Microbiology Mini workshop - AAU

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Microbiology Mini workshop

1st lecture: Chapter 3 + 11

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Tools of the Laboratory

Sources of Microorganisms

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The 5 I’s of Culturing Microbes

1. Inoculation – introduction of a sample into a container of media to produce a culture of observable growth

2. Incubation – under conditions that allow growth

3. Isolation –separating one species from another

4. Inspection

5. Identification

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Inoculation - Producing a culture

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Tools of the LaboratoryIncubation

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Isolation – Separating one species from another

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Inspection + Identification

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Isolation – Separating one species from another

If an individual bacterial cell is separated from other cells and has space on a nutrient surface, it will grow into a mound of cells - a colony.

A colony consists of one species.

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Tools of the LaboratoryMethods for Isolation of Bacteria

Isolation techniques include:• streak plate technique• pour plate technique• spread plate technique

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Tools of the LaboratoryMedia -> Providing nutrients

-> Media can be classified according to three properties:

Physical state – liquid, semisolid and solid

Chemical composition – synthetic (chemically defined) and nonsynthetic (complex)

Functional type – general purpose, enriched, selective, differential, anaerobic, transport, assay

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Tools of the LaboratoryMedia -> Physical states

Liquid – broth; does not solidify -> nutrient broth

Semisolid – clot-like consistency; contains solidifying agent (agar or gelatin)

Solid – firm surface for colony formation -> nutrient agar

• contains solidifying agent

• liquefiable and nonliquefiable

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-> Most commonly used solidifying agent is agar

A complex polysaccharide isolated from red algae

• solid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oC

• provides framework to hold moisture and nutrients

• not digestible for most microbes

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Liquid media

semisolid media

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Tools of the LaboratoryMedia -> Chemical composition

Synthetic – contains pure organic and inorganic compounds in an exact chemical formula -> defined media

Complex or nonsynthetic – contains at least one ingredient that is not chemically definable

General purpose media- grows a broad range of microbes, usually nonsynthetic

Enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes

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Enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes

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Selective media- contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes

Differential media –allows growth of several types of microbes and displays visible differences among desired and undesired microbes

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Differential media

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Tools of the LaboratoryIncubation, Inspection, and Identification

1. Incubation – temperature-controlled chamber at appropriate temperature and atmosphere

• microbe multiplies and produces macroscopically observable growth

2. Inspection – observation; macroscopic and microscopic

• pure culture – grows only single known species of microorganisms

• mixed cultures – hold two or more identified species or microorganisms

• contaminated culture – once pure or mixed culture that has unwanted microbes growing

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Tools of the LaboratoryIncubation, Inspection, and Identification

3. Identification – macroscopic and microscopic appearance, biochemical tests, genetic characteristics, immunological testing

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Tools of the LaboratoryMaintenance and disposal of cultures

-> Potentially hazardous cultures and specimens are usually disposed of in two ways:

• steam sterilization

• Incineration (burning)

-> Culture collections :

American Type Culture Collection (ATCC)German Collection of Microorganisms and Cell Cultures (DSMZ)

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Tools of the LaboratoryThe Microscope

Key characteristics of a reliable microscope are:

Magnification – ability to enlarge objects

Resolving power – ability to show detail

Magnification in most microscopes results from interaction between visible light waves and curvature of the lens.

• angle of light passing through convex surface of glass changes –refraction

• Depending on the size and curvature of the lens, the image appears enlarged.

• extent of enlargement - magnification

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Tools of the LaboratoryThe Microscope

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Tools of the LaboratoryResolution

Resolution defines the capacity to distinguish or separate two adjacent objects – resolving power

• function of wavelength of light that forms the image along with characteristics of objectives

Visible light wavelength is 400 nm – 750 nm.

Numerical aperture of lens ranges from 0.1 to 1.25.

Oil immersion lens requires the use of oil to prevent refractive loss of light.

Shorter wavelength and larger numerical aperture will provide better resolution.

Oil immersion objectives resolution is 0.2 μm.

Magnification between 40X and 2000X

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Tools of the LaboratoryTypes of Light Microscopes

Bright-field – most widely used; specimen is darker than surrounding field; live and preserved stained specimens

Dark-field – brightly illuminated specimens surrounded by dark field; live and unstained specimens

Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures

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Tools of the LaboratoryFluorescence Microscope

Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye

Uses dyes that emit visible light when bombarded with shorter UV rays -fluorescence

Useful in diagnosing infections

Confocal

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Tools of the LaboratoryElectron Microscopy

Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds

Electron waves are 100,000 times shorter than the waves of visible light.

Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength.

Magnification between 5,000X and 1,000,000X

Transmission electron microscopes (TEM)

Scanning electron microscopes (SEM)

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Tools of the LaboratoryElectron Microscopy

Transmission electron microscopes (TEM) – transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts.

Scanning electron microscopes (SEM)– provide detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.

TEMSEM

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Tools of the LaboratoryProbing Microscopes

-> Scanning tunneling microscope (STM)-> Atomic force microscope (AFM)

Carbon monoxide man-> STM

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Tools of the LaboratoryStaining

Simple stains – one dye is used; reveals shape, size, and arrangement

Differential stains – use a primary stain and a counterstain to distinguish cell types or parts (examples: gram stain, acid-fast stain and endospore stain)

Special stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains

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Microbial Control

Physical, chemical, and mechanical methods to destroy or reduce undesirable microbes in a given area

Primary targets are microorganisms capable of causing infection or spoilage:

• vegetative bacterial cells and endospores

• fungal hyphae and spores, yeast

• protozoan trophozoites and cysts

• worms

• viruses

• prions

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Microbial Control

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Microbial ControlRelative Resistance of Microbes

Highest resistance

• bacterial endospores, prions

Moderate resistance

• Pseudomonas sp.

• Mycobacterium tuberculosis

• Staphylococcus aureus

• protozoan cysts

Least resistance

• most bacterial vegetative cells

• fungal spores and hyphae, yeast

• enveloped viruses

• protozoan trophozoites

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Microbial ControlTerminology and Methods of Control

Sterilization – a process that destroys all viable microbes, including viruses and endospores; microbicidal

Disinfection – a process to destroy vegetative pathogens, not endospores; inanimate objects

Antiseptic – disinfectants applied directly to exposed body surfaces

Sanitization – any cleansing technique that mechanically removes microbes

Degermination – reduces the number of microbes

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Microbial Control

Microbial death

Permanent loss of reproductive capability, even under optimum growth conditions

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Microbial ControlFactors that affect death rate

The effectiveness of a particular agent is governed by several factors:

• Number of microbes• Nature of microbes in the population• Temperature and pH of environment• Concentration or dosage of agent• Mode of action of the agent• Presence of solvents, organic matter, or inhibitors

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Microbial ControlFactors that affect death rate

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Microbial ControlMethods of Physical Control

1. Heat – moist and dry

2. Cold temperatures

3. Desiccation (dry)

4. Radiation

5. Filtration

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Microbial ControlMethods of Physical Control

Moist heat – lower temperatures and shorter exposure time; coagulation and denaturation of proteins

Dry heat – moderate to high temperatures; dehydration, alters protein structure; incineration

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Microbial ControlHeat Resistance and Thermal Death

Bacterial endospores most resistant – usually require temperatures above boiling

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Microbial Control

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Microbial Control

Steam under pressure – sterilization

Autoclave 15 psi/121oC/10-40min

Steam must reach surface of item being sterilized

Item must not be heat or moisture sensitive

Mode of action – denaturation of proteins, destruction of membranes and DNA

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Microbial Control

Pasteurization – heat is applied to kill potential agents of infection and spoilage without destroying the food flavor or value

63°C - 66°C for 30 minutes (batch method)

71.6°C for 15 seconds (flash method)

Not sterilization - kills non-spore-forming pathogens and lowers overall microbe count; does not kill endospores or many nonpathogenic microbes

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Microbial Control

Dry heat using higher temperatures than moist heat

Incineration – flame or electric heating coil

• ignites and reduces microbes and other substances

Dry ovens – 150-180oC- coagulate proteins

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Microbial Control

• Ionizing radiation – deep penetrating power that has sufficient energy to cause electrons to leave their orbit, breaks DNA,

• gamma rays, X-rays, cathode rays

• used to sterilize medical supplies and food products

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Microbial ControlFiltration

Physical removal of microbes by passing a gas or liquid through filter

Used to sterilize heat sensitive liquids and air in hospital isolation units and industrial clean rooms

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Microbial Control

Microbes on a normal unwashed hand

Microbiology Mini workshop - AAU

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