MBIO 3812 Fundamentals of Microbiology

1

Guidelines for the Environmental Isolate Report The overall goal of this assignment is for the student to isolate and identify a microorganism from the environment. At the completion of this semester long assignment the student should be able to:

i.) Apply aseptic techniques when handling microorganisms ii.) Understand the meaning and necessity of obtaining and maintaining a pure

microbial culture for an extended period of time iii.) Demonstrate proficiency in the isolation and sub-culturing of pure microbial

cultures iv.) Utilize data obtained from a wide variety of biochemical tests, growth conditions

and staining methods in order to obtain a presumptive identification of an unknown environmental isolate

v.) Maintain a formal laboratory notebook

This assignment begins with Exercise 2-1 “Ubiquity of Microorganisms” and continues throughout the semester. The following is a general outline of what will be required for the presumptive identification of the environmental isolate (EI). 1. During Ex 2-1 “Ubiquity of Microorganisms” each student will sample an object

outside of the building and inoculate this sample onto a TSA plate in order to allow any microbes present in the sample to grow.

2. After the plate has been incubated for ~24 hours at 37°C each student will choose 3 DISTINCT WELL-ISOLATED bacterial colonies from their plate and subculture each to a new TSA plate in order to obtain a pure culture of each (done in conjunction with the Supplemental Exercise “Sub-culturing Bacterial Isolates”).

3. Once pure cultures of the 3 isolates have been obtained the student will choose 1 of

the isolates for further characterization. An important consideration in the choice of the EI is the microbe’s ability to grow rapidly enough to produce easily visible colonies after 24 hr incubation. Isolates that do not fit this criterion are not recommended for further characterization because of laboratory exercise time constraints. PURE CULTURES OF AT LEAST ONE ISOLATE SHOULD BE OBTAINED BY FEBRUARY 11th.

4. Once the EI has been chosen, the student should transfer the microbe to a TSA slant that will serve as the EI master stock. This master stock will be kept at 4°C for the remainder of the semester and is to be used only if a problem arises. From the master stock the student will inoculate a TSA plate and this will serve as the working stock (i.e., the stock that will be used in all tests and sub-cultured weekly).

5. In order to ensure viability of the EI the student will sub-culture the microbe to a new TSA plate once a week for the remainder of the semester regardless of whether the EI is to be used in tests that week or not.

MBIO 3812 Fundamentals of Microbiology

2

6. Once the EI has been selected, verified to be a pure culture and stock cultures produced each student will begin to perform a variety of tests on their EI in order to permit a presumptive identification to be made at the end of the semester.

7. Over the semester, the EI will be evaluated in many different laboratory exercises alongside the control strains used in each exercise. The exercises that the EI will be examine in include: (1) Ex 2-2 Colony Morphology (description of EI colony features) (2) Ex 3-7 Gram Stain (3) Ex 3-8 Acid-Fast Stain (4) Ex 3-9 Capsule Stain (5) Ex 3-10 Endospore Stain (6) Ex 5-28 Motility Test (7) Ex 2-6 Agar Deep Stabs (8) Ex 2-7 Fluid Thioglycollate Medium (9) Ex 2-8 Anaerobic Jar (10) Ex 2-9 The Effect of Temperature on Microbial Growth (11) Ex 2-10 The Effect of pH on Bacterial Growth (12) Ex 2-11 The Effect of Osmotic Pressure on Microbial Growth (13) Ex 5-2 Oxidation-Fermentation Test (14) Ex 5-3 Phenol Red Broth (3 different carbohydrates) (15) Ex 5-4 Methyl Red & Voges Proskauer Tests (16) Ex 5-5 Catalase Test (17) Ex 5-6 Oxidase Test (18) Ex 5-7 Nitrate Reduction Test (19) Ex 5-8 Citrate Test (20) Ex 5-10 Decarboxylation Tests (3 different amino acids) (21) Supplemental Exercise Indole Test (22) Ex 5-12 Starch Hydrolysis (23) Ex 5-13 Urea Hydrolysis (24) Ex 5-14 Casein Hydrolysis (25) Ex 5-15 Gelatin Hydrolysis (26) Ex 5-16 DNA Hydrolysis Test (27) Ex 5-21 Triple Sugar Iron Agar (TSI) (28) Ex 5-27 Coagulase Test (29) Ex 5-24 Bacitracin, Novobiocin and Optochin Susceptibility Tests (30) Ex 5-25 Blood Agar (31) Beta-Lactamase Test (supplemental exercise) (32) Ex 7-3 Antimicrobial Susceptibility Test (Kirby-Bauer Method)

Three times during the semester laboratory notebooks will be collected and 2-3 pre-determined exercises graded (see laboratory syllabus for further details on notebook grading). At least one of the exercises chosen for grading will have included the examination of the EI for its presumptive identification.

MBIO 3812 Fundamentals of Microbiology

3

A final EI report will be handed in at the end of the semester. This formal report is worth 75 points and should consist of 7 sections: Title, Abstract, Introduction, Materials & Methods, Results, Conclusions and References. Guidelines for the report are detailed below. Title (2 points)

• Concise and short (the take-home message of the report in one clear sentence) Abstract (8 points)

• A brief summary of the entire report (no more than 150 words) • From the abstract the reader should know such things as the purpose of the report,

the nature of the environmental sample (i.e., where it came from), the important tests and results that lead to your presumptive identification and your overall conclusion(s)

Introduction (10 points)

• Should provide the reader with the reasons/goals/importance for conducting this exercise, information on how bacteria can be identified and on the place you chose to sample and what type of microorganisms might be found there

• This is NOT the place to discuss what you believe your EI to be • This is a section where references would be appropriate and EXPECTED

Materials & Methods (10 points) • The material and procedures used for each exercise do not need to be written out

in full if the correct reference to the laboratory manual exercise or a D2L handout is cited; HOWEVER, any deviations or changes in a laboratory exercise need to be indicated.

• Consider breaking the laboratory exercises/tests into categories such as morphological (i.e., staining), physiological (i.e, growth under various environmental conditions) and biochemical.

• Consider listing in sentence form (and properly referenced) the tests performed in each category.

• Because of the amount of information (i.e., all the different procedures and materials used) this section must be CLEAR WRITTEN/REFERENCED and WELL ORGANIZED.

Results (20 points) • The results should be presented in tabular form. • Each table should be numbered and given an overall titled (e.g. Table 1 Results of

Morphological Tests) as well as column and row headings. The table should appear professional (e.g., the data fits in the appropriate box etc…).

MBIO 3812 Fundamentals of Microbiology

4

• The contents of the table should allow all the tests results on the EI to be easily located and examine.

• The table should be completely filled out. If a test was not done it should be noted as a footnote below the table with the reason for omission included.

• Consider breaking the results down into sections such as morphology, physiology and biochemical.

• There MUST also be a written results section that summarizes the salient/important characteristics of the EI (consider breaking the written text into sections that correspond with the different result table sections). All tables MUST be referred to within the text.

• If problems were encountered with a test or the EI (e.g., lost viability or no growth) they should be mentioned in this section.

• Because this section contains a large amount of information and important/critical results used in the identification of the EI it MUST be CLEARLY WRITTEN and WELL ORGANIZED.

Conclusions (15 points) • The EI must be identified AT LEAST to the level of the GENUS. • The critical characteristics that led to this identification must be discussed (i.e.,

how did you come up with the identification of the EI?) • Walk the reader through the logic used to come up with identification • Remember this is the section where you convince/justify to the reader the validity

of your identification based on the results you obtained. • Based on the literature/information about this organism discuss whether you

would have expected this organism to be found in the environment you sampled. • This is a section where references would be appropriate and EXPECTED

References (10 points) • A minimum of 3 references (NOT including the laboratory manual) is required

and must be cited within the text of the report. • Wikipedia and eHOW ARE NOT legitimate references and points will be

deducted if used. • References must be properly formatted according to the American Society for

Microbiology guidelines (this information will be posted separately on D2L).