2.1: Appendix A

Equipment, Supplies, and Cultures

The following is a list of equipment, supplies, cultures, and media needed to support this laboratory experience. Specific bacteria may be replaced with other cultures according to individual lab preferences or requirements. Amounts are not provided, because numbers will vary by section size. Best outcomes will be achieved if students work alone for labs in which new skills are introduced.

General Lab Equipment

• Microscopes and immersion oil
• Bunsen burners or sterilizers
• Inoculating loops and needles (metal or sterile disposable plastic)
• Metal forceps
• Pipette pumps
• Incubators set for body temperature and room temperature incubation
• Biohazard disposal container(s)
• Autoclave
• Refrigerator

Maintained Stock of Laboratory Supplies

The following should be readily available to students during lab and open lab times:

• Glass slides
• Clear plastic flexible laboratory metric rulers
• Kimwipes and/or lens paper
• Sterile 10 ml glass pipettes
• Sterile swabs
• Sterile wood applicator sticks

Replaced and refilled as needed each week:

• Staining “kits,” each kit containing stains in dropper bottles for Gram and Endospore staining procedures (Crystal violet, Gram’s iodine, 95% ethanol, Safranin, and Malachite Green)
• Spray bottles with laboratory disinfectant
• 70% ethanol for disinfecting forceps and spreaders, with beakers
• 4–6 racks (160–240 tubes) of sterile glass culture tubes, empty
• 2 racks (80 tubes) of tubes with sterile distilled water for making smears
• 2 sleeves (80 plates) of TSA plates
• 1 rack (40 tubes) of TSA slants
• 2 bottles (100 ml) of TSB

The recommended nonselective growth medium for routine culture is Tryptic Soy (TS), but other types could easily be substituted.

Lab 1: Biosafety Practices and Procedures

• Principles of Biosafety Evaluation and Student Affirmation form to assess understanding and document biosafety training.

Lab 2: The Microscopic World

Prepared slides (purchased, stained and mounted):

• Rectal (or fecal) smear (Gram stain)
• Mouth smear (Gram stain)
• Yogurt smear (Gram stained)

Cultures:

• Yogurt, store bought, any brand
• Yogurt, freshly made (prepared within a week of lab)

To make yogurt: Combine a large spoonful of yogurt (or a starter culture such as YoGourmet) with steamed milk in a glass pint canning jar with a lid. Stir vigorously to mix, then incubate at 38–40°C overnight. Refrigerate when milk has thickened into yogurt.

Lab 3: Bacteriological Culture Methods

Cultures:

• Overnight plate cultures of Micrococcus luteus and Enterococcus faecalis
• Overnight broth culture containing two bacteria

For broth cultures: Grow broth cultures of bacteria separately, combine in one tube right before the lab begins.

• For BSL-1 only: M. luteus, Micrococcus roseus in equal proportions
• For BSL-2: any combination of bacteria. Try to use bacteria which grow at a similar rate, or proportions added to the mixed culture can be adjusted to account for growth rate (for example, 2x as much Gram-positive to Gram-negative ratio)

Culture Media:

• Tryptic Soy Broth (or other nonselective general growth medium) in bottles
• Tryptic Soy Agar plates
• Tryptic Soy Agar slants in culture tubes
• Tryptic Soy deeps in culture tubes (semisolid agar: use 3.5 g agar per liter TSB)

Lab 4: The Environmental Isolate Project

Cultures:

• Bacteria cultured from skin and isolated in pure culture

Culture media (available in lab for the duration of the project):

• TSA plates and TSA slants for primary culture and pure culture maintenance

The media necessary to identify the genus and species of the isolated bacteria will vary each week. See media requirements and supplies for Growth Characteristics and Food Safety labs.

Lab 5: Differential Staining Techniques

Prepared slides (purchased already stained and mounted):

• Flavobacterium capsulatum, capsule stained (or other capsule-stained smear)
• Corynebacterium diphtheriae, methylene blue stain (or other stain for metachromatic granules)
• Mycobacterium tuberculosis, acid-fast stain

Cultures:

• Overnight TSA plate cultures of a Gram-positive bacterium and a Gram-negative bacterium (suggested are Escherichia coli and Staphylococcus saprophyticus)
• Overnight TSA plate cultures of two species of Bacillus, each with a different endospore appearance: suggested are B. subtilis OR B. cereus (with oval, central endospores) AND B. sphaericus OR B. globisporus (with spherical, terminal endospores)

Lab 7: Metabolism, Physiology, and Growth Characteristics of Cocci

Prepared slides (purchased, stained and mounted):

• Neisseria gonorrhoeae, Gram stain

Cultures:

• Overnight TSA plate cultures of Staphylococcus aureus, S. epidermidis, and S. saprophyticus, Micrococcus luteus, Enterococcus faecalis, Streptococcus pyogenes

Media and Test Reagents:

• Nitrate broth, in bottles
• Nitrate reagents A and B, stored at 4°C
• Urease broth, in bottles
• Triple Sugar Iron (TSI) agar slants
• Mannitol Salt Agar (MSA) plates
• Blood Agar Plates (BAP)
• Bile Esculin (BE) agar plates
• 3% hydrogen peroxide in dropper bottles (for catalase test)
• DrySlide Oxidase test cards (for oxidase test)
• Staphaurex (or other coagulase test kit) cards and reagent

Lab 8: Metabolism, Physiology, and Growth Characteristics of Bacilli

Cultures:

• Overnight TSA plate cultures of a Bacillus spp., Corynebacterium xerosis, Pseudomonas aeruginosa, Escherichia coli, a Salmonella spp., Citrobacter freundii, Enterobacter aerogenes

Media and Test Reagents:

• Nitrate broth, in bottles
• Nitrate reagents A and B, stored at 4°C
• MR-VP broth, in bottles
• Methyl Red reagent, and VP (Barritt’s) reagents A and B, stored at 4°C
• Triple Sugar Iron (TSI) agar slants
• Sulfide-Indole-Motility (SIM) agar stabs
• Indole (Kovac’s) reagent, stored at 4°C
• Simmon’s Citrate Agar plates or slants
• 3% hydrogen peroxide in dropper bottles (for catalase test)
• DrySlide Oxidase test cards (for oxidase test)

Lab 9: Microbiological Food Safety

Cultures:

• A selection of food items, such as ground beef, bagged lettuce, eggs, chicken, etc. (which may be “spiked” with bacteria). Students may be encouraged to include items of interest that they transport to lab with them.

Media and Test Reagents:

• Plastic disposable culture tubes with caps (to facilitate cleanup and disposal)
• Tryptic Soy Broth (TSB) in bottles
• MacConkey Agar (MAC) plates
• Sorbitol-MacConkey Agar (SMAC) plates
• Mannitol Salt Agar (MSA) plates
• TSI agar slants
• Staphaurex (or other coagulase test kit) cards and reagent

Lab 10: Germ Warfare

Cultures:

• Overnight TSA plate cultures of Staphylococcus aureus, S. saprophyticus, Escherichia coli, and Pseudomonas aeruginosa

Media and other supplies:

• 0.85% saline, sterile, in bottles
• 0.5 McFarland turbidity standards
• Mueller Hinton Agar (MHA) plates
• Antibiotic disks with: novobiocin, cefixime, azithromycin, ciprofloxacin, tetracycline
• Sterile blank filter paper disks
• Samples of a variety of known and potential disinfectants and antiseptics, such as a commercial home product, hand sanitizer, antibacterial dish soap, mouthwash, products made with essential oils, honey, etc.

Lab 11: Epidemiology and Public Health

Supplies:

• Calculators
• Access to the internet

Lab 12: Blood: the Good, the Bad, and the Ugly

Prepared slides and other supplies:

• Wrights-Giemsa stained blood smears (no pathology)
• Wrights-Giemsa or Giemsa stained blood smears of Anaplasma spp., Babesia spp., and Dirofilaria (microfilarial form)
• Blood cell counters (optional)
• Sure-Vue Mono Test kit (or other commercial test kit for Infectious Mononucleosis)