You must have a pure culture in order to get results that you can use. The Gram stain will provide another check of the purity of the culture. All identifications begin with the Gram stain. The eubacterial world is divided into four parts: Gram positive or negative, cocci or bacilli. In reality, there are a number of families that don't Gram stain well, including mycobacteria, myxobacteria and actinomycetes. You should try to stick to those that do Gram stain well. If you are unable to get a consistent Gram result, you should probably choose a different organism.
Unless we have taken trouble to culture anaerobically, we will not have any strict anaerobes. That is, if your organism will grow on a regular TSA plate in the incubator or in a drawer, you are unlikely to have any of these organisms.
When you do tests for metabolic characters, or staining techniques, you really must take the time to run controls. Although we have pictures in the lab manuals of what positive and negative reactions look like, it is not the same as having your own controls. Use our known cultures. We won't have controls for everything, but we do for most tests.
Gram positive rods
Among the Gram positive rods are some spore formers and the mycobacteria. The mycobacteria are distinguished by the acid fast stain; you can find that procedure in any lab manual and it is relatively easy to perform. The genus Bacillus is composed of sporulating rods. The spore test is also easy to perform and is described in the lab procedures at this site. There are a few other sporulating Gram positive rods, but most of them are in this genus. Some form capsules, which can be detected by negative staining, for instance, with India ink. Corynebacteria are also gram positive rods, but they are neither acid-fast nor do they form spores. They have very short rods which can sometimes be mistaken for cocci. They often have inclusion bodies that appear "metachromatic" under simple stains, like methylene blue. Distinguishing among these organisms can usually be done with the help of SIM agar and various carbohydrate fermentation broths.
Gram positive cocci
Gram positive cocci include many free-living organisms, but also commensals and parasites of humans and other mammals. Although few are strict anaerobes, many are facultative or have only fermentative metabolism (the Streptococci, especially). Usually catalase and oxidase activities are determined. Growth at relatively high salt concentrations (5-10%) can help distinguish organisms, as well. Usually mannitol-salt plates are used for this because you can simultaneously test for mannitol fermentation among salt-tolerant microbes. If you happen to have Staph. aureus, mannitol-salt will usually make that clear. There are a number of other tests that are helpful depending on the results of these early tests. Hemolysis on blood agar, fermentation tests (including MR-VP), bile solubility and coagulase tests are also used.
Gram negative rods
One large group of Gram negative rods is the Enterobacteria. This includes the coliforms, as well as some others. These organisms are usually catalase(+) and oxidase(-). They are distinguished by a number of differential media, including SIM agar, TSI agar, EMB agar, blood agar, Simmons citrate agar, MR-VP tests, and a variety of carbohydrate fermentations. A number of Gram negative rods are strict aerobes living in the soil. The SIM test can tell you if you have a strict aerobe. Fermentation tests are usually not useful on these organisms. Catalase and oxidase tests, as well as ability to use a variety of carbon sources can help in distinguishing these. Even the way the cells associate in the Gram stain can be helpful. For example, Streptococcus tend to form pairs and chains; Staphylococcus and Micrococcus tend to form irregular clusters, 2 dimensional sheets.
Gram negative cocci
This is a relatively small group, often found on mammalian mucosal surfaces.
The major genera are Neisseria, Branhamella, and Moraxella. They are usually oxidase positive and are often distinguished by carbohydrate fermentation (glucose, sucrose, maltose).
Sulfur-indole-motility agar. This is a semisolid medium in a test tube which is stab-inoculated with a needle. Strict aerobes grow only at the top. Strict anaerobes grow only at the bottom. Facultative anaerobes grow throughout. And so on. Growth only along the stab indicates a non-motile organism. Growth spreading out from the stab indicates motility. The medium turns black if sulfate is reduced to sulfide (iron sulfide precipitation - what biochemical process is responsible?). Indole testing requires a special reagent described in the lab manuals. It is not often needed.
Organisms in a number of groups are distinguished by the mode of fermentation of various substances, usually carbohydrates. The possible results are no fermentation, or fermentation with acid production, or fermentation with acid and gas production. The broth is made from a commercial base (phenol red broth base) and an added carbohydrate (glucose, sucrose, mannitol, etc.). A small test tube is inverted in the broth (and filled with broth) to trap any gas that is formed. Acid production is indicated by a red to yellow color change.
These are simple tests performed on already grown cultures. A substrate is added and a reaction is observed. It is essential to do positive and negative controls with these tests. The catalase test is usually done on a slide; the oxidase test is usually done on a plate or a piece of filter paper.
This is a pair of tests to distinguish the type of glycolytic fermentation taking place: mixed acid or butanediol. A pair of tubes of the same medium are inoculated together. After incubation for 24-48 h, methyl red (MR) is added to one; the VP reagent to the other. MR turns deep red if the pH is low enough, indicating mixed acid fermentation. The VP reagent also turns color. Controls are needed.
Eosin methylene blue agar. This is prepared as plates and is used to distinguish various gram negative rods. Lactose fermenters have a dark, almost black center. Coliforms have a metallic sheen. Gram positive organisms are inhibited. MacConkey's agar performs a similar function.
This is usually done on an agar slant. Organisms that are able to use citrate as a carbon source will turn the medium blue. Generally for enteric bacteria.
This test requires the use of a blood agar plate. For economy, try to run two organisms on a half-plate each. Streak or swab for moderate growth, and incubate overnight. There are two types of reaction: a hemolysis is due to the production of H2O2 and produces a greenish area around the colonies; b hemolysis is due to the lysis of the red blood cells and results in an area of clearing around the colonies. No change in the medium around the colonies indicates no hemolysis, sometimes called g hemolysis.
Remember that these are living organisms, and not always 100% predictable. It is by the preponderance of the evidence that they are identified. Be aware that they will, from time to time, show phenotypic traits that supposedly they don't have.