Federated Department
of Biological Sciences
120:335 General Microbiology Laboratory/Fall 2014
rd
Required Lab Manual: Leboffe MJ, Pierce BE (2010) Microbiology: Laboratory Theory and Application, 3 Edition, Morton
Publishing Company, Englewood, Colorado. ISBN 978-0-89582-830-9. Please note: student’s version is loose leaf for 3-ring binder.
Required laboratory coat: For your own safety you are required to wear a lab coat while working at the lab. A list of suggested
providers can be found on the next page.
Lab Instructors: Pavan Vedula ([email protected]), sections 1 and 4 ; Chaitali Saqcena ([email protected]),
sections 2 and 3 .
Technician: Mr. Kenneth Feraudo
Schedule per Section: 1 Tue, 2:30-5:20 P.M.;
2
Mon, 1-3:50 P.M.;
3
Wed,10-11:20 A.M.;
4
Thu, 11:30 A.M.-2:20 P.M.
Room: Boyden 123
Calendar of Experiments
Experiments to be
finished
N°
1
Date
Sep 8-11
Pre-Lab activities
Read pp. 1-9 of the manual
Read Exp. 1-2 (for experiment
titles, see lab manual table of
contents below)
Experiments to be performed
Check in. Orientation. Use of the
autoclave Exp. 1-3, 1-4, and 1-5 Exp. 2-15
nd
(from 2 edition; work in groups of 2).
Filter-sterilization demo, brushes
2
Sep 15-18
—
Exp. 2-2 (observations) Exp. 2-3, 2-4, 2-6,
and 2-7 Please note: 2 organisms will be
provided to inoculate 2 slants and 2
broths).
Exp. 1-3, 1-4, and 1-5
3
Sep 22-25
Read Exp. 3-1, 3-2 and 3-3
Read pp. 95-99 Handout on
microscope handling (posted
on Blackboard)
Exp. 3-1, 3-2, 3-3
Exp. 2-6, and 2-7
4
Sep 29-Oct 2
—
Exp. 3-5, 3-6, and 3-7
Exp. 3-1, 3-2, 3-3
5
Oct 6-9
—
Exp. 3-8, 3-9 and 3-10 Exp. 3-14
Exp. 3-5, 3-6, and 3-7
6
Oct 13-16
Read pp. 129, 130, 135, 149,
and 150
Exp. 4-1, 4-3, 4-4, 4-5, and 4-6.
Exp. 5-2, 5-3, and 5-20 (modified SIM)
Exp. 3-8, 3-9, 3-10, and 314 (microscopic
observation)
—
Organization of students in groups of 2 (unless otherwise indicated)
Laboratory Midterm Examination
7
Oct 20-23
Read p. 243, 250-251.
Handout for alternative
cellometer is posted on
Blackboard. Please note: For
dilutions, use procedure on pp.
449-450 instead (Eppendorf
tubes).
Exp. 6-1 (alternative dilution procedure)
and 6-3
Exp. 4-1, 4-4, 4-5, and 4-6
Exp. 5-2, and 5-3
8
Oct 27-30
Read Exp. 5-31 before starting
work on unknown mixed
sample
Exp. 6-6 (groups of 4) Start identification
of unknown mixed sample.
Exp. 6-1
9
Nov 3-6
Read pp. 161-164, 177-178,
184-186, 190, 194, 197
Exp. 5-4, 5-9, 5-12, 5-14, 5-15, 5-16, 5-17,
Exp. 6-6.
Continue identification of
unknown mixed sample
10
Nov 10-13
Read pp. 268-273.
Read E-test strips hand-out
and probiotics
Exp. 7-3 and 7-4 (modified), E-test strips.
Enumeration of strains from commercial
probiotics.
Exp. 5-4, 5-9, 5-12, 5-14, 515, 5-16 and 5-17. Continue
identification of unknown
mixed sample
11
Nov 17-20
Read hand-outs for
chromogenic plates and
EnteroPluri-Test System
Use of chromogenic plates in the
identification of bacteria. EnteroPluri-Test
System demonstration
Exp. 7-3 and 7-4
Commercial probiotics
Finish identification of
unknown mixed sample
12
Nov 24-30
Thanksgiving Break—No Laboratory
13
Dec 1-4
Laboratory final examination. Finish identification of unknown sample, if necessary.
Check out.
Version of 4Sep14
Page 2
Grading: The various elements of your laboratory performance and examinations will be evaluated as follows:
Quizzes
10%
First report
10%
Midterm
exam
20%
Second
report
20%
Final lab
exam
20%
Unknown
project
20%
Total
100%
The laboratory grade will count as 25% of the final course grade.
Where to purchase lab coats in Northern New Jersey:
•
New Jersey Books, 167 University Avenue, at Bleeker St., Newark, NJ. Phone: (973) 624-5383. Mon-Thu 9:00A.M.-6 P.M., Sat 10 A.M.-2 P.M., Sun closed, except for the first two weeks of each semester.
•
Life Uniforms, West 158, Route 4 East, Paramus, NJ (near Paramus Road). Phone: (201) 843-2288. Hours:
Mon-Fri 10 A.M.-9:30 P.M., Sat 10 A.M.-8 P.M., Sun Closed.
•
Scrubs and Beyond, Jersey Gardens Mall, 651 Kapkowski Road, Elizabeth, NJ. Phone: (908) 558-1661. Hours:
Mon-Sat 10 A.M.-9 P.M., Sun 11 A.M.-7 P.M.
•
Atlantic Uniform, 444 Washington Avenue, Belleville NJ, Phone: (973) 751-1242. Hours: Mon-Fri 9:30 A.M.-5 P.M.,
Sat 10 A.M.-5 P.M., Sun 11 A.M.-4 P.M.
Lab Manual Table of Contents
rd
Leboffe MJ, Pierce BE (2010) Mictrobiology: Laboratory Theory & Application, 3 Edition, Morton
Publishing Company, Englewood, Colorado.
1-1
1-2
1-3
1-4
1-5
1. Fundamental Skills for the Microbiology Laboratory
Glo-Germ Hand Wash Education System
Nutrient Broth and Nutrient Agar Preparation
Common Aseptic Transfers and Inoculation Methods
Streak Plate Methods of Isolation
Spread Plate Method of Isolation
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2-8
2-9
2-10
2-11
2-12
2-13
2-14
2. Microbial Growth
Ubiquity of Microorganisms
Colony Morphology
Growth Patterns on Slants
Growth Patterns in Broth
Evaluation of Media
Agar Deep Stabs
Fluid Thioglycolate Medium
Anaerobic Jar
The Effect of Temperature on Microbial Growth
The Effect of pH on Microbial Growth
The Effect of Osmotic Pressure on Microbial Growth
Steam Sterilization
The Lethal Effects of Ultraviolet Radiation on Microbial Growth
Chemical Germicides: Disinfectants and Antiseptics
3-1
3-2
3-3
3-4
3-5
3-6
3-7
3-8
3-9
3-10
3-11
3-12
3-13
3-14
3. Microscopy and Staining
Introduction to the Light Microscope
Calibration of the Ocular Micrometer
Microscopic Examination of Eukaryotic Microbes
Microscopic Examination of Pond Water
Simple Stains
Negative Stains
Gram Stain
Acid-fast Stain
Capsule Stain
Endospore Stain
Parasporal Crystal Stain
Wet Mount and Hanging Drop Preparation
Flagella Stain
Morphological Unknown
Page 3
4-1
4-2
4-3
4-4
4-5
4-6
4-7
4-8
4. Selective Media
Phenylethyl Alcohol Agar
Columbia CNA With 5% Sheep Blood Agar
Bile Esculin Test
Mannitol Salts Agar
MacConkey Agar
Eosin Methylene Blue Agar
Hektoen Enteric Agar
Xylose Lysine Desoxycholate Agar
5-24
5-25
5-26
5-27
5-28
5-29
5-30
5-31
5. Differential Tests
Reduction Potential
Oxidation-Fermentation Test
Phenol Red Broth
Methyl Red and Voges-Proskauer
Catalase Test
Oxidase Test
Nitrate Reduction Test
Citrate Test
Malonate Test
Decarboxylation Test
Phenylalanine Deaminase Test
Starch Hydrolysis
Urea Hydrolysis
Casein Hydrolysis Test
Gelatin Hydrolysis test
DNA Hydrolysis Test
Lipid Hydrolysis Test
ONPG Test
PYR Test (In Gram-positive coccus section of 2e)
SIM Medium
Triple Sugar Iron Agar/Kligler Iron Agar
Lysine Iron Agar
Litmus Milk Medium
Bacitracin, Novobiocin, and Optochin Susceptibility Tests (In Gram-positive coccus section of
second edition)
Blood Agar
CAMP Test (In Gram-positive coccus section of second edition)
Coagulase Tests
Motility Test
API 20 E identification System for Enterobacteriaceae and Other Gram-Negative Rods
Enterotube® II
Bacterial Unknowns Project
6-1
6-2
6-3
6-4
6-5
6-6
6. Quantitative Techniques
Standard Plate Count
Urine Culture
Direct Count (Petroff-Hausser)
Closed-System Growth
Plaque Assay of Virus Titre
Thermal Death Time Versus Decimal Reduction Value
7-1
7-2
7-3
7-4
7-5
7-6
7-7
7-8
7-9
7. Medical Microbiology
Snyder Test
Lysozyme Assay
Antimicrobial Susceptibility Test (Kirby-Bauer Method)
Clinical Biofilms
MMWR Report
Epidemic Simulation
Identification of Enterobacteriaceae
Identification of Gram-positive Cocci
Identification of Gram-positive Rods
5-1
5-2
5-3
5-4
5-5
5-6
5-7
5-8
5-9
5-10
5-11
5-12
5-13
5-14
5-15
5-16
5-17
5-18
5-19
5-20
5-21
5-22
5-23
Page 4
8-1
8-2
8-3
8-4
8-5
8-6
8-7
8-8
8-9
8-10
8-11
8-12
8-13
8. Environmental Microbiology
Winogradsky Column
Nitrogen Fixation
Nitrification: The Production of Nitrate
Ammonification
Denitrification: Nitrate Reduction
Photosynthetic Sulfur Bacteria
Chemolithotrophic Sulfur-Oxidizing Bacteria
Sulfur-Reducing Bacteria
Bioluminesence
Soil Slide Culture
Soil Microbial Count
Membrane Filter Technique
Multiple Tube Fermentation Method for Coliform Determination
9-1
9-2
Methylene Blue Reductase Test
Making Yogurt
10-1
10-2
10-3
10-4
10-5
10-6
10-7
10. Microbial Genetics
Extraction of DNA from Bacterial Cells
Restriction Digest
Bacterial Transformation: The pGLO System
Polymerase Chain Reaction
Ultraviolet Damage and Repair
Ames Test
Phage Typing of E. coli Strains
11-1
11-2
11-3
11-4
11-5
11-6
11-7
11. Hematology and Serology
Differential Blood Cell Count
Precipitin Ring Test
Radial Immunodiffusion
Slide Agglutination
Blood Typing
Mononucleosis Hemagglutination Test
Quantitative Indirect ELISA
12-1
12-2
12-3
12-4
12. Eukaryotic Microbes
The Fungi - Common Yeasts and Molds
Fungal Slide Culture
Examination of Common Protozoans of Clinical Importance
Parasitic Helminths
9. Food Microbiology
A
B
C
D
E
F
G
H
Appendices
Biochemical Pathways
Miscellaneous Transfer Methods
Transfer from a Broth Culture Using a Glass Pipette
Transfer from a Broth Culture Using a Digital Pipette
The Spectrophotometer
Alternative Procedures for Section 6
Aragose Gel Electrophoresis
Media, Reagent, and Stain Recipes