 15 seconds

• Ultrahigh temp pasteurization, 150C for 1-3 seconds

Biological Monitors of Sterilization

• Bacterial spores

• Spore strips

• Glass vials

What are chemical indicators to monitor sterilization?

• Rapid-change indicator

• Slow-change indicator

Do chemical indicators prove that the instruments are sterilized?

NO.

They ensure the machine worked properly but do not ensure sterilization.

Sterilization Cycle Components

• Heat up period

• Exposure interval

• Cool down period

Chemiclave Temperatures

131C, 20psi, 30mins