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Tools of the Laboratory:. The Methods for Studying Microorganisms Chapter 3. The 5 I’s of culturing microbes. I noculation – introduction of a sample into a container of media I ncubation – under conditions that allow growth I solation –separating one species from another I nspection
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Tools of the Laboratory: The Methods for Studying Microorganisms Chapter 3
The 5 I’s of culturing microbes • Inoculation – introduction of a sample into a container of media • Incubation – under conditions that allow growth • Isolation –separating one species from another • Inspection • Identification
Inoculation & Incubation • Prior to inoculation the specimen must be obtained. How and where it is obtained is extremely important to the process. It is is obtained improperly or from the wrong site, the experiment or analysis can fail. For instance, if you did a throat swab for someone with a diarrheal infection, you would probably not isolate the causative agent. Unfortunately, a rectal swab or a stool sample would be dictated in this case. Likewise, if the specimen is not kept alive, the experiment will fail. For this reason, specimens are often taken and immediately placed in rich media or on ice. It is also necessary to protect them from outside contamination prior to inoculation so they are placed in sterile tubes/containers immediately after they are collected.
The media that is used for inoculation is also important because many bacteria have very explicit growth factor requirements and will not grow in anything that does not have this growth factor. In some cases we know these and use special media or special hosts. For instance, viruses are often inoculated into embryonated eggs. When we don’t know the growth requirements of an organism (or don’t know the organism) we use growth media that is very rich, containing many different growth factors. In some cases this won’t even work because the concentration of salt of some other substance in the rich medium, inhibits growth. The fact is that there are more than 4,000 individual bacterial species in a gram of soil (rich soil from the Midwest). We can only grow in culture about 1% of these because the rest have media/growth requirements that we cannot duplicate. They are termed “unculturable organisms”.
The characteristics of incubation are also very important. For instance, bacteria have optimum growth temperatures. Bacteria isolated from humans tend to grow best at 37°C, whereas bacteria isolated from the soil tend to grow better at 25 °C. Why do you think this is? In fact, some soil organisms die when incubated at 37 °C. Oxygen requirements are also important during incubation. Some bacteria can only grow in air (presence of oxygen). They are termed “strict aerobes”. Other bacteria can only grow in the absence of oxygen. They are termed “strict anaerobes”. Other bacteria can grow in either the presence or absence of oxygen. They are “facultative anaerobes” (sometimes called facultative aerobes). So before you incubate, you must know something about the organism. The collection site can tell you a lot.
at what temperature would you incubate an organism if you collected it from a deep-sea thermal vent? • what aerobic conditions would you use if you isolated an organism from someone’s face? • how about if you isolated an organisms from the mud found at the bottom of the salt river?
Isolation Why? In order to study a species you must have it isolated (in pure culture) before you can attribute specific traits. If you had the Mormon tabernacle choir sing to you, could you tell who were the best singers? Not likely.
Isolation • If an individual bacterial cell is separated from other cells & has space on a nutrient surface, it will grow into a mound of cells- a colony • A colony consists of one species
The same type of isolation can be done using the pour plate dilution method, or by simply making dilutions of a sample and plating them out (spread plate technique).
Media – providing nutrients in the laboratory • Most commonly used: • nutrient broth – liquid medium containing beef extract & peptone • nutrient agar – solid media containing beef extract, peptone & agar • agar is a complex polysaccharide isolated from red algae • solid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oC (like Jello) • provides framework to hold moisture & nutrients • not digestible for most microbes
Types of media • liquid – tubes, flasks, fermentors. Nutrient broth (peptone and beef extract) • semisolid – fluid thioglycolate. Has a low percentage of solidifying agent in it so it is viscous, but not solid. Usually tubes. • solid – converted to liquid. Nutrient agar. Usually plates, slants, or stabs. • solid – cannot be liquefied. potato slices, rice grains, etc.
Types of media • synthetic – contains pure organic & inorganic compounds in an exact chemical formula • 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
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
Selective media It is important to note that selective media is usually not totally selective, but also takes into account the normal flora of the collection site.
Differential media TSI slant – no growth, growth but no acid, acid only in bottom, acid througout, acid and H2S production Chromagar
Miscellaneous media • reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria • carbohydrate fermentation medium- contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi
When analyzing characteristics of bacteria, it is usually very important that you have a pure culture. Otherwise, you will not be able to attribute your results to a single species. Fortunately, bacterial colonies can be quite diverse in their appearance and the establishment of a pure culture can be determined by macroscopic observation. To insure that a pure culture is obtained, new plates are made by inoculating from a single colony.
magnification – ability to enlarge objects • resolving power – ability to show detail
The basis for the resolution of the electron microscope: D = /2 x N.A. The numerical aperture of a light microscope and an electron microscope are virtually the same. However, electron waves are 100,000X shorter than the waves of visible light. So what does that do to the equation? If resolution of light microscope is 0.2 micrometers, then resolution of electron microscope is about 0.000002 micrometers, or 0.002 nm or 2 picometers. (I personally think that this is pushing it a bit…. globular proteins are about 5 nm in width (average) so you can actually see molecules)
Types of light microscopes • Bright-field – most widely used, specimen is darker than surrounding field • Dark-field – brightly illuminated specimens surrounded by dark field • Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures
Fluorescence 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. • Useful in diagnosing infections
Electron 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 100,000X 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
2 types of electron microscopes • Transmission electron microscopes (TEM) – transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts • Scanning electron microscopes (SEM)– provides detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.
Specimen preparation • wet mounts & hanging drop mounts – allow examination of characteristics of live cells: motility, shape, & arrangement • fixed mounts are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.
Staining • cationic dyes - basic, with positive charges on the chromophore • anionic dyes - acidic, with negative charges on the chromophore • surfaces of microbes are negatively charged and attract basic dyes – positive staining. • negative staining – microbe repels dye & it stains the background
Staining • simple stains –one dye is used • 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: capsule and flagellar stains
