MicrobesAND CULTURING TECHNIQUES

Image Source: dhmh.Maryland.gov

Microbes

Image Sources: nuigalway.ie; clordisys.com

Microbes• Have been around for at least 3.5 billion years

• Rise of the Cyanobacteria

• Most abundant life form on Earth

• Affect every aspect of life on earth

• Variety of shapes and sizes

• Can exist in a wide range of habitats

Image Source: nuigalway.ie

Microbes• Include:

• All bacteria

• All archaea

• Almost all protozoa

• Some fungi

• Some algae

• Certain animals and plants

• Juvenile stages of macro-animals and macro-plants

• And (depending on who you ask) viruses

Microbe Examples

Image Source: Mined from Google image search

Microbiology•The study of microorganisms (‘microbes’) is called Microbiology• Discovery in 1674

• Antonie van Leeuwenhoek

• Robert Hooke

Image Sources: micro.magnet.fsu.edu; lacienciaexplica.blogspot.com

Importance of Microbes

“I make no apologies for putting microorganisms on a

pedestal above all other living things. For if the last

blue whale choked to death on the last panda, it would

be disastrous but not the end of the world. But if we

accidentally poisoned the last two species of ammonia-

oxidisers, that would be another matter. It could be

happening now and we wouldn’t even know...”

- Tom Curtis

July 2006 in Nature Reviews Microbiology

Microbes & Human Health• Microbes outnumber somatic cells in the human body• Human digestive system

• Skin and internal organs

• A relatively small number of microbes are harmful to human health • Pathogenic

• Examples: Ebola virus, Malaria, HIV, Strep throat, Staph infection, influenza, common cold, measles, chickenpox, tuberculosis, food poisoning, anthrax, etc.

Mycobacterium tuberculosisImage Source: nuigalway.ie; louisville.edu

• A pathogen is a microorganism that has the potential to cause disease.

• An infection is the invasion and multiplication of pathogenic microbes in an individual or population.

• Disease is when the infection causes damage to the individual’s vital functions or systems.

To make us ill microbes have to:• Reach & attach to their target site in

the body• Multiply rapidly• Obtain nutrients from the host• Survive attack by the host’s immune

systemSource: microbiologyonline.org.uk

Microbes & Ecosystems• Microbes are important decomposers

• Bacteria, fungi, and algae all participate in breaking down waste material

• Decomposition is important because:• Releases nutrients

• Cycles important compounds in and between ecosystems

• Some microbes employ photosynthesis• Use radiant energy (sunlight) to convert CO2 into O2

Image Source: nuigalway.ie

Microbes & Food• Food “spoilers”

• Food poisoning

Penicillium mold growing on bread

Image Source: microbiologyonline.org.uk

The bacterium Campylobacter jejuni is a common cause of food poisoning

Microbes & Food

• Eight major pathogens responsible for most foodborne illnesses in U.S.:

Image Source: microbiologyonline.org.uk

Microbes & Food• Microbes are used in the production of many foods:

• Yogurt

• Cheese

• Bread

• Beans

• Chocolate

• Wine

• Beer

Image Source: Mined from several Google image searches

Microbes & Medicine• Vaccines

• Antibiotics

• Probiotics (and prebiotics)

• Development & production of medical drugs

Image Source: Mined from Google image search

Microbes & Industrial Applications• Wastewater treatment plants

• Composting facilities & landfills

• Methane gas as an energy source

• Biofuel

• GMOs

Image Source: 2013.igem.org

Microbes & Natural Waters• Clean Water Act of 1972 (CWA)

• Protection of surface water for drinking water, recreational, and aquatic food source uses

• Safe Drinking Water Act of 1974 (SDWA)• Regulation of contamination of finished drinking water and protection

of source waters

• US EPA Water Quality Standards• Recreational waters

• Drinking water

ContaminantMCLG(mg/L)

Potential Health Effects from Long-Term Exposure Above the MCL

Sources of Contaminant in Drinking Water

Cryptosporidium zeroGastrointestinal illness (such as diarrhea, vomiting, and cramps)

Human and animal fecal waste

Giardia lamblia zeroGastrointestinal illness (such as diarrhea, vomiting, and cramps)

Human and animal fecal waste

Legionella zero Legionnaire's Disease, a type of pneumoniaFound naturally in water; multiplies in heating systems

Total Coliforms (including fecal coliform and E. Coli)

zeroNot a health threat in itself; it is used to indicate whether other potentially harmful bacteria may be present

Coliforms are naturally present in the environment; as well as feces; fecal coliforms and E. coli only come from human and animal fecal waste.

Turbidity n/a

Higher turbidity levels are often associated with higher levels of disease-causing microorganisms. These organisms can cause symptoms such as nausea, cramps, diarrhea, and associated headaches.

Soil runoff

Viruses (enteric) zeroGastrointestinal illness (such as diarrhea, vomiting, and cramps)

Human and animal fecal waste

US EPA Drinking Water Standards

Class Freshwater Marine

Shellfishing 14 FC 14 FC

Based on median value. No more than 10% of FC samples may exceed 43 in the areas most probably exposed to fecal contamination.

Recreational (swimming)

200 FC 200 FC

No more than 20% of FC samples may exceed 400. Violations are expected immediately following periods of rainfall in areas where uncontrollable nonpoint source pollution prevents attainment.

US EPA Recreational Water Standards

North Carolina

Culturing MicrobesLABORATORY TECHNIQUES

Image Source: kemira.com

Culture Media• Contains nutrients needed to sustain microbe growth and reproduction

• Can be liquid (broth) or gelatinous (gelatin or agar)

• Media can vary widely in ingredients

• Common ingredients include:• Glucose or glycerol – carbon sources

• Ammonium salts or nitrates – inorganic nitrogen sources

• Animal extracts (broth) – additional nutrient sources

• Animal proteins or plant carbohydrates – solidifiers

Selective & Differential Media• Selective Media:

• Assists the growth of a desired organism

• Stops the growth of (or kills) non-desired organisms

• Differential Media:• Creates a visible change when growth of target organisms are present

(e.g., color change)

MacConkey Agar

• Most common

• Selective and differential

• Isolates enteric, gram-negativebacteria based on their ability to ferment lactose.

Gram-positive vs.

Gram-negative

• Gram-positive bacteria:• retain crystal violet dye

• thick layer of peptidoglycan

• Lack outer membrane

• Gram-negative bacteria:• do not retain the violet dye

• Possess outer membrane (cell wall)

• more resistant against antibodies because of their impenetrable cell wall Image Source: vetsci.co.uk

Aseptic Technique

•Mindset and procedures that maintain a contamination-free lab space:

• Good hand washing technique

• Mindfulness of sources of contaminants

• Gathering all materials & staying at the lab bench until all culture work is complete

• Avoid coughing, sneezing, or breathing on samples

• Keeping sample covered at all times

• Maintaining a ‘sterile’ environment around the immediate work area

Microbe Control Methods

• Sanitize: Make safe to use or eat from

• Disinfect: Kill pathogens present

• Sterilize: Kill or remove all life present

Ways to Sterilize & Disinfect• STERILIZE

• Burning

• Boiling

• Autoclave (steam)

• Filtration

• Irradiation

• Ozone

• Nitrogen dioxide

• Formaldehyde

• Hydrogen Peroxide

• Alcohols

• Chlorine

• Iodine

• DISINFECT

• Detergents/Soaps

• Acids & Alkalis

• Alcohols

• Chlorine

• Aldehydes

• Iodophors

• Heavy metals (i.e. mercury and silver)

• Cresols/Phenols

• Hydrogen peroxide

• Antibiotics

• Heating

• Pasteurization

Flame Sterilization

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