BIOT 120
Cleanroom Microbiology
Laboratory Preparation
Guide
Copyright ©2009 by Gary Fromert, M.S.
PETRI Project, Northampton Community College
Table of Contents
Lab 1: Introduction to Cleanroom Microbiology and Contamination Control
1. Introduction to the Microscope
2. Viewing Prepared Slides of Microorganisms (Optional; if time)
Page
3
Lab 2: Introduction to Microorganisms
1. Introduction to Bacteria and Viewing of Prepared Slides of Bacteria
2. Microscopic Comparison and Identification of Yeast, Molds, Protozoa,
and Algae
4
Lab 3: Aseptic Handling and Transfer of Bacteria
1. Aseptic Transfer of Bacteria
2. Isolation Techniques: Streak Plates
5
Lab 4: Basic Classification of Bacteria
1. Gram Stain
2. Spore Stain and Capsule Stain
6
Lab 5: Basic Characterization and Identification of Bacteria
1. Oxygen and Growth of Bacteria
2. Basic Biochemical Characterization and Identification of Bacteria
7
Lab 6: Control of Microbial Growth – Physical Methods
1. Physical Methods of Control - Heat
8
Lab 7: Control of Microbial Growth – Biochemical Methods
1.
Testing Antiseptics and Disinfectants: Disk Diffusion Technique
Lab 8: Personnel Control and Hygiene in Contamination Control
1. Effectiveness of Hand Washing
2. Gloving and Gowning Practicum
7
Lab 9: Environmental Sampling
1. Static vs. Dynamic Environments
2. Water Analysis - Membrane Filtration Technique
3. Air and Surface Sampling
9
Lab 10: Sterility and Product Integrity
1. Product Sterilization - Membrane Filtration Technique
2. Evaluation of Compromised Product
10
Lab 11: Evaluation of a Controlled Environment
1. Aseptic Setup of Isolator
2. Media Fills
11
Lab 12: Documentation Basics and Summary of Evaluations
12
2
Lab 1: Introduction to Cleanroom Microbiology and the Microscope
Activity 1. Introduction to the Microscope
Materials (per student)
□ Compound Light Microscope
□ Prepared slides: Letter “e” and Colored Threads
□ Lens paper
Activity 2. Viewing Prepared Slides of Microorganisms (if time with proficiency of students)
Materials (per student)
□ Compound Light Microscope
□ Immersion Oil
□ Lens Paper
□ Prepared slides of various types of microorganisms as follows:
□ Bacteria - Coccus Clumps/Coccus Chains and Typical Bacillus/Typical Spirillum
□ Fungi - Penicillium, Aspergillus, and Rhizopus
3
Lab 2: Introduction to Microorganisms
Activity 1. Viewing Prepared Slides of Microorganisms
Materials (per student)
□ Compound Light Microscope
□ Immersion Oil
□ Prepared slides of various types of microorganisms as follows:
□ Bacteria - Coccus Clumps/Coccus Chains and Typical Bacillus/Typical Spirillum
□ Fungi - Penicillium, Aspergillus, and Rhizopus
Activity 2. Microscopic Comparison of Fresh Slides of Different Microorganisms
Materials in Classroom:
□ Posters: POND I and POND III, Bacteria, The Microscope, and Microscope Magnification
□ Demo slide, posters or other information on viruses.
Materials (per student):
□ Microscope supplies from Activity 1 (first 2 listed above)
□ China marker or Sharpie
□ Clean microscope slides
□ Clean coverslips
□ Small dropper bottle filled with water
□ Bibulous Paper or Paper Towel
□ Kimwipes
□ Methylene Blue or I2KI (Iodine) Solution
□ Sterile Transfer Pipettes
□ Organisms as follows (Optional – Treat as Unknowns):
A. Yeast - Either a Sabourand agar culture of Saccharomyces cerevisiae (Baker's
Yeast) or an activated suspension of S. cerevisiae.
Optional: Label as Unknown "A"
B. Mold (Filamentous Fungi) – use mounted slides of Apergillus or Phenicillium
Optional: Cover over the name on slide and label as Unknown "B"
C. Protozoa - Mixed Protozoa (Carolina Biological) in specialized viewing tubes.
Optional: Label as Unknown "C"
D. A survey mix of Green Algae (Carolina Biological)
Optional: Label as Unknown "D"
4
Lab 3: Aseptic Handling and Transfer of Bacteria
Activity 1. Aseptic Transfer of Bacteria
Materials (per table)
□ Prepared Pure Culture Slant of Proteus vulgaris or Serratia marcescens
□ Prepared Pure Culture Slant of Micrococcus luteus or Enterococcus faecalis
Materials (per student)
□ 2 TSA or Nutrient Agar Slants
□ 1 Inoculating/Transfer Loop
□ China Marker or Sharpie
□ Bunsen Burner
Activity 2. Isolation Techniques: Streak Plates
Materials (per table)
□ Prepared Pure Culture Slant of Proteus vulgaris or Serratia marcescens
□ Prepared Pure Culture Slant of Micrococcus luteus or Enterococcus faecalis
Materials (per student)
□ 2 TSA or Nutrient Agar Plates
□ 1 Inoculating/Transfer Loop
□ China Marker or Sharpie
□ Bunsen Burner
5
Lab 4: Basic Classification of Bacteria
Activity 1. Gram Stain
Materials (per table)
□ Prepared Pure Culture Slant of Proteus vulgaris or Serratia marcescens
□ Prepared Pure Culture Slant of Micrococcus luteus or Enterococcus faecalis
Materials (per student)
□ 1 Inoculating/Transfer Loop
□ 2 Glass Microscope Slides
□ 2 Glass Cover Slips
□ Gram Stain Reagents
□ 1 Compound Light Microscope with Oil Immersion Lens
□ Immersion Oil
□ Bibulous Paper or Paper Towel
□ Kimwipes
□ Lens Paper
□ China Marker or Sharpie
□ Bunsen Burner
Activity 2. Spore Stain and Capsule Stain
Materials (per table)
□ Pure Culture Slant of 48 hour and 5 day Bacillus subtilis or Bacillus cereus
□ Pure Culture Slant of Enterobacter aerogenes or Serratia marcescens
Materials (per student)
□ 1 1 Inoculating/Transfer Loop
□ 4 Glass Microscope Slides
□ 3 Glass Cover Slips
□ Spore Stain Reagents (Schaeffer-Fulton Method)
□ Capsule Stain Reagents (Congo red or Nigrosin and Maneval's stain)
□ 1 Compound Light Microscope with Oil Immersion Lens
□ Immersion Oil
□ Bibulous Paper or Paper Towel
□ Kimwipes
□ Lens Paper
□ China Marker or Sharpie
□ Bunsen Burner
6
Lab 5: Basic Characterization and Identification of Bacteria
Activity 1. Oxygen and Growth of Bacteria
Materials (per table)
□ 1 Pure Culture Broth of Pseudomonas fluorescens or Staphylococcus epidermidis
□ 1 Pure Culture Broth of Escherichia coli or Proteus vulgaris
Materials (per student or per pair of students)
□ 2 Fluid Thioglycollate Broth Tubes
□ 1 Inoculating/Transfer Loop
□ China Marker or Sharpie
□ Bunsen Burner
Activity 2. Basic Biochemical Characterization and Identification of Bacteria
Day 1 – Inoculation of Enterotubes – Materials (per student or per pair of students)
□ 1 Enterobacterizceae Unknown Culture:
Note: Each student will get only one unknown, label as Unknown "A", "B", "C", & "D".
A - Bacillus cereus
B - Enterobacter aerogenes
C - Escherichia coli
D - Proteus vulgaris
□ 1 Enterotube™ II
Day 2- Reading the Tubes & Follow-up – Materials (per class)
□ Kovac’s Reagent
□ Voges-Proskauer Reagents (20% KOH and 5% alpha-naphthol)
7
Lab 6: Growth Control of Microorganisms
Activity 1. Physical Methods of Control - Heat
Materials (per group)
□ 1 Pure Culture Broth of Serratia marcescens
□ 2 Pure Culture Broth of 48 to 72 hour Escherichia coli
□ 3 TSA or Nutrient Agar Slants
□ 10 TSA or Nutrient Broth Tubes
□ 2 Sterile 0.1 mL or 1 mL Pipettes
□ China Marker or Sharpie
□ Hot Plate
□ Dry Oven Set to 70°C
□ 1 400 mL Beaker
□ 1 Glass Thermometer
□ Inoculating/Transfer Loop
□ Bunsen Burner
Activity 2. Testing Antiseptics and Disinfectants: Disk Diffusion Technique
Materials (per group)
□ 24 Blank Diffusion Disks
□ 1 Pure Culture Broth of 24 hour Pseudomonas fluorescens
□ 1 Pure Culture Broth of 24 hour Staphylococcus epidermidis
□ 1 Pure Culture Broth of 24 hour Bacillus subtilis
□ 4 TSA or Nutrient Agar Plates
□ 3-5% Solution Bleach
□ 1-2% Solution Household Disinfectant Cleaner (e.g. Lysol®)
□ 0.5-1% Solution Silver Nitrate
□ 100% Antiseptic Mouthwash (e.g. Listerine®)
□ 100% Liquid Antibacterial Soap
□ 1-2% Solution of a Pine Cleaner (e.g. Pine-sol®)
□ 1 bottle of antibacterial soap
□ Sterile Cotton Swabs
□ Forceps (tweezers)
□ Alcohol for Flaming
□ China Marker or Sharpie
□ Inoculating/Transfer Loop
□ Bunsen Burner
8
Lab 7: Personnel Control and Hygiene in Contamination Control
Activity 1. Effectiveness of Hand Scrubbing
Materials (per group of 2 students)
□ 2 TSA or Nutrient Agar Plates
□ Liquid Hand Soap (preferably non-antimicrobial)
□ Timer
□ China Marker or Sharpie
Activity 2. Gloving and Gowning Practicum
Gowning Demonstration/Practicum
Materials (per group)
A Set of Cleanroom Garments
A Long Bench
Full Length Mirror
Gloving Practicum
Materials (per student)
1 Package of Sterile Gloves
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7
Lab 8: Evaluation of Environments and Contamination Control
Activity 1. Static vs. Dynamic Environments
Materials (per student)
3 Nutrient Agar or Trypticase Soy Agar (TSA) Plates
3 Sabouraund's Agar Plates
Timer
China Marker or Sharpie
Activity 2. Mechanical Barriers in Contamination Control
Materials (per student)
8 Nutrient Agar or Trypticase Soy Agar (TSA) Plates
1 Package Sterile Gloves
1 Sterile Procedure Mask
China Marker or Sharpie
10
8
Lab 9: Environmental Sampling
Activity 1. Water Analysis - Membrane Filtration Technique
Materials (per group of students)
5 Different Water Samples of Various Purity and Sterility 100mL each
Examples:
1. Sterilized water
2. Nonsterilized DI water
3. Chlorinated tap water
4. Nonchlorinated tap (well) water
5. Pond, lake or stream water
6 Nutrient Agar or Trypticase Soy Agar (TSA) Plates
6 Sterile Membrane Filtration Units 0.45 um (Note: units must be of the type that have
removable membranes)
50 mL Beaker
95% Ethanol
China Marker or Sharpie
Bunsen Burner
Büchner Vacuum Flask and No. 8 stopper with 9/16 in. single-hole opening (Note: the
flask and stopper may or may not be used in this lab exercise depending on the design
of the filtration unit used)
Activity 2. Air and Surface Sampling
Materials (per student)
4 Nutrient Agar or Trypticase Soy Agar (TSA) Plates
4 Sabouraund's Agar Plates
4 Nutrient Agar Contact or RODAC Plates
2 Sterile Cotton or Polyester Swabs (Plastic Handles)
2 Nutrient Broth Tubes
Timer
China Marker or Sharpie
11
9
Lab 10: Sterility and Product Integrity
Activity 1. Product Sterilization - Membrane Filtration Technique
Materials (per student)
1 Sterile Trypticase Soy or Nutrient Broth Tube
1 Trypticase Soy or Nutrient Broth Suspension of Escherichia coli
4 Nutrient Agar or Trypticase Soy Agar (TSA) Plates
2 Sterile Vacuum Filter Units 0.22 um (preferred) or 0.45 um (Note: units must be of the
type that allow for access to the filtrate)
2 Empty Sterile Broth Tubes with Caps (for filtrate if using filter units with pour spout)
Sterile 0.1 mL Pipettes
Glass Spreader or Sterile Disposable Plastic Spreaders
95% Ethanol (for flame sterilization if using glass spreader)
200 mL Beaker (for ethanol if flame sterilizing glass spreader)
China Marker or Sharpie
Bunsen Burner
Activity 2. Evaluation of Compromised Product
Label the outer package of two sterile vacuum filter units respectively "A" and "B".
To compromise one of the units, take a thin rigid piece of sharp wire or a new straight inoculating
needle and inconspicuously pierce the outer package material and work the wire down inside the
funnel and pierce a few holes into the filters membrane.
Label the outer package of two sterile cotton swabs respectively "A" and "B".
To compromise one of the swabs, take a single edge razor blade and inconspicuously cut the package
approximately 1" exactly where the head of the swab is located and stick the swab head out of the
pakage and rub it with your fingers or on some contaminated surface then work the head back into the
slit on the package. Try not to rip the package at the slit as it will make it more obvious.
Label two sterile TS or nutrient broth tubes respectively "Swab A" and "Swab B".
Label two sterile TS or nutrient broth tube respectively "Media A" and "Media B".
To compromise one of the media tubes, inoculate it with a small amount of E. coli.
Materials (per student)
2 Trypticase Soy or Nutrient Broth Suspension of Escherichia coli
2 Nutrient Agar or Trypticase Soy Agar (TSA) Plates
2 Sterile Vacuum Filter Units 0.2 um (preferred) or 0.45 um (Note: units must be of the
type that allow for access to the filtrate) marked "A" and "B"
2 Empty Sterile Broth Tubes with Caps (for filtrate if using filter units with pour spout)
2 Trypticase Soy or Nutrient Broth Tubes marked "Media A" and "Media B"
2 Trypticase Soy or Nutrient Broth Tubes marked "Swab A" and "Swab B"
12
Sterile 0.1 mL Pipettes
Glass Spreader or Sterile Disposable Plastic Spreaders
95% Ethanol (for flame sterilization if using glass spreader)
200 mL Beaker (for ethanol if flame sterilizing glass spreader)
2 Sterile Cotton Swabs marked "A" and "B"
2 Tubes of Sterile DI Water
China Marker or Sharpie
10
Lab 11: Evaluation of a Controlled Environment
Activity 1. Aseptic Setup of Isolator and Activity 2. Media Fills
1. Glove Bag Inflatable Glove Chambers Cole Parmer Cat.#EW-04408-30 (I2R Glove Bag model S20-20H)
2. Cotton Gloves for Inflatable Glove Chamber Cole Parmer Cat.#EW-04408-50
or
White Cotton Inspection Gloves, Fisher Scientific Womens Med. 19-013-538 6 PK of 12 Pair
Mens Large 19-013-540 PK of 12 Pair
3. I2R Handy-Lok closures 13" or I2R Quick closing clamps 13"
To seal off the large opening there is the Quick Closing Clamp that can be purchased from Glas-Col
but they are pricey,
http://www.glascol.com/index.php?sid=27&cid=238
108D AC-13 $75.00 for use with S, SS and X-17-17 I2R Glove Bags.
or Kitchen bag clips or Bulldog clips
4. Dialysis Tubing Clamps, Flinn Scientific Inc. Cat. No. AP4349
5. Coleman Electric Quickpump 120VAC Model# 5999A426 (Walmart) Note: you only need one or
two of these units as they inflate the bags quickly and can be shared by the students.
6. Double-sided carpet tape or double-sided heavy duty tape
13
7. Whatman HEPA-Cap Disposable Air Filtration Capsules Fisher Sci Cat.#09-744-12 Note: you will
need one of these to be taped on to the end of each of the Coleman Quickpump inflation hose using
electrical tape.
8. Roll of electrical tape
Materials (per student)
□ 1 Glove Bag (I2R Model 520-20 or equivalent)
□ 1 Pair "Outer" Cotton Gloves
□ 1 Pair "Prep" Exam Gloves
□ 1 Pair "Inner" Sterile Gloves
□ 1 Large Plastic Glove Bag Clip
□ 1 Small Plastic Glove Bag Clip
□ 4 12" Strips of Double Sided Tape
□ 2 Sterile Cotton or Polyester Swabs (Plastic Handles)
□ 2 Nutrient Agar or Trypticase Soy Agar (TSA) Plates
□ 2 Nutrient Agar Contact or RODAC Plates
□ 2 Nutrient Broth Tubes
□ 2 Prepared Bulk Sterile Media Broth
□ 6 Sterile Product Vials
□ 2 Sterile 3 mL Transfer Pipets
□ Alcohol Prep Pads or Alcohol and Sterile Wipe Pads
□ China Marker or Sharpie
Lab 12: Summary of Evaluations and Documentation Basics
Activity 1. Determination of Bioburden
Activity 2. Establishment of Alert and Action Limits
Activity 3. Validation and Documentation Practices
Materials:
None required
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