There are always things mentioned in lab manuals which may not be expressly defined or discussed there. Careful note-taking during lab lectures (as always!) and/or consultation of outside resources can help to understand them more fully. This page is a review of some of these terms and concepts, each linked from the following list:
- Basic chemistry terms. You should already be familiar with the basic terminology, having had chemistry as a prerequisite. Many times we find that the terms solution and suspension become misused! Remember that bacterial cells (which are insoluble particles) in liquid are a suspension, not a solution.
- Definition of colony-forming unit (CFU) – and its differentiation from the terms cell and colony. (In Experiment 9, we make use of the completely analogous terms plaque-forming unit (PFU), phage particle and plaque, respectively.) These terms should be defined adequately enough in the introduction to Experiment 1, but unfortunately the terms colony and colony-forming unit are too-often used interchangeably. A colony-forming unit is simply one cell or a group of two or more cells which – when plated on a medium whose nutrients the cell(s) can utilize (under the incubation conditions given) – give rise to a colony. A colony is defined in bacteriology as a visible (to the naked eye) mass of cells which have arisen from a common source (the colony-forming unit). Thus the term colony-forming unit is not a strictly quantitative term; one colony arises from one colony-forming unit whether or not anything is being counted. See our "CFU Page" for more.
- Definition of cell envelope. We define the cell envelope as the cell wall and the underlying cell membrane. The capsule (a coherent, extracellular slime layer produced by some species) would not be considered a layer of the cell envelope.
The cell wall of Mycobacterium we have generally considered to be a somewhat-modified gram-positive type with a coating of mycolic acids, but modern thinking tends to separate this arrangement as a distinct type of cell wall containing a discrete mycolic acid layer; this is discussed here.
- Definition of extracellular enzyme. This is an enzyme produced by a bacterial cell which is transported out of the cell to catalyze a reaction occuring outside of the cell. An example of such an enzyme is amylase which catalyzes the extracellular breakdown of starch. There is more about amylase here. Another example is coagulase, an enzyme produced by Staphylococcus aureus which coagulates plasma around the cell, should the cell find itself in the blood stream (or a tube of rabbit plasma in the coagulase test).
- Growth curves and their formulas. We expect you to be familiar already with the concept and use of logarithms and why an exponential axis on a graph should be important and relevant here. See our growth curve page.
- Respiration. When we just casually say "respiration," we generally mean aerobic respiration – where oxygen is reduced to water – and we tend to treat anaerobic respiration as something exceptional. In Experiment 5.1, we can tell by the growth patterns in the Thioglycollate Medium whether an organism can respire (aerobically) or ferment, and we deal with anaerobic respiration later when we do the test for nitrate reduction in Experiment 7. See our page on catabolism.
- Classifying bacteria according to oxygen relationships and/or mode of energy generation. A classification system that is more inclusive than one based on our test for oxygen relationships is one which groups organisms according to mode of energy generation. Note the table below. These concepts are further explained here. Reasons why an organism can grow under anaerobic conditions (if it were capable of doing so with the appropriate medium and/or incubation conditions provided) are discussed here.
|Chemotrophs||Respirers||Derive energy by oxidative phosphorylation.|
Most respirers use oxygen; this is aerobic respiration. Some respirers may also use nitrate or some other "oxygen substitute" in the process of anaerobic respiration.
Certain organisms can only perform anaerobic respiration – for example, the methane producers and many sulfate reducers.
|Fermenters||Derive energy by substrate-level phosphorylation.|
|Phototrophs||Derive energy by photophosphorylation.|
Phototrophs may be oxygenic (oxygen-evolving) or anoxygenic (not oxygen-evolving).
- Definitions of genotype and phenotype. According to the manual on page 21: "Genotype is defined as the entire array of genes possessed by a cell, i.e., the sum of the genetic constitution of the organism, a 'blueprint' in code. The characteristics of an organism which are based on the genotype but expressed within a given environment make up the phenotype of the organism. Thus, the genotype represents the potential of the organism, and the phenotype describes what the organism actually is and does." Sometimes one hears these terms applied only to mutants, but one should stick with the general definitions as given herein.
Genotype is what is in the genes – for example, whether or not an organism has the ability to make functional flagella and be motile. Genotype can be changed by mutation and also by recombination. Phenotype is what one actually observes – such as what one sees regarding motility in a wet mount or motility medium. Whether or not an organism is seen to be motile not only depends on what is in the genes (genotype) but also environmental conditions, as some motile bacteria cease being motile at high temperatures or (for especially strict aerobes) when oxygen is low and they can't respire.
- Definitions of antibiotic and other kinds of antimicrobial agents. The following discussion is taken from the new 4th edition of the manual. (Previous editions tended to "mix apples and oranges.")
The control of microbial growth is necessary in many practical situations, and some of the most important advances in medicine, agriculture and food science have been made by applications of microbiological knowledge. Control can be effected either by killing organisms or by inhibiting their growth. The killing of organisms can be brought about by use of heat, radiation and chemicals. Methods for inhibiting growth may involve drying (recall from Appendix A that all organisms require water for growth), low temperatures and chemicals. An antimicrobial agent is a chemical which kills or inhibits the growth of microorganisms. Such a substance may be either a synthetic chemical or a natural product.
Agents which kill organisms are called "cidal" agents, with a prefix indicating the kind of organisms killed. Thus, we have bactericidal and fungicidal agents. Chemicals which do not kill but only inhibit growth are called "static" agents, and they may be bacteriostatic and/or fungistatic. The distinction between cidal and static is somewhat arbitrary, since an agent that is cidal at high concentrations may be only static at low concentrations. To be effective, a static agent must be present continuously with the product, and if it is removed or its activity neutralized, the organisms present in the product will initiate growth.
Following is a list of the types of antimicrobial agents:
- Antiseptics are microbiocidal agents harmless enough to be applied to the skin and mucous membranes, but they should not be taken internally. Examples include mercurials, silver nitrate, iodine solution, alcohols and detergents.
- Disinfectants are agents that kill microorganisms, but not necessarily their spores. They are not safe for application to living tissues. They are used on inanimate objects such as tables, floors, utensils, etc. Examples: chlorine, hypochlorites, chlorine compounds, lye, copper sulfate and quaternary ammonium compounds.
- Preservatives are static agents used to inhibit the growth of microorganisms, most often in foods. If eaten, they should be nontoxic. Examples: calcium propionate, sodium benzoate, formaldehyde, nitrate, and sulfur dioxide.
- Antibiotics are defined as chemical agents produced by microorganisms which are able to kill or inhibit certain other (i.e., "sensitive") microorganisms. Among the molds, the notable antibiotic producers are Penicillium and Cephalosporium which are the main source of the beta-lactam antibiotics (penicillin and its relatives). In the Bacteria, the Actinomycetes, particularly many species of Streptomyces, produce a variety of types of antibiotics including the aminoglycosides (e.g., streptomycin), macrolides (e.g. erythromycin), and the tetracyclines. Certain species of the endospore-forming genus Bacillus produce polypeptide antibiotics such as polymyxin and bacitracin.
In Experiment 10.2, you will follow the general procedure for isolating potential antibiotic-producing bacteria from soil, subsequently testing the pure isolates for inhibitory effect on selected bacterial species ("test organisms"). New antibiotics may still be discovered by this procedure, although much emphasis currently is on chemical modification of existing antibiotics and genetic manipulation of microorganisms in order to increase their yield.
An organism that is sensitive (susceptible) to a particular antibiotic is affected by the agent at a "target site" which can be a specific structure or essential biochemical reaction in the cell. As an example, you learned in Experiment 8.1 that Staphylococcus epidermidis is sensitive to streptomycin, an antibiotic that inhibits cells at the ribosomes, stopping translation (protein synthesis). Mutant cells may arise in the population which have "resistant ribosomes," and such a cell is then resistant to streptomycin. It should be mentioned that many species of bacteria are naturally resistant to various antibiotics and would never be affected by them. Other target sites affected by antibiotics include cell membrane synthesis (polymyxin) and cell wall synthesis (penicillin).
Gram-positive bacteria are usually more sensitive to antibiotics than are gram-negative bacteria, although, conversely, some antibiotics act only on gram-negative cells. An antibiotic which acts upon a wide variety of gram-positive and negative bacteria is called a broad-spectrum antibiotic. In general, such an antibiotic will find wider medical usage than one which is narrow-spectrum, although the latter may be quite valuable for the control of specific disease-causing organisms. The physician needs and utilizes a wide variety of antibiotics and must select carefully the one that is needed for a particular patient with a particular kind of infection. Unfortunately, the inappropriate use of some antibiotics has allowed for the selective enrichment of antibiotic-resistant mutant bacteria in the human population! An antibiotic must always be tested on the infectious agent in such a way that it can be found to be truly inhibitory at the target site of that organism. Any indication that resistant mutants will arise will necessitate the consideration of another antibiotic! In Experiment 10.1, we will perform the commonly-used disc sensitivity test to evaluate the usefulness of selected antibiotics on several bacterial species.
- Chemotherapeutic agents are antimicrobial agents of synthetic origin useful in the treatment of microbial or viral disease and used like antibiotics. Examples include the sulfonilamides, isoniazid, AZT, nalidixic acid and chloramphenicol. Sulfonamides such as sulfadiazine can substitute for a reactant in an essential biochemical process, and thus growth of the organism can be halted after several generations. The process of DNA replication is the target site of nalidixic acid.
- The concept of antibiotic resistance and how misuse of antibiotics can lead to the spread of antibiotic-resistant strains of infectious bacteria. For any given antibiotic, some species of bacteria are naturally resistant to it (including those bacteria that produce that antibiotic!), and other species are sensitive to it – being hit at a certain target site by that antibiotic. For a given population of cells of a species which would normally be considered sensitive to a certain antibiotic, there can be some cells which mutate such that they happen to become resistant to that antibiotic, passing along this characteristic to their descendants as the cells continue to reproduce. This is the basis of Experiment 8.1 where we select for colonies of such mutants (each potentially being the source of an "antibiotic-resistant strain") and also consider how a mutation can cause a cell to become antibiotic-resistant. Remember that these are "spontaneous mutants" – i.e., the mutation is a random happening and does not occur because of any effect of the streptomycin.
Consider the human body as a "medium" for bacteria – including pathogens. If a person has an infection caused by a certain species of pathogenic bacteria, that person would be given an antibiotic which is supposed to wipe out the population of that pathogen. If some of the cells of that pathogen happen to be resistant to the antibiotic (as we may determine in Experiment 10.1), that person can become a "selective enrichment medium" where the antibiotic-resistant cells have the potential to "take over"!
- Definitions of mutation frequency and recombination frequency. By frequency, we mean the rate at which something occurs. In Experiment 8 we determine frequency of mutation or recombination in a given population of cells as simply the "number who did" divided by the "total number who could" – expressing the result as a fraction, decimal or percentage. Click here for more about the "mechanics" involved in Experiment 8.
In Experiment 8.1, we inoculated each of our three plates (Nutrient Agar, NA+low streptomycin, NA+high streptomycin) with approximately 1X109 CFUs of Staphylococcus epidermidis, an organism which is normally sensitive to streptomycin – i.e., streptomycin hits the cell at the ribosome and stops protein synthesis. (See Materials for Period 1 for the concentration of CFUs per ml of the original S. epidermidis suspension.) On the plates containing streptomycin, we expected no colony formation; however, we saw colonies appear of those cells in the suspension which were streptomycin-resistant mutants. So, to determine mutation frequency, here's an example: If we count 100 colonies on a plate of NA+streptomycin, that means that 100 of the 1X109 CFUs that we originally inoculated onto the plate were resistant mutants, and the mutation frequency would then be determined by dividing 100 by 1X109.
In Experiment 8.2, we made dilution plates of a mixture of Hfr and F– cells to determine the number of recombinant cells (i.e., those which underwent recombination) per ml of the mixture. Only F– cells can undergo recombination. As the F– cells constituted half of the population of the mixture, and as the total cell concentration was 1X108/ml, the number of F– cells would therefore be half that number or 5X107/ml. So, to determine recombination frequency, we would first determine the number of recombinant cells/ml of the mixture; this comes from our dilution plating results. Then we would divide that number by the total number of F– cells/ml of the mixture which is 5X107. Be sure to see the practice problems mentioned in Experiment 8.2; solutions are here. (In Exp. 8.2, we treated "cells" and "CFU's" as equivalents, just for convenience.)
- Taxonomic terms such as family, genus and species. We expect you to have had a full dose of the taxonomy concept in a previous biology course. Keep in mind that species of plants and animals are relatively easy to identify when compared to the situation we have with bacteria. Many hundreds of species of bacteria can look alike microscopically, so we often have to use many physiological tests in the identification process. Taxonomic groups of bacteria (such as genus and species) are becoming defined genotypically as discussed on our pages concerning bacterial identification.
- Definition of strain – and its differentiation from related terms such as isolate and species. A complete discussion of the subject is found here.
Essentially, "strain" is not a taxonomic term such as "species" but is simply a designation applied to a particular isolated culture – whether or not the organisms which constitute the culture are "typical" for the species to which they belong. Indeed, many strains can be studied and become well-known before anything is determined about their genus or species identity. From my early studies in the Dept. of Bacteriology way back in the 1960's, Dr. William B. Sarles' definition comes to mind: A "strain" is simply a pure culture of which some properties are known.
One may recall how this term was used in the news back in Fall, 2001 concerning anthrax infections acquired from handling letters received in the mail. In such an outbreak, whether or not such infections are caused by organisms of the same strain – in which case they could be traceable back to a common source – can be determined with considerable accuracy by genetic testing of the organisms isolated from the infected individuals. Determining the source and dispersal of any organism that causes an outbreak or epidemic is the essence of epidemiology. Attempting to find what was behind the 2001 "outbreak" of anthrax amounted to an unprecedented challenge.
- The concept of coliforms and the procedure to enrich for, detect, isolate and identify them. This discussion has been expanded (with clarification) and is now found on its own page here.
- Definition of aliquot. Many times this term is carelessly misused to refer in the general sense to dispensed or replicated materials (inocula, cultures, etc.) of a volume unrelated in any way to the total volume. If something is divided into identically-sized portions, an aliquot is defined as being one of those portions. Various dictionaries show this word as a noun, adjective or verb – or as more than one of these parts of speech. Funk & Wagnall's Standard Desk Dictionary (1984) gives the word as an adjective: "Dividing evenly into another number: 4 is an aliquot part of 12." Note the appropriate use of aliquot in the discussion of the MPN method here and also in the manual (page 91).
- More to come!