Experiment 1 and 2

Where should you discard pipette tips?

Orange biohazard bag on benchtop

If you break a test tube containing a bacterial sample, where would the TA throw away the broken glass?

In the red biohazard bin for contaminated sharps/broken glass

What should you do with your test tube at the end of the day?

Wipe off the sharpie using 95% ethanol and place it in the bin by the sinks

What should you do at the start and end of the lab?

Wipe down the lab bench with 70% ethanol and wash your hands

microbiology

study of microscopic organisms

• includes: bacteria, archaea, viruses, fungi, and protozoa

taxonomy

science of classifying microbes into various groups based on their shared characteristics

nomenclature

• system of naming microorganisms

  • Binomial name

    • Genus species

      
      

microbial systematics

• aims to reconstruct the evolutionary history of microorganisms and create a hierarchical classification system that reflects evolutionary relatedness

• 16S ribosomal RNA (rRNA) gene sequences

broth is a liquid culture media while agar is _________ .

a solid

Petri dishes + media

plates (type of culture media)

Test tubes + upright media

deep (type of culture media)

Test tubes + angled media

slant (type of culture media)

complex media

Consists of a variety of ingredients (extracts from animal or plant tissue) to provide a broad range of nutrients and support the growth of a wide range of microbes, including those with complex nutritional requirements

• a medium whose organic carbon, energy, and nitrogen sources are often supplied via various extracts and peptones (not exact composition)

defined media

Consists of precisely known defined components and concentrations to allow for a more controlled and specific environment that only allows certain microbes to grow

• a medium in which the exact chemical composition is known

sterilization

Process of killing or removal of all viable microbes from equipment, media, and other laboratory materials

types of sterilization

• autoclaving

• dry heat (flaming)

• filtration

• cold sterilization

• electromagnetic radiation (UV and ionizing radiation)

microbial ecology

Studies the relationships microorganisms have with their environments, plants and animals, as well as with one another

Role of Microbial ecologists

look at biogeochemical cycles and study the roles that microbes play (ex: carbon and nitrogen cycles)

How do microbes have a mutualistic relationship with plants?

• interact with the roots of plants and can promote growth, provide protection from pathogens, allow for stress tolerance, etc.

• Bacteria receive nutrients from the plant and are capable of fixing nitrogen, etc. for the plant to utilize from the soil

  • Nutrient acquisition

human microbiome

Normal flora on/in the human body

• The urogenital tract, respiratory tract and skin are all examples of places where microbial diversity can be found!

• Over 1,000 different species of microbes in the gut; prevalence and type vary from individual to individual

  
  

Many of the microbes in and on our bodies _________ .

keep us healthy and are mutualistic

spontaneous generation

Theory that matter could arise spontaneously from non-living matter

• Antonie van Leeuwenhoek, Francesco Redi, Lazzaro Spallanzani, and Louis Pasteur contributions helped disprove this theory

Leeuwenhoek

• constructed microscopes which could magnify up to 30 - 100X

• Described microorganisms that he observed in teeth scrapings & rainwater

• one of the first people to observe microbes

• “Animalcules”

Redi

Contributed to disproven this theory by exposing a piece of meat to flies and keeping another piece covered (people thought maggots would spontaneous appear but this is not how this works in actuality)

Spallanzani

• Utilized heat to kill microorganisms within flasks containing broth; found that sterilized broth infusion flasks could only become contaminated by leaving it exposed to air

• Critics of his experiment argued that air was necessary for spontaneous generation

• Led to Pasteur’s experiments

Pasteur

• Utilized heat to kill microorganisms within a swan neck flask

  • The neck would capture particulates entering and prevent contamination of broth

  • If the broth was exposed to those particulates, it would get cloudy indicating contamination

• Developed sterilization procedures still utilized today in the food industry, clinical medicine, and research

• Vaccine development

Robert Koch

• Original experiments were using mice to study anthrax infections in cattle (found that giving infected cattle blood to other animals gave them anthrax)

• Found ways to grow bacteria outside of the host within a lab setting

• Obvious cause and effect for infectious disease

• Developed techniques for culturing microorganisms within a lab

• validated germ theory of disease (microorganisms can cause diseases)

pure cultures

• Consist of only one species of microorganism

• Needed to characterize and identify microbes

We can tell from a plate whether or not we have a ______ .

pure culture

A pure culture will have _________ .

Colonies of a single morphological type are all that are present upon our streak plate

If you have several different morphologies (different sizes, shapes, colors, etc.) then you have a ________.

mixed population (not a pure culture)

The five I’s

1. inoculation

2. incubation

3. isolation

4. inspection

5. identification

inoculation

the transfer of the bacterium or sample to a medium (liquid, solid, semi-solid)

incubation

inoculated media gets moved into a temperature-controlled location to allow growth to occur

isolation

methods that allow for isolation of spaced-out growth and ability to grow a specific bacterium

identification

• Utilizes biochemical tests (you will get into this in future labs)

• Genotypic testing

• Immunologic testing

• Test examples: ELISA, PCR, rapid tests, agglutination, etc.

Ignaz Semmelweis

• identified a correlation between autopsies and incidence of puerperal sepsis (childbirth fever)

• Introduced handwashing in chlorinated lime solutions

• Documented decrease in incidence of puerperal fever upon implementation of handwashing protocol

  • Noted differences when midwives delivered babies as opposed to the doctors which were coming from autopsies without sanitation in between!

  • was ridiculed for this and ironically died by this same infection in an asylum

joseph lister

• Father of Antisepsis

• focused on infections that followed compound fractures; in his time they all resulted in amputations or death

• Cleaned hands and surgical instruments in carbolic acid solution (phenol) before and during surgeries

• Noted a decreased in infections following surgeries and less amputations required

normal microbiota (normal flora)

• Microorganisms that are permanent residents of our body for the duration of our lifetime

• “Bacterial fingerprint”

transient microbiota

• AKA transient flora

• Microorganisms which may be present on the body temporarily

nosocomial

• Hospital-acquired infections

How handwashing works

• soap molecules have two ends: a hydrophilic end and an hydrophobic one

• hydrophobic end dissolved nonpolar molecules like oil and dirt

• emulsification helps remove insoluble matter and rubbing action helps

hand sanitizers are not more effective than soap and water based cleansing because _______ .

it is the mechanical action of hand washing that is antimicrobial ( it aids in the effectiveness of hand washing)

independent variable

part of the experiment that is intentionally changed by the researcher

dependent variable

part of the experiment that will change as a result of the changes made to the independent variable (can measure this)

constants

parts of the experiment that are kept the same throughout all testing

controls

sets a baseline for reading the results of the dependent variable

replicates

number of test subject or groups that are treated the same within an experiment

How to properly label petri dishes

• write on back or agar side of dish

• write on edge

• write initials, lab section, experiment name/number, and name of sample

requirements for microbial growth

• suitable environment with essential nutrients

• pH levels

• moisture

culture media

provide the necessary nutrients and conditions to support growth and proliferation of microorganisms

By cultivating microorganisms in media we can __________ .

study their morphology, physiology, metabolism, and perform tests to understand their characteristics

Defined media are useful when ________ .

studying specific nutritional requirements or metabolic pathways of microorganisms

Media can be considered __________ .

selective, differential, both, or neither

broth

liquid culture media that provide a homogeneous growth environment, allowing microorganisms to grow and multiply in suspension

• commonly used for the growth of a large number of cells or to facilitate production of microbial metabolites

solid medium

solid culture media which contains a solidifying agent (agar)

agar

extracted from red algae; a good solidifying agent

solid media allow for ________ .

isolation and enumeration of individual microbial colonies, making them suitable for colony morphology, observation, pure culture isolation, and antimicrobial susceptibility testing

plates

when media is allowed to solidify in petri dishes

• surface of plates used for growth and isolation of microbes

deep

media is allowed to solidify in a test tube in an fully upright position

• commonly used for anaerobic growth

slant

media is allowed to solidify in test tubes at an angle

• used for maintenance of stock cultures of microbes

effective sterilization

essential to preventing the introduction and proliferation of unwanted contaminants that could interfere with experimental results and comprise the safety of researchers

autoclaving

utilizes high pressure steam in a sealed device called an autoclave to achieve sterilization; widely used and effective in killing both vegetative cells and endospores (highly-heat resistant) of microorganisms

• standard conditions 151 degrees Celsius at 15 psi for 15 mins (solid media takes longer to reach 151 degrees Celsius than liquid media)

dry heat sterilization

direct flaming (putting inoculation loops and needles over Bunsen burner until red hot; one of the simplest methods)

• kills by oxidation effects

filtration

involves passing a liquid or gas through a filter with pore sizes too small for the passage of microbes, but large enough to allow passage of the liquid or gas

• the physical removal of bacteria or other contaminants

Autoclaving is great for sterilizing _________ .

liquid media and other items that are heat resistant

filtration is often used for __________ .

sterilizing heat-sensitive solutions or for removing microbes from the air in laminar flow hoods

cold sterilization

• uses no heat but gas chemosteriliziers which penetrate packaging and destroy microbial cells by damaging their genetic material (example: ethylene oxide)

• used by NASA

Cold sterilization is useful for sterilizing ________ .

heat-sensitive or moisture-sensitive materials (ex: plastic petri dishes, plastic syringes, paper, leather, wood, metal, and rubber products)

electromagnetic radiation

a way of sterilization; includes microwave, ultraviolet radiation, x-rays, gamma rays, ionizing radiation, etc.

ionizing radiation

used in the food industry and for sterilization/decontamination of medical supplies

Ionizing radiation causes ions and other reactive molecules to be produced and these can _________ .

degrade or later DNA and proteins

Ultraviolet radiation cannot penetrate solid, opaque, or light absorbing surfaces and is useful only to __________ .

sterilize exposed surfaces such as bench tops, biosafety cabinets, and laminar flow hoods

UV radiation can _______ .

damage DNA and kill organisms

three forms of solid culture media

agar plates, deep, slant

Robert Koch’s experiments on anthrax became the basis for __________ .

koch’s postulates

Pasteur’s experiments and Koch postulates led to the _________ .

germ theory of disease (microbes can cause disease)

aseptic techniques

transfer of microorganisms from one type of medium to another without contamination or self infection

Medium must contain ___________ for culturing.

macronutrients (carbon, nitrogen, phosphorous, sulfur), an energy source, and any necessary growth factors

Minority of microbes are _________ .

pathogenic

opportunistic pathogens

nonpathogenic bacteria that become infectious in an weakened host

why is condensation on agar surface of plates undesirable?

• source of contamination

• introduce unwanted microbes

• interfere with colonial growth

we all have a __________ .

unique bacterial flora “fingerprint”

• we leave bacterial organisms present on our skin behind

• if someone comes into contact with it and don’t have these microbes they could get sicks