CONSTRUCTING AN IDENTIFICATION KEY

Transcription

1 CONSTRUCTING AN IDENTIFICATION KEY The following pages show you an example of how to make an identification key. Every student should make his/her own key from the information in the Bergey's Manual, course textbook and other material. Although two different keys may look different as far as sequence of the events is concerned, they may accomplish the same thing. Suppose we have been given an unknown sample and we want to identify the microorganisms in it. Also suppose that we are told that the organisms present belong to one or more of the species listed below: Streptococcus mitis Micrococcus ureae Neisseria subflava Bacillus licheniformis Serratia marcescens Enterobacter intermedium Streptococcus equi Neisseria sicca Corynebacterium diphtheriticum Bacillus pumilus Salmonella gallinarum Pseudomonas stutzeri The first step before using any diagnostic tests is to construct an identification key or a dichotomous key by consulting different sources and finding the characteristics of the above species. The best source that we have is the Bergey's Manual. Thus, we go to the index or table of contents of this manual, and finding the genera names, determine if they are cocci, rods or spirilla and also find their reaction to the Gram stain. We can then group the genera of our organisms as follows: Group I Group II Group III Group IV Positive Cocci Negative Positive Rods Negative Rods Cocci Streptococcus Neisseria Corynebacterium Enterobacter Micrococcus Bacillus Serratia Salmonella Pseudomonas In this manner we have thus far classified our organisms according to cell shape and Gram reaction and have obtained four groups. Next, we have to further subdivide the organisms within each group. Let us start with Group I that contains Streptococcus and Micrococcus. To be able to find tests that we can perform to differentiate between these two genera, we have to once again consult Bergey's Manual and read the general descriptions concerned with each genus. For example, if we use the index again and go to the starting page describing the family Streptococcaceae we can note down the main characteristics of this family as follows: 1

2 - Cells in pairs or chains of varying length or in tetrads - Non-motile or rarely motile - Endospore not formed - Facultative anaerobes Next we can look under the genus Streptococcus and record the following characteristics: - Ferment glucose - Cells in pairs or chains and in one plane - Catalase negative So, our summary for the genus Streptococcus would be: Gram positive, pairs or chains, non-motile, facultative anaerobes, catalase negative, ferment glucose. Since our task is to differentiate this genus from Micrococcus, it is sufficient to find one or two characteristics in the Micrococcus genus that are different from those of the Streptococcus. Consulting the Bergey's again, we find that the family Micrococcaceae has the following main characteristics: - Cells in clusters or packets dividing in more than one plane - Ferment glucose but do not produce gas - Catalase positive - Aerobes or facultative anaerobes. To find which characteristics are specific to the genus Micrococcus, we read on and note that: - Cells in pairs or chains dividing in more than one plane to form clusters, tetrads or cubical packets - Usually non-motile - Aerobes Thus our summary for the Micrococcus genus is: cells in clusters and dividing in more than one plane, catalase positive, aerobes, usually non-motile. It is now obvious that to be completely certain in our differentiation of these two genera and also to use the simplest and fastest possible test, we can either use a thioglycollate tube (to see what the oxygen requirement is; i.e., aerobe, anaerobe, facultative anaerobe, etc.) or a catalase test. Any one of these two tests would subdivide the two genera. However, looking at our original list, we find that we have to next find a way to differentiate between Streptococcus mitis and Streptococcus equi. If we read the descriptions of these two species and write down the main characteristics and especially those for which we are equipped to do a test, we will have: Streptococcus equi: ovoid or spherical cells µm in diameter; occur in pairs, short or long chains; very long chains in broth; ferments glucose, maltose, 2

3 sucrose but not arabinose, lactose, trehalose, inulin, glycerol, mannitol or sorbitol; wide zone of beta hemolysis on blood agar; on agar, colonies are small and watery at first but later dry out and leave flat, glistening colonies. Streptococcus mitis: spherical or ellipsoidal cells mm in diameter; long chains in broth; ferments glucose, maltose, sucrose and usually lactose but not inulin, mannitol, sorbitol, glycerol, arabinose or xylose; some strains ferment trehalose; strong alpha reaction on blood agar. Looking at the above two descriptions, it is evident that the best diagnostic test to separate the two species would be growth on blood agar where equi produces a β while mitis produces an α hemolytic reaction. Since there is only a single genus in Group II and it can be differentiated from all other genera by microscopic observations (cell shape and Gram reaction), we just need to find one or two tests to differentiate between Neisseria sicca and Neisseria subflava. This time, we do not need to note the family (Neisseriaceae) characteristics since we want to separate the two species within the same family and genus. The characteristics that we find for these two are as follows: Neisseria sicca: dry, wrinkled, adherent colonies; spontaneous agglutination in saline; some strains produce a yellowish pigment. Neisseria subflava: smooth and opaque colonies; yellow pigment. It is seen that the colony characteristics are the important criteria for the separation of the above two species. Thus if grown on agar, we would be able to differentiate between the two because the colonies of N. sicca are dry and wrinkled while those of N. subflava are smooth and opaque. Next, we work on Group III genera, namely Corynebacterium and Bacillus. Again, after consulting the Bergey's Manual, we summarize the important characteristics of these genera as follows: Corynebacterium: straight or curved rods, Gram positive; non-motile; generally aerobic; catalase positive; generally ferment glucose. Bacillus: straight rods; Gram positive; majority motile; form endospores; catalase positive; strict aerobes or facultative anaerobes. The above characteristics that we have extracted from Bergey's seem to be very similar. The only promising characteristic that could differentiate the species may be the motility. It is thus a good idea to check the motility of the three species in Group III. Doing this, we find that Corynebacterium diphtheriticum is definitely non-motile while Bacillus pumilus and Bacillus licheniformis are motile (since there is no mention of them being non-motile in the species descriptions). 3

4 To separate the two Bacillus species, let us note down some of their more important characteristics: Bacillus pumilus: colonies on agar smooth and slightly yellowish; can not grow anaerobically; can not hydrolyze starch; can not reduce nitrate. Bacillus licheniformis: colonies on agar opaque with dull to rough surface; hairlike outgrowths common; slimy lobes; can grow anaerobically; liquefies gelatin; hydrolyzes starch; reduces nitrates. It is evident that there is quite a large number of differences between the two Bacillus species that can be used in their separation (colony morphology on agar and /or α-amylase, thioglycollate or nitratase tests). The last group that we finally need to tackle is the group of negative rods. Let us write down the genus characteristics: Enterobacter: straight rods, mm wide, mm long; conforms to the general definition of the Enterobacteriaceae, facultative anaerobes; ferments glucose ("+"g); liquefies gelatin; DNase positive. Serratia: straight rods, mm wide, mm long; colonies white, pink or red; conforms to the general definition of the Enterobacteriaceae; facultative anaerobes; strong catalase reaction; ferments glucose, maltose, mannitol and trehalose; DNase positive; does not liquefy starch. Salmonella: straight rods, mm wide, mm long; conforms to the general definition of the Enterobacteriaceae; motile; facultative anaerobes; reduces nitrate; ferments glucose ("+"g); indole negative; DNase negative. Pseudomonas: straight or curved rods, mm wide, mm long; motile; aerobes; catalase positive; mostly ferment glucose. Since Enterobacter, Serratia and Salmonella conform to the general definition of the family Enterobacteriaceae, referring to this family shows that they are all oxidase negative and facultative anaerobes. However, if we check the characteristics of Pseudomonas stutzeri, we find it to be oxidase positive. Thus we first need to use the oxidase test to separate Pseudomonas from the rest. Then all Enterobacteriaceae can be separated by the use of Enterotube. The overall dichotomous key would then look as shown in the next drawing. Remember that this was just an example of how to make a key for a certain number of microbial species. If you have a different group of species from which your unknown sample was taken or if you do not know to which group your unknown belongs, then you have to make your own key. The suggestions and techniques shown above will help you in such an endeavor. 4

5 Use of any section of this Lab Manual without the written consent of Dr. Eby Bassiri, Dept. of Biology, University of Pennsylvania is strictly prohibited. 5

6 Organisms Listed MICROSCOPY GRAM REACTION Positive cocci Negative cocci Streptococcus Micrococcus Neisseria CATALASE GROWTH ON AGAR "-" "+" Dry, wrinkled colonies Smooth, opaque colonies Streptococcus M. ureae N. sicca N. subflava SBA α-hemolysis β-hemolysis Negative Rods Strep. mitis Strep. equi Enterobacter Serratia Salmonella Pseudomonas Positive Rods OXIDASE TEST Corynebacterium Bacillus "-" "+" Others P.stutzeri MOTILITY TEST ENTEROTUBE II Separated by codes Motile Non-motile Bacillus C. diphtheriae NITRATASE TEST "-" "+" B. pumilus B. licheniformis 6