Laboratory Safety, Microbiology Concepts, and Microscopy Essentials
Safety in the Microbiology Laboratory
Microbiology labs work with living infectious microorganisms. Rare laboratory infections have occurred due to handling these organisms. Proper technique and safety practices are essential to protect lab personnel and the environment.
Biosafety levels (BSL) define the required containment and practices for different organisms. CSN microbiology labs operate at BSL-1 and BSL-2.
CSN Safety Sheet and institutional rules apply to all lab activities.
CSN Safety Policy and General Lab Rules
CSN Safety Sheet must be reviewed and signed by students; follow the lab book and instructor instructions; when in doubt, ASK.
Personal belongings must be in lockers; no food, gum, drinks, including water, or water bottles in the lab.
Smoking/vaping, eating, and drinking are prohibited in laboratories.
Children and/or guests are not allowed in the laboratory.
No unauthorized laboratory experiments; students must work under instructor supervision.
Personal Protective Equipment (PPE) and Conduct
Students must wear/properly use all protective clothing/equipment designated by the instructor; failure to do so may result in removal from the lab.
All materials must be cleaned up and the work area left clean and dry at the end of the session.
All waste must be disposed of as directed by the instructor.
No unauthorized removal of lab materials or equipment.
Know the location and proper use of all safety equipment in the classroom and surroundings.
Safety Procedures and Emergency Information
In case of fire: inform the instructor, evacuate, pull the fire alarm, and notify CSN campus security on duty.
Report all classroom accidents/injuries to the instructor immediately.
Read and understand the labels on laboratory materials and posted safety information; the information is available at a designated site in the department.
Emergency procedures are listed in the Hazardous Materials Right to Know Safety Training Manual (Section XIII); students are responsible for reading and understanding these procedures.
Lab Hygiene, Cleanliness, and Equipment Use
Students are responsible for the location and proper use of all safety equipment; use equipment as instructed.
PPE (lab coats, goggles, gloves) are required in accordance with the procedural requirements (e.g., BSL-2 work requires gloves, lab coat, goggles, and BSC for aerosols/splashes).
Wash hands before and after the lab session; disinfect benches before/after work.
Do not touch face, apply cosmetics, adjust contact lenses, or bite nails while in the microbiology lab; no mouth contact with materials.
Fire Safety, Accidents, and Safety Documentation
Fire safety: if you discover a fire, inform the instructor, evacuate, pull the alarm, and contact campus security.
All accidents/injuries must be reported immediately to the instructor.
Read and understand the posted safety information for all materials in the lab; access is provided via a designated site in the department.
Laboratory Dress Code and Equipment Care
Lab coats are required and should be worn buttoned up; store autoclaved coats in a labeled plastic bag with your name; coats are autoclaved and returned at the end of the lab.
Goggles are required; store in a sealed plastic bag labeled with your name after use; goggles are cleaned with disinfectant and returned at the end of the semester.
Disposable gloves provided by CSN; use gloves during staining or working with microbial cultures; remove by turning inside out and dispose in glove disposal.
Use the Biosafety Cabinet for BSL-2 work and liquid cultures.
Closed-toe shoes required; tie back long hair.
Hand Hygiene and Flora of the Skin
Handwashing is the most important procedure to prevent the spread of infectious disease, especially in healthcare settings, where hands can transmit pathogens and cause nosocomial infections.
Nosocomial infections exceed 1.7\times 10^{6} cases per year, with roughly 9.9\times 10^{4} deaths.
Hand microbiota consists of transient flora (temporary) and resident (permanent) flora.
Resident flora: normal microflora (e.g., Staphylococcus epidermidis, Staphylococcus aureus in nares), Corynebacterium, Propionibacterium, Bacillus spp., yeasts, molds; they help protect by occupying space and producing antimicrobial compounds.
Transient flora: easily acquired, can cause nosocomial infections and cross-contamination; includes MRSA, VRE, Clostridium difficile, E. coli, Salmonella.
Skin conditions: slightly hypertonic, slightly acidic (pH ~5–6); sebaceous oils limit growth for many microbes; keratinocytes form a metabolically inactive barrier with low nutrients.
Hand Hygiene: Techniques and Products
Handwashing technique (proper procedure): Wet hands, apply soap, wash for 15\text{--}20\text{ seconds}, rub palm to palm, interlace fingers and thumbs, rub tops and bottoms of hands and fingernails, rinse, dry thoroughly, and use a towel to turn off the water.
Hand sanitizers: Alcohol-based, typically 60\%-70\% ethanol or isopropanol, gel or foam.
Mechanism: alcohol denatures proteins and disrupts plasma membranes, giving broad-spectrum activity.
Effective against vegetative bacteria and fungi and enveloped viruses; less effective against non-enveloped viruses and bacterial spores/ protozoan cysts.
Glo-Germ Handwashing Exercise
Purpose: demonstrate effectiveness of handwashing in removing microbes; helps identify missed areas.
Procedure: apply Glo-Germ lotion, observe under UV light to see fluorescence before washing; wash hands; re-examine under UV light; any fluorescence indicates missed areas.
Safety: wear goggles for UV protection; work in pairs (one washes, one observes).
Repeat roles so both students perform the exercise.
Document results on the provided table and switch roles.
Microbial Flora on Hands: Normal vs Transient
Normal flora (resident) live on skin and hair follicles; protect against pathogens; are difficult to remove; include Gram-positive bacteria like Staphylococcus epidermidis, Staphylococcus aureus (nares), Corynebacterium, Propionibacterium; molds and yeasts are present to a lesser extent; keratinocytes form a barrier with limited nutrients.
Transient flora are acquired via contact with contaminated surfaces (fomites); can cause nosocomial infections and cross-contamination; easier to remove with proper handwashing.
Microbiology Culture Media: Growth Fundamentals
Microbes are grown by sampling from sources, inoculating onto culture media, and observing growth to demonstrate ubiquity.
A culture medium provides nutrients for growth.
Broth: liquid medium; turbidity indicates bacterial growth.
Solid media (agar): grows as colonies; colonies are visible masses of microbes with characteristic shapes, colors, and sizes.
Incubation: plates are incubated at 25^{\circ}\mathrm{C} (room temperature) for molds and at 37^{\circ}\mathrm{C} (body temperature) for many bacteria; typical incubation is 24\text{--}48\ \text{hours} and plates may be stored in the refrigerator to arrest growth; some may incubate up to a week.
Types of Culture Media and Their Uses
General purpose media: support growth of many microorganisms; examples: Nutrient broth, Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA).
Enrichment media: contain growth factors that some organisms require (e.g., blood components).
Blood agar (BAP) contains sheep red blood cells; microbes may lyse these cells to obtain heme/nutrients.
Selective media: inhibit some groups while supporting others; e.g., Sabouraud's Dextrose Agar (SDA) with high dextrose favors fungi; suppresses most bacteria.
Differential media: reveal differences between colonies; Blood Agar (BAP) is also differential via patterns of hemolysis.
Three Specific Culture Media Used Today
Tryptic Soy Agar (TSA): general purpose medium for many microbes.
Sabouraud’s Dextrose Agar (SDA): selective for fungi (inhibits most bacteria due to high glucose).
Blood Agar (BAP): enrichment and differential; provides growth factors and allows observation of hemolysis patterns.
Inoculation, Incubation, and Colony Observation
Inoculate using the same environmental source for TSA and SDA plates when indicated; each student uses one TSA plate (37°C) and one SDA plate (25°C).
Materials: sterile cotton swabs, sterile saline (0.7%), parafilm, plates inverted during incubation, label with initials and specimen source.
Human samples: use BAP with throat or the same environmental source as used on TSA.
Growth and Colony Characteristics
After inoculation, incubate as described; colonies appear as distinct masses with varying shapes, colors, and sizes.
Colony shape may be round, irregular, or filamentous; color and size vary among species.
Inoculation Techniques and Environmental Sampling Details
Use sterile swabs for sampling; avoid pressing into agar too hard.
Parafilm-seal plates; invert plates before incubating to avoid condensation falling onto media.
When collecting environmental samples, inoculate TSA and SDA from the same source.
Microscopy: Brightfield and Components
Brightfield microscopy transmits light through a specimen; image appears dark on a light background.
Light microscope components include:
Ocular lenses (eyepieces) magnify ~$15\times$ each.
Objective lenses: 4x (scanner), 10x (low power), 40x (high dry), 100x (oil immersion).
Nosepiece (rotating); condenser under the stage; iris diaphragm to regulate light; stage with clips; illumination (lamp); rheostat (brightness control); mechanical stage.
Focusing controls: coarse adjustment knob and fine adjustment knob.
Magnification and Resolution in Light Microscopy
Magnification vs. resolution:
Magnification is the size increase of the image; resolution is the ability to distinguish two objects as separate.
Maximum resolution of a light microscope is d_{max} = 0.2\ \mu m; objects smaller than this are not resolvable.
The light wavelength limits resolution; blue light (shorter wavelength) improves resolution.
Total magnification:
\text{Total Magnification} = (\text{ ocular magnification}) \times (\text{ objective magnification})
With ocular = 15\times and objectives of 4\times, 10\times, 40\times, 100\times, total magnifications are 60\times, 150\times, 600\times, 1500\times respectively.
Viruses cannot be seen with light microscopy; electron microscopy is required.
Parfocal, Focusing, and Working Distance
Parfocal: once an object is focused at a lower power, it remains roughly in focus when switching to a higher power objective; fine focus is used to sharpen.
Working distance: distance between the objective lens and the slide when in focus; it decreases with higher magnification (e.g., very small with 40x/100x).
Only the fine adjustment knob is used at high magnifications (40x and 100x).
Anatomy of a Microscope and Care
Core components include: head, ocular lens, objective lenses, nosepiece, stage, condenser, iris diaphragm, lamp, rheostat, base, mechanical stage.
Oil immersion (100x) uses immersion oil that matches glass refractive index to reduce bending and improve resolution.
Care steps after use:
Remove slides, turn off light, set rheostat to minimum.
Lower stage with coarse adjustment, wipe oil from 100x objective with Kimwipes and cleaner, ensure no oil on high-dry lens.
Wipe stage and condenser; lock scanner 4x objective in place; wrap cord, and return to cabinet per instructor.
Microscope Objective Lenses: Working Distances and Magnifications
Common objective lenses and total magnifications when combined with a 15x ocular:
4x objective → total magnification 60\times
10x objective → total magnification 150\times
40x objective → total magnification 600\times
100x (oil) objective → total magnification 1500\times
Working distances are longer for lower magnification and shorter for higher magnification (e.g., 40x and 100x have very small working distances).
Eukaryotes and Prokaryotes: Cell Types and Domains
There are two main cell types:
Prokaryotic cells: small, single-celled, lack a true nucleus and membrane-bound organelles; include Bacteria and Archaea.
Eukaryotic cells: larger, may be single-celled or multicellular, possess a nucleus and membrane-bound organelles; belong to the domain Eukarya.
Domains:
Archaea, Bacteria (both prokaryotes), and Eukarya (eukaryotes).
Within Eukarya, kingdoms include Protista, Fungi, Plantae, and Animalia.
Microbial Eukaryotes: Protista, Fungi, and Animalia
Kingdom Protista: diverse group including protozoa and algae; locomotion-based classification for protozoa (cilia, pseudopodia, flagella, or non-motile); algae may be unicellular or multicellular.
Kingdom Fungi: non-photosynthetic heterotrophs; includes:
Molds: multicellular, filamentous (hyphae/mycelium), reproduce sexually via spores.
Yeasts: unicellular, round, lack hyphae, reproduce sexually via budding (asexual reproduction common).
Dimorphic fungi: can exist in both mold and yeast forms depending on environment.
Kingdom Animalia: multicellular; include parasitic worms and helminths.
The presentation includes an overview diagram of the eukaryotic kingdoms and mentions of Polyphyletic groups (e.g., Volvox as an example in algae).
Notable Concepts and Practical Implications
Hand hygiene saves lives in clinical settings; nosocomial infections are a major concern.
Use of PPE, proper waste disposal, containment, and proper cleaning routines minimize risk.
Culture media selection (general, enrichment, selective, differential) guides what organisms can be recovered and identified.
Microscopy fundamentals (resolution vs magnification) determine what can be observed; understanding oil immersion and parfocal lenses improves observation quality.
Ethical and practical implications include ensuring patient and worker safety, responsible handling of pathogens, and compliance with institutional safety policies.
Quick References and Formulas
Handwash duration: 15\text{--}20\ \text{s}
Nosocomial infections per year: 1.7\times 10^{6}
Deaths from nosocomial infections: 9.9\times 10^{4}
Alcohol-based hand sanitizer concentration: 60\%\text{--}70\%
Incubation temperatures: 25^{\circ}\mathrm{C} (environment/molds) and 37^{\circ}\mathrm{C} (human pathogens)
Maximum light microscope resolution: d_{max} = 0.2\ \mu\mathrm{m}
Total magnification examples: 15\times\times 4\times=60\times,\ 15\times\times 10\times=150\times,\ 15\times\times 40\times=600\times,\ 15\times\times 100\times=1500\times
Immersion oil index similarity to glass improves resolution at 100x
Notable Examples and Nomenclature Notes
The handwashing exercise uses Glo-Germ to visualize missed areas under UV light; fluorescence intensity categories include "+++" (heavy), "+" (light), and "0" (no fluorescence).
Aqueous and saline samples are used for environmental swabbing; plates should be inverted to prevent condensation from dripping onto media.
When discussing microbes, terms such as commensals, normal microflora, opportunists, and pathogens describe relationships to the host and disease potential.
The slides and diagrams in this deck include depictions of typical microbes (e.g., Staphylococcus spp., Malassezia spp., Propionibacterium acnes) to illustrate ubiquity and diversity, though exact species lists are not exhaustive for exam purposes.
Epilogue: Summary of Core Themes
Microbes are ubiquitous and can exist in benign, commensal, or pathogenic relationships.
Laboratory safety is foundational: BSL-1/BSL-2 practices, PPE, correct waste handling, and emergency procedures.
Hand hygiene and surface disinfection are critical to preventing disease spread.
Culture techniques (media choice, inoculation, incubation) enable recovery and study of microbes from environmental and clinical sources.
Microscopy relies on understanding magnification, resolution, and proper focusing to observe microbial cells; oil immersion enhances resolution.
Eukaryotic microbial life includes Protista, Fungi, and Animalia; prokaryotes (Archaea and Bacteria) are distinct in cell structure and organization.
Practical skills practiced in this course (Glo-Germ, sample handling, plate labeling, and safe microscopy) build a foundation for safe and effective microbiology work.