Bio 316L                                        Microbiology Lab                                          2007

 

Instructor:  R. Pinette                   Office:  Folsom 307  MWF 11-12 or by appointment

 

Photo Atlas: Leboffe, M. and B. Pierce.  Photographic Atlas for the Microbiology 

                                 Laboratory. 3rd.  Englewood, CO. 216 pp.

Lab Manual    Pinette, R.  2005.  with 2007 corrections.  Laboratory Exercises for

                                Microbiology.  Presque Isle, ME. 89 pp.

 

I.  Tentative Lab Schedule.   Lab meets Tuesday and Thursday 1:40 to 3:40 in F305

Week of

Tuesday

Thursday

1/15

  No lab meeting

     Lab Safety (ii-iii & syllabus)

     Hazardous Waste Plan (88-89)

1.  Microscope Familiarization      

1/22

  2.  Sampling for Microbes

15  Sampling for Fungi

  3. Microscope Calibration

4.      Preparing Isolates

5.      Smears, Simple & Negative Stains           

       Quiz on Lab Safety & Waste Plan

1/29

16. Survey of the Fungi

17. Protozoa

 

2/5

6.       Differential Staining (Gram)

 

6.   Differential Staining (Spore )

7.       Capsule stain

Quiz  Exercises 1-5 & 16

2/12

9        Testing for oxygen utilization

10 Streak/Pour Plate Methods

8        Fluorescent Acid Fast Stain

Follow up on streak/pour plates

     Turn in lab notebooks

                                                     Winter Break Feb 17-25

2/26

11 Motility Determination

 

      Follow up motility tube test

13. Media Preparation

3/5

14.  Bacterial Unknowns

       (Streak Plates/ Gram Stain)

 

     Preparing isolates of each component     

     Quiz on Exercises 6-10 & 17

3/12

Gram stain of isolates.

Inoculate another slant plus a broth and streak plate of each isolate.

 

Morphology Study ,

Label Stock Cultures

Start Methyl Red Test

3/19

Bacterial Identification--inoculation of media for identifying unknowns. 15.  Isolating Fungi from Soil

Complete any morphology work

Read results from physiological tests.

3/26

Prepare plates, slant and slides to turn in. Transfer fungi to sterile PDA plates.

Turn in Cultures, Plates and Slides of bacterial unknown and rationale. 

                                         SPRING BREAK  March 31-April 8

4/9

18 . Helminths & Roundworms

19. Bacterial Conjugation

Follow up on Conjugation/ Finish Ex 18

4/16

Streak-Stab Procedure for Staph.

21.  Fermentation of Milk

        Follow up on Exercise 21

22.  Latex Agglutination Test   

 

4/23

12. Standard Plate Count

23. Membrane Filter Method

 

      Follow up on Exercises 12 & 23

 

 

4/30

 Review for lab Practical

 Course Evaluation

Lab Practical (6-12, 16- 19 & 21-23)   

Turn in Notebooks

 

II.  Course Grade.   The laboratory contributes approximately 40% of the course grade distributed approximately as follows.

 

                        3 Quizzes                                                        12%

                        Lab Practical                                                    11%

                        Unknown work products and write up                7%

                        Notebook                                                          10%                              

                                                           

Points earned from lab will be combined with the lecture to arrive at your final grade. See lecture syllabus and general course policies for further information on grades.

 

III. Lab Tests.  Tests will consist of two written quizzes.   Lab practical questions will consist of a series of power point slides accompanied by questions based on recognition of microbial structures (spores, capsules, shape) and recognition of major taxonomic groups (divisions, phylums). 

 

IV. Unknown Project.  Students will isolate a Gram negative bacterium from a mixture and identify it to species based on morphological evidence.  Your grade will be based on work products, including a pure culture slant, streak plate, Gram stain, capsule stain.  This is an opportunity to show off your skills in preparing these materials so take care in preparing good quality products.  Pay attention to details such as information and placement of labels on slides and cultures given in below in item V.   Included with this will be a 2-3 page write up discussing your results, a comparison with information in Bergey’s Manual and a conclusion explaining the basis of your determination. 

 

V.  Labeling. Labeling is an important part of doing microbiological work.  All slides, tubes, plates and other materials used in culturing organisms must be labeled with the genus of the organism, the date and your last name spelled out.   Materials prepared as part of laboratory exercises should include the exercise number.   The type of label used varies with the container:

·         Plates should be labeled with wax markers on the plate bottom--do not use tape.

·         Tubes will be labeled with a piece of tape no longer than 2 inches and placed immediately below the plastic cap  with the writing beginning on the end nearest the cap.

·         Slides will be labeled with self-adhesive labels.  When placed in a box, the labels should be on the left side of the slide with the writing right side up when one looks in the box.

 

VI. Laboratory Notebook.   Using a composition type notebook, students will record all laboratory observations and data gathered during the semester.  More information is available on this in a separate handout.

 

General Laboratory Safety Guidelines

 

I. Emergency Equipment and Telephone

Emergency Number:   8-9-1-1 Phone is located on wall by the refrigerator.

 

Be prepared to give your name, address (181 Main Street, Presque Isle), building name, room number and explain the nature of the emergency.   The dispatcher is able to send an ambulance, police or fire.

 

II. Location of Safety Equipment in Laboratory

 

Eyewash and shower:  End of center table by windows. 

Fire extinguisher:  Hanging on partition near refrigerator by windows.

First aid kit:  On opposite side of refrigerator on south wall

Fire blanket:  On wall along right side of microscope cabinets with dry boards.

Emergency gas valve:  On south wall near main laboratory exit.

 

III.  General Lab Safety Guidelines

 

1.      Wear appropriate clothing and shoes in the laboratory.  If you own a lab coat, bring it to lab.

2.      Avoid loose fitting items of clothing.  Coats and jackets should be placed on bench designated for this purpose.             

3.      Food and drinks are not allowed in the laboratory. 

4.      Keep your workspace free of all unnecessary materials.   Backpacks should be place on the floor near your feet, but not in the aisle.

5.      Shoulder length hair must be tied back in a pony tail to prevent injury from Bunsen burners and to avoid contamination of laboratory materials.

6.      Report spills and accidents immediately to your instructor.

7.      Be sure to follow the instructor’s directions in disposal of all chemicals.

8.      Only aqueous solutions of inorganic chemicals can be poured into sinks.  Organic wastes must be disposed of in appropriate containers marked for this purpose. 

9.      Do not return unused chemicals to the stock bottles.

10. Dispose of broken glass in yellow receptacle marked for this purpose.

11. Dispose of razor blades and sharp metals objects in red biohazard receptacle.

12. Wash your hands with disinfectant soap provided at the sink before leaving lab. 

 

IV. Emergency Procedures for a Laboratory Fire

 

·        Shut off emergency gas valve in case of gas leak or fire involving gas spigots on laboratory benches.

·        In case of a small fire at your desk, smother with a book or piece of cloth.  Do not use your hands.

·        If your clothes are on fire such as a sleeve run water over it. Otherwise use the fire blanket.  It is likely that a student will need help with this, be prepared to help someone with the wrap into the fire blanket should their clothes or hair catch on fire.

·        If a large fire occurs such as in a waste basket, smother the fire with a jacket or other piece of cloth.

·        If the fire is larger, leave the laboratory.   Follow the evacuation plan.  If the alarm has not yet sounded, pull the fire alarm (left side of hall before fire doors leading to the north stairway and right side of hall before fire doors leading to the south stairway). Continue to gathering area on the other side of the parking lot on the west side of the building.

 

V. Electrical Equipment

 

·        Do not spray or splash water in the vicinity of electrical equipment which is plugged in.

·        If equipment crackles, snaps or begins to smoke, do not attempt to disconnect it, call the instructor immediately.

·        In case of any frayed or broken electrical cords, notify the instructor.

 

VI. First Aid for Minor Injuries.

 

·        Band aids and some basic first aid materials are available in the lab.

·        For spills in the eyes, use the eyewash stations.

·        For burns, chill the affected part with cold water and ice as soon as possible.

 

VII. Evacuation Plan

 

Should conditions in the laboratory require evacuation due to an accident, hazardous chemical spill, fire or due to the sound of the fire alarm in the building, students will immediately leave the building.  Turn left at the laboratory exit and proceed to the North stairway.  Upon exiting the building turn left and proceed down the sidewalk on the west side of Folsom Hall.  Stop here at this point and wait for your instructor to confirm that the entire class has safely vacated the building.