Lab 15 Bacterial Unknowns FA23

Bacterial Unknown OBJECTIVES: Students should be able to 1. identify an unknown sample of bacteria using a combination of selective & differential media, staining techniques, and biochemical tests. 2. create a dichotomous key for bacterial organisms 3. properly follow a flowchart/dichotomous key through the steps of identifying the unknown INTRODUCTION: In this exercise you will asked to assume the role of a clinical microbiologist, in a search for the identity of an unknown potential pathogen. The identification of microorganisms has always been at the heart of the understanding of infectious diseases and you will be using the skills you have learned in this course to solve the mystery presented to you. The key to the solution of this puzzle is to isolate and identify the characteristics of your unknown sample and to construct a “profile” of your organism that can be used to compare to a known list of organisms. This will be done by a process of elimination using a sort of dichotomous key to arrive at a final possible outcome. MATERIALS: Each student will have: 1 unknown bacterial sample, staining supplies, TSA plate, TSA slant, selective & differential media, biochemical tests. METHODS : Before obtaining your unknown: 1) Create a dichotomous key for all possible unknown bacterial organisms. This flowchart will guide you through the results of each step and help identify the next step in the identification process. Please take a look of the flowchart that has been started for you on the next page. You will continue to complete this flowchart by placing ALL of the possible organisms list into the appropriate groups at the bottom of the flowchart. 2) Make sure to have the key/flowchart checked and signed off before you continue with your unknown. After obtaining your unknown: 3) Be sure to write the unknown # into your lab notebook. Remember, no # = no points. 4) Perform a Gram stain of your unknown to obtain its cell morphology & Gram reaction.

5) Streak a plate for isolation; you must obtain a pure culture before you can do any other biochemical tests. 6) After you have obtained a pure culture you will need to prepare a reference culture by streaking an agar slant so that you can keep you organism alive while you are testing it. 7) Based on your flowchart/key, perform the necessary diagnostic tests to determine the identity of your bacterium. 8) Each and every test or reference tube you use should be labeled with your name, unknown #, and your lab section so that they are not lost or confused with others. RESULTS : All of your tests and results need to be recorded in order in your notebook with enough information so that your processes could be repeated with the same results by someone else. It is of the utmost importance that you keep good notes in your lab notebook and that you organize your work in such a way that you will not miss any necessary steps or duplicate your work by doing repeated or unnecessary tests. Please use the FOV circles to draw any staining results with notes on cell shape & arrangement and record any media plate results.

Bacterial Unknowns Organism List Alcaligenes faecalis Bacillus polymyxa Bacillus megaterium Citrobacter freundii Clostridium sporogenes Corynebacterium xerosis Enterobacter aerogenes Enterococcus faecalis Escherichia coli Klebsiella pneumoniae Lactobacillus casei Micrococcus luteus Micrococcus roseus Morganella morganii Mycobacterium smegmatis Neisseria sicca Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Pseudomonas fluorescens Serratia marcescens Streptococcus mutans Staphylococcus aureus Staphylococcus epidermis

Gram Positive Bacteria carbohydrate fermentation Group Genus and species ideal growth temp pigment on TSA glucose sucrose lactose mannitol glycerol nitrate reduction MR VP citrate H 2 S coagulase starch gelatin BACILLACEAE Bacillus cereus 37 white A A K K A + V + (+) - NA + + Bacillus megaterium 37 white A A K V+ V+ V- V + (+) - NA + + Bacillus polymyxa 37 white A A (A) A A + V - - - NA + - Bacillus subtilis 37 white A A K A V + V + + - NA + + Clostridium sporogenes 37 white NA NA NA NA NA NA NA NA NA NA NA NA NA MYCOBACTERIACEAE Mycobacterium smegmatis 37 white NA NA NA NA NA NA NA NA NA NA NA NA NA Mycobacterium phlei 37 white NA NA NA NA NA NA NA NA NA NA NA NA NA OTHER GENERA Corynebacterium xerosis 37 white A A NA NA NA - - NA NA NA NA NA NA Corynebacterium diphtheriae 37 white A K NA NA NA + + NA NA NA NA NA NA LACTOBACILLACEAE Lactobacillus acidophilus 37 white NA NA NA NA NA NA NA NA NA NA NA NA NA Lactobacillus casei 37 white NA NA NA NA NA NA NA NA NA NA NA NA NA MICROCOCCACEAE MSA Micrococcus luteus 37 yellow K K K V K - - - NA - - - - NG Micrococcus roseus 37 pink V K K V V + - - NA - - - + NG Staphylococcus aureus 37 ale yellowA A V A K + V V NA + + - + growth/A Staphylococcus epidermidis 37 white A V V K A + V V NA - - - + growth/K STREPTOCOCCACEAE 6.5% NaCl broth SF Enterococcus faecalis 10, 45 white A A A V A NA NA V NA NA NA NA NA + + Lactococcus lactis 37 white A A A A A NA NA V NA NA NA NA NA - - Streptococcus mutans 37 white A A A K A NA NA A NA NA NA NA NA - - + positive reaction NG no growth ( ) slow reaction , a week or more - negative reaction V variable results ? unknown results A acid reaction NA not applicable results given after 48 hrs. growth K alkaline reaction G growth special growth conditions